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Review Article

Molecular Imaging and Therapy of Atherosclerosis

With Targeted Nanoparticles
Samuel A. Wickline, MD,1,2* Anne M. Neubauer,2 Patrick M. Winter, PhD,1,2
Shelton D. Caruthers, PhD,2,3 and Gregory M. Lanza, MD, PhD1,2
Advances in bionanotechnology are poised to impact the
eld of cardiovascular diagnosis and therapy for decades to
come. This review seeks to illustrate selected examples of
newly developed diagnostic and therapeutic nanosystems
that have been evaluated in experimental atherosclerosis,
thrombosis, and vascular biology. We review a variety of
nanotechnologies that are capable of detecting early cardiovascular pathology, as well as associated imaging approaches and conjunctive strategies for site-targeted treatment with nanoparticle delivery systems.
Key Words: nanotechnology; contrast agents; imaging;
drug therapy; atherosclerosis
J. Magn. Reson. Imaging 2007;25:667 680.
2007 Wiley-Liss, Inc.


and/or vulnerable/unstable plaques are often diagnosed only after an acute, sometimes fatal event. Of the
700,000 cardiac deaths reported per year in America,
approximately 60% are sudden deaths that occur
without any advance warning of pathology (1). Predicting if and when a plaque might rupture and cause acute
infarction or sudden death is an uncertain business.
Atherosclerotic plaques grow in discrete stages that involve repeated episodes of rupture, hemorrhage, thrombosis, and healing, which lead inevitably to a nal rupture event and complete vascular obstruction (2).
Exposure of the lipid core, even through a small, localized rupture, can induce a clotting cascade through the
interaction of serum clotting factors with locally ex-

Department of Medicine, Washington University, St. Louis, Missouri,
Department of Biomedical Engineering, Washington University, St.
Louis, Missouri, USA.
Philips Medical Systems, Best, The Netherlands.
Contract grant sponsor: NIH; Contract grant numbers: HL-42950; HL59865; EB-01704; HL-073646; NO1-CO-07121; U54-CA-119342; Contract grant sponsors: Philips Medical Systems; Olin Foundation; Edith
and Alan Wolf Charitable Trust.
*Address reprint requests to: S.A.W., Washington University School of
Medicine, Campus Box 8086, 660 South Euclid Ave., St. Louis, MO
63110. E-mail: saw@wuphys.wustl.edu
Received December 23, 2005; Accepted October 5, 2006.
DOI 10.1002/jmri.20866
Published online 8 March 2007 in Wiley InterScience (www.interscience.

2007 Wiley-Liss, Inc.

pressed tissue factor (TF) (3). The brin matrix and

hemorrhagic components are incorporated into the
plaque mass and extend the dimensions of the lesion,
which means that vulnerable plaques will likely rupture
(i.e., be unstable or disrupted) during their life cycle (4).
The accumulation of macrophages and other inammatory cells that secrete high levels of metalloproteinases
(MMPs) also undermines the brous cap, potentially
exposing the thrombotic lipid core (5,6). Up-regulation
of angiogenesis can lead to erosion of the extracellular
matrix and replacement with physically fragile neovascular beds, weakening the brous cap and promoting
plaque rupture (7,8). These and other cellular processes are suitable targets for molecular imaging and
targeted drug therapy.
Recent developments in the elds of cellular and molecular imaging promise to allow noninvasive detection
of the molecular components of pathologic processes,
such as image-based identication of specic molecules associated with inammation or angiogenesis.
Molecular and cellular imaging techniques are available for most imaging modalities, including nuclear
(9,10), optical (10,11), ultrasound (US) (12,13), and
magnetic resonance imaging (MRI) (14,15). These methods are nondestructive in vivo analogs of traditional
immunocytochemical techniques (16). We review a selection of the advanced imaging methods and new targeted nanoparticle contrast agents for early characterization of atherosclerosis and cardiovascular pathology
at the cellular and molecular level that may represent
the next frontier for combining imaging and rational
drug delivery (14).
Investigators in the eld of nanotechnology seek to
develop and combine new materials by precisely engineering atoms and molecules to yield new molecular
assemblies on the scale of individual cells, organelles,
or even smaller components. These components have
been practically classied as ranging from 1 to 100 nm,
although somewhat larger agents are often included
under the rubric of nanoparticle because of their potential utility and in accordance with the traditional
scientic denition of the nanoscale regime (14,15,17).
The specic organization of such nanoscale materials is
anticipated to confer unique chemical and biological
properties based on interactions that occur at their
surfaces. These materials may mimic or substitute for



many existing features of cell behavior that already

operate at the nanoscale level. The synthesis of such
materials may occur from a top-down approach entailing the miniaturization of existing microscopic materials, or more likely from a bottom-up approach
involving self assembly of molecules into reproducible
and well-dened nanoscale constructs (18). To achieve
clinically effective cellular and molecular imaging, targeted nanoscale contrast agents must be designed to
accomplish a long circulating half-life, sensitive and
selective binding to the epitope of interest, prominent
contrast-to-noise ratio (CNR) enhancement, acceptable
toxicity, ease of clinical use, and applicability with standard commercially available imaging systems. The use
of nanoparticles or targeted nanoemulsions as carriers
is advantageous because large payloads of imaging or
therapeutic agents can be selectively delivered to the
tissue site.
The array of nanomaterials that can be used as contrast
agents for molecular imaging and drug delivery is
broad, and the following description is intended to be
illustrative rather than comprehensive. Size considerations dictate both the mechanism and rate of clearance, as well as access to molecular targets. For example, for agents larger than 50 nm, intravascular targets
appear to be the most appropriate, whereas smaller
particles may penetrate the intact or leaky vascular
endothelium by endothelial permeability and retention
(EPR) mechanisms, and thus directly target tissues.
Functionalization (or preparation of the particle surface for binding targeting ligands or drug delivery ligands) can be achieved by various chemical means
(e.g., provision of reactive moieties on the surface) and
through avidin biotin or electrostatic interactions (e.g.,
DNA binding to cationic lipids in particle membranes).
To improve particle stability and permit adequate circulation times, some types of particles (such as liposomes and or polymers) may require surface-component cross-linking to enhance structural integrity,
and/or incorporation of polyethylene glycol to avoid
immediate sequestration by the reticuloendothelial system.
One can achieve multifunctional activity by incorporating combinations of one or more targeting ligands,
imaging agents, and/or drugs into the formulation simultaneously. Materials can be covalently or noncovalently linked to the particle surface, dissolved in the
coating (e.g., lipophilic drugs deposited in lipid membrane layers), or carried in the particle interiors for
cellular deposition and activation. For most types of
particles, the reticuloendothelial system is the predominant clearance pathway. Toxicity is inuenced not only
by chemical composition and dose of the agent, but also
by additional factors such as its size and shape, and the
route of administration.
Targeting Ligands and Specicity
A variety of different types of targeting ligands can be
utilized, including antibodies or antibody fragments,

