REVIEW

Clinical applications of perfluorocarbon nanoparticles

for molecular imaging and targeted therapeutics

Trung D Tran 1 Abstract: Molecular imaging is a novel tool that has allowed non-invasive diagnostic imaging
Shelton D Caruthers 1,2 to transition from gross anatomical description to identication of specic tissue epitopes and
Michael Hughes 1 observation of biological processes at the cellular level. This technique has been conned to
John N Marsh 1 the eld of nuclear imaging; however, recent advances in nanotechnology have extended this
Tillmann Cyrus 1 research to include ultrasound (US) and magnetic resonance (MR) imaging. The exploitation
of nanotechnology for MR and US molecular imaging has generated several candidate contrast
Patrick M Winter 1
agents. One multimodality platform, targeted peruorocarbon (PFC) nanoparticles, is useful for
Anne M Neubauer 1
noninvasive detection with US and MR, targeted drug delivery, and quantication.
Samuel A Wickline 1
Keywords: nanoparticles, peruorocarbon, molecular imaging, ultrasound, magnetic resonance
Gregory M Lanza 1 imaging
1
Division of Cardiology, Washington
University Medical School, 660 South
Euclid Blvd, St Louis, Missouri, USA; Introduction
2
Philips Medical Systems, Cleveland,
Ohio, USA Recent advances in genomics, proteomics and molecular biology have created an
unprecedented opportunity to identify clinical pathology in their pre-disease states.
Unfortunately, detection of small aggregates of dysplastic cells and their biochemi-
cal signatures are beyond the resolution and sensitivity of conventional acoustic and
magnetic resonance imaging techniques. Recognition of these molecular epitopes
requires target-specic probes, a robust signal amplication strategy and a sensitive
high-resolution imaging modality.
Numerous nano- or micro-particle systems are in development for targeted diagnos-
tic imaging and drug delivery, which have been recently reviewed (Cyrus et al in press).
The peruorocarbon (PFC) nanoparticle is a unique platform technology, which may
be applied to clinically relevant modalities and illustrates many of the key principles
found in other agents. Ligand-directed, lipid-encapsulated, liquid PFC nanoparticles
(250 nm nominal diameter) have inherent physicochemical properties, which provide
acoustic contrast when the agent is bound in aggregate to a surface. High surface area of
the nanoparticle accommodates 50 to 500 homing ligands, which imparts high avidity
and gives the agent a robust stick and stay quality (see Figure 1). Surface incorpo-
ration of large payloads of lipid-gadolinium chelate conjugates further extends the
utility of the agent to detect sparse concentrations of cell surface biochemical markers
with MRI (Lanza, Lorenz et al 1998). Moreover, the high uorine signal from the
nanoparticle core allows noninvasive quantication of ligand-bound particles, which
will permit clinicians to conrm tissue concentrations of drugs when functionality of
Correspondence: Gregory M Lanza
Associate Professor of Medicine and the nanoparticles is extended to include targeted therapy (see Figure 2).
Bioengineering, Division of Cardiology, The denition of nanoparticle is varied based on the perspective and historical time-
Washington University Medical School,
660 S. Euclid Ave, Campus Box 8086, Saint frame. In the materials section of the NIH and NSF, the size of nanoparticles dened
Louis, MO 63110, USA by a change in the properties of structures, particularly with respect to their quantum
Tel +1 314 454 8813
Fax +1 314 454 5265
features for optical imaging. The NCI and NHLBI typically refer to nanoparticles in
Email greg@cvu.wustl.edu their RFAs for biomedical applications at 300 nm and the Europeans use a 500 nm

International Journal of Nanomedicine 2007:2(4) 515526 515

2007 Dove Medical Press Limited. All rights reserved
Tran et al

Figure 1 Scanning electron micrographs (x30 000) of control fibrin clot (A) and fibrin-targeted paramagnetic nanoparticles bound to the clot surface (B). Arrows indicate
(A) fibrin fibril; (B) fibrin-specific nanoparticles-bound to fibrin epitopes. Copyright 2001. Reprinted with permission from Flacke S, Fischer S, et al. 2001. Novel MRIM
contrast agent for molecular imaging of fibrin: implications for detecting vulnerable plaques. Circulation, 104:12805.

cut-off. In biomedical applications, the surface to volume that encouraging the NIH to include engineered viruses as
ratio and the pharmacokinetics and biodistribution features nanoparticles! Thus in reality, the denition of nanoparticle
of nanoparticles are the important qualities. Although in the biological sciences is dynamic, referring to particles
the majority of nanoparticles are synthetic using natural generally less than 500 nm and typically, though not exclu-
products or polymers, there are contingents of scientists sively, chemically synthesized or fabricated.

Payloads
Paramagnetic Ions (eg, Gd3+)
Fluorophores
Radionuclides
Lipid Capsule
Drugs/Genes

19F
19F

Molecular Targeting System

Monoclonal Antibodies
Aptamers
Peptidomimetics
Polysaccharides ~250 nm
etc.

Figure 2 Paradigm for targeted liquid perfluorocarbon-based nanoparticle contrast agents. This example has a payload of Gd3+ chelates and monoclonal antibodies. This
platform is extremely versatile and applicable to almost any imaging modalities and capable of carrying other payloads such as drugs or genes.

