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 Ͼ50 000 Gd 3ϩ 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 ⅐ s Ϫ1 · pmol Ϫ1 (10% Gd-DTPA nanoparticles) to 0.54 mL ⅐ s Ϫ1 · pmol Ϫ1 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 Ϸ118Ϯ21, and that between the targeted clot and the control clot was 131Ϯ37. Conclusions-These

These data provide the first in vivo demonstration of a site-specific ultrasonic contrast agent and have potential for improved sensitivity and specificity for noninvasive diagnosis of thrombi and other pathological diseases.

Unstable atherosclerotic plaques exhibit microdeposits of fibrin that may indicate the potential for a future rupture. However, current methods for evaluating the stage of an atherosclerotic lesion only involve characterizing the level of vessel stenosis, without delineating which lesions are beginning to rupture. Previous work has shown that fibrin-targeted, liquid perfluorocarbon nanoparticles, which carry a high payload of gadolinium, have a high sensitivity and specificity for detecting fibrin with clinical 1 H MRI. In this work, the perfluorocarbon content of the targeted nanoparticles is exploited for the purposes of 19 F imaging and spectroscopy to demonstrate a method for quantifiable molecular imaging of fibrin in vitro at 4.7 T. Additionally, the quantity of bound nanoparticles formulated with different perfluorocarbon species was calculated using spectroscopy. Results indicate that the high degree of nanoparticle binding to fibrin clots and the lack of background 19 F signal allow accurate quantification using spectroscopy at 4.7 T, as corroborated with proton relaxation rate measurements at 1.5 T and trace element (gadolinium) analysis. Finally, the extension of these techniques to a clinically relevant application, the evaluation of the fibrin burden within an ex vivo human carotid endarterectomy sample, demonstrates the potential use of these particles for uniquely identifying unstable atherosclerotic lesions in vivo.

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Noninvasive magnetic resonance (MR) molecular imaging and targeted drug delivery systems, often referred to as theranostic agents, are being developed to enable improved detection, patient risk stratification, site-specific treatment, and longitudinal monitoring. 1 One example of these agents, a gadolinium-based perfluoro-carbon nanoparticle, has been used to detect, characterize, treat, and follow angiogenesis in preclinical models of cancer and atherosclerosis. Despite the preclinical success of this and related nanotechnology platforms, the recent discovery of nephrogenic systemic fibrosis (NSF), a serious and unexpected side effect of gadolinium blood pool agents observed in some patients with renal disease or following liver transplant, has cast a shadow on currently approved MR contrast agents. 2 Patients with NSF develop thickening of the skin and connective tissues that can inhibit arm and leg movements and even lead to bone fractures. Approximately 5% of patients experience a rapidly progressive course, which may result in death due to widespread fibrosis. The cause of NSF is unknown and there is no effective treatment of this condition. Although gadolinium has been the dominant paramagnetic metal for MR contrast agents, the issue of NSF has induced consideration of alternative approaches.Manganese was one of the first reported examples 3,4 of paramagnetic contrast material studied in cardiac and hepatic MRI because of its efficient R 1 enhancement. Similar to Ca 2+ and unlike the lanthanides, manganese is a natural cellular constituent, and often a cofactor for enzymes and receptors. Manganese blood pool agents, such as mangafodipir trisodium, have been approved as a hepatocyte-specific contrast agent with transient side-effects due to dechelation of manganese from the linear chelate. Nontargeted liposomal agents have included MnSO 4 3c or Mn-DTPA. 3d Release of Mn caused by disruption of the vesicles allowed MR detection of sites where the vesicles were non-specifically entrapped.Manganese(III)-labeled nanobialys (1) are a potential targeted MR theranostic nanoparticle produced by molecular self-assembly of amphiphilic branched polyethylenimine, which has a toroidal shape, tunable particle size, and low polydispersity. The "bialy" shape affords increased stability and presents kinetically stable, porphyrin coupled Mn(III) complexes directly to the surrounding water. In a typical synthesis, commercially available branched polyethylenimines (MW = 10 kDa) are hydrophobically modified (nominal 55% conjugation of the 1° amine) with linoleic acid by activating the carboxylic acid groups with 1-(3′-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (1.2 equiv) and allowing the reaction overnight at ambient temperature. Supramolecular self-assembly of the amphiphilic polymer in anhydrous chloroform, assumes inverted micellar 5 structures (2) that are able to transfer a water soluble new candidate contrast agent Mn(III)-protoporphyrin chloride (Mn-PPC, 4) into chloroform. Synergistic self-assembly of the ag...