Wickline et al.

small peptides, peptidomimetics, polysaccharides, and

aptamers. To complex these elements with particles,
one can functionalize the particle surfaces by including
standard linking groups. Common methods for associating targeting ligands with particles include 1) noncovalent avidin biotin interactions, 2) covalent complexation via reactive groups, or 3) nonspecic surface
adsorption. Avidin biotin interactions are extremely
useful, high-afnity, noncovalent targeting systems
that have been incorporated into many biological and
analytical systems and selected in vivo applications.
Additionally, avidin, which has four independent biotin-binding sites, provides signal amplication and
improves detection sensitivity. One can employ avidin
biotin interactions to create a one-step system by performing the avidin biotin conjugations in vitro prior to
injection. Investigators have used this approach to successfully target vascular epitopes in vivo (19,20).
For in vivo use, targeting ligands are preferably attached chemically to the contrast agent by a variety of
methods depending on the nature of the particle surface (21). Conjugations may be performed before or
after the particle is created, depending on the ligand
employed and its tolerance to the chemical processing
conditions required. Direct chemical conjugation of ligands to proteinaceous agents often takes advantage of
numerous amino groups (e.g., lysine) that are inherently present within the surface. Alternatively, functionally active chemical groups, such as pyridyldithiopropionate, maleimide, amino, and aldehyde, may be
incorporated into the surface as chemical hooks for
ligand conjugation after the particles are formed. Another common postprocessing approach is to activate
surface carboxylates with carbodiimide prior to addition of the ligand. The selected covalent linking strategy
is primarily determined by the chemical nature of the
Multiple copies of ligands can be incorporated depending on the size of the particle, which serves to
enhance avidity and target detectability by reducing the
particle dissociation rate and securing the agent at the
intended site. Specicity is conferred by the targeting
ligand itself and generally should be in the nanomolar
range, although high-avidity agents may in part overcome this limitation by multivalent interactions. To ensure high ligand binding integrity and maximize targeted particle avidity, exible polymer spacer arms
(e.g., polyethylene glycol or simple caproate bridges)
can be inserted between an activated surface functional
group and the targeting ligand. These extensions can be
10 nm or longer, and minimize interference of ligand
binding by particle surface interactions. Regardless of
the targeting, it is clear that most such agents will
exhibit a modicum of nonspecic targeting related either to nonspecic ligand attachment, the EPR effect, or
sequestration in immature vasculature (angiogenesis).
Liposomes (50 700 nm, uni-or multilammelar vesicles
that consist of lipid bilayer membranes surrounding an
aqueous interior) have been approved to enhance the
efcacy and safety of various drugs, such as doxorubi-

Molecular Imaging With Nanoparticles

cin (e.g., Doxil; ALZA Corp., Tibotec Therapeutics, NJ,

USA). Applications of liposomal technology as molecular imaging agents for both US and MRI have been
reported (20,22).
Emulsions, which are chemically distinct from liposomes, are oil-in-water-type mixtures that are stabilized with surfactants to maintain their size and shape.
Peruorocarbon core emulsions (200 400 nm) have
been used for molecular imaging with MRI, US, uorescence, nuclear, and computed tomography imaging
(CTI) (1315,23). For example, when vast numbers of
paramagnetic gadolinium (Gd) complexes (50,000)
are incorporated onto emulsion particles, the possible
signal enhancement for each binding site is magnied
dramatically, by a factor of 106 over conventional
paramagnetic extracellular contrast agents (24,25).
Modied micellar particles, such as high-density lipoprotein (HDL) or low-density lipoprotein (LDL) particles, have been utilized as molecular imaging agents for
MRI (26,27).
Polymers (40 200 nm) are used in a wide variety of
exible designer approaches to construct molecular
imaging agents and therapeutic delivery devices (18).
They may be linear, branched, or globular, and comprise single or multiple molecular components (copolymers). Their size and shape can be tightly controlled,
and functionalization of their surface permits binding
of myriad targeting and therapeutic moieties for imaging, as well as drug and gene delivery. Polymers made
from polyhydroxy acids, such as the copolymer of poly(lactic acid) (PLA) and poly(D,L-lactide-co-glycolide)
(PLGA), have been investigated for localized drug and
gene delivery. Dendrimers, or cascade polymers, are
highly branched polymeric structures that are globular
in conguration. Their cores are varied, and the
branches are sequentially assembled in covalent interactions that produce layers referred to as generations.
Paramagnetic polyamidoamine (PAMAM) and diaminobutane (DAB) dendrimers have been employed for
MRI applications (28,29). The multivalent surface comprises a number of functional sites that can undergo
reactions to add drugs, imaging agents, and targeting
Metallic particles, such as iron oxide nanoparticles
(15 60 nm), generally comprise a class of superparamagnetic agents that can be coated with dextran, phospholipids, or other compounds to inhibit aggregation
and enhance stability for use as passive or active targeting agents. The iron in monocrystalline iron oxide
nanoparticles (MIONs), small particles of iron oxide
(SPIO, 50 500 nm), and ultrasmall particles of iron
oxide (USPIO, 10 50 nm) produces strong local disruptions in the magnetic eld of MRI scanners, which leads
to increased T2* relaxation and hence a decrease in
image intensity in areas with iron particle accumulation (susceptibility effects). These particles exhibit a
very long circulating half-life (24 hours) and may be
sequestered by tissue macrophages. These properties
have allowed dextran-coated USPIO nanoparticles to be
employed for passive targeted imaging of pathological
inammatory processes, such as unstable atherosclerotic plaques, by MRI (30). Alternatively, other similar
types of particles (e.g., cross-linked iron oxide (CLIO)