516 International Journal of Nanomedicine 2007:2(4)

 Clinical applications of perfluorocarbon nanoparticles

Targeted versus non-targeted transiently enhance the blood pool signal, which is otherwise
contrast agents weakly echogenic. When insonied by an ultrasound wave,
Localization of non-targeted contrast agents depends greatly microbubbles improve gray scale images and Doppler signal
upon the bodys innate system for clearance of foreign par- via two distinct mechanisms (Dalla Palma and Bertolotto
ticles, ie, the reticuloendothelial system (RES). Macrophages 1999; McCulloch et al 2000; Correas et al 2001). First, at
of the RES are responsible for the removal of these contrast lower acoustic power, microbubbles are highly efcient scat-
agents from the circulation, which occurs in a size-dependent ters due to their large differences in acoustic impedance (Z)
fashion from the lung, spleen, liver and bone marrow. Fur- compared with surrounding tissue or blood. With increasing
thermore, phagocytosis and accumulation within specic acoustic energies, microbubbles begin non-linear oscilla-
sites can be enhanced by opsonization (ie, biologic tagging) tions and emit harmonics of the fundamental (incidence)
with blood proteins such as immunoglobins, complement frequencies; thus behaving as a source of sound, rather than
proteins or nonimmune serum factors. In general, liver as a passive reector. Biological tissue does not display this
sequestration appears to be complement mediated, while degree of harmonic generation, thus the contrast generated
the spleen removes foreign particulate matter via antibody signal can be exploited to preferentially image microbubbles
Fc receptors (Moghimi and Patel 1989). This process of and improve signal-to-noise ratios. To emphasize these
nonspecic and nondirected uptake of particles is generally strong echogenic properties, it has been shown that even one
referred to as passive targeting. microbubble can be detected with medical ultrasound systems
Ligand-directed (targeted) contrast agents are designed (Klibanov et al 2004). Interestingly, destruction and cavitation
to identify specic pathological tissue that otherwise might of microbubbles by ultrasound waves have been suggested as
be difcult to distinguish from surrounding normal tissue. A a means to facilitate drug delivery by sonoporating mem-
wide variety of ligands can be utilized including monoclonal branes and allowing drugs and gene therapy to enter the cell
antibodies and fragments, peptides, polysaccharides, aptamers (Shohet et al 2000; Blomley et al 2001). However, the long
and drugs. These ligands may be attached covalently (ie, direct term effects on cell viability have yet to be assessed. When
conjugation) or non-convalently (ie, indirect conjugation) to this process occurs in capillary beds, permeability increases
the contrast agent. Further surface modication, such as the allowing a subset of particles access to surrounding tissue for
incorporation of polyethylene glycol, are used to delay or further drug deposition et al 1998).
avoid rapid systemic removal of the agents and allow ligand- The wide use of microbubbles in everyday clinical appli-
to-target binding to occur. To demonstrate the effectiveness cations highlight its effectiveness as a blood pool agent. For
of this concept of targeting contrast agents, we need only example, microbubbles enhance the blood-tissue boundary
look at the application of paramagnetic MRI contrast agents. of the left ventricular cavity allowing for better diagnostic
Paramagnetic agents only inuence protons in their immedi- yield in resting as well as stress echocardiograms (Cheng
ate vicinity and removal of these contrast agents by the RES et al 1998). Improved Doppler signals are benecial in the
during passive targeting may decrease their effectiveness via diagnosis of valvular stenosis and regurgitation (Terasawa
two mechanisms: (1) accumulation of contrast agent in specic et al 1993). Additionally, microbubbles are removed from cir-
organs that are distal to region of interest, and (2) endocytosis culation via the RES and accumulate in the liver and spleen,
further decreases their exposure to free water protons. By ie, passive targeting. This mechanism can be employed for
targeting the contrast agent, the paramagnetic ions can be the detection of focal liver lesions and malignancies (Harvey
brought in close proximity to the region of interest with sig- et al 2000; Blomley et al 2001). For use as targeted contrast
nicant accumulation to overcome the partial dilution effect agents, microbubbles have been conjugated with ligands for a
that plagues some MR contrast agents. Its efcacy is further variety of vascular biomarkers including integrins expressed
enhanced with some targeting platforms by delivering multiple during angiogenesis, the glycoprotein IIb/IIIa receptor on
contrast ions per particle (Lanza, Lorenz et al 1998). activated platelets in clots and L-selectin for the selective
enhancement of peripheral lymph nodes in vivo (Schumann
et al 2002; Leong-Poi et al 2003; Hauff et al 2004). One
Basic principles of ultrasound contrast disadvantage to the targeting of microbubbles is the tether-
agents ing of these particles to a surface. This interaction with a
Commercially available ultrasound contrast agents are based solid structure limits the ability of insonied microbubbles
on gas-lled encapsulated microbubbles (~2.55 microns) that to oscillate and dampens their echogenicity.

