nanomedicina1

Nanomedicine Opportunitiesin Cardiology

GREGORY LANZA,a PATRICK WINTER,a TILLMANN CYRUS,a

SHELTON CARUTHERS,a,b JON MARSH,a MICHAEL HUGHES,a

AND SAMUEL WICKLINEa

aWashington University School of Medicine, St. Louis, Missouri 63110, USAbPhilips Medical Systems, Cleveland, Ohio 44143, USA

ABSTRACT: Despite myriad advances, cardiovascular-related diseasescontinue 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 eventthrough 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 thesepatients, the diagnostic and therapeutic options are woefully inadequate.Nanomedicine offers options to each of these challenges. Antiangiogenicparamagnetic nanoparticles may be used to serially assess the severity ofatherosclerotic disease in asymptomatic, high-risk patients by detectingthe development of plaque neovasculature, which reflects the underlyinglesion activity and vulnerability to rupture. The nanoparticles can locallydeliver 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 proceedingto the catheterization laboratory for revascularization, nanoparticles in-corporating antirestenotic drugs can be delivered directly into the wallof lesions not amenable to DES placement. Targeted nanoparticles couldhelp ensure that antirestenotic drugs are available for all lesions. More-over, displacement of antiproliferative agents from the intimal surfaceinto 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.

e-mail: [email protected]

Ann. N.Y. Acad. Sci. 1080: 451–465 (2006). C© 2006 New York Academy of Sciences.doi: 10.1196/annals.1380.034

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452 ANNALS NEW YORK ACADEMY OF SCIENCES

INTRODUCTION

Cardiovascular disease (CVD), principally heart disease and stroke, contin-ues to be the nation’s leading killer for both men and women across all racialand ethnic groups. Nearly 1 million or 42% of all American deaths are due toCVD, and these victims were not simply the elderly. Approximately 160,000individuals between the ages of 35 and 64 years died.1 Current techniquesfor early medical detection and treatment are limited and their effectivenessin actually preventing heart attacks is debatable. In one retrospective study,86 of 326 individuals received physical examinations within a 7-day periodprior to death from heart attack, and their physicians predicted none to have amyocardial infarction. As tragic as this death toll is, even more grievous arethe 57 million American survivors who daily struggle with the complicationsof CVD. Moreover, the direct medical and lost productivity costs to societyare 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 todetect and treat CVD in asymptomatic patients are needed in order to preventthe first presentation of symptoms from being the last. Improved and saferapproaches 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, rapidevolution of molecular biology, cell biology, genomics, and proteomics com-bined with discoveries in material sciences and bioengineering have createdmany new cadres of “nanotools” to address these challenges. Pharmaceuticalnanoparticles have emerged as multifaceted systems capable of identifyingand characterizing early disease before the gross anatomical manifestationsare easily apparent with a variety of clinically relevant imaging modalities.Moreover, targeted particles can deliver therapeutics preferentially to sites ofpathologic disease by recognizing and binding to unique biochemical signa-tures. The synergy of biomarker imaging and therapy is a powerful adjunctiveparadigm to current medical practice, which offers a rich palette of approachesto 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 coreof the emulsion particle (98 vol%) comprises perfluorochemicals, whichhave twice the specific gravity of water and offer excellent safety pro-files in pharmaceutical formulations. 2 The fluorine-carbon bonds of thesecompounds render them both chemically and biologically inert. Chemicallystable, nonmetabolizable, and intrinsically nontoxic, perfluorochemicals haveseen 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, ultrasoundimaging, and percutaneous transluminal cardiac angioplasty (PTCA), withmany products approved or in development.

For imaging, the perfluorocarbon core of the nanoparticles provides inherentacoustic contrast relative to blood and tissues due primarily to a speed-of-soundthat 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 byheating.5 For traditional proton MR imaging, the high surface area of nanopar-ticles increases the ionic relaxivity of each atom of gadolinium by three- tosixfold due to the slowed rotational effects, while increasing the payloads ofparamagnetic metals from a few to 100,000 per particle greatly amplifies thesignal, that is, the molecular or particular relaxivity.6–8 As with ultrasound, theperfluorocarbon core of the particle can contribute to the MR signal through19F imaging and spectroscopy.9–11 The high concentration of 19F at sites tar-geted with nanoparticles in combination with the negligible amount of fluorinein the surrounding tissues creates a unique and inherent second marker. In ad-dition, the fluorine signal provides a confirmation of nanoparticle delivery aswell as the quantity of particles delivered within a voxel or region independentof 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 withoutthe addition of surface polyethylene glycol groups or surfactant cross-linking,which frequently complicate targeting efforts, interfere with drug transport, ormask surface components such as metal chelates or bioactive agents.