particles complexed with retroviral tat protein ligands) have been used for localization and transcellular
deposition (31).
Other metal-based agents, such as gold shell nanoparticles (120 nm), have been used for both imaging
and therapy (3234). Carbon nanotubes and fullerenes
(4 nm) have been utilized as particulate systems whose
surfaces also can be functionalized for tissue binding
(35,36). Native uorescent properties have been reported (37,38). Quantum dots (2 8 nm) are constructed
from semiconductor materials (e.g., cadmium selenide)
that manifest stable (nonquenching) uorescent properties at various wavelengths depending on the exact
composition of the materials (39 42). These properties
allow registration of multiple simultaneous signals
from distinct particles that bear unique spectra. For
use in vivo, they must be coated with materials (polymers) that both allow solubilization and prevent leaching of the toxic heavy metals.
Synthetic viral capsid structures (e.g., cowpea virus)
that self-assemble as protein cage particles were originally developed by Allen et al (43). These types of nanoparticles can be manipulated under certain chemical
conditions to create pores that permit encapsulation of
imaging agents or drugs. Approximately 180 binding
sites are available for Gd, as compared to 50,000 or
more for other active particle systems in which payloads are critical for performance (44). Calcium and Gd
compete for the same sites that exhibit approximately
micromolar dissociation constants, which raises the
issue of undesirable transmetallation potential and free
Gd release.
Smart Imaging Agents
So-called smart contrast agents represent an effort to
develop selectively excitable agents that only show signal upon some specic form of activation, such that
background signal does not interfere with detection of
the molecular target. The problem is exemplied by the
use of iron oxide particulates with a serum half-life of
many hours to days. This creates negative contrast in
the blood pool that can interfere with detection of vascular wall targets, such as macrophages that ingest the
particles. In this case, imaging generally must be performed more than 24 hours after contrast injection to
allow blood-pool clearance in order to avoid confounding susceptibility artifacts produced by the circulating
Examples of the various smart agents available include agents that 1) are selectively activated by targeted
enzymic processes, 2) become apparent only through
selective spectral activation, 3) exhibit little appreciable
signal until they accumulate in high concentrations at
specic targets of interest, and 4) exhibit multifunctionality to enhance the specicity of diagnosis. The rst
type was initially proposed by Louie et al (45) and involves uncaging a sequestered Gd ion by the action of
-galactosidase action on a galactopyranose capping
molecule, which then permits free water access to the
lanthanide, a tripling of proton relaxation, and detection of the presence of a transfected lacZ gene that
produces the enzyme. The second type includes chem-


Wickline et al.

Table 1
Examples of Nanosystems Reported for Cardiovascular Imaging and/or Therapy
Nanoparticle class and

Imaging modality



SPIO (50-500 nm)

USPIO (5-50 nm)


CLIO (40 nm)



PFC emulsions (200300 nm)

PM, uorine

Liposomes (50-300 nm)


Micelles (50-150 nm)





Cardiovascular target


Stem cell labeling

Macrophages, ischemic
Macrophages, VCAM,
selectins, stem cell
labeling, apoptosis
Plaque angiogenesis,
tissue factor,
Integrins, brin
thrombi, matrix
collagen, stem cell
Angiogenesis, adhesion


Plaque targeting


A, P

A, P

Doxorubicin, paclitaxel

A, P

SPIO superparamagnetic iron oxide particles, SP superparamagnetic, USPIO ultrasmall superparamagnetic iron oxides, PM
paramagnetic, CLIO cross-linked iron oxides, NIR near infrared uorescent, PFC peruorocarbon, F uorescent, HDL high
density lipoproteins, Nuc nuclear imaging, A active targeting, LDL low density lipoproteins, P passive targeting.

ical exchange saturation transfer (CEST) agents. With

these agents, saturation of exchangeable protons using
alternative lanthanide chelates, such as europium, allow bulk water signal suppression after selective excitation at proton resonances that are removed from the
bulk water frequency (46). Another example is uorine
imaging, which allows selective spectral excitation of
various uorine moieties incorporated into nanoparticles (47). The third type is represented by agents that
have weak signals in circulating blood but enhance
upon accumulation (e.g., multivalent targeting of paramagnetic ions, and polymerization of iron oxide compounds) at a selected site (15,48,49). The fourth type is
typied by agents that can simultaneously produce signals that are detectable by more than one imaging modality (e.g., uorescence and superparamagnetic effects
(50) or selected combinations of nuclear, US, and paramagnetic effects (44)).
Nuclear/PET Imaging
The role of nanoparticles in imaging cardiovascular pathology is diverse and varies among the available imaging modalities (Table 1). In the case of nuclear (gamma/
SPECT) imaging or positron emission tomography
(PET), for example, the general approach has been to
utilize very small tracer quantities of contrast agents
(e.g., radionuclide-labeled antibodies, peptides, or
small molecules) rather than large payload particles.
However, the high sensitivity, unique spectral signatures of the tracer elements, and potential for local
quantication of these tracers based on the emission
count rate enhance their role in early detection and
serial evaluation of pathology. For example, folate receptor-targeted polymeric shell cross-linked nanoparticles containing 64Cu were recently used for PET imaging of tumors (51). More typical approaches for

characterizing atherosclerosis include imaging of apoptosis by annexin-phosphatidyl serine targeting (52),

unstable carotid plaque imaging with metabolic (FDG)
readouts (53), and macrophage chemotaxis imaging (9).
Optical Imaging
Optical approaches are promising, especially for more
localized detection of pathologies. Quantum dots that
home to vascular endothelial targets have been reported to be useful for identifying selected tissue zipcodes (39,40). Gold nanoshells directly injected into
tumors have been used to evaluate and treat tumors
with application of exogenous thermal energy (3234).
Carbon nanotubes also emit detectable uorescence
that varies depending on the composition of the local
environment (3538). In some cases, multifunctionality
has been designed into nanoparticles for combined imaging (54). Tissue autouorescence can obscure diagnosis at some wavelengths; however, near-infrared
wavelengths appear to be useful for enhanced sensitivity and specicity in this regard (55,56). The lack of
larger-scale noninvasive imaging systems for patients
is a drawback.
Given the large installed base of imaging equipment
worldwide and the ease of use of CTI and US, the incorporation of nanoparticle-based imaging agents in these
techniques hypothetically shows great potential. A major challenge for CT researchers is to produce robust
and potent contrast agents. Because X-ray absorption
depends directly on the potency of the material used as
contrast agent (the Z value) and exponentially on the
thickness of the layer of material deposited, sensitivity
is expected to be only modest for nanoparticles that
accumulate in small concentrations when used as molecular imaging tools. Nevertheless, lipid emulsion

