Published on: Mar 3, 2016
Transcripts - Nanomedicina1
GREGORY LANZA,a PATRICK WINTER,a TILLMANN CYRUS,a
SHELTON CARUTHERS,a,b JON MARSH,a MICHAEL HUGHES,a
AND SAMUEL WICKLINEa
a Washington University School of Medicine, St. Louis, Missouri 63110, USA
b Philips Medical Systems, Cleveland, Ohio 44143, USA
ABSTRACT: Despite myriad advances, cardiovascular-related diseases
continue to remain our greatest health problem. In more than half of pa-
tients with atherosclerotic disease, their first presentation to medical at-
tention becomes their last. Patients often survive their first cardiac event
through acute revascularization and placement of drug-eluting stents
(DES), but only select coronary lesions are amenable to DES placement,
resulting in the use of bare metal or no stent, both of which lack the bene-
fit of antirestenotic therapy. In other patients, transient ischemic attacks
(TIAs) and stroke constitute the initial presentation of disease. In these
patients, the diagnostic and therapeutic options are woefully inadequate.
Nanomedicine offers options to each of these challenges. Antiangiogenic
paramagnetic nanoparticles may be used to serially assess the severity of
atherosclerotic disease in asymptomatic, high-risk patients by detecting
the development of plaque neovasculature, which reflects the underlying
lesion activity and vulnerability to rupture. The nanoparticles can locally
deliver antiangiogenic therapy, which may acutely retard plaque progres-
sion, allowing aggressive statin therapy to become effective. Moreover,
these agents may be useful as a quantitative marker to guide atheroscle-
rotic management in an asymptomatic patient. In those cases proceeding
to the catheterization laboratory for revascularization, nanoparticles in-
corporating antirestenotic drugs can be delivered directly into the wall
of lesions not amenable to DES placement. Targeted nanoparticles could
help ensure that antirestenotic drugs are available for all lesions. More-
over, displacement of antiproliferative agents from the intimal surface
into the vascular wall is likely to improve rehealing of the endothelium,
improving postprocedural management of these patients.
KEYWORDS: nanoparticle; angiogenesis; restenosis; thrombolysis
Address for correspondence: Prof. Gregory M. Lanza, M.D., Ph.D., Med and Biomed Engineering,
WUSTL, 4003 Kingshighway Bldg., St. Louis, MO 63130. Voice: 314-454-8813; fax: 314-454-5265.
Ann. N.Y. Acad. Sci. 1080: 451–465 (2006). C 2006 New York Academy of Sciences.
452 ANNALS NEW YORK ACADEMY OF SCIENCES
Cardiovascular disease (CVD), principally heart disease and stroke, contin-
ues to be the nation’s leading killer for both men and women across all racial
and ethnic groups. Nearly 1 million or 42% of all American deaths are due to
CVD, and these victims were not simply the elderly. Approximately 160,000
individuals between the ages of 35 and 64 years died.1 Current techniques
for early medical detection and treatment are limited and their effectiveness
in actually preventing heart attacks is debatable. In one retrospective study,
86 of 326 individuals received physical examinations within a 7-day period
prior to death from heart attack, and their physicians predicted none to have a
myocardial infarction. As tragic as this death toll is, even more grievous are
the 57 million American survivors who daily struggle with the complications
of CVD. Moreover, the direct medical and lost productivity costs to society
are staggering, approximately $274 billion each year and growing annually.
Although changes in environmental exposures, reduction in tobacco use, ad-
justments in diet, and increased physical activity can all improve patient health,
the progression of CVD is relentless in Western societies. New paradigms to
detect and treat CVD in asymptomatic patients are needed in order to prevent
the first presentation of symptoms from being the last. Improved and safer
approaches to coronary and intracranial revascularization are still required,
despite the myriad of advances in the last 10 years.
No single technology offers a solution for all problems. However, rapid
evolution of molecular biology, cell biology, genomics, and proteomics com-
bined with discoveries in material sciences and bioengineering have created
many new cadres of “nanotools” to address these challenges. Pharmaceutical
nanoparticles have emerged as multifaceted systems capable of identifying
and characterizing early disease before the gross anatomical manifestations
are easily apparent with a variety of clinically relevant imaging modalities.