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Tran et al

As mentioned previously, we have developed a novel dephasing, which is a process of nuclei to nuclei interactions
multidimensional targeted nanoparticle platform that is a (ie, spin-spin or transverse relaxation) characterized by the
ligand-targeted, lipid encapsulated, nongaseous peruo- time constant T2. T1 and T2 relaxation times, as well as the
rocarbon emulsion. It is robustly stable to handling, pres- density of the nuclei of interest, determine signal intensity
sure, atmospheric exposure, heat and shear forces. Unlike of various types of tissue in MR imaging.
microbubble formulations that are naturally echogenic, these MR contrast agents work by shortening T1 and T2. The
nanoparticles have poor inherent acoustic reectivity and most commonly used non-targeted MR contrast agents uti-
have been shown to exhibit backscattering levels 30 dB below lize paramagnetic ions (eg, gadolinium chelates) and they
that of whole blood (Hughes et al 2005). However, when predominantly shorten T1 relaxation resulting in a bright
collective deposition occurs on the surfaces of tissues or a signal on T1 weighted images. T1 agents chiey inuence
cell in a layering effect, these particles create a local acoustic protons proximate to themselves and are highly dependent on
impedance mismatch that produces a strong ultrasound sig- local water ux (Lanza et al 2004). Superparamagnetic and
nal without a concomitant increase in the background level ferromagnetic compounds have a high magnetic susceptibil-
(Lanza, Trousil et al 1998). The echogenicity of nanopar- ity and produce local disturbances in the eld (Nelson and
ticles does not depend upon the generation of harmonics and Runge 1995). These disturbances induce signal dephasing
therefore is not affected by binding of nanoparticles with in tissue and result in loss of signal through apparent T2
molecular epitopes. Due to their small size and inherent in decay (ie, T2*). Contrary to T1 contrast agents, superpara-
vivo stability, peruorocarbon nanoparticle emulsions have magnetic agents have a net effect far beyond their immediate
a long circulatory half-life compared to microbubble contrast vicinity.
agents. This is accomplished without modication of their For use as a T1 weighted paramagnetic contrast agent, PFC
outer lipid surfaces with polyethylene glycol or incorporation nanoparticles can be functionalized by surface incorporation
of polymerized lipids, which can detract from the targeting with homing ligands and typically 100,000 gadolinium che-
efcacy. Data suggest that the PFC nanoparticles persist lates (Gd3+) per particle (Flacke et al 2001). In addition, all of
when bound to tissue for hours and possibly days. Addi- the paramagnetic ions are present in the outer surface where
tionally, nongaseous nanoparticles do not easily deform or they optimally inuence the local water for maximum effect
cavitate with ultrasound imaging. on T1 relaxation (Winter, Caruthers et al 2003). The result is
PFC nanoparticles that are capable of overcoming the diluting
Basic principles of MR contrast partial volume effects that plague most MR contrast agents
agents (Gupta and Weissleder 1996). To illustrate the potency of this
In addition to acoustic imaging, this nanoparticle platform contrast agent platform, we need only examine its relaxivity.
has been coupled to paramagnetic ions for MR imaging. As The efciency of an MR contrast agent can be described by
a dual-modality contrast agent, the strengths of ultrasound its relaxivity (mM1sec1), which is simply calculated as the
imaging, ie, exibility, portability and ease of use are com- change in relaxation rate (1/T1 or T2) divided by the concen-
bined with the high spatial resolution of MR imaging. To tration of the contrast agent. The relaxivity of Gd3+ in saline
understand how nanoparticles can be employed as an MR (4.5 mM1sec1) (Stanisz and Henkelman 2000) is lower when
contrast agent, an understanding of the NMR phenomenon compared to Gd3+ bound to the surface of a PFC nanoparticles
is crucial. The nuclei of elements such as hydrogen (1H) or (33.7 mM1sec1) (Winter, Caruthers et al 2003) at 1.5T. Since
uorine (19F), when exposed to a strong magnetic eld will each nanoparticle bears high payloads of Gd ions, the overall
change from their random orientation to adopt either a paral- relaxivity of the construct, also referred to as the particle-based
lel or anti-parallel alignment. Absorption of radiofrequency relaxivity, has been measured at over 2,000,000 mM1sec1
(RF) energy at a characteristic resonance frequency excites (Winter, Caruthers et al 2003). This allows for detection and
nuclei into a higher energy state. Upon termination of the quantication of tissue biomarkers at low nanomolar concen-
RF excitation, nuclei return to the previous lower energy trations (Morawski et al 2004).
state in an exponential fashion with a characteristic rate time
constant known as T1 (ie, spin-lattice or longitudinal relax- PFC nanoparticles for fluorine (19F)
ation). This process of excitation and relaxation results in a MR spectroscopy and imaging
changing magnetic eld that is detectable with an external RF The intensity of an MR signal is directly proportional to
antenna. This detected signal strength is diminished due to the gyromagnetic ratio () and density (number of nuclei