ASSESSING AND TREATING ATHEROSCLEROSIS INASYMPTOMATIC PATIENTS WITH PERFLUOROCARBON

NANOPARTICLES

Perhaps one of the most active areas of cardiovascular research of immediateclinical significance is the quest to identify, quantify, and treat vulnerable andunstable plaque. For some time it has been recognized that thrombosis asso-ciated with plaque rupture is the principal cause of acute coronary syndromesand strokes, and that these events occur more often than not in asymptomaticvascular regions with approximately 50% diameter stenosis. Until recently, thedogma has been that a single complex lesion was responsible for the clinicalevent. But the diffuse nature of arterial tree inflammation renders many le-sions within a vascular bed equally susceptible to extrinsic mechanical forcesmodulated by sympathetic tone or direct proteolytic degradation of the fibrouscap. Although multiple sites of rupture are uncommon as a cause of suddencoronary death, luminal fibrin from multiple ruptures are frequent and asso-ciated with plaque hemorrhage and superficial macrophages.12–14 These sitesof intimal fissuring, demarcated by accumulated surface fibrin, are suggested


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 fibrinby nanoparticle technology may define important strategies for the preven-tion of plaque progression and its sequellae. We7 have previously reported anddemonstrated 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 diseaseearlier in its natural progression in order to achieve any meaningful clinicalimpact.

One signature of atherosclerosis is the proliferation of an angiogenic vas-culature, which frequently develops disproportionately from the vasa vasorumin response to the metabolic activity of plaque cellular constituents.18–22 Ex-tensive neovascular proliferation has been spatially localized to atheroscleroticplaque, and in particular, to “culprit” lesions clinically associated with unstableangina, myocardial infarction, and stroke. In addition, plaque angiogenesis hasbeen suggested to promote plaque growth, intraplaque hemorrhage, and lesioninstability. The interplay between angiogenesis and plaque development wasexplored by Moulton et al.23 in Apo E −/− mice treated with antiangiogenictherapy for 4 months (20 to 36 weeks): TNP-470, a water-soluble fumagillinanalogue, 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 noteddespite persistent elevation of total cholesterol levels. TNP-470 and its parentcompound, fumagillin, directly inhibit endothelial cell proliferation by cova-lently binding to methionine aminopeptidase 2 specifically, which catalyzesthe cleavage of N-terminal methionine from nascent polypeptides.24–26 Un-fortunately, chronic, high doses of TNP-470 administered systemically havecaused neurocognitive side effects in humans.27,28

Site-targeted nanoparticles offer the opportunity for local drug delivery incombination 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 initiallydemonstrated in vitro using doxorubicin and paclitaxel nanoparticles to inhibitthe proliferation of vascular smooth muscle cells.29 At that time, we proposedthat targeted perfluorocarbon nanoparticles could deliver chemotherapeuticagents through a novel mechanism we called “contact facilitated lipid ex-change.” Subsequent studies using confocal microscopy have illustrated theexchange of fluorescent-labeled phospholipids from the outer surfactant layerof the particle to the target cell membrane.30

Using a hyperlipidemic New Zealand White rabbit model, we initiallydemonstrated the antiangiogenic effectiveness of �v�3-targeted fumagillinnanoparticles administered as a single dose,31,32 which was several ordersof magnitude less than used systemically in the ApoE model.23 In that study,


hyperlipidemic rabbits (∼80 days on diet) were injected via the ear vein with�v�3-targeted fumagillin nanoparticles (n = 5), �v�3-targeted nanoparticleswithout fumagillin (n = 6), or nontargeted fumagillin nanoparticles (n = 6)at 1.0 mL/kg. Four hours after nanoparticle injection, rabbits were reimagedto assess the magnitude and distribution of signal enhancement. Multislice,T1-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, numberof signals averaged = 8) were acquired. After treatment, all rabbits wereconverted to normal rabbit chow (Purina Mills). One week later, the extentof �v�3-integrin expression in each animal was reassessed by injection ofintegrin-targeted paramagnetic nanoparticles (1.0 mL/kg; no drug) and nonin-vasive imaging as described earlier. MRI signal enhancement from the aorticwall was averaged over all imaged slices using a custom, semiautomated seg-mentation program previously described.33 Signal enhancement in the aorticwall was measured for each individual animal using all properly segmentedslices. The percentage enhancement in MRI signal was calculated slice-by-slice in the 4-h postinjection images relative to the average preinjection MRIsignal.

