Shelton D. Caruthers

Molecular Imaging of Angiogenesis in Early-Stage Atherosclerosis With αvβ3-Integrin-Targeted Nanoparticles

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. &agr;v&bgr;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 &agr;v&bgr;3-targeted, paramagnetic nanoparticles. Pretreatment of atherosclerotic rabbits with &agr;v&bgr;3-targeted, nonparamagnetic nanoparticles competitively blocked specific contrast enhancement of the &agr;v&bgr;3-targeted paramagnetic agent. MRI revealed a pattern of increased &agr;v&bgr;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.

Endothelial ανβ3 Integrin–Targeted Fumagillin Nanoparticles Inhibit Angiogenesis in Atherosclerosis

Objective—Angiogenic expansion of the vasa vasorum is a well-known feature of progressive atherosclerosis, suggesting that antiangiogenic therapies may stabilize or regress plaques. &agr;&ngr;&bgr;3 Integrin–targeted paramagnetic nanoparticles were prepared for noninvasive assessment of angiogenesis in early atherosclerosis, for site-specific delivery of antiangiogenic drug, and for quantitative follow-up of response. Methods and Results—Expression of &agr;&ngr;&bgr;3 integrin by vasa vasorum was imaged at 1.5 T in cholesterol-fed rabbit aortas using integrin-targeted paramagnetic nanoparticles that incorporated fumagillin at 0 &mgr;g/kg or 30 &mgr;g/kg. Both formulations produced similar MRI signal enhancement (16.7%±1.1%) when integrated across all aortic slices from the renal arteries to the diaphragm. Seven days after this single treatment, integrin-targeted paramagnetic nanoparticles were readministered and showed decreased MRI enhancement among fumagillin-treated rabbits (2.9%±1.6%) but not in untreated rabbits (18.1%±2.1%). In a third group of rabbits, nontargeted fumagillin nanoparticles did not alter vascular &agr;&ngr;&bgr;3-integrin expression (12.4%±0.9%; P>0.05) versus the no-drug control. In a second study focused on microscopic changes, fewer microvessels in the fumagillin-treated rabbit aorta were counted compared with control rabbits. Conclusions—This study illustrates the potential of combined molecular imaging and drug delivery with targeted nanoparticles to noninvasively define atherosclerotic burden, to deliver effective targeted drug at a fraction of previous levels, and to quantify local response to treatment.

19F magnetic resonance imaging for stem/progenitor cell tracking with multiple unique perfluorocarbon nanobeacons

MRI has been employed to elucidate the migratory behavior of stem/progenitor cells noninva‐sively in vivo with traditional proton (1H) imaging of iron oxide nanoparticle‐labeled cells. Alternatively, we demonstrate that fluorine (19F) MRI of cells labeled with different types of liquid perfluorocarbon (PFC) nanoparticles produces unique and sensitive cell markers distinct from any tissue background signal. To define the utility for cell tracking, mononuclear cells harvested from human umbilical cord blood were grown under proendothelial conditions and labeled with nanoparticles composed of two distinct PFC cores (perfluorooctylbromide and perfluoro‐15‐crown‐5 ether). The sensitivity for detecting and imaging labeled cells was defined on 11.7T (research) and 1.5T (clinical) scanners. Stem/progenitor cells (CD34+CD133+CD31+) readily internalized PFC nanoparticles without aid of adjunctive labeling techniques, and cells remained functional in vivo. PFC‐labeled cells exhibited distinct 19F signals and were readily detected after both local and intravenous injection. PFC nanoparticles provide an unequivocal and unique signature for stem/progenitor cells, enable spatial cell localization with 19F MRI, and permit quantification and detection of multiple fluorine signatures via 19F MR spectroscopy. This method should facilitate longitudinal investigation of cellular events in vivo for multiple cell types simultaneously.—Partlow, K. C., Chen, J., Brant, J. A., Neubauer, A. M., Meyerrose, T. E., Creer, M. H., Nolta, J. A., Caruthers, S. D., Lanza, G. M., Wickline, S. A. 19F magnetic resonance imaging for stem/progenitor cell tracking with multiple unique perfluorocarbon nanobeacons. FASEB J. 21, 1647–1654 (2007)

Targeted nanoparticles for quantitative imaging of sparse molecular epitopes with MRI