518 International Journal of Nanomedicine 2007:2(4)

 Clinical applications of perfluorocarbon nanoparticles

in the volume of interest) (Bushong 2003). Though there RES and excreted primarily through the lungs and in small
are many medically relevant nuclei, the 1H proton is the amounts through the skin (Joseph et al 1985). Tissue half-
most commonly imaged nuclei in clinical practice because life ranges from 4 days for peruorooctylbromide up to 65
of its high gyromagentic ratio, high natural abundance and days for peruorotripropylamine. The prolonged systemic
high concentrations in biological tissue (see Table 1). Fluo- half-life of PFC nanoparticles in conjunction with the local
rine-19, whose gyromagnetic ratio is close to 1H and has concentrating effect produced by ligand-directed binding,
a natural abundance of virtually 100%, is a theoretically permit 19F spectroscopy and imaging studies at clinically
attractive nucleus for MR imaging (Longmaid et al 1985). relevant magnetic eld strengths (Yu et al 2000).
Its sensitivity is 83% when compared to 1H at a constant eld
strength and with equivalent number of nuclei. In biological Fibrin-imaging for detection
tissue, low 19F concentrations (in the range of micromoles) of unstable plaque and thrombus
makes MR imaging ineffective without 19F rich contrast Of the over 720,000 cardiac related deaths per year in the
agents (McFarland et al 1985). PFC nanoparticles are 98% United States, roughly 63% are classied as sudden car-
peruorocarbon by volume, which equates for peruorooc- diac death (SCD) (Zheng et al 2001). Unfortunately for
tylbromide (1.98 g/ml, 498 daltons) to approximately 100M many patients, this is the rst and only symptom of their
concentration of uorine within a nanoparticle. The paucity atherosclerotic heart disease (ASHD) (Kuller et al 1966).
of endogenous uorine in biological tissue allows the use of Atherosclerosis begins as a fatty streak and without proper
exogenous PFC nanoparticles as an effective 19F MR con- treatment, it can progress to a vulnerable plaque character-
trast agent without interference from signicant background ized by a large lipid core, thin brous cap and macrophage
signal. When combined with local drug delivery, detection inltrates (Naghavi 2003). These vulnerable plaques are
of the 19F signal serves as a highly specic marker for the prone to rupture which can lead to thrombosis, vascular
presence of nanoparticles that would permit quantitative occlusion and subsequent myocardial infarction (Davies and
assessment of drug dosing. Thomas 1985) or stroke. Routine angiography is the most
19
F has seven outer-shell electrons rather than a single common method of diagnosing ASHD with identication
electron as is the case with hydrogen; as a result, the range of high grade lesions (70% stenosis) being referred for
and sensitivity of chemical shifts to the details of the local immediate therapeutic intervention. Ironically, most ruptured
environment are much higher for uorine than hydrogen. plaques originate from coronary lesions classied as non-
Distinct spectra from different peruorocarbon species can stenotic (Naghavi et al 2003). Even nuclear and US based
be obtained and utilized for simultaneously distinguishing stress test are only designed to detect ow limiting lesions.
multiple biochemical markers via MR spectroscopy or imag- Because the most common source of thromboembolisms
ing (Caruthers et al 2006). come from atherosclerotic plaques with 50%60% stenosis
For use as a clinically applicable contrast agent, the (Ambrose et al 1988), diagnosis by traditional techniques
biocompatibility of PFC nanoparticles must be reviewed. remains elusive. In addition, there appears to be a window
Liquid PFC were previously developed for use as a blood of opportunity that exists between the detection of a vulner-
substitute (Sloviter and Mukherji 1983) and no toxicity, car- able or ruptured plaque and acute myocardial infarction (a
cinogenicity, mutagenicity or teratogenic effects have been few days to months) (Ojio et al 2000) in which intervention
reported for pure uorocarbons within the 460 to 520 MW could prove to be benecial.
range. PFCs are inert biologically and removed through the Acoustic enhancement of thrombi using brin targeted
nanoparticles was rst demonstrated in vitro as well as in
Table 1 MRI properties of clinically important nuclei vivo in a canine model at frequencies typically used in clini-
Nuclei Gyromagnetic Spin Natural Relative cal transcutaneous scanning (Lanza et al 1996). Detection of
ratio quantum abundance sensitivity thrombi was later expanded to MRI in a study by Flacke et al
(MHz/T) number (%) (2001). Fibrin clots were targeted in vitro with paramagnetic
1
H 42.6 1/2 99 1.0 nanoparticles and imaged using typical low resolution T1
13
C 10.7 1/2 1.1 0.016
proton imaging protocols (gradient and spin echo) at 1.5T. Low
17
O 5.8 5/2 0.1 0.029
19
F 40.0 100 0.83 resolution images show enhancement of MR signal by the PFC
23
Na 11.3 3/2 100 0.093 nanoparticles and appear to completely ll the clot volume (see
31
P 17.2 1/2 100 0.07 Figure 3). However, at higher in-plane resolution, the same clot

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Tran et al

0.7 x 0.7mm 0.1 x 0.1mm

Figure 3 (Left) Low-resolution images (3D gradient and spin echo) of control and (Middle) fibrin-targeted clot with paramagnetic nanoparticles presenting a homogenous,
T1-weighted enhancement. (Right) High resolution scans of fibrin clots (3D T1-weighted gradient recalled echo sequence), revealing that image B results from a thin layer
of paramagnetic nanoparticles along the surface. Copyright 2001. Reprinted with permission from Flacke S, Fischer S, et al. 2001. Novel MRI contrast agent for molecular
imaging of fibrin: implications for detecting vulnerable plaques. Circulation, 104:12805.

reveals that the nanoparticles were bound only at the surface and 19
F signal. Bound NP was calculated from the 19F signal and the
were excluded from penetration into the dense brin matrix (see calibration curve described above and compared with the mass
Figure 1). In the same study, in vivo MR images were obtained of Gd3+ tracer as determined by neutron activitation analysis.
of brin clots in the external jugular vein of dogs. Enhancement As expected, there was excellent agreement between measured
with brin targeted PFC nanoparticles produced high signal Gd3+ mass and number of bound nanoparticles (calculated from
intensity in treated clots (1780 327), whereas the control clot the 19F signal) (see Figure 5c).
had a signal intensity (815 41) similar to that of the adjacent In addition, clots were treated with fibrin targeted
muscle (768 47). This method was extended to the detection nanoparticles with two distinct peruorocarbon cores, crown
of ruptured plaque in human carotid artery endarterectomy ether and PFOB (Morawski et al 2004). They both exhibited
specimens resected from a symptomatic patient (see Figure 4).
Fibrin deposition was localized to the shoulders of the ruptured
plaque in the targeted vessel but this was not appreciated in the
control. Further investigation in the molecular imaging of brin in Fibrin NP
ruptured plaque may someday detect this silent pathology sooner
in order to preempt stroke or myocardial infarction. Lumen
The high uorine content of brin-targeted PFC nanopar- Plasma
ticles as well as the lack of background signal were exploited
for 19F MR imaging and spectroscopy. In a recent study,
Morawski et al (2004) describe several methods for quantifying
the number of nanoparticles bound to a brin clot using the
19
F signal. First, brin-targeted paramagnetic peruoro-crown-
Calcium
ether nanoparticles and trichlorouoromethane 19F spectra
were obtained (Figure 5a). The relative crown ether signal
intensity (normalized to the external trichlorouoromethane Targeted Control
reference) from known emulsion volumes provided a calibra-
tion curve for nanoparticle quantication (Figure 5b). The PFC Figure 4 Color enhanced magnetic resonance imaging of fibrin-targeted and con-
(crown ether) nanoparticles were then mixed in titrated ratios trol carotid endarterectomy speciments revealing contrast enhancement (white) of
a small fibrin deposit on a symptomatic ruptured plaque. Calcium deposit (black).
with brin-targeted nanoparticles containing safower oil and 3D, fat-suppressed, T1-weighted fast gradient echo. Copyright 2001. Reprinted
with permission from Flacke S, Fischer S, et al. 2001. Novel MRI contrast agent for
bound to plasma clots in vitro. As the competing amount of molecular imaging of fibrin: implications for detecting vulnerable plaques. Circulation,
safower agent was increased, there was a linear decrease in 104:12805.