Consistent with the early stage of atherosclerosis in this animal model,T1-weighted, black-blood images showed no gross evidence of plaque devel-opment in terms of luminal narrowing or wall thickening when compared toprevious experiments using age-matched, nonatherosclerotic rabbits.33 MRIsignal enhancement in the aortic wall following injection of ��3-targetednanoparticles, 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 theneovasculature at much lower levels with a similar heterogeneous distribution,consistent with previous reports. The average MRI signal enhancement perslice integrated across the entire aortic wall was identical for ��3-targetednanoparticles 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 thetortuous microvasculature. 34

One week after nanoparticle treatment, the residual expression of ��3-integrin was assessed as a marker of angiogenic activity within the aorticwall. Preinjection scans were collected, followed by injection of ��3-targetedparamagnetic nanoparticles (no drug) and contrast enhancement imaging 4 hpost injection. The preinjection aortic wall signal intensities for all groupsat treatment and at the 1-week follow-up were identical, confirming that theparamagnetic nanoparticles administered 1 week prior were no longer de-tectable. MRI aortic wall signal enhancement 1 week following ��3-targetedfumagillin nanoparticle treatment was markedly reduced (2.9 ± 1.6%; P <

0.05) in both spatial distribution as well as intensity. By comparison, MRI


FIGURE 1. MRI aortic wall signal enhancement with ��3-targeted paramagneticnanoparticle (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). Treatmentwith 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 oraldose of TNP-470 reported by Moulton et al. Reduced ��3-integrin expressionas determined by MRI molecular imaging and corroborated by decreased ��3-integrin positive vessel density supported the potential reduction in dosage andincrease in efficacy of chemotherapeutic agents using targeted nanomedicineapproaches. Incorporation of fumagillin into paramagnetic nanoparticles al-lowed both aortic expression of ��3-integrin and local drug delivery to beassessed and quantified concomitantly and noninvasively. Moreover, the initialmagnitude of the aortic signal response among hyperlipidemic rabbits receiv-ing ��3-targeted paramagnetic nanoparticles was correlated with the degreeof change in MRI enhancement measured 7 days later with ��3-targetedparamagnetic nanoparticles (without drug). These data illustrate the conceptof “rational drug dosing,” which provides noninvasive measures of treatmentdosimetry and allows follow-up of response.

For asymptomatic patients, nanomedicine approaches offer the ability toquantitatively interrogate the severity and distribution of disease more directly,to locally treat pathology with minimal doses, and to follow up response to


treatment noninvasively when there are no clinical symptoms or meaningfulmeasures with which to titrate treatment efficacy.

NANOMEDICINE APPROACH TO CORONARYREVASCULARIZATION

Far too often the progression of atherosclerosis to acute coronary syndromespresents the need for acute revascularization. Fortunately, continuous advancesfrom balloon angioplasty, bare-metal stents, and drug-covered stents such asheparin-coated stents, to the more recent, new class of drug-eluting stents(DES), have expanded our armamentarium for reopening stenotic vessels whilepreventing 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 tosuppress neointimal proliferation. Current DES have reduced the rate of an-giographic restenosis to below 9% and diminished the frequency for repeatrevascularization to below 5%.38,39 Unfortunately, DES cannot be routinelyused for all lesions. In some situations, vessel tortuosity or the distal locationof lesions prevents manipulation of the relatively inflexible DES. In other cases,the vessel diameter at the culprit lesion is too small for stent placement. Asa result many lesions, in whole or part, do not receive the local antirestenotictherapy after revascularization.