Molecular Imaging With Nanoparticles


Figure 1. US molecular imaging with nanoparticles. a: Fibrin-targeted nanoparticles enhance contrast in thrombi formed in the
carotid arteries of pigs with the use of clinical 7.5-MHz linear-array transducers. Top: Carotid artery lumen with an echogenic
anode (arrowhead) to induce brin-platelet thrombus, which remains invisible at 7.5 MHz. Bottom: After brin-targeted
nanoparticles bind to the thrombus, backscatter is augmented (brighter) throughout and along the extent of the clot (multiple
arrows) (reprinted from Lanza GM, Wallace KD, Scott MJ, et al. A novel site-targeted ultrasonic contrast agent with broad
biomedical application. Circulation 1996;95:3334 3340, with permission). b: TF imaging after balloon injury to a porcine carotid
artery. Top: Scanning electron micrograph of TF expression in vitro on smooth muscle cells targeted with nanoparticles
containing mAb ligands to TF. Bottom: 30-MHz intravascular ultrasound imaging of TF induced in medial smooth muscle cells
by balloon stretch injury. Note the contrast enhancement (brighter) heterogeneously distributed throughout the media of the
vessel representing binding of TF-targeted nanoparticles (see arrows in targeted site) (reprinted from Lanza GM, Abendschein
DR, Hall CS, et al. In vivo molecular imaging of stretch-induced tissue factor in carotid arteries with ligand-targeted nanoparticles. J Am Soc Echocardiography 2000;13:608 614, with permission). c: Fibrin-targeted echogenic liposomes binding to and
enhancing contrast (brighter) from LV thrombi in four-chamber (top) and parasternal (bottom) views (left: preinjection; right:
postinjection) (reprinted from Hamilton A, Huang SL, Warnick D, et al. Left ventricular thrombus enhancement after intravenous
injection of echogenic immunoliposomes: studies in a new experimental model. Circulation 2002;105:27722778, with permission).

nanoparticles that contain radio-opaque iodinated triglycerides have been described for passive hepatic targeting (57), and work in this eld is ongoing (58). An
unavoidable drawback for serial use remains the considerable radiation dose required.
US Imaging
US offers many advantages, such as benign imaging
energy (i.e., compressional waves), exibility, high
throughput, low cost, and excellent patient tolerance.
However, it is more highly operator-dependent than
other tomographic methods and cannot image all areas
of the body. Available US contrast agents include sizable shell-stabilized gas-lled microbubbles, and many
have been developed that can be targeted to vascular
epitopes (57). In the regime of smaller particles, acoustically active emulsion nanoparticles for both imaging
and therapy, and reective liposomes for imaging
(12,59,60) have received the most attention (Fig. 1).
Although US is exquisitely sensitive for detecting microbubbles, it is less so for nanoparticles because of the
size dependency (r6) and relative incompressibility of
liquid particles, which preclude the use of available
harmonic resonance-based imaging techniques that
are typically applied to microbubble detection. A balancing consideration is the unique potential for precisely depositing large amounts of highly focused energy in a convenient manner that can facilitate
nanoparticle-based imaging and therapeutics with exogenous US activation, as was recently described by
several groups (61 63). The advent of mathematical
models to characterize the fundamental scattering behavior from newer classes of nanoparticles (e.g., emul-

sions) raises the potential for extracting more quantitative information from the reected signals (64,65).
MRI offers several advantages over other modalities,
including high resolution, high anatomical contrast,
high signal-to-noise ratios (SNRs), widespread clinical
availability, and lack of ionizing radiation (15,66). However, the comparatively modest MR contrast enhancement achievable with targeted contrast agents for molecular imaging necessitates the delivery of higher
payloads of contrast materials, which can be provided
by novel nanotechnologies. Because molecular epitopes
of interest may reside on or inside cells in very sparse
quantities at low nanomolar or picomolar concentrations, one can considerably amplify the local contrast
effect by incorporating large amounts of paramagnetic
or superparamagnetic materials as the payload. For
paramagnetic agents (e.g., in T1-weighted imaging), the
simple attachment of a few Gd atoms to an antibody for
use as a targeting ligand may not provide enough signal
if micromolar concentrations of the lanthanide are required to elicit contrast enhancement based on conventional T1 relaxation mechanisms (see below) (67). In the
case of T1-weighted imaging, the surface of the particle
can be decorated with numerous copies of Gd chelates
(up to 100,000) to achieve the micromolar concentrations required per voxel (Fig. 2). Imaging with superparamagnetic agents takes advantage of the fact that
enough material can be packed into the core of the
nanoparticle to exert a prominent T2* effect and produce a localized signal reduction that can be detected
with potentially greater sensitivity than is possible with


Wickline et al.

Figure 2. MR image of thrombi with paramagnetic nanoparticles targeted to brin. a: Thrombus formed in vivo in a canine
jugular vein imaged at 1.5T. Note the hot spot at the site of nanoparticle binding. b: Disrupted carotid endarterectomy
specimens incubated with brin-targeted nanoparticles binding to small amounts of brin at the shoulder regions (note the hot
spots indicated by yellow arrows) of ruptured plaque cap imaged ex vivo at 1.5T (reprinted from Flacke S, Fischer S, Scott MJ,
et al. Novel MRI contrast agent for molecular imaging of brin: implications for detecting vulnerable plaques. Circulation
2001;104:1280 1285, with permission).