520 International Journal of Nanomedicine 2007:2(4)

 Clinical applications of perfluorocarbon nanoparticles

the luminal surface due to binding of targeted paramagnetic

nanoparticles to brin deposits (not shown in Figure 6). 19F
projection images of the artery, taken in approximately 5
min, showed an asymmetric distribution of brin-targeted
nanoparticles around the vessel wall corroborating the signal
enhancement observed with 1H MRI. Concomitant visualiza-
25 0 25 50 75 100 125
tion of 1H and 19F images would permit the visualization of
a PPM anatomical and pathological information in a single image.
In theory, atherosclerotic plaque burden could be visualized
with paramagnetic PFC contrast enhanced 1H images while
Relative 19F Signal

300
19
F could locally identify plaques with high levels of brin
200 and thus prone to rupture. This simultaneous information
could improve treatment strategies.
100
Detection of angiogenesis
0
0 2 4 6 8 10 and vascular injury
b Volume of emulsion (L) As described previously, ligand-directed PFC nanoparticles
are well suited to detect very sparse biomarkers, such as inte-
12 grins involved in the process of angiogenesis. Angiogenesis
Bound NP ( 1011)

is a critical component in wound healing, inammation and

8 development; but also contributes to the pathology of many
disease processes such as diabetic retinopathy, rheumatoid
4 arthritis, cancer and atherosclerosis. The process of angio-
genesis depends upon the adhesion interactions of vascular
0 cells and the integrin 3 has been identied as playing a
0 10 20 30 vital role in angiogenic vascular tissue. The function of 3
Mass of Gd (g) 3+ includes smooth muscle cell (SMC) migration and prolifera-
c
tion, vascular cell apoptosis, and vascular remodeling (Sajid
Figure 5 (a) Representative spectrum taken at 4.7 T of crown ether emulsion
and Stouffer 2002) and it is expressed on the luminal surface
(~90 ppm) and trichlorofluormethane (0 ppm) used as a reference. (b) The calibra-
tion curve for crown ether emulsion has a slope of 28.06 with an r2 of 0.9968. (c) of activated endothelial cells but not on mature quiescent
The calculated number of bound nanoparticles (mean standard error, n = 3) as
calculated from 19F spectroscopy versus the mass of total gadolinium (Gd3+) in the
cells. These ndings support that the role of 3 in patho-
sample as determined by neutron activation analysis show excellent agreement as logical conditions characterized by neovascularization may
independent measures of fibrin-targeted nanoparticles binding to clots. The linear
regression line has an r2 of 0.9997. Copyright 2004. Reprinted with permission be an important diagnostic and therapeutic target. In fact, the
from Morawski AM, Winter PM, et al. 2004. Targeted nanoparticles for quantitative use of a monoclonal antibody against 3 has demonstrated
imaging of sparse molecular epitopes with MRI. Magnetic Resonance in Medicine,
51:4806. inhibition of angiogenesis without affecting mature vessels
(Brooks et al 1994).
PFC nanoparticles have been developed to detect the
two distinct 19F spectra at 4.7T and the binding of each respec- sparse expression of the 3 integrin on the neovascula-
tive nanoparticles was highly related to the ratio of PFOB and ture (Anderson et al 2000) and to deliver anti-angiogenic
crown ether emulsion applied. These ndings demonstrate therapy. This has been utilized in New Zealand white rabbits
the possibility of simultaneous imaging and quantifying two bearing Vx-2 tumors (1.0 cm) at 1.5T (Winter, Caruthers
separate nanoparticles and hence two distinct biomarkers et al 2003). MRI signal 2 h post injection of 3-targeted
(Caruthers et al 2006). nanoparticles revealed signal enhancement of 126% pre-
The clinical exercise of these techniques was applied to dominantly in an asymmetrical distribution along the tumor
the analysis of human carotid endarterectomy samples (see border. These results paralleled the immunohistochemical
Figure 6). An optical image of the carotid revealed extensive staining results. Moreover, in vivo competitive blocking
plaques, wall thickening and luminal irregularities. Multi-slice with 3 targeted non-paramagnetic nanoparticles resulted
1
H images showed high levels of signal enhancement along in decreased signal enhancement to a level attributable to

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Tran et al

Concentration of
Nanoparticles (nM)

>1.5

1.0

0.5

a b c 0
Optical Image 19
F MRI Nanoparticle Concentration Map
Figure 6 (a) Optical image of a 5-mm cross-section of a human carotid endarterectomy sample. This section showed moderate luminal narrowing as well as several
atherosclerotic lesions. (b) A 19F projection image acquired at 4.7 T through the entire carotid artery sample shows high signal along the lumen due to nanoparticles bound
to fibrin. (c) Concentration map of bound nanoparticles in the carotid sample. Copyright 2004. Reprinted with permission from Morawski AM, Winter PM, et al. 2004.
Targeted nanoparticles for quantitative imaging of sparse molecular epitopes with MRI. Magnetic Resonance in Medicine, 51:4806.