Moreover, despite the clear success of DES, the incidence of late instentthrombosis has arisen as an infrequent but serious complication of delayedendothelial healing.40 To avoid acute thrombosis, aggressive dual (and occa-sionally triple) antiplatelet therapy is employed for 6 months to a year. Wenow recognize that some patients are nonresponders to one or more of thedrugs.41–43 In other instances, thrombosis presents when antithrombotic drugsare withheld secondary to bleeding complications or the need for emergentsurgery. Late instent thrombosis has been linked to fatal outcomes,40 and therisk can persist up to 30 months after DES implantation.42,43 We anticipate thattargeted local delivery of antirestenotic drugs such as paclitaxel or rapamycininto the stretch-injured arterial wall rather than the intimal surface will permitbetter healing and recovery of the endothelium. More rapid endothelial repairof the injured wall should substantially diminish the incidence of thrombosisand reduce the long-term requirement for aggressive antiplatelet therapy.

Moreover, DES are now known to elicit unwanted effects on vessel healingand 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 inanimal models and patients alike. Swine coronary artery segments exposedto sirolimus for 48 h showed severe impairment of endothelial function.45

In patients, exercise-induced coronary vasoconstriction was noted in vessel


segments adjacent to DES but not bare-metal stents.46 The mechanism forthis effect is unclear. Higher rates of restenosis proximal to stent placementcompared with the distal edge support an asymmetric downstream effect.

We use a nanomedicine approach to address restenosis in lesions notamenable to current DES stent technology by intramural targeting and an-choring of rapamycin nanoparticles to the �v�3-integrin, present on smoothmuscle and other plaque components (e.g., macrophages, T cells). In previousstudies, we demonstrated that ligand-directed perfluorocarbon nanoparticlescould penetrate balloon-injured vessel walls and target intramural biomark-ers, including tissue factor,47 collagen III, and integrins.48 We have recentlyreported that integrin-targeted PFC nanoparticles can provide effective intra-mural delivery of rapamycin and inhibit vascular stenosis following balloonoverstretch injury. In these studies, femoral arteries of 12 rabbits on athero-genic diets for 3 weeks were subjected to balloon stretch injury via a catheterapproach from the left common carotid artery. Using a double-balloon tech-nique, paramagnetic ��3-nanoparticles with rapamycin were administered toone artery while the contralateral vessel received targeted nanoparticles with-out drug or saline. Two weeks after nanoparticle treatment, plaque developmentwas determined by MR angiography and by microscopic morphometric quan-tification. Routine MR angiograms were indistinguishable between controland targeted-vessel segments. Microscopic analysis of serial vascular sections2 weeks after injury revealed that the intimal plaque to lumen area ratio ofvessels treated with ��3-targeted rapamycin nanoparticles were significantly(P < 0.05) less (∼50%) than arteries receiving targeted nanoparticles withoutdrug or saline (FIG. 2). Scanning electron microscopy of the intima performed24 h after injury and treatment with ��3-targeted rapamycin nanoparticlesdemonstrated their binding to the underlying matrix and cells. Immunofluo-rescent imaging of the vessel wall ∼2 h after treatment demonstrated that thenanoparticles had penetrated into the media and adventitia, consistent withthe MR images previously obtained. Although early, the results suggest that��3-integrin-targeted nanoparticles can provide effective intramural therapyand may be a tool to extend the use of antirestenotic drugs to all revascularizedsites with or without adjunctive stent placement.

NANOMEDICINE APPROACH TO THROMBOLYSIS

Stroke is the third leading cause of death in the United States and oftenresults in functional impairment and long-term disability among survivors.49

Clinical trials have demonstrated that thrombolytic treatment such as tissueplasminogen activator (t-PA) can reduce or reverse ischemia in patients treatedwithin 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 receiptof aggressive thrombolytic therapy until intracranial bleeding can be ruled out


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-targetedrapamycin nanoparticles were significantly (P < 0.05) less than arteries receiving targetednanoparticles without drug or saline.

by CT study of the head. As a result of these delays, the window of opportunityto ameliorate neural damage is lost, and the personal and societal losses aremagnified.

The advent of perfluorocarbon nanoparticles to specifically deliver drugpayloads 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 effectivenessof thrombolytic therapy. We have previously demonstrated targeting of liquidperfluorocarbon nanoparticle emulsions to thrombi in vitro and in vivo,51 withconcomitant enhancement of acoustic reflectivity from the targeted surfaces.Acoustic reflectivity enhancement of surfaces targeted with the nanoparticlesarises because of the acoustic impedance mismatch between the adherent layerof nanoparticles and the surrounding media. We have recently demonstratedthat nanoparticles modified with thrombolytic enzyme (streptokinase) can betargeted onto plasma clots and effect rapid dissolution in the presence of plas-minogen. In this series of experiments, acellular thrombi were produced fromcitrated human plasma combined with 500 mM calcium chloride and thrombin.Since this study was conducted in vitro, targeting of the nanoparticles to fibrinwas accomplished using a three-step process in which biotinylated antifibrinantibody (NIB 1H10 52), avidin, and biotinylated nanoparticle emulsions (withor without streptokinase, depending on treatment group) were combined se-quentially with interval washings of unbound reagents. Acoustic microscopywas performed on targeted and control samples using a broadband, 25-MHzimmersion transducer (Panametrics V324, Waltham, MA, USA) operated in