Moreover, targeted particles can deliver therapeutics preferentially to sites of
pathologic disease by recognizing and binding to unique biochemical signa-
tures. The synergy of biomarker imaging and therapy is a powerful adjunctive
paradigm to current medical practice, which offers a rich palette of approaches
to address cardiovascular problems from a new perspective.
LIGAND-DIRECTED PERFLUOROCARBON NANOPARTICLES
Perfluorocarbon (PFC) nanoparticles are unusual lipid-encapsulated col-
loidal emulsions with nominal sizes between 200 nm and 250 nm. The core
of the emulsion particle (98 vol%) comprises perfluorochemicals, which
have twice the specific gravity of water and offer excellent safety pro-
files in pharmaceutical formulations. 2 The fluorine-carbon bonds of these
compounds render them both chemically and biologically inert. Chemically
stable, nonmetabolizable, and intrinsically nontoxic, perfluorochemicals have
seen use in varied human applications including blood replacement, liquid
LANZA et al.: NANOMEDICINE OPPORTUNITIES IN CARDIOLOGY 453
breathing, ocular fluid replacement, MR imaging, CT imaging, ultrasound
imaging, and percutaneous transluminal cardiac angioplasty (PTCA), with
many products approved or in development.
For imaging, the perfluorocarbon core of the nanoparticles provides inherent
acoustic contrast relative to blood and tissues due primarily to a speed-of-sound
that is one-half to one-third that of water.2–4 Moreover, this echo contrast ef-
fect can be augmented by further decreases in the speed-of-sound imparted by
heating.5 For traditional proton MR imaging, the high surface area of nanopar-
ticles increases the ionic relaxivity of each atom of gadolinium by three- to
sixfold due to the slowed rotational effects, while increasing the payloads of
paramagnetic metals from a few to 100,000 per particle greatly amplifies the
signal, that is, the molecular or particular relaxivity.6–8 As with ultrasound, the
perfluorocarbon core of the particle can contribute to the MR signal through
F imaging and spectroscopy.9–11 The high concentration of 19 F at sites tar-
geted with nanoparticles in combination with the negligible amount of fluorine
in the surrounding tissues creates a unique and inherent second marker. In ad-
dition, the fluorine signal provides a confirmation of nanoparticle delivery as
well as the quantity of particles delivered within a voxel or region independent
of the local tissue environment.
As site-targeted agents for medical applications, in vivo stability and pro-
longed circulatory clearance offers many advantages. Liquid PFC nanoparti-
cles minimize rapid systemic destruction, clearance, and coalescence without
the addition of surface polyethylene glycol groups or surfactant cross-linking,
which frequently complicate targeting efforts, interfere with drug transport, or
mask surface components such as metal chelates or bioactive agents.
ASSESSING AND TREATING ATHEROSCLEROSIS IN
ASYMPTOMATIC PATIENTS WITH PERFLUOROCARBON
Perhaps one of the most active areas of cardiovascular research of immediate
clinical significance is the quest to identify, quantify, and treat vulnerable and
unstable plaque. For some time it has been recognized that thrombosis asso-
ciated with plaque rupture is the principal cause of acute coronary syndromes
and strokes, and that these events occur more often than not in asymptomatic
vascular regions with approximately 50% diameter stenosis. Until recently, the
dogma has been that a single complex lesion was responsible for the clinical
event. But the diffuse nature of arterial tree inflammation renders many le-
sions within a vascular bed equally susceptible to extrinsic mechanical forces
modulated by sympathetic tone or direct proteolytic degradation of the fibrous
cap. Although multiple sites of rupture are uncommon as a cause of sudden
coronary death, luminal fibrin from multiple ruptures are frequent and asso-
ciated with plaque hemorrhage and superficial macrophages.12–14 These sites
of intimal fissuring, demarcated by accumulated surface fibrin, are suggested
454 ANNALS NEW YORK ACADEMY OF SCIENCES
to be responsible for the rapid angiographic progression of vascular steno-
sis in patients.15 In fact, accumulated surface fibrin may be a critical hall-
mark of lesion instability, and the sensitive and specific detection of fibrin
by nanoparticle technology may define important strategies for the preven-
tion of plaque progression and its sequellae. We7 have previously reported and
demonstrated the use of fibrin-specific paramagnetic nanoparticles for detect-
ing fibrin with MRI, while others have used small paramagnetic peptides.16,17