local extravasation. In an analogous study, athymic nude mice nanoparticle that can target 3 integrin exposed on smooth
bearing human melanoma tumors (C32, ATCC; ~33mm3) muscle cells by arterial overstretch injury and provide new
were injected with 3 targeted PFC nanoparticles and prognostic data relating the extent and severity of balloon
imaged at 2 h (Schmieder, Winter et al. 2005). MR enhance- injury as well as facilitate the delivery of therapeutic agents
ment was apparent within 0.5 h and increased by 173% at 2 h from within the damaged arterial wall (Cyrus et al 2003).
(see Figure 7). Again, MR imaging results were corroborated
with histological results. In both studies, in vivo competitive
blocking with 3 targeted non-paramagnetic nanoparticles
inhibited the evolution of the targeted signal. These ndings
speak to the high specicity achievable with 3 targeted
nanoparticles.
Angiogenesis also plays a crucial and early role in the
pathogenesis of atherosclerosis and the inhibition of angiogen-
esis has been shown to decrease plaque progression (Moulton
A
et al 1999, 2003). Unfortunately, detection of atherosclerosis
generally does not occur until late stages of the disease when
a clinical event is caused by a plaque rupture. Conventional
noninvasive imaging techniques are not sensitive or specic
enough to detect the sparse biomarkers associated with
early atherosclerosis. However, 3-targeted paramagnetic
nanoparticles have been demonstrated to spatially localize
Baseline Image 30 min
and quantify neovascularization associated with early ath-
erosclerosis in hyperlipidemic New Zealand White rabbits
(see Figure 8) (Winter, Morawski et al 2003).
In addition to angiogenesis, 3 has an important role in
the development of restenosis following balloon injury from
angioplasty. It is expressed following vascular injury and
facilitates smooth muscle migration and proliferation, cell B 60 min 120 min
adhesion to the extracellular matrix as well as induction of
Figure 7 (A) T1-weighted MR image (axial view) of an athymic nude mouse before
extracellular metalloproteinase expression. Multiple animal injection of paramagnetic 3-targeted nanoparticles. Arrow indicates a C32
studies have shown that IV administration of 3 antagonism tumor that is difficult to detect (Ref = Gd3+ DTPA in 10 cc syringe). (B) Enlarged
section of an MR image showing T1-weighted signal enhancement of angiogenic
following balloon angioplasty results in decreased neointimal vasculature of early tumors over 2 h as detected by 3-targeted nanoparticles.
Copyright 2005. Reprinted with permission from Schmieder AH, Winter PM,
growth and lumen stenosis (Srivatsa et al 1997; Bishop et al et al. 2005. Molecular MR imaging of melanoma angiogenesis with alphanubeta3-
2001). We have developed a high avidity 3 integrin specic targeted paramagnetic nanoparticles. Magnetic Resonance in Medicine, 53:6217.

522 International Journal of Nanomedicine 2007:2(4)

 Clinical applications of perfluorocarbon nanoparticles

Slice

1 10 20 30

Renal Diaphragm

100

80

60

40

20

Pre Post Segmented Enhancement

Figure 8 In vivo spin-echo image reformatted to display long axis of aorta from renal arteries to diaphragm of a cholesterol-fed rabbit (top) and at single transverse level
(bottom) before (Pre) and after (Post) treatment, after semi-automated segmentation (Segmented, grayish ring), and with color-coded signal enhancement (Enhancement)
above baseline (in percent). Copyright 2003. Reprinted with permission from Winter PM, Morawski AM, et al. 2003. Molecular imaging of angiogenesis in early-stage
atherosclerosis with alpha(v)beta3-integrin-targeted nanoparticles. Circulation, 108:22704.

Similar to 3, collagen III is omni-present within the vascular Contact facilitated drug delivery
wall and accessible immediately after balloon injury for tar- Besides detecting sparse epitopes for non-invasive imaging,
geted delivery of therapeutic paramagnetic nanoparticles. We PFC nanoparticles are capable of specically and locally deliv-
have covalently coupled a monoclonal Fab fragment specic ering drugs and other therapeutic agents through a novel process
for collagen III to target nanoparticles into carotid extracellular known as contact facilitated drug delivery (Lanza et al 2004).
matrix following balloon overstretch injury in pigs, analogous Direct transfer of lipids and drugs from the outer surfactant
to the studies previously described for 3-targeted paramag- layer to the cell membrane of the targeted cell is usually a slow
netic nanoparticles (Cyrus et al 2003). and inefcient process. Through ligand directed targeting, this

Contact-Facilitated Drug Delivery

A B C

Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y

Circulate to Target Bind to Biomarker Lipid-Drug Exchange

Figure 9 Schematic representation illustrating contact-facilitated drug delivery. Phospholipids and drug within the nanoparticles surfactant exchange with lipids of the target
membrane through a convection process, rather than diffusion, as is common among other targeted systems. Copyright 2004. Reprinted with permission from Lanza GM,
Winter PM, et al. 2004. Magnetic resonance molecular imaging with nanoparticles. Journal of Nuclear Cardiology, 11:73343.

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Tran et al

Figure 10 In vitro targeting of FITC-labeled nanoparticles (white arrows) targeted to ab3-integrin expressed by C-32 melanoma cells, which illustrate the delivery of
FITC-labeled surfactant lipids into target cell membranes (yellow arrows). Scale bar = 2 microns. Copyright 2004. Reprinted with permission from Lanza GM, Winter PM,
et al. 2004. Magnetic resonance molecular imaging with nanoparticles. Journal of Nuclear Cardiology, 11:73343.