pulse-echo mode. A computer-controlled pulser receiver was used to generateinsonifying pulses and amplify the received echoes. The transducer was affixedto a three-axis, computer-controlled, motorized gantry. Radiofrequency (RF)data were acquired, digitized to 8 bits at 500 MHz for 2,048 point records, andstored to disk at every site as the transducer was scanned over each sample ina rectangular grid with 100-�m resolution. In preparation for scanning, eachsample was sealed within a chamber having a cellophane acoustic window, andwhich was filled with phosphate buffered saline (PBS). The sample chamberwas submerged within a 37◦C water bath, and the sample was scanned to yielda baseline measurement. The chamber was then emptied of PBS through aninjection port and refilled with either plasminogen in PBS buffer (3 U/mL) orPBS alone. Scans were then performed at 15-min intervals for 3 h, and spatialregistration was maintained at all times.

RF data were analyzed to assess temporal changes in clot morphology andbackscatter. A sliding Hamming window (0.2 �sec duration) was applied andmoved over the data in 2-nsec steps. The sum of the squared values within eachsegment, a quantity proportional to the reflected energy, was used as input toa peak-detection algorithm (implemented in LabVIEW, National InstrumentsCorp., Austin, TX, USA) and used to determine the arrival time of the echofrom the thrombus surface. A similar technique was used to detect the echofrom the nitrocellulose substrate in the same waveform. The difference betweenthe echo arrival times of the clot surface and substrate determined the profile ofthe clot, and these values were used to generate surface plots for visualization ofthe sample volume. Backscatter was quantified by first applying a rectangularwindow to each waveform to isolate the reflection from the targeted surface,and then by calculating the log spectral difference with respect to the reflectionfrom 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 generatea C-scan image of the integrated backscatter from the sample surface.

The detected clot volume was dramatically decreased (P < 0.05) forclots treated with fibrin-targeted streptokinase nanoparticles and exposed toplasminogen in buffer (FIG. 3). Treatment with fibrin-targeted streptokinasenanoparticles incubated in saline or fibrin-targeted nanoparticles without strep-tokinase incubated with plasminogen in buffer had no thrombolytic effect. Thetime to complete lysis varied with small changes in the synthesis process ofthe streptokinase nanoparticle formulations. Initial conjugates had to be leftovernight for complete dissolution while more optimized agents formed latercompletely dissolved clots in vitro within an hour, often less than 15 min.Fibrin clot dissolution occurred from inside to outside. In some replicates, theclot measured at 1 h was a hollow fibrin shell, which immediately collapsedwith slight motion. None of the control clots revealed morphologic or acousticchanges.

The measurements presented here suggest that fibrin-targeted streptokinasenanoparticles could be used to promote local thrombolysis of plasma clotsin vivo. We have previously shown that fibrin-targeted nanoparticles can


FIGURE 3. Mean normalized clot volume following 2-h treatment with fibrin-targetednanoparticles streptokinase-modified or control perfluorocarbon nanoparticles incubatedwith plasminogen or saline.

penetrate and acoustically enhance acute intravascular thromboses in dogs.Moreover, we have found that perfluorocarbon nanoparticles are constrainedto the vasculature due to their nominal size, even in “leaky” vascular bedssuch as tumor neovasculature. Collectively, those results suggest that fibrin-targeted streptokinase nanoparticles could be used early in acute stroke orunstable angina with limited extravascular effects.

SUMMARY

Nanomedicine is a new evolving field referred to by many names, whichpromises to significantly enhance the tools available to clinicians to addresssome of the serious challenges responsible for profound mortality, morbidity,and numerous societal consequences. Unlike the simple pharmaceutics of thepast, nanomedicine agents are typically three-dimensional, multicomponentsystems, which require interdisciplinary expertise to produce and use. In thisreview, we have briefly introduced the opportunities associated with targetedperfluorocarbon nanoparticles in early atherosclerosis, in acute revasculariza-tion, and in thrombolytic therapy. The potential impact of these three conceptsis enormous but pales in comparison with the advancements likely to evolvein this field over the coming decades.

