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Background Molecular imaging of thrombus within fissures of vulnerable atherosclerotic plaques requires sensitive detection of a robust thrombus-specific contrast agent. In this study, we report the development and characterization of a novel ligand-targeted paramagnetic molecular imaging agent with high avidity for fibrin and the potential to sensitively detect active vulnerable plaques. Methods and Results The nanoparticles were formulated with 2.5 to 50 mol% Gd-DTPA-BOA, which corresponds to >50 000 Gd 3+ atoms/particle. Paramagnetic nanoparticles were characterized in vitro and evaluated in vivo. In contradistinction to traditional blood-pool agents, T1 relaxation rate as a function of paramagnetic nanoparticle number was increased monotonically with Gd-DTPA concentration from 0.18 mL · s −1 · pmol −1 (10% Gd-DTPA nanoparticles) to 0.54 mL · s −1 · pmol −1 for the 40 mol% Gd-DTPA formulations. Fibrin clots targeted in vitro with paramagnetic nanoparticles presented a highly detectable, homogeneous T1-weighted contrast enhancement that improved with increasing gadolinium level (0, 2.5, and 20 mol% Gd). Higher-resolution scans and scanning electron microscopy revealed that the nanoparticles were present as a thin layer over the clot surface. In vivo contrast enhancement under open-circulation conditions was assessed in dogs. The contrast-to-noise ratio between the targeted clot (20 mol% Gd-DTPA nanoparticles) and blood was ≈118±21, and that between the targeted clot and the control clot was 131±37. Conclusions These results suggest that molecular imaging of fibrin-targeted paramagnetic nanoparticles can provide sensitive detection and localization of fibrin and may allow early, direct identification of vulnerable plaques, leading to early therapeutic decisions.

Introduction : MR molecular imaging and anti-angiogenic treatment with integrin-targeted nanoparticles offer an attractive nanomedicine tool against atherosclerosis. Objective: The objective was to synthesize targeted colloidal iron oxide nanoparticles (CION), which allow high-resolution T1 w images within 2hrs of injection without T2* artifacts or latent blood pool interference. Method: Oleate-coated magnetite nanoparticles were suspended in vegetable oil and emulsified with phospholipid surfactants. With MR (1.5T & 3T), CION (140 –160 nm) were characterized in solution, on fibrin clot phantoms and targeted to angiogenesis in vivo . Blood pool signal was measured over time following IV injection of CION. Result: CION r1 ionic and particulate relaxivities were 1.1±0.1 mmol[Fe] −1 s −1 and 18466±1739 mmol[CION] −1 s −1 at 1.5T and 0.7±0.06 mmol[Fe] −1 s −1 and 10966±1062 mmol[CION] −1 s −1 at 3T. CION targeted to fibrin clots produced marked T1w signal enhancement (SNR=26) versus control clots (SNR=10). T2-weighted images of the bound agent was notable for signal dropout. In atherosclerotic rabbits, the T1 & T2* effects on blood signal ( i.e. , background interference) were resolved in < 1hr. In vivo T1w molecular imaging of the atherosclerotic neovasculature 2h post-injection, was analyzed with a semiautomatic algorithm and demonstrated the potential of T1w neovascular imaging. Conclusion: Targeted CION are a new class of vascular constrained T1w molecular imaging agent with potential for detection and characterization of ruptured plaque and atherosclerotic neovasculature, which overcome the temporal and spatial imaging challenges of previous iron oxide agents.

1.5T MRI with α v β 3 -integrin targeted gadolinium nanoparticle contrast agents (NP) can detect stage I atherosclerosis in experimental animals by localizing specifically to inflammatory plaque components within minutes after injection. We hypothesized that NP could serve as an early surrogate biomarker of therapeutic efficacy for benfluorex treatment of metabolic syndrome in diabetic JCR-LA:cp rats on a 3T clinical scanner. Six week old JCR-LA:cp rats (n=6) received benfluorex in their feed (0.069% wt/wt) for 16 weeks, while control animals (n=5) received regular diet. The abdominal aorta was imaged (T1w spin echo) in 4 cross-sections at baseline and 2-hour post-injection (IV, 1.0 ml/kg NP) every 4 wks with a 3.0 T whole-body clinical MRI scanner, and total aortic wall signal was computed across all sections. Treatment of diabetic JCR-LA:cp rats with benfluorex reduced food consumption and body weight (16.0%; p , *p This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).