process is accelerated by minimizing the separation of the lipids 4.7T revealed the image intensity of the targeted VSCMs was
surfaces and increasing the frequency and duration of the lipid 2-fold higher as compared with nontargeted cells. In addition,
surface interactions (see Figures 9 and 10). Spatial localization the uorine signal amplitude at 0.47T was linearly correlated to
(via high resolution 1H MR imaging) and quantication of the the peruorocarbon concentrations, which by direct inference
nanoparticles (via 19F spectroscopy) permits the local therapeu- could be related to nanoparticles number.
tic concentrations to be estimated. Thus, PFC nanoparticles can
be used for detection, therapy and treatment monitoring. Conclusion
As an example, in vitro vascular smooth muscle cells were Ligand-directed liquid PFC nanoparticles are an extremely
treated with tissue-factor targeted PFC nanoparticles containing versatile imaging platform that permit noninvasive assess-
0, 0.2, or 2.0 mole % doxorubicin or paclitaxel or an equivalent ment of clinically important sparce biomarkers, such as
amount of drug in buffer solution alone (Lanza, Yu et al 2002). integrins in angiogenesis as well as high density epitopes,
With only targeting for 30 minutes, proliferation measured such as brin. The unique properties of targeted nanopar-
at 72 hours was inhibited. High resolution MR imaging with ticles present the opportunity to diagnose, deliver therapy

524 International Journal of Nanomedicine 2007:2(4)

 Clinical applications of perfluorocarbon nanoparticles

and predict therapeutic response with a single platform. Lanza GM, Lorenz CH, et al. 1998. Enhanced detection of thrombi with
a novel brin-targeted magnetic resonance imaging agent. Academic
Furthermore, they allow image based serial monitoring. Radiology, 5(Suppl 1):S1736; discussion S1834.
Their impact in the elds of cardiology and oncology are Lanza GM, Trousil RL, et al. 1998. In vitro characterization of a novel,
tissue-targeted ultrasonic contrast system with acoustic microscopy.
predicted to be profound. Journal of the Acoustical Society of America, 104:366572.
Lanza GM, Wallace KD, et al. 1996. A novel site-targeted ultrasonic contrast
agent with broad biomedical application. Circulation, 94:333440.
References [Erratum appears in Circulation 1997 May 20;95:2458.]
Ambrose JA, Tannenbaum MA, et al. 1988. Angiographic progression of Lanza GM, Winter PM, et al. 2004. Magnetic resonance molecular imaging
coronary artery disease and the development of myocardial infarction. with nanoparticles. Journal of Nuclear Cardiology, 11:73343.
Journal of the American College of Cardiology, 12:5662. Lanza GM, Yu X, et al. 2002. Targeted antiproliferative drug delivery
Anderson SA, Rader RK, et al. 2000. Magnetic resonance contrast enhance- to vascular smooth muscle cells with a magnetic resonance imaging
ment of neovasculature with alpha(v)beta(3)-targeted nanoparticles. nanoparticle contrast agent: implications for rational therapy of reste-
Magnetic Resonance in Medicine, 44:4339. nosis. Circulation, 106:28427.
Bishop GG, McPherson JA, et al. 2001. Selective alpha(v)beta(3)-receptor Leong-Poi H, Christiansen J, et al. 2003. Noninvasive assessment of angio-
blockade reduces macrophage inltration and restenosis after balloon genesis by ultrasound and microbubbles targeted to alpha(v)-integrins.
angioplasty in the atherosclerotic rabbit. Circulation, 103:190611. Circulation, 107:45560.
Blomley MJ, Cooke JC, et al. 2001. Microbubble contrast agents: a new Longmaid HE 3rd, Adams DF, et al. 1985. In vivo 19F NMR imaging of
era in ultrasound. BMJ, 322:12225. liver, tumor, and abscess in rats. Preliminary results. Investigative
Brooks PC, Clark RA, et al. 1994. Requirement of vascular integrin alpha Radiology, 20:1415.
v beta 3 for angiogenesis. Science, 264:56971. McCulloch M, Gresser C, et al. 2000. Ultrasound contrast physics: A series
Bushong SC. 2003. Magnetic resonance imaging: physical and biological on contrast echocardiography, article 3. Journal of the American Society
principles. Saint Louis: Mosby. of Echocardiography, 13:95967.
Caruthers SD, Neubauer AM, et al. 2006. In Vitro Demonstration Using McFarland E, Koutcher JA, et al. 1985. In vivo 19F NMR imaging. Journal
19F Magnetic Resonance to Augment Molecular Imaging with Para- of Computer Assisted Tomography, 9:815.
magnetic Peruorocarbon Nanoparticles at 1.5 Tesla. Investigative Moghimi SM, Patel HM. 1989. Serum opsonins and phagocytosis of satu-
Radiology, 41:30512. rated and unsaturated phospholipid liposomes. Biochimica et Biophysica
Cheng SC, Dy TC, et al. 1998. Contrast echocardiography: review and future Acta, 984:3847.
directions. American Journal of Cardiology, 81:41G48G. Morawski AM, Winter PM, et al. 2004. Targeted nanoparticles for quan-
Correas JM, Bridal L, et al. 2001. Ultrasound contrast agents: properties, titative imaging of sparse molecular epitopes with MRI. Magnetic
principles of action, tolerance, and artifacts. European Radiology, Resonance in Medicine, 51:4806.
11:131628. Morawski AM, Winter PM, et al. 2004b. Quantitative magnetic resonance
Cyrus T, Caruthers S, et al. In Press. Nanoparticles for magnetic resonance immunohistochemistry with ligand-targeted (19)F nanoparticles.
imaging of cancer. In Challa S, Kumar SR. Nanomaterials for cancer Magnetic Resonance in Medicine, 52:125562.
therapy and diagnosis. WILEY ~ VCH 6. Moulton KS, Heller E, et al. 1999. Angiogenesis inhibitors endostatin or
Cyrus T, Winter P, et al. 2003. Molecular imaging of avB3-integrin and TNP-470 reduce intimal neovascularization and plaque growth in apoli-
collagen-III targeted nanoparticles in pig carotids following balloon- poprotein E-decient mice [see comment]. Circulation, 99:172632.
ination injury. Molecular Imaging, 2:281. Moulton KS, Vakili K, et al. 2003. Inhibition of plaque neovasculariza-
Dalla Palma L, Bertolotto M. 1999. Introduction to ultrasound contrast agents: tion reduces macrophage accumulation and progression of advanced
physics overview. European Radiology, 9(Suppl 3):S33842. atherosclerosis. Proceedings of the National Academy of Sciences of
Davies MJ, Thomas AC. 1985. Plaque ssuring the cause of acute myocar- the United States of America, 100:473641.
dial infarction, sudden ischaemic death, and crescendo angina. British Naghavi M, Libby P, et al. 2003. From vulnerable plaque to vulnerable
Heart Journal, 53(4):36373. patient: a call for new denitions and risk assessment strategies: Part
Flacke S, Fischer S, et al. 2001. Novel MRI contrast agent for molecular I. Circulation, 108:166472.
imaging of brin: implications for detecting vulnerable plaques. Nelson KL, Runge VM. 1995. Basic principles of MR contrast. Topics in
Circulation, 104:12805. Magnetic Resonance Imaging, 7:12436.
Gupta H, Weissleder R. 1996. Targeted contrast agents in MR imaging. Ojio S, Takatsu H, et al. 2000. Considerable time from the onset of plaque
Magnetic Resonance Imaging Clinics of North America, 4:17184. rupture and/or thrombi until the onset of acute myocardial infarction in
Harvey CJ, Blomley MJ, et al. 2000. Hepatic malignancies: improved detec- humans: coronary angiographic ndings within 1 week before the onset
tion with pulse-inversion US in late phase of enhancement with SH U of infarction [see comment]. Circulation, 102:20639.
508A-early experience. Radiology, 216:9038. Price RJ, Skyba DM, et al. 1998. Delivery of colloidal particles and red
Hauff P, Reinhardt M, et al. 2004. Molecular targeting of lymph nodes with blood cells to tissue through microvessel ruptures created by targeted
L-selectin ligand-specic US contrast agent: a feasibility study in mice microbubble destruction with ultrasound. Circulation, 98:12647.
and dogs.[see comment]. Radiology, 231:66773. Sajid M, Stouffer GA. 2002. The role of alpha(v)beta3 integrins in vascular
Hughes MS, Marsh JN, et al. 2005. Acoustic characterization in whole healing. Thrombosis & Haemostasis, 87:18793.
blood and plasma of site-targeted nanoparticle ultrasound contrast Schmieder AH, Winter PM, et al. 2005. Molecular MR imaging of melanoma
agent for molecular imaging. Journal of the Acoustical Society of angiogenesis with alphanubeta3-targeted paramagnetic nanoparticles.
America, 117:96472. Magnetic Resonance in Medicine, 53:6217.
Joseph PM, Yuasa Y, et al. 1985. Magnetic resonance imaging of Schumann PA, Christiansen JP, et al. 2002. Targeted-microbubble binding
uorine in rats infused with articial blood. Investigative Radiology, selectively to GPIIb IIIa receptors of platelet thrombi. Investigative
20:5049. Radiology, 37:58793.
Klibanov AL, Rasche PT, et al. 2004. Detection of individual microbubbles Shohet RV, Chen S, et al. 2000. Echocardiographic destruction of albumin
of ultrasound contrast agents: imaging of free-oating and targeted microbubbles directs gene delivery to the myocardium. Circulation,
bubbles. Investigative Radiology, 39:18795. 101:25546.
Kuller L, Lilienfeld A, et al. 1966. Epidemiological study of sudden and Sloviter HA, Mukherji B. 1983. Prolonged retention in the circulation of
unexpected deaths due to arteriosclerotic heart disease. Circulation, emulsied lipid-coated peruorochemicals. Progress in Clinical and
34:105668. Biological Research, 122:1817.