ACKNOWLEDGMENTS

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


Medical Systems (Cleveland, OH, USA) provided valuable equipment, soft-ware, and engineering support.

REFERENCES

1. ANONYMOUS. 2005. American Heart Association, Vol. 2005.2. LANZA, G., K. WALLACE, M. SCOTT, et al. 1996. A novel site-targeted ultra-

sonic contrast agent with broad biomedical application. Circulation 94: 3334–3340.

3. MATTREY, R.F. 1994. The potential role of perfluorochemicals (pfcs) in diagnosticimaging. Artif. Cells Blood Substit. Immobil. Biotechnol. 22: 295–313.

4. HALL, C.S., G.M. LANZA, J.H. ROSE, et al. 2000. Experimental determination ofphase velocity of perfluorocarbons: applications to targeted contrast agents. IEEETrans. Ultrason. Ferroelec. Freq. Contr. 47: 75–84.

5. HALL, C., J. MARSH, M. SCOTT, et al. 2001. Temperature dependence of ultrasonicenhancement with a site-targeted contrast agent. J. Acous. Soc. AM. 110: 1677–1684.

6. LANZA, G., C. LORENZ, S. FISCHER, et al. 1998. Enhanced detection of thrombiwith a novel fibrin-targeted magnetic resonance imaging agent. Acad. Radiol.5(Suppl 1): s173–s176.

7. FLACKE, S., S. FISCHER, M. SCOTT, et al. 2001. A novel MRI contrast agent formolecular imaging of fibrin: implications for detecting vulnerable plaques. Cir-culation 104: 1280–1285.

8. WINTER, P., S. CARUTHERS, X. YU, et al. 2003. Improved molecular imagingcontrast agent for detection of human thrombus. Mag. Reson. Med. 50: 411–416.

9. YU, X., S.-K. SONG, J. CHEN, et al. 2000. High-resolution MRI characterization ofhuman thrombus using a novel fibrin-targeted paramagnetic nanoparticle con-trast agent. Mag. Reson. Med. 44: 867–872.

10. LANZA, G.M., P.M. WINTER, A.M. NEUBAUER, et al. 2005. 1H/19Fmagnetic reso-nance molecular imaging with perfluorocarbon nanoparticles. Curr. Top. Dev.Biol. 70: 57–76.

11. CARUTHERS, S.D., A.M. NEUBAUER, F.D. HOCKETT, et al. 2006. In vitro demon-stration using 19f magnetic resonance to augment molecular imaging with para-magnetic perfluorocarbon nanoparticles at 1.5 tesla. Invest. Radiol. 41: 305–312.

12. KOLODGIE, F.D., R. VIRMANI, A.P. BURKE, et al. 2004. Pathologic assessment ofthe vulnerable human coronary plaque. Heart 90: 1385–1391.

13. VIRMANI, R., F.D. KOLODGIE, A.P. BURKE, et al. 2005. Atherosclerotic plaque pro-gression and vulnerability to rupture: angiogenesis as a source of intraplaquehemorrhage. Arterioscler. Thromb. Vasc. Biol. 25: 2054–2061.

14. SCHAAR, J.A., J.E. MULLER, E. FALK, et al. 2004. Terminology for high-riskand vulnerable coronary artery plaques. Report of a meeting on the vulnera-ble plaque, June 17 and 18, 2003, Santorini. Greece. Eur. Heart J. 25: 1077–1082.

15. OJIO, S., H. TAKATSU, T. TANAKA, et al. 2000. Considerable time from the onsetof plaque rupture and/or thrombi until the onset of acute myocardial infarctionin humans: coronary angiographic findings within 1 week before the onset ofinfarction. Circulation 102: 2063–2069.


16. BOTNAR, R.M., A. BUECKER, A.J. WIETHOFF, et al. 2004. In vivo magnetic reso-nance imaging of coronary thrombosis using a fibrin-binding molecular magneticresonance contrast agent. Circulation 110: 1463–1466.