However, plaque rupture is a late manifestation of atherosclerotic plaque pro-
gression and further techniques are required to assess and treat the disease
earlier in its natural progression in order to achieve any meaningful clinical
One signature of atherosclerosis is the proliferation of an angiogenic vas-
culature, which frequently develops disproportionately from the vasa vasorum
in response to the metabolic activity of plaque cellular constituents.18–22 Ex-
tensive neovascular proliferation has been spatially localized to atherosclerotic
plaque, and in particular, to “culprit” lesions clinically associated with unstable
angina, myocardial infarction, and stroke. In addition, plaque angiogenesis has
been suggested to promote plaque growth, intraplaque hemorrhage, and lesion
instability. The interplay between angiogenesis and plaque development was
explored by Moulton et al.23 in Apo E −/− mice treated with antiangiogenic
therapy for 4 months (20 to 36 weeks): TNP-470, a water-soluble fumagillin
analogue, or endostatin (30 mg/kg every other day, 1.68 g/kg total dose).23 Re-
duction in plaque angiogenesis and diminished atheroma growth were noted
despite persistent elevation of total cholesterol levels. TNP-470 and its parent
compound, fumagillin, directly inhibit endothelial cell proliferation by cova-
lently binding to methionine aminopeptidase 2 specifically, which catalyzes
the cleavage of N-terminal methionine from nascent polypeptides.24–26 Un-
fortunately, chronic, high doses of TNP-470 administered systemically have
caused neurocognitive side effects in humans.27,28
Site-targeted nanoparticles offer the opportunity for local drug delivery in
combination with molecular imaging, which can provide noninvasive confir-
mation of targeting, spatial localization of drug distribution, and quantifica-
tion of therapeutic payload accumulated at the site. This concept was initially
demonstrated in vitro using doxorubicin and paclitaxel nanoparticles to inhibit
the proliferation of vascular smooth muscle cells.29 At that time, we proposed
that targeted perfluorocarbon nanoparticles could deliver chemotherapeutic
agents through a novel mechanism we called “contact facilitated lipid ex-
change.” Subsequent studies using confocal microscopy have illustrated the
exchange of fluorescent-labeled phospholipids from the outer surfactant layer
of the particle to the target cell membrane.30
Using a hyperlipidemic New Zealand White rabbit model, we initially
demonstrated the antiangiogenic effectiveness of v 3 -targeted fumagillin
nanoparticles administered as a single dose,31,32 which was several orders
of magnitude less than used systemically in the ApoE model.23 In that study,
LANZA et al.: NANOMEDICINE OPPORTUNITIES IN CARDIOLOGY 455
hyperlipidemic rabbits (∼80 days on diet) were injected via the ear vein with
v 3 -targeted fumagillin nanoparticles (n = 5), v 3 -targeted nanoparticles
without fumagillin (n = 6), or nontargeted fumagillin nanoparticles (n = 6)
at 1.0 mL/kg. Four hours after nanoparticle injection, rabbits were reimaged
to assess the magnitude and distribution of signal enhancement. Multislice,
T 1 -weighted, spin-echo, fat-suppressed, black-blood images of the entire ab-
dominal aorta from the renal arteries to the diaphragm (TR = 380 msec, TE =
11 msec, 250 × 250 m inplane resolution, 5 mm slice thickness, number
of signals averaged = 8) were acquired. After treatment, all rabbits were
converted to normal rabbit chow (Purina Mills). One week later, the extent
of v 3 -integrin expression in each animal was reassessed by injection of
integrin-targeted paramagnetic nanoparticles (1.0 mL/kg; no drug) and nonin-
vasive imaging as described earlier. MRI signal enhancement from the aortic
wall was averaged over all imaged slices using a custom, semiautomated seg-
mentation program previously described.33 Signal enhancement in the aortic
wall was measured for each individual animal using all properly segmented
slices. The percentage enhancement in MRI signal was calculated slice-by-
slice in the 4-h postinjection images relative to the average preinjection MRI
Consistent with the early stage of atherosclerosis in this animal model,
T 1 -weighted, black-blood images showed no gross evidence of plaque devel-
opment in terms of luminal narrowing or wall thickening when compared to
previous experiments using age-matched, nonatherosclerotic rabbits.33 MRI
signal enhancement in the aortic wall following injection of 3 -targeted
nanoparticles, both with and without fumagillin, displayed a patchy distribu-
tion, with typically higher levels of angiogenesis occurring near the diaphragm.