International Journal of Nanomedicine 2007:2(4) 525

Tran et al

Srivatsa SS, Fitzpatrick LA, et al. 1997. Selective alpha v beta 3 integrin Winter PM, Caruthers SD, et al. 2003. Improved molecular imaging con-
blockade potently limits neointimal hyperplasia and lumen stenosis fol- trast agent for detection of human thrombus. Magnetic Resonance in
lowing deep coronary arterial stent injury: evidence for the functional Medicine, 50:4116.
importance of integrin alpha v beta 3 and osteopontin expression during Winter PM, Morawski AM, et al. 2003. Molecular imaging of angiogenesis
neointima formation. Cardiovascular Research, 36:40828. in early-stage atherosclerosis with alpha(v)beta3-integrin-targeted
Stanisz GJ, Henkelman RM. 2000. Gd-DTPA relaxivity depends on macro- nanoparticles. Circulation, 108:22704.
molecular content. Magnetic Resonance in Medicine, 44:6657. Yu X, Song SK, et al. 2000. High-resolution MRI characterization of human
Terasawa A, Miyatake K, et al. 1993. Enhancement of Doppler ow signals thrombus using a novel brin-targeted paramagnetic nanoparticle con-
in the left heart chambers by intravenous injection of sonicated albumin. trast agent. Magnetic Resonance in Medicine, 44:86772.
Journal of the American College of Cardiology, 21:73742. Zheng ZJ, Croft JB, et al. 2001. Sudden cardiac death in the United States,
Winter PM, Caruthers SD, et al. 2003. Molecular imaging of angiogenesis 1989 to 1998 [see comment]. Circulation, 104:215863.
in nascent Vx-2 rabbit tumors using a novel alpha(nu)beta3-targeted
nanoparticle and 1.5 tesla magnetic resonance imaging. Cancer
Research, 63:583843.

526 International Journal of Nanomedicine 2007:2(4)