17. BOTNAR, R.M., A.S. PEREZ, S. WITTE, et al. 2004. In vivo molecular imagingof acute and subacute thrombosis using a fibrin-binding magnetic resonanceimaging contrast agent. Circulation 109: 2023–2029.

18. KAHLON, R., J. SHAPERO & A.I. GOTLIEB. 1992. Angiogenesis in atherosclerosis.Can. J. Cardiol. 8: 60–64.

19. FAN, T.P., R. JAGGAR & R. BICKNELL. 1995. Controlling the vasculature: angiogen-esis, anti-angiogenesis and vascular targeting of gene therapy. Trends Pharmacol.Sci. 16: 57–66.

20. SUEISHI, K., Y. YONEMITSU, K. NAKAGAWA, et al. 1997. Atherosclerosis and angio-genesis. Its pathophysiological significance in humans as well as in an animalmodel induced by the gene transfer of vascular endothelial growth factor. Ann.N. Y. Acad. Sci. 811: 311–322; 322–324.

21. MOULTON, K. 2002. Plaque angiogenesis: its functions and regulation. Cold SpringHarb. Symp. Quant. Biol. 67: 471–482.

22. MOULTON, K.S. 2001. Plaque angiogenesis and atherosclerosis. Curr. Atheroscler.Rep. 3: 225–233.

23. MOULTON, K.S., E. HELLER, M.A. KONERDING, et al. 1999. Angiogenesis inhibitorsendostatin or tnp-470 reduce intimal neovascularization and plaque growth inapolipoprotein e-deficient mice. Circulation 99: 1653–1655.

24. KLEIN, C.D. & G. FOLKERS. 2003. Understanding the selectivity of fumagillin forthe methionine aminopeptidase type II. Oncol. Res. 13: 513–520.

25. RODRIGUEZ-NIETO, S., M.A. MEDINA & A.R. QUESADA. 2001. A re-evaluation offumagillin selectivity towards endothelial cells. Anticancer Res. 21: 3457–3460.

26. LIU, S., J. WIDOM, C.W. KEMP, et al. 1998. Structure of human methionineaminopeptidase-2 complexed with fumagillin. Science 282: 1324–1327.

27. TRAN, H.T., G.R. BLUMENSCHEIN JR., C. LU, et al. 2004. Clinical and pharmacoki-netic study of TNP-470, an angiogenesis inhibitor, in combination with paclitaxeland carboplatin in patients with solid tumors. Cancer Chemother. Pharmacol. 54:308–314.

28. HERBST, R.S., T.L. MADDEN, H.T. TRAN, et al. 2002. Safety and pharmacokineticeffects of TNP-470, an angiogenesis inhibitor, combined with paclitaxel in pa-tients with solid tumors: evidence for activity in non-small-cell lung cancer. J.Clin. Oncol. 20: 4440–4447.

29. LANZA, G.M., X. YU, P.M. WINTER, et al. 2002. Targeted antiproliferative drugdelivery to vascular smooth muscle cells with a magnetic resonance imagingnanoparticle contrast agent: implications for rational therapy of restenosis. Cir-culation 106: 2842–2847.

30. CROWDER, K.C., M.S. HUGHES, J.N. MARSH, et al. 2005. Sonic activation ofmolecularly-targeted nanoparticles accelerates transmembrane lipid delivery tocancer cells through contact-mediated mechanisms: implications for enhancedlocal drug delivery. Ultrasound Med. Biol. 31: 1693–1700.

31. LANZA, G.M., D.R. ABENDSCHEIN, X. YU, et al. 2002. Molecular imaging andtargeted drug delivery with a novel, ligand-directed paramagnetic nanoparticletechnology. Acad. Radiol. 9(Suppl 2): S330–S331.

32. LANZA, G.M., P. WINTER, S. CARUTHERS, et al. 2004. Novel paramagnetic contrastagents for molecular imaging and targeted drug delivery. Curr. Pharm. Biotech-nol. 5: 495–507.


33. WINTER, P.M., A.M. MORAWSKI, S.D. CARUTHERS, et al. 2003. Molecular imag-ing of angiogenesis in early-stage atherosclerosis with alpha(v)beta3-integrin-targeted nanoparticles. Circulation 108: 2270–2274.

34. WILSON, M.W., J.M. LABERGE, R.K. KERLAN, et al. 2002. MR portal venography:preliminary results of fast acquisition without contrast material or breath holding.Acad. Radiol. 9: 1179–1184.