paramagnetic agents. Recently, quantitative approaches have been described for molecularly targeted
paramagnetic emulsions that allow the concentration
of bound nanoparticles to be computed under certain
circumstances (68).
Recent advances in imaging techniques have enabled
hot-spot detection of iron oxide-based particles. Exploiting the same inherent dipole of magnetic particles
that causes signal dropout on typical MRI, Cunningham et al (69) and Stuber et al (70) developed techniques for off-resonance imaging that can instead
produce bright signals in regions surrounding the accumulation of particles. These techniques require specialized excitation pulses that image the water molecules in close proximity to an accumulation of particles.
Once optimized, these techniques offer potential for localizing sources of extraneous magnetic dipoles (i.e.,
superparamagnetic particles) and, via their signal in-

tensity, tracking their size and distribution and providing a method for relative quantication.
Alternatively, the signal generated by the uorine atoms in the peruorocarbon core of peruorocarbonbased nanoparticles has been introduced as a unique
signature for molecular MRI (16,71). Because biological
tissues contain little endogenous uorine, measurement of the uorine component of targeted particles
may allow denitive conrmation of nanoparticle deposition at the site. This recent approach has been demonstrated for imaging and spectroscopy of brin at
4.7T, and for quantifying the concentration of nanoparticle binding to a selected site based on localized uorine spectroscopy (16) and subsequently for stem cell
imaging (see below) in vivo (72).
Another use for uorine imaging and spectroscopy of
peruorocarbon particles is to identify different targeted moieties on the same sample. Due to differences

Figure 3. VCAM-1 imaging in apoE / mice with peptide-targeted CLIO nanoparticles (reprinted from Kelly KA, Allport JR,
Tsourkas A, Shinde-Patil VR, Josephson L, Weissleder R. Detection of vascular adhesion molecule-1 expression using a novel
multimodal nanoparticle. Circ Res 2005;96:327336, with permission). a: Twenty-four hours after injection, MRI signal loss
occurs where the nanoparticles localize to aortic plaque in vivo (arrows). Note the loss of signal resulting from the susceptibility
effect of accumulated iron oxide particles. (b) Before and (c) 24 hours after injection of targeted nanoparticles showing aortic
cross sections with signal loss (darker) at the plaque cap. d: Ex vivo MR image of signal loss (darker) in the aorta after
nanoparticle binding. e: Matched epiuorescent image of dual-function particles containing uorescent probe.

Molecular Imaging With Nanoparticles

in their local nuclear environments (e.g., electron

shielding, J-coupling, etc.), different uorine atoms resonate at slightly different frequencies and thus are often readily distinguishable on an NMR spectrum (73).
This means that by using peruorocarbon nanoparticles formulated with different peruorocarbon species, one can target them to the same sample and quantify their presence separately with one spectroscopic
scan (16). While spectroscopy is ultimately the most
useful for quantication, other techniques allow imaging of the different peruorocarbon particles as well.
Such techniques include frequency-selective excitation
so that only the peruorocarbon species of interest produces a signal, and other forms of chemical shift imaging (CSI) that have been developed to differentiate fat
from water in clinical imaging (74,75).
The sensitivity of MRI for detecting paramagnetic or
superparamagnetic nanoparticulate imaging agents
depends on the specic eld strength, pulse sequence,
coil sensitivities, epitope prevalence, and contrast
agent concentration used. In general, the MR signal
strength is modest compared to nuclear imaging applications. For example, at clinical imaging eld
strengths, micromolar concentrations of paramagnetic
agents (e.g., Gd and CEST agents) are required. For
superparamagnetic agents that elicit susceptibility artifacts, nanomolar concentrations may sufce. CNRs of
5 or better generally produce readily identiable (diagnostic) qualitative signal enhancement. Examples of
optimizing pulse sequences and paramagnetic and uorinated nanoparticle concentrations to enhance sensitivity have been presented by Morawski et al (16,68)
with MR signal modeling approaches.

Atherosclerotic Plaques
Rupturing atherosclerotic plaques are frequently manifested at various stages in arteries with only modest
(40 60%) stenosis (76), and they remain diagnostically
elusive with routine clinical imaging techniques. Serum
biomarkers may offer information about the general
state of the vasculature, but provide no useful information about the propensity for any given lesion to rupture. Accordingly, one major motivation for molecular
imaging is the recognition and localization of telltale
molecular elements of unstable or disrupted plaques
that might provide a window of opportunity extending
from days to weeks or months to intervene before more
serious clinical sequelae ensue (2). A sine qua non of
disrupted plaque is brin deposition. Not only is brin
deposition one of the earliest signs of plaque rupture or
erosion, it also accounts for (along with intraplaque
hemorrhage) a considerable part of the core of growing
lesions (77). The ability to diagnose disrupted plaque by
detecting small deposits of brin in erosions or microfractures could allow characterization of a potential
culprit lesion before a high-grade stenosis has been
formed that is detectable by cardiac catheterization.
The possibility of performing nanoparticle targeted
brin imaging with US or paramagnetic MR contrast
agents was rst demonstrated by Lanza and Wickline


(12) and Lanza et al (78) as early as 1996. In this case,

the ligand comprises an antibody fragment that is
highly specic for certain cross-linked brin peptide
domains, which can be complexed to the particle either
through avidin biotin linkages, or covalently to the
functionalized nanoparticle, as has been shown for TF
targeting (71,79). For US imaging, thrombi formed in
situ in canine carotid arteries were detectable within 30
minutes with commercially available 7.5 MHz lineararray imaging transducers (Fig. 1) (78).
TF is a prothrombotic transmembrane glycoprotein
that is expressed within plaques, is upregulated following vascular injury or stent placement, and contributes
as a mitogen to restenosis (80). TF in the core of plaques
is exposed during plaque rupture and is the proximate
cause of local thrombosis that leads to vessel occlusion
or distal embolization. TF imaging has been demonstrated in vivo for molecular imaging with US (Fig. 1)
and in vitro with MRI (12,68). These observations illustrate the potential use of the rst reported molecular
imaging agent for US to delineate molecules that are
involved in plaque instability and restenosis. In fact,
the ability to image TF-targeted paramagnetic nanoparticles bound to smooth muscle cell monolayers in cell
culture at 1.5T attests to the potency of nanoparticles
agents that carry 50,000 or more Gd chelates.
Echogenic liposomes (ELIPS), in contrast to nanoparticles or emulsions, are composed of alternating layers
of aqueous uid and lipid bilayers that are formulated
to produce a US signal. Hamilton et al (59,81) used
these liposomes to target thrombi (Fig. 1) and various
vascular signatures associated with atheroma development in injured vessels of miniswine for intravascular
US imaging (22). By targeting intercellular adhesion
molecule-1 (ICAM-1), vascular cell adhesion molecule-1
(VCAM-1), brin, brinogen, and TF, they were able to
produce targeted enhancement in the vessel walls ve
minutes after intravenous administration of the liposomes.
In MRI studies, peruorocarbon particles loaded with
50 90,000 Gd atoms per particle yielded a substantial
amplication of signal from brin clots at 1.5T both in
vitro and in vivo (15,64). Furthermore, the detection of
disrupted plaque was illustrated in actual human carotid endarterectomy specimens obtained from patients
symptomatic with transient ischemic attacks, stroke,
or bruits (Fig. 2) (24). These data provided early evidence that disrupted dangerous atherosclerotic
plaques can be detected noninvasively with MRI. MRI of
VCAM-1 was recently performed with the use of peptide-targeted superparamagnetic nanoparticles in the
aortas of cholesterol-fed ApoE null mice by Kelly et al
(54) (Fig. 3), indicating that early molecular mechanisms that are important in the evolution of atherosclerosis can be dened noninvasively with MRI.
EPIX Pharmaceuticals (Cambridge, MA, USA) utilized
phage display methods to produce a peptide ligand specic for brin (EP-2104R), which may be useful for
imaging thrombi in various body locations such as the
left atrium, pulmonary arteries, and coronary arteries
in experimental preparations (82 84). It contains four
Gd-DTPA chelates per peptide moiety and thus provides signal enhancement on an MR image. Despite the