Although angiogenesis plays a critical role in the development of atherosclerosis, its participation in early valvular disease remains speculative. To delineate the presence of angiogenesis and its potential responsiveness to medical therapy in experimental valve disease, we used unique signaling ( 19 F), background-free molecular MR nanobeacons as simultaneous diagnostic and therapeutic (theranostic) tools to quantify valvular angiogenesis in 24 cholesterol-fed NZW rabbits (0.25% cholesterol, 6 mo.) and its response to nanoparticle-borne antiangiogenic therapy with an aminopeptidase-2 inhibitor, fumagillin. Perfluorocarbon nanobeacons (~250 nm) targeted to neovascular α v β 3 integrins and loaded with 0.2% fumagillin were given i.v. at 1 mL/kg for 4 weekly doses (n=8; 30 μg/kg fumagillin/dose). Controls included treatment with nontargeted drug-bearing nanoparticles (n=7) and saline (n=9). MR spectroscopy (470 MHz) for detection of 19 F signal from bound nanoparticles was performed after valve excision. Immunocytochemistry confirmed angiogenesis in all valves. In the targeted drug treatment group, both 50% fewer vessels and 50% lower 19 F signal were observed vs nontargeted or saline groups (p ). Across all animals, cholesterol levels exerted a strong linear effect on the extent of angiogenesis (r=0.52; p Figure 1: (Left) Least squares means of 19 F signal from the saline (S), targeted fumagillin (TF), and untargeted fumagillin (UF) treated groups, normalized to the saline control. Note 50% reduction of 19 F signal in the group treated with targeted fumagillin nanoparticles compared to the untreated and untargeted controls. (Right) LS means of vessel count on stained valve sections. Note 50% fewer vessels in targeted fumagillin group corresponding to reduction in 19 F signal. (* p

Metabolic syndrome elicits widespread inflammatory changes in vascular structures leading to early atherosclerosis that cannot be detected with classical imaging methods. Because pronounced early angiogenesis within the arterial wall typically accompanies activated inflammatory cells in standard models of atherosclerosis, we hypothesized that the expression of α v β 3 -integrins on neovascular endothelium would provide an abundant and sensitive biomarker of the metabolic vasculopathy. The mildly diabetic JCR:LA-cp rat was used as a model for MR molecular imaging of angiogenesis with α v β 3 -targeted gadolinium nanoparticles (NP). 5 month-old male JCR:LA-cp lean (control) and obese (affected) rats (n=5 each) were studied. The abdominal aorta was imaged with a 3.0 T clinical MRI scanner and a T1w, fat-suppressed, black-blood imaging sequence. Imaging was performed before (BSL) and 2 hours (2 HR) after NP injection (IV, 1.0 ml/kg). Fat suppressed imaging revealed large abdominal fat deposits in the obese rats but not in controls, concordant with elevated cholesterol levels (~2 times higher) in the obese rats (p : top, arrow) in the obese rats was significantly higher than that in the lean rats (bottom, *p This research has received full or partial funding support from the American Heart Association, AHA Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota & Wisconsin).