35. JAEGERE PD, P., R. DOMBURG RV, H. NATHOE, et al. 1999. Long-term clinicaloutcome after stent implantation in coronary arteries. Int. J. Cardiovasc. Interv.2: 27–34.

36. KIMURA, T., T. TAMURA, H. YOKOI, et al. 1994. Long-term clinical and angiographicfollow-up after placement of palmaz-schatz coronary stent: a single center ex-perience. J. Interv. Cardiol. 7: 129–139.

37. KEANE, D., A.J. AZAR, P. DE JAEGERE, et al. 1996. Clinical and angiographic out-come of elective stent implantation in small coronary vessels: an analysis of thebenestent trial. Semin. Interv. Cardiol. 1: 255–262.

38. SERRUYS, P.W., P.A. LEMOS & B.A. VAN HOUT. 2004. Sirolimus eluting stent im-plantation for patients with multivessel disease: rationale for the arterial revas-cularisation therapies study part II (ARTS II). Heart 90: 995–998.

39. SERRUYS, P.W., E. REGAR & A.J. CARTER. 2002. Rapamycin eluting stent: the onsetof a new era in interventional cardiology. Heart 87: 305–307.

40. ONG, A.T., E.P. MCFADDEN, E. REGAR, et al. 2005. Late angiographic stent throm-bosis (last) events with drug-eluting stents. J. Am. Coll. Cardiol. 45: 2088–2092.

41. WENAWESER, P. & O. HESS. 2005. Stent thrombosis is associated with an impairedresponse to antiplatelet therapy. J. Am. Coll. Cardiol. 46: CS5–CS6.

42. MCFADDEN, E.P., E. STABILE, E. REGAR, et al. 2004. Late thrombosis in drug-eluting coronary stents after discontinuation of antiplatelet therapy. Lancet 364:1519–1521.

43. RODRIGUEZ, A.E., J. MIERES, C. FERNANDEZ-PEREIRA, et al. 2006. Coronary stentthrombosis in the current drug-eluting stent era: insights from the eraci iii trial.J. Am. Col. Cardiol. 47: 205–207.

44. HOFMA, S.H., W.J. VAN DER GIESSEN, B.M. VAN DALEN, et al. 2006. Indicationof long-term endothelial dysfunction after sirolimus-eluting stent implantation.Eur. Heart J. 27: 166–170.

45. JEANMART, H., O. MALO, M. CARRIER, et al. 2002. Comparative study of cy-closporine and tacrolimus on coronary endothelial function. J. Heart Lung Trans-plant. 21: 990–998.

46. TOGNI, M., S. WINDECKER, R. COCCHIA, et al. 2005. Sirolimus-eluting stents as-sociated with paradoxic coronary vasoconstriction. J. Am. Coll. Cardiol. 46:231–236.

47. LANZA, G., D. ABENDSCHEIN, C. HALL, et al. 2000. Molecular imaging of stretch-induced tissue factor expression in carotid arteries with intravascular ultrasound.Invest. Radiol. 35: 227–234.

48. CYRUS, T., D.R. ABENDSCHEIN, S.D. CARUTHERS, et al. 2006. MR three-dimensionalmolecular imaging of intramural biomarkers with targeted nanoparticles. J. Car-diovasc. Magn. Reson. 8: 535–541.

49. HINCHEY, J.A. & C. BENESCH. 2000. Thrombolytic therapy in patients with acuteischemic stroke. Arch. Neurol. 57: 1430–1436.

50. GROUP, T.N.I.O.N.D.A.S.R.-P.S.S. 1995. Tissue plasminogen activator for acuteischemic stroke. The National Institute of Neurological Disorders and Strokert-PA Stroke Study Group. N. Engl. J. Med. 333: 1581–1587.


51. LANZA, G.M., K.D. WALLACE, M.J. SCOTT, et al. 1996. A novel site-targeted ultra-sonic contrast agent with broad biomedical application. Circulation 95: 3334–3340.

52. RAUT, S. & P. GAFFNEY. 1996. Evaluation of fibrin binding profile of two antifibrinmonoclonal antibodies. Thromb. Haemost. 76: 56–64.

nanomedicina1

Health & Medicine

broad biomedical application

sonic contrast agent

american heart association

atherosclerotic plaque pro

targeted paramagnetic nanoparticles

accumulated surface fibrin

targeted streptokinase nanoparticles

targeted nanoparticles