Wickline et al.

Figure 4. Fluorine imaging with brin-targeted peruorocarbon-based nanoparticles (reprinted from Morawski AM, Winter PM,
Yu X, et al. Quantitative magnetic resonance immunohistochemistry with ligand-targeted 19F nanoparticles. Magn Reson Med
2004;52:12551262, with permission). a: Optical image of an excised human carotid endarterectomy sample shows asymmetrical plaque distribution, with areas of intimal fat deposition (yellow). b: Matched uorine projection image at 4.7T shows
heterogeneous nanoparticle binding where brin is present (bright signal with no background) on the plaque. c: With NMR
spectroscopy the uorine image can be converted into a false color map of the nanoparticle binding, and coregistered and
overlaid on the proton image (shown in grayscale), corresponding to the intravoxel concentration of nanoparticles bound to brin
epitopes. d: Quantitative false color mapping scale illustrating quantication of signal enhancement as a nanoparticle concentration expressed in nanomolar per voxel.

low Gd load per binding site, the excess of brin

epitopes in fresh or chronic clots allows the accumulation of contrast agent concentrations that are sufcient
to achieve micromolar levels of the lanthanide, which
enables ready detection of the clot after the signal in the
blood pool is sufciently decreased (one to two hours).
Alternatively, the uorine component of peruorocarbon-based nanoparticles can be utilized to advantage
for molecular imaging. Our group originally demonstrated the concept of targeted nanoparticle uorine
imaging at 1.5T or 4.7T for detection of experimental
thrombi or small brin deposits in disrupted human
carotid arteries using particles made with peruoroctylbromide (PFOB), crown ether (CE), or other PFC core
materials (Fig. 4) (16,71). We recently demonstrated the
use of rapid steady-state free precession (SSFP) 19F
imaging of combined PFOB and CE brin-targeted
nanoparticles on brin clots and endarterectomy specimens in vitro at 1.5T (47). This approach uses spectralselective excitation to delineate various types of nanoparticles that contain different peruorocarbon cores,

and produces no appreciable confounding background

While brin and TF can be utilized to delineate unstable
cardiovascular diseases, the 3-integrin is a general
marker of angiogenesis and plays an important role in a
wide variety of disease states, including atherosclerosis
(85) and cancer. The 3-integrin is a well-characterized heterodimeric adhesion molecule that is widely expressed by endothelial cells, monocytes, broblasts,
and vascular smooth muscle cells. In particular, 3integrin plays a critical part in smooth muscle cell migration and cellular adhesion (86,87), both of which are
required for the formation of new blood vessels. The
3-integrin is expressed on the luminal surface of
activated endothelial cells, but not on mature quiescent
cells (88). We have demonstrated the utility of 3integrin targeted nanoparticles for the detection and
characterization of angiogenesis associated with

Figure 5. Detection of plaque neovascularization in cholesterol-fed rabbits (reprinted from Winter PM, Morawski AM, Caruthers
SD, et al. Molecular imaging of angiogenesis in early-stage atherosclerosis with alpha(v)beta(3)-Integrin-targeted nanoparticles.
Circulation 2003;108:2270 2274, with permission). a: Aortic cross sections imaged at 1.5T with v3-integrin targeted nanoparticles. Note the heterogeneous distribution in aortic cross sections (false colored contrast enhancement), but little enhancement in nontargeted rabbits (v3-) or rabbits on a standard diet (Chol-). b: MRI signal modeling illustrates that picomolar (100
pM) intravoxel concentrations of peruorocarbon-based paramagnetic nanoparticles are required to achieve a diagnostic CNR
ratio of 5 (blue dashed line). c: Immunochemical staining for v3-integrin at the media-adventitia border of aortic segments.
Note the abundant red-brown vascular segments indicating the presence of integrin.

Molecular Imaging With Nanoparticles


Figure 6. Macrophage imaging in cholesterol-fed rabbits with untargeted superparamagnetic nanoparticles (reprinted from
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). a: SPIO nanoparticles taken up by plaque
macrophages depict atherosclerosis in cholesterol-fed rabbits according to magnetic susceptibility effects (dark regions) that are
seen distributed along the aorta in T2-weighted images acquired more than 24 hours after injection. b: Iron stain of plaque
demonstrating uptake of particles by intimal macrophages (blue stain).