Nontargeted nanoparticles produced less extensive MRI enhancement of the
neovasculature at much lower levels with a similar heterogeneous distribution,
consistent with previous reports. The average MRI signal enhancement per
slice integrated across the entire aortic wall was identical for 3 -targeted
nanoparticles with (16.7 ± 1.1%) and without (16.7 ± 1.6%) fumagillin. Non-
targeted nanoparticles, however, provided less signal enhancement, presum-
ably representing nonspecific accumulation and/or delayed washout within the
tortuous microvasculature. 34
One week after nanoparticle treatment, the residual expression of 3-
integrin was assessed as a marker of angiogenic activity within the aortic
wall. Preinjection scans were collected, followed by injection of 3 -targeted
paramagnetic nanoparticles (no drug) and contrast enhancement imaging 4 h
post injection. The preinjection aortic wall signal intensities for all groups
at treatment and at the 1-week follow-up were identical, confirming that the
paramagnetic nanoparticles administered 1 week prior were no longer de-
tectable. MRI aortic wall signal enhancement 1 week following 3 -targeted
fumagillin nanoparticle treatment was markedly reduced (2.9 ± 1.6%; P <
0.05) in both spatial distribution as well as intensity. By comparison, MRI
456 ANNALS NEW YORK ACADEMY OF SCIENCES
FIGURE 1. MRI aortic wall signal enhancement with 3 -targeted paramagnetic
nanoparticle (no drug) 1 week following treatment with 3 -targeted fumagillin or control
(no drug) nanoparticles.
signal enhancement 1 week after treatment with 3 -targeted nanoparti-
cles lacking fumagillin was undiminished (18.1 ± 2.1%) (FIG. 1). Treatment
with nontargeted fumagillin nanoparticles did not significantly diminish 3-
integrin levels as determined by MRI signal enhancement 1 week after treat-
ment, although a numerical decrease was observed (12.4 ± 0.9%).
In this study, the total dose of fumagillin administered as a single injection in
3 -targeted nanoparticles was >10,000 times lower than the cumulative oral
dose of TNP-470 reported by Moulton et al. Reduced 3 -integrin expression
as determined by MRI molecular imaging and corroborated by decreased 3-
integrin positive vessel density supported the potential reduction in dosage and
increase in efficacy of chemotherapeutic agents using targeted nanomedicine
approaches. Incorporation of fumagillin into paramagnetic nanoparticles al-
lowed both aortic expression of 3 -integrin and local drug delivery to be
assessed and quantified concomitantly and noninvasively. Moreover, the initial
magnitude of the aortic signal response among hyperlipidemic rabbits receiv-
ing 3 -targeted paramagnetic nanoparticles was correlated with the degree
of change in MRI enhancement measured 7 days later with 3 -targeted
paramagnetic nanoparticles (without drug). These data illustrate the concept
of “rational drug dosing,” which provides noninvasive measures of treatment
dosimetry and allows follow-up of response.
For asymptomatic patients, nanomedicine approaches offer the ability to
quantitatively interrogate the severity and distribution of disease more directly,
to locally treat pathology with minimal doses, and to follow up response to
LANZA et al.: NANOMEDICINE OPPORTUNITIES IN CARDIOLOGY 457
treatment noninvasively when there are no clinical symptoms or meaningful
measures with which to titrate treatment efficacy.