To determine whether rotational blood flow or chiral asymmetry exists in the human descending thoracic aorta, we established the ability of color Doppler ultrasound to detect rotational flow in a tornado tube model of a vortex descending fluid column. In a model of the human aortic arch with a pulse duplicator, color Doppler was then used to demonstrate that rotational flow occurs first in the transverse arch and then in the proximal descending thoracic aorta. With the use of color Doppler esophageal echocardiography, 53 patients (age range, 25-78 years; mean age, 56.4 years) were prospectively examined for rotational flow in the descending thoracic aorta. At 10 cm superior to retro-left ventricular position, 22 of 38 patients (58%) revealed rotational flow with obvious diastolic counterclockwise rotation but less obvious systolic clockwise rotation. At 5 cm superior to retro-left ventricular position, 29 of 46 patients (63%) revealed rotational flow with a tendency toward systolic clockwise and diastolic counterclockwise rotation. At the retro-left ventricular position, 47 of 53 patients (89%) revealed rotational flow, usually of a clockwise direction, occurring in systole. Our data suggest that aortic flow is not purely pulsatile and axial but has a rotational component. Rotational flow begins in the aortic arch and is carried through to the descending thoracic aorta, where flow is chirally asymmetric with systolic clockwise and diastolic counterclockwise components. These data demonstrate an aortic rotational flow component that may have physiological implications for organ perfusion.

Background— Angiogenesis is a critical feature of plaque development in atherosclerosis and might play a key role in both the initiation and later rupture of plaques that lead to myocardial infarction and stroke. The precursory molecular or cellular events that initiate plaque growth and that ultimately contribute to plaque instability, however, cannot be detected directly with any current diagnostic modality. Methods and Results— Atherosclerosis was induced in New Zealand White rabbits fed 1% cholesterol for ≈80 days. α v β 3 -Integrin–targeted, paramagnetic nanoparticles were injected intravenously and provided specific detection of the neovasculature within 2 hours by routine magnetic resonance imaging (MRI) at a clinically relevant field strength (1.5 T). Increased angiogenesis was detected as a 47±5% enhancement in MRI signal averaged throughout the abdominal aortic wall among rabbits that received α v β 3 -targeted, paramagnetic nanoparticles. Pretreatment of atherosclerotic rabbits with α v β 3 -targeted, nonparamagnetic nanoparticles competitively blocked specific contrast enhancement of the α v β 3 -targeted paramagnetic agent. MRI revealed a pattern of increased α v β 3 -integrin distribution within the atherosclerotic wall that was spatially heterogeneous along both transverse and longitudinal planes of the abdominal aorta. Histology and immunohistochemistry confirmed marked proliferation of angiogenic vessels within the aortic adventitia, coincident with prominent, neointimal proliferation among cholesterol-fed, atherosclerotic rabbits in comparison with sparse incidence of neovasculature in the control animals. Conclusions— This molecular imaging approach might provide a method for defining the burden and evolution of atherosclerosis in susceptible individuals as well as responsiveness of individual patients to antiatherosclerotic therapies.

Background— Restenosis is a serious complication of coronary angioplasty that involves the proliferation and migration of vascular smooth muscle cells (VSMCs) from the media to the intima, synthesis of extracellular matrix, and remodeling. We have previously demonstrated that tissue factor–targeted nanoparticles can penetrate and bind stretch-activated vascular smooth muscles in the media after balloon injury. In the present study, the concept of VSMC-targeted nanoparticles as a drug-delivery platform for the prevention of restenosis after angioplasty is studied. Methods and Results— Tissue factor–targeted nanoparticles containing doxorubicin or paclitaxel at 0, 0.2, or 2.0 mole% of the outer lipid layer were targeted for 30 minutes to VSMCs and significantly inhibited their proliferation in culture over the next 3 days. Targeting of the nanoparticles to VSMC surface epitopes significantly increased nanoparticle antiproliferative effectiveness, particularly for paclitaxel. In vitro dissolution studies revealed that nanoparticle drug release persisted over one week. Targeted antiproliferative results were dependent on the hydrophobic nature of the drug and noncovalent interactions with other surfactant components. Molecular imaging of nanoparticles adherent to the VSMC was demonstrated with high-resolution T 1 -weighted MRI at 4.7T. MRI 19 F spectroscopy of the nanoparticle core provided a quantifiable approach for noninvasive dosimetry of targeted drug payloads. Conclusions— These data suggest that targeted paramagnetic nanoparticles may provide a novel, MRI-visualizable, and quantifiable drug delivery system for the prevention of restenosis after angioplasty.