growth factor expression (19), tumor growth (89,90),

and atherosclerosis (91).
Angiogenesis plays a critical role in plaque growth
and rupture (92,93). In regions of atherosclerotic lesions, angiogenic vessels proliferate from the vasa vasorum to meet the high metabolic demands of plaque
growth (94,95). Inammatory cells within the lesion
stimulate angiogenesis through local molecular signaling, which in turn promotes neovascular growth and
provides an avenue for more inammatory cells to enter
the plaque (92).
Molecular imaging of expanded vasa vasorum in atherosclerotic lesions in cholesterol-fed rabbits was rst
demonstrated for MRI by Winter et al (91) with the use
of paramagnetic nanoparticles targeted to 3-integrin
expressing endothelial cells (Fig. 5). Animals on a control diet exhibited no increased signal, and background
was minimal. Expression of 3-integrins in the adven-

titial layer and beyond was conrmed by co-localized

histological staining of 3-integrin and PECAM, a
general endothelial marker. This work was the rst to
demonstrate the potential of MRI for the noninvasive
detection and quantication of angiogenesis in atherosclerotic plaque.
Cyrus et al (96) recently employed 3-integrin targeted and collagen-III targeted paramagnetic nanoparticles to image arteries subjected to balloon stretch injury (angioplasty). They observed a high degree of
binding associated with the exposure of native smooth
muscle cell integrins after injury, and the upregulation
of integrins as a component of the inammatory response. The molecular targeting extended far beyond
the length of the actual balloon itself, indicating the
potential for extensive yet occult injury beyond the connes of angioplasty and potential stent deployment,
which may have consequences for restenosis. Both col-

Figure 7. Stem-cell labeling and imaging with iron-oxide nanoparticles (reprinted from Kraitchman DL, Heldman AW, Atalar E,
et al. In vivo magnetic resonance imaging of mesenchymal stem cells in myocardial infarction. Circulation 2003;107:2290 2293,
with permission). Left: Stem cells incubated with nontargeted iron oxide nanoparticles that undergo endocytosis are injected
near the apex in a porcine heart and imaged at 1.5T. Note the dark spot due to the susceptibility effect (arrow). Right: Iron stain
(blue) of heart tissue illustrating stem cells containing abundant nanoparticles in the cytoplasmic compartment.


lagen-III and 3-integrins were detectable specically,

although the MRI integrin signal exceeded that of the
collagen signal.
Other Plaque Components
Macrophage imaging was rst performed with nontargeted USPIO by Schmitz et al (30) in Watanabe rabbits,
and by Rheum et al (97) in cholesterol-fed atherosclerotic rabbits (Fig. 6). These are among the rst illustrations of passive targeting of important early cell types
involved in atherosclerosis. Because macrophages are
abundant in plaques throughout the vascular tree, and
they are known to ingest particulate matter, the use of
superparamagnetic agents to delineate macrophages
and foam cells has been pursued in both animal models
and clinical trials (98). The demonstration of macrophage targeting in vivo in rabbits required a waiting
period of one to three days to allow for both passive
uptake of sufcient numbers of particles and bloodstream clearance of the long-circulating particles. In
general, the susceptibility artifacts produced extended
beyond the connes of the plaque macrophages and
appeared as heterogeneously distributed signal voids
up and down the aorta.
In similar clinical trials conducted by Kooi et al (99)
and Trivedi et al (100) with patients undergoing carotid
endarterectomy, USPIO particles accumulated in the
macrophages in plaques and were optimally imaged as
signal reductions 24 hours after injection. Kooi et al
(99) also noted that more contrast change was observed
for ruptured than for stable plaques. USPIO-labeled
macrophages have been imaged and localized to unstable and ruptured plaques (75% demonstrating uptake),
but not in stable lesions (only 7% showing USPIO uptake).
Frias et al (26) recently reported the development of
recombinant paramagnetic HDL-like particles that can
enhance atherosclerotic regions in apoE-decient mice.
These particles are formed through the delipidation of
normal isolated human HDL particles, followed by reconstitution with phospholipids and the addition of a
phospholipid-based conjugate of Gd-DTPA (1520 molecules of Gd included in each 9-nm particle) for signal
enhancement. Nonselective accumulation in atherosclerosis has been demonstrated.
This group also demonstrated the use of conventional
nontargeted agents, such as gadouorine, that appear
to preferentially label the fatty cores of plaques (101).
Gadouorine is a lipophilic chelate of Gd (Gd-DO3A
derivative) with a uorinated side-chain that forms
5-nm-sized micelles in aqueous solution. The small size
and lipophilic nature of this contrast agent allows it to
accumulate in lipid-rich areas of plaque in cholesterolfed rabbits.
The Weissleder group (102) has developed other MRI
susceptibility agents to image plaque components. Magnetic switches (iron oxide particles) that contain numerous copies of high-afnity ligands have been used to detect myeloperoxidase activity in plaques. This enzyme has
been implicated as a product of inammatory cells that
may incite plaque instability and lead to myocardial infarction. Components of thrombi important for stabilizing

Wickline et al.

clots (Factor 13) have been detected with targeted iron

oxide probes consisting of a dextran-coated caged iron
oxide particle (CLIO) conjugated to a2AP peptide fragments that are recognized by activated Factor 13 (103). In
vitro experiments with human plasma thrombi incubated
with F13-CLIO revealed thrombus contrast enhancement
as compared with control probes.
Stem-Cell Imaging Methods Relevant to
Stem-cell imaging with MRI is another emerging area
that might t under the rubric of molecular imaging
with targeted nanoparticle contrast agents. Stem cells
loaded with superparamagnetic nanoparticles in vitro
can be engrafted into the selected location by local injection. The stem cells are induced in vitro to ingest
nanoparticles through endocytosis by various strategies, such as coating the particles with dendrimers,
transfection agents, or antibodies/peptides (104 106).
This results in the intracellular accumulation of significant amounts of intact nanoparticles, which can then
exert a local susceptibility effect for detection in vivo.
Original work by Frank et al (105), Bulte and Kraitchman (107), and others has demonstrated the use of MRI
methods for stem-cell tracking based on detection of
susceptibility artifacts created by the superparamagnetic nanoparticles. Using this approach, Kraitchman
et al (108) were able to detect and track mesenchymal
stem cells injected into necrotic regions of a pig heart at
1.5T (Fig. 7). This group more recently used a combination of SPECT/CT to track stem-cell migration in the
heart after local injections (109). Additional applications likely include detecting and tracking stem-cell
activity in vascular inammation. These approaches
offer sensitive and robust detection of important cellular vehicles for cardiovascular tissue regeneration.
Ahrens et al (72) recently followed the uorine molecular imaging approaches demonstrated originally by Yu
et al (25) and Morawski et al (16) by employing a crown
ether preparation of nanoparticles to load dendritic immune cells with no loss of viability, and demonstrated
an extension of the use of uorine imaging at research
eld strengths (11.7T) to track cells after local and systemic injections (Fig. 8). Again, the advantage to this
approach is that no background signal exists because
there is no appreciable amount of uorine in the body to
confound the signal from the targeted cells. Recent
methods for labeling of proangiogenic endothelial precursor cells with multiple types of peruorocarbon
nanoparticles and rapid imaging at clinical (1.5T) and
research (11.7T) eld strengths have been reported
The potential dual use of nanoparticles for both imaging and targeted delivery of therapeutic agents to sites
of cardiovascular disease offers great promise for individualizing therapeutics. Image-based therapeutics
with site-selective agents should enable conclusive assurance that the drug is reaching the intended target
and a molecular effect is occurring. Traditional phar-