NANOMEDICINE APPROACH TO CORONARY
Far too often the progression of atherosclerosis to acute coronary syndromes
presents the need for acute revascularization. Fortunately, continuous advances
from balloon angioplasty, bare-metal stents, and drug-covered stents such as
heparin-coated stents, to the more recent, new class of drug-eluting stents
(DES), have expanded our armamentarium for reopening stenotic vessels while
preventing vascular reocclusion. Within the last few years, the use of conven-
tional balloon angioplasty and bare-metal stent implantation, which were asso-
ciated with clinical restenosis rates of 32–42% and 19–30%, respectively,35–37
have been improved with the local deposition of a pharmacologic agent to
suppress neointimal proliferation. Current DES have reduced the rate of an-
giographic restenosis to below 9% and diminished the frequency for repeat
revascularization to below 5%.38,39 Unfortunately, DES cannot be routinely
used for all lesions. In some situations, vessel tortuosity or the distal location
of lesions prevents manipulation of the relatively inflexible DES. In other cases,
the vessel diameter at the culprit lesion is too small for stent placement. As
a result many lesions, in whole or part, do not receive the local antirestenotic
therapy after revascularization.
Moreover, despite the clear success of DES, the incidence of late instent
thrombosis has arisen as an infrequent but serious complication of delayed
endothelial healing.40 To avoid acute thrombosis, aggressive dual (and occa-
sionally triple) antiplatelet therapy is employed for 6 months to a year. We
now recognize that some patients are nonresponders to one or more of the
drugs.41–43 In other instances, thrombosis presents when antithrombotic drugs
are withheld secondary to bleeding complications or the need for emergent
surgery. Late instent thrombosis has been linked to fatal outcomes,40 and the
risk can persist up to 30 months after DES implantation.42,43 We anticipate that
targeted local delivery of antirestenotic drugs such as paclitaxel or rapamycin
into the stretch-injured arterial wall rather than the intimal surface will permit
better healing and recovery of the endothelium. More rapid endothelial repair
of the injured wall should substantially diminish the incidence of thrombosis
and reduce the long-term requirement for aggressive antiplatelet therapy.
Moreover, DES are now known to elicit unwanted effects on vessel healing
and local endothelium-dependent vasomotor responses 6 months after implan-
tation in the vessel distal to the intervened segment.44 Although still limited,
data on the effect of sirolimus on vasomotor response are accumulating in
animal models and patients alike. Swine coronary artery segments exposed
to sirolimus for 48 h showed severe impairment of endothelial function.45
In patients, exercise-induced coronary vasoconstriction was noted in vessel
458 ANNALS NEW YORK ACADEMY OF SCIENCES
segments adjacent to DES but not bare-metal stents.46 The mechanism for
this effect is unclear. Higher rates of restenosis proximal to stent placement
compared with the distal edge support an asymmetric downstream effect.
We use a nanomedicine approach to address restenosis in lesions not
amenable to current DES stent technology by intramural targeting and an-
choring of rapamycin nanoparticles to the v 3 -integrin, present on smooth
muscle and other plaque components (e.g., macrophages, T cells). In previous
studies, we demonstrated that ligand-directed perfluorocarbon nanoparticles
could penetrate balloon-injured vessel walls and target intramural biomark-
ers, including tissue factor,47 collagen III, and integrins.48 We have recently
reported that integrin-targeted PFC nanoparticles can provide effective intra-
mural delivery of rapamycin and inhibit vascular stenosis following balloon
overstretch injury. In these studies, femoral arteries of 12 rabbits on athero-
genic diets for 3 weeks were subjected to balloon stretch injury via a catheter
approach from the left common carotid artery. Using a double-balloon tech-
nique, paramagnetic 3 -nanoparticles with rapamycin were administered to
one artery while the contralateral vessel received targeted nanoparticles with-
out drug or saline. Two weeks after nanoparticle treatment, plaque development
was determined by MR angiography and by microscopic morphometric quan-
tification. Routine MR angiograms were indistinguishable between control
and targeted-vessel segments. Microscopic analysis of serial vascular sections
2 weeks after injury revealed that the intimal plaque to lumen area ratio of
vessels treated with 3 -targeted rapamycin nanoparticles were significantly
(P < 0.05) less (∼50%) than arteries receiving targeted nanoparticles without
drug or saline (FIG. 2). Scanning electron microscopy of the intima performed
24 h after injury and treatment with 3 -targeted rapamycin nanoparticles
demonstrated their binding to the underlying matrix and cells. Immunofluo-
rescent imaging of the vessel wall ∼2 h after treatment demonstrated that the
nanoparticles had penetrated into the media and adventitia, consistent with
the MR images previously obtained. Although early, the results suggest that
3 -integrin-targeted nanoparticles can provide effective intramural therapy
and may be a tool to extend the use of antirestenotic drugs to all revascularized
sites with or without adjunctive stent placement.