Molecular imaging of microthrombus within fissures of unstable atherosclerotic plaques requires sensitive detection with a thrombus-specific agent. Effective molecular imaging has been previously demonstrated with fibrin-targeted Gd-DTPA-bisoleate (BOA) nanoparticles. In this study, the relaxivity of an improved fibrin-targeted paramagnetic formulation, Gd-DTPAphosphatidylethanolamine (PE), was compared with Gd-DTPA-BOA at 0.05-4.7 T. Ion-and particle-based r 1 relaxivities (1.5 T) for Gd-DTPA-PE (33.7 (s*mM) -1 and 2.48 ؋ 10 6 (s*mM) -1 , respectively) were about twofold higher than for Gd-DTPA-BOA, perhaps due to faster water exchange with surface gadolinium. Gd-DTPA-PE nanoparticles bound to thrombus surfaces via anti-fibrin antibodies (1H10) induced 72% ؎ 5% higher change in R 1 values at 1.5 T (⌬R 1 ‫؍‬ 0.77 ؎ 0.02 1/s) relative to Gd-DTPA-BOA (⌬R 1 ‫؍‬ 0.45 ؎ 0.02 1/s). These studies demonstrate marked improvement in a fibrin-specific molecular imaging agent that might allow sensitive, early detection of vascular microthrombi, the antecedent to stroke and heart attack. The acute formation of thrombus on ruptured atherosclerotic plaques is well recognized as the source of unstable angina, myocardial infarction, transient ischemic attacks, and stroke (1). Although myriad medical advances in the detection and treatment of advanced carotid and coronary artery disease have emerged, early detection of the most common source of thromboembolism-rupturing atherosclerotic plaques in arteries with modest 40-60% stenosis (2)-remains diagnostically elusive with the use of routine angiography or duplex ultrasound techniques.A variety of approaches have been proposed to improve detection of vulnerable plaques, including intravascular ultrasound elastography (3), radionuclide imaging (4), and thermography (5). Magnetic resonance imaging (MRI) also is emerging as a particularly sensitive modality to noninvasively visualize thromboses within the carotid artery (6). However, the proximate cause of heart attacks and strokes-rupture of the vulnerable plaque-cannot be reliably detected with any nondestructive imaging modality.Recent studies reveal that vulnerable plaque rupture and microthrombus formation precedes acute myocardial infarction by days to months (7), providing a window of opportunity to intercede and prevent serious sequelae. Sensitive molecular imaging and detection of microthrombi along the intimal surface of vulnerable plaques will require a high-avidity, target-specific probe with robust signal amplification compatible with a sensitive, high-resolution imaging modality. Until recently, the signal amplification required to detect and visualize important molecular or cellular moieties present in nano-and picomolar concentrations in vivo was obtainable only with nuclear imaging modalities. However, more recently, molecular imaging with magnetic resonance has shown promise (8,9).A new approach entails the use of a fibrin-specific paramagnetic molecular imaging agent to improve detection and quantification of these...

The incidence of vein-graft occlusion associated with myocardial infarction and thrombosis following the use of the plasmin inhibitor, aprotinin, to reduce blood loss during vascular surgery has prompted the isolation of an alternative kinetically distinct inhibitor of plasmin from the venom of Pseudonaja textilis. This inhibitor has been called textilinin (Txln) and two distinct forms have been isolated from the Brown-snake venom (molecular weight, 6688 and 6692). A comparison of plasmin inhibitor constants for aprotinin and the Txlns 1 and 2 indicated that the former bound very tightly (inhibitor constant, Ki approximately 10(-11) mol/l), while both of the latter bound less tightly (Ki approximately 10(-9) mol/l). Homogeneity of Txlns 1 and 2 was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and mass spectrometry. A sequence difference of six amino acids was observed between the two forms of Txln. Txln 1 and 2 showed, respectively, 45 and 43% homology with aprotinin, while there was 58 and 55% homology, respectively, with a plasmin inhibitor from the venom of eastern Taipan, Oxyuranus scutellatus. Both Txlns have six cysteines, like other inhibitors of this group, and homology was determined by alignment of these cysteines. Both have been shown to reduce blood loss by about 60% in a murine tail vein bleeding model. It is proposed that the kinetic profiles of Txln 1 and 2 for plasmin allow the arrest of haemorrhage without the possible threat of thrombosis.

Recent advances in the design of fluorinated nanoparticles for molecular magnetic resonance imaging (MRI) have enabled specific detection of 19 F nuclei, providing unique and quantifiable spectral signatures. However, a pressing need for signal enhancement exists because the total 19 F in imaging voxels is often limited. By directly incorporating a relaxation agent, gadolinium (Gd), into the lipid monolayer that surrounds the perfluorocarbon (PFC), a marked augmentation of the 19 F signal from 200-nm nanoparticles was achieved. This design increases the magnetic relaxation rate of the 19 F nuclei fourfold at 1.5 T and effects a 125% increase in signal-an effect that is maintained when they are targeted to human plasma clots. By varying the surface concentration of Gd, the relaxation effect can be quantitatively modulated to tailor particle properties. This novel strategy dramatically improves the sensitivity and range of 19