Molecular Imaging With Nanoparticles


Figure 8. In vivo MR image of peruorocarbon-labeled dendritic cells in a mouse (reprinted from Ahrens ET, Flores R, Xu H,
Morel PA. In vivo imaging platform for tracking immunotherapeutic cells. Nat Biotechnol 2005;23:983987, with permission). a:
Electron micrograph of dendritic cells labeled with PFC nanoparticles. Note the clear spherical nanoparticles scattered throughout the cytoplasm. b: The 19F intensity is displayed on a hot-iron intensity scale, and the 1H images are shown in grayscale.
c: Mouse quadriceps after intramuscular injection of peruorocarbon-labeled dendritic cells (asterisk indicates injection site).
Shown (from left to right) are 19F and 1H images and a composite 19F/1H image.

macokinetic and pharmacodynamic analyses used to

predict drug efcacy and toxicity are rooted in monitoring serum concentrations as inputs to linked differential equations to describe the transport of drug from one
compartment (e.g., serum) to another (i.e., the extracellular space where the drug binds to its target). In the
case of particulate agents, however, the mechanisms of

drug delivery become more complicated and hence serum concentrations are not necessarily indicative of the
amount of drug that is accessible to the desired site
(112). Furthermore, when the carrier is targeted to the
tissue of interest, the drug release is also localized to
that area, resulting in a much higher effective drug
concentration (or area under the curve (AUC)) at the site

Figure 9. Augmenting therapeutics with US applied to bound targeted nanoparticles (reprinted from Crowder KC, Hughes MS,
Marsh JN, et al. Sonic activation of molecularly-targeted nanoparticles accelerates transmembrane lipid delivery to cancer cells
through contact-mediated mechanisms: implications for enhanced local drug delivery. Ultrasound Med Biol 2005;31:1693
1700, with permission). a: Peruorocarbon-based nanoparticles can be loaded with lipophilic drug in the outer lipid monolayer
and delivered to the cell by lipid mixing and/or lipid vesicle-cell membrane fusion. As an example, rhodamine-labeled lipids (red
stream from the nanoparticle) rapidly mix into the cell membrane (see arrow in inset) upon interaction with C32 melanoma cells
(cells are transfected with GFP to label endosomes), and then rapidly distribute into the cytoplasm without requiring endocytosis
of the intact particles. b: US potentiation of lipid uptake by cells in culture. FITC-labeled nanoparticles targeted to C32 cells
demonstrate lipid mixing and fusion of particles and cells, and cytoplasmic delivery (note the green lipid-conjugated uorophores
distributed in the cytoplasm). c: After a ve-minute insonication of cells in culture with a 2.5-MHz clinical phased-array
transducer at medium power, a marked increase in cytoplasmic lipid delivery is achieved with no untoward effects on the
viability of targeted cells.


than is indicated by serum levels alone. The unique

ability to image these particles could be of great benet
for estimating local drug concentrations and developing
new pharmacokinetic and dynamic paradigms to describe this new class of agents.
As an example of this new paradigm for drug delivery,
Lanza et al (23,71) treated smooth muscle cells in culture with TF-targeted nanoparticles that were loaded
with paclitaxel. The smooth muscle cells were harvested from pig aorta and constitutively expressed TF
epitopes in vitro. Binding of the drug-free nanoparticles
to the cells yielded no alterations in growth characteristics of the cultured cells. However, when paclitaxelloaded nanoparticles were applied to the cells, specic
binding elicited a substantial reduction in smooth muscle cell proliferation. Nontargeted paclitaxel-loaded particles applied to the cells (i.e., with no binding of nanoparticles to cells) resulted in normal cell proliferation,
indicating that selective targeting may be a requirement
for effective drug delivery for these emulsions. Similar
behavior has been demonstrated for doxorubicin-containing particles (71). Recent reports indicate that intravenous delivery of fumagillin-loaded nanoparticles
(an antiangiogenic agent) targeted to v3-integrin
epitopes on the vasa vasorum in growing plaques results in marked inhibition of plaque angiogenesis in
cholesterol-fed rabbits (113). Kolodgie et al (114) also
utilized Taxol-containing albumin nanoparticles to
limit the restenotic response after angioplasty and stent
placement in experimental animals.
The unique mechanism of drug delivery for highly
lipophilic agents (e.g., paclitaxel) contained within
emulsions depends on close apposition between the
nanoparticle carrier and the targeted cell membrane,
and has been described as contact facilitated drug
delivery (71). In contrast to liposomal drug delivery,
which generally requires endocytosis, the mechanism
of drug transport in this case involves lipid exchange or
lipid mixing between the emulsion vesicle and the targeted cell membrane (Fig. 9) (61,62), which depends on
the extent and frequency of contact between two lipidic
surfaces (61,62,71). The rate of lipid exchange and drug
delivery can be greatly increased by the application of
clinically safe levels of US energy. This increases the
propensity for fusion or enhanced contact between the
nanoparticles and the targeted cell membrane by stimulating these interactions between nanoparticles and
cell membranes (Fig. 9) (62). These methods offer additional mechanisms for facilitating targeted drug delivery with the application of exogenous, safe US energy in
conjunction with therapeutic targeted nanoparticles.
In conclusion, the combination of targeted drug delivery and molecular imaging with MRI has the potential
to revolutionize the detection and treatment of cardiovascular disease. Drug-delivery agents that are also
quantiable at the targeted site based on imaging readouts may ultimately permit serial characterization of
the molecular epitope expression and conrmation of
therapeutic efcacy. Rapid developments in genomics,
molecular biology, and nanotechnology are contributing to the multidisciplinary eld of molecular imaging,
and clinical trials are beginning.

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