NANOMEDICINE APPROACH TO THROMBOLYSIS
Stroke is the third leading cause of death in the United States and often
results in functional impairment and long-term disability among survivors.49
Clinical trials have demonstrated that thrombolytic treatment such as tissue
plasminogen activator (t-PA) can reduce or reverse ischemia in patients treated
within the first 3 h of onset.50 However, intravenously administered throm-
bolytic agents are associated with an increased incidence of intracerebral hem-
orrhage and expanding stroke. This serious risk frequently delays the receipt
of aggressive thrombolytic therapy until intracranial bleeding can be ruled out
LANZA et al.: NANOMEDICINE OPPORTUNITIES IN CARDIOLOGY 459
FIGURE 2. Microscopic analysis of serial vascular sections 2 weeks after injury re-
vealed that the intimal plaque to lumen area ratio of vessels treated with 3 -targeted
rapamycin nanoparticles were significantly (P < 0.05) less than arteries receiving targeted
nanoparticles without drug or saline.
by CT study of the head. As a result of these delays, the window of opportunity
to ameliorate neural damage is lost, and the personal and societal losses are
The advent of perfluorocarbon nanoparticles to specifically deliver drug
payloads to intravascular sites of interest presents a unique opportunity to tar-
get clot-dissolving therapeutics to cerebral sites of embolism while decreas-
ing the risk of hemorrhagic complications and increasing the effectiveness
of thrombolytic therapy. We have previously demonstrated targeting of liquid
perfluorocarbon nanoparticle emulsions to thrombi in vitro and in vivo,51 with
concomitant enhancement of acoustic reflectivity from the targeted surfaces.
Acoustic reflectivity enhancement of surfaces targeted with the nanoparticles
arises because of the acoustic impedance mismatch between the adherent layer
of nanoparticles and the surrounding media. We have recently demonstrated
that nanoparticles modified with thrombolytic enzyme (streptokinase) can be
targeted onto plasma clots and effect rapid dissolution in the presence of plas-
minogen. In this series of experiments, acellular thrombi were produced from
citrated human plasma combined with 500 mM calcium chloride and thrombin.
Since this study was conducted in vitro, targeting of the nanoparticles to fibrin
was accomplished using a three-step process in which biotinylated antifibrin
antibody (NIB 1H10 52 ), avidin, and biotinylated nanoparticle emulsions (with
or without streptokinase, depending on treatment group) were combined se-
quentially with interval washings of unbound reagents. Acoustic microscopy
was performed on targeted and control samples using a broadband, 25-MHz
immersion transducer (Panametrics V324, Waltham, MA, USA) operated in
460 ANNALS NEW YORK ACADEMY OF SCIENCES
pulse-echo mode. A computer-controlled pulser receiver was used to generate
insonifying pulses and amplify the received echoes. The transducer was affixed
to a three-axis, computer-controlled, motorized gantry. Radiofrequency (RF)
data were acquired, digitized to 8 bits at 500 MHz for 2,048 point records, and
stored to disk at every site as the transducer was scanned over each sample in
a rectangular grid with 100- m resolution. In preparation for scanning, each
sample was sealed within a chamber having a cellophane acoustic window, and
which was filled with phosphate buffered saline (PBS). The sample chamber
was submerged within a 37◦ C water bath, and the sample was scanned to yield
a baseline measurement. The chamber was then emptied of PBS through an
injection port and refilled with either plasminogen in PBS buffer (3 U/mL) or
PBS alone. Scans were then performed at 15-min intervals for 3 h, and spatial
registration was maintained at all times.
RF data were analyzed to assess temporal changes in clot morphology and
backscatter. A sliding Hamming window (0.2 sec duration) was applied and
moved over the data in 2-nsec steps. The sum of the squared values within each
segment, a quantity proportional to the reflected energy, was used as input to
a peak-detection algorithm (implemented in LabVIEW, National Instruments
Corp., Austin, TX, USA) and used to determine the arrival time of the echo
from the thrombus surface. A similar technique was used to detect the echo
from the nitrocellulose substrate in the same waveform. The difference between
the echo arrival times of the clot surface and substrate determined the profile of
the clot, and these values were used to generate surface plots for visualization of
the sample volume. Backscatter was quantified by first applying a rectangular
window to each waveform to isolate the reflection from the targeted surface,
and then by calculating the log spectral difference with respect to the reflection
from a steel plate. The average value within the usable bandwidth (10–30 MHz)
was recorded in dB for each point in the scan, and this value was used to generate
a C-scan image of the integrated backscatter from the sample surface.
The detected clot volume was dramatically decreased (P < 0.05) for
clots treated with fibrin-targeted streptokinase nanoparticles and exposed to
plasminogen in buffer (FIG. 3). Treatment with fibrin-targeted streptokinase
nanoparticles incubated in saline or fibrin-targeted nanoparticles without strep-
tokinase incubated with plasminogen in buffer had no thrombolytic effect. The
time to complete lysis varied with small changes in the synthesis process of
the streptokinase nanoparticle formulations. Initial conjugates had to be left
overnight for complete dissolution while more optimized agents formed later
completely dissolved clots in vitro within an hour, often less than 15 min.
Fibrin clot dissolution occurred from inside to outside. In some replicates, the
clot measured at 1 h was a hollow fibrin shell, which immediately collapsed
with slight motion. None of the control clots revealed morphologic or acoustic
The measurements presented here suggest that fibrin-targeted streptokinase
nanoparticles could be used to promote local thrombolysis of plasma clots
in vivo. We have previously shown that fibrin-targeted nanoparticles can
LANZA et al.: NANOMEDICINE OPPORTUNITIES IN CARDIOLOGY 461
FIGURE 3. Mean normalized clot volume following 2-h treatment with fibrin-targeted
nanoparticles streptokinase-modified or control perfluorocarbon nanoparticles incubated
with plasminogen or saline.
penetrate and acoustically enhance acute intravascular thromboses in dogs.
Moreover, we have found that perfluorocarbon nanoparticles are constrained
to the vasculature due to their nominal size, even in “leaky” vascular beds
such as tumor neovasculature. Collectively, those results suggest that fibrin-
targeted streptokinase nanoparticles could be used early in acute stroke or
unstable angina with limited extravascular effects.
Nanomedicine is a new evolving field referred to by many names, which
promises to significantly enhance the tools available to clinicians to address
some of the serious challenges responsible for profound mortality, morbidity,
and numerous societal consequences. Unlike the simple pharmaceutics of the
past, nanomedicine agents are typically three-dimensional, multicomponent
systems, which require interdisciplinary expertise to produce and use. In this
review, we have briefly introduced the opportunities associated with targeted
perfluorocarbon nanoparticles in early atherosclerosis, in acute revasculariza-
tion, and in thrombolytic therapy. The potential impact of these three concepts
is enormous but pales in comparison with the advancements likely to evolve
in this field over the coming decades.
This research was supported by the NIH grants (HL-42950, HL-59865,
HL-78631, NO1-CO-37007, and EB-01704), SCAI/Bracco Diagnostics, Inc.-
ACIST Fellowship Program, and the American Heart Association. Philips
462 ANNALS NEW YORK ACADEMY OF SCIENCES
Medical Systems (Cleveland, OH, USA) provided valuable equipment, soft-
ware, and engineering support.
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