Before molecular imaging with MRI can be applied clinically, certain problems, such as the potential sparseness of molecular epitopes on targeted cell surfaces, and the relative weakness of conventional targeted MR contrast agents, must be overcome. Accordingly, the conditions for diagnostic conspicuity that apply to any paramagnetic MRI contrast agent with known intrinsic relaxivity were examined in this study. A highly potent paramagnetic liquid perfluorocarbon nanoparticle contrast agent (∼250 nm diameter, >90000 Gd3+/particle) was imaged at 1.5 T and used to successfully predict a range of sparse concentrations in experimental phantoms with the use of standard MR signal models. Additionally, we cultured and targeted the smooth muscle cell (SMC) monolayers that express “tissue factor,” a glycoprotein of crucial significance to hemostasis and response to vascular injury, by conjugating an anti‐tissue factor antibody fragment to the nanoparticles to effect specific binding. Quantification of the signal from cell monolayers imaged at 1.5 T demonstrated, as predicted via modeling, that only picomolar concentrations of paramagnetic perfluorocarbon nanoparticles were required for the detection and quantification of tissue factor at clinical field strengths. Thus, for targeted paramagnetic agents carrying high payloads of gadolinium, it is possible to quantify molecular epitopes present in picomolar concentrations in single cells with routine MRI. Magn Reson Med 51:480–486, 2004.

Practical Value of Cardiac Magnetic Resonance Imaging for Clinical Quantification of Aortic Valve Stenosis Comparison With Echocardiography

Background—Valvular pathology can be analyzed quickly and accurately through the use of Doppler ultrasound. For aortic stenosis, the continuity equation approach with Doppler velocity-time integral (VTI) data is by far the most commonly used clinical method of quantification. In view of the emerging popularity of cardiac magnetic resonance (CMR) as a routine clinical imaging tool, the purposes of this study were to define the reliability of velocity-encoded CMR as a routine method for quantifying stenotic aortic valve area, to compare this method with the accepted standard, and to evaluate its reproducibility. Methods and Results—Patients (n=24) with aortic stenosis (ranging from 0.5 to 1.8 cm2) were imaged with CMR and echocardiography. Velocity-encoded CMR was used to obtain velocity information in the aorta and left ventricular outflow tract. From this flow data, pressure gradients were estimated by means of the modified Bernoulli equation, and VTIs were calculated to estimate aortic valve orifice dimensions by means of the continuity equation. The correlation coefficients between modalities for pressure gradients were r =0.83 for peak and r =0.87 for mean. The measurements of VTI correlated well, leading to an overall strong correlation between modalities for the estimation of valve dimension (r =0.83, by means of the identified best approach). For 5 patients, the CMR examination was repeated using the best approach. The repeat calculations of valve size correlated well (r =0.94). Conclusions—Velocity-encoded CMR can be used as a reliable, user-friendly tool to evaluate stenotic aortic valves. The measurements of pressure gradients, VTIs, and the valve dimension correlate well with the accepted standard of Doppler ultrasound.

Molecular MR imaging of melanoma angiogenesis with ανβ3-targeted paramagnetic nanoparticles

Neovascularization is a critical component in the progression of malignant melanoma. The objective of this study was to determine whether ανβ3‐targeted paramagnetic nanoparticles can detect and characterize sparse ανβ integrin expression on neovasculature induced by nascent melanoma xenografts (∼30 mm3) at 1.5T. Athymic nude mice bearing human melanoma tumors were intravenously injected with αvβ3‐integrin‐targeted paramagnetic nanoparticles, nontargeted paramagnetic nanoparticles, or αvβ3‐targeted‐nonparamagnetic nanoparticles 2 hr before they were injected with αvβ3‐integrin‐targeted paramagnetic nanoparticles (i.e., in vivo competitive blockade) and imaged with MRI. Contrast enhancement of neovascularity in animals that received ανβ3‐targeted paramagnetic nanoparticles increased 173% by 120 min. Signal contrast with nontargeted paramagnetic nanoparticles was approximately 50% less than that in the targeted group (P < 0.05). Molecular MRI results were corroborated by histology. In a competitive cell adhesion assay, incubation of ανβ3‐expressing cells with targeted nanoparticles significantly inhibited binding to a vitronectin‐coated surface, confirming the bioactivity of the targeted nanoparticles. The present study lowers the limit previously reported for detecting sparse biomarkers with molecular MRI in vivo. This technique may be employed to noninvasively detect very small regions of angiogenesis associated with nascent melanoma tumors, and to phenotype and stage early melanoma in a clinical setting. Magn Reson Med 53:621–627, 2005.

Improved molecular imaging contrast agent for detection of human thrombus

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‐bis‐oleate (BOA) nanoparticles. In this study, the relaxivity of an improved fibrin‐targeted paramagnetic formulation, Gd‐DTPA‐phosphatidylethanolamine (PE), was compared with Gd‐DTPA‐BOA at 0.05‐4.7 T. Ion‐ and particle‐based r1 relaxivities (1.5 T) for Gd‐DTPA‐PE (33.7 (s*mM)‐1 and 2.48 × 106 (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 R1 values at 1.5 T (ΔR1 = 0.77 ± 0.02 1/s) relative to Gd‐DTPA‐BOA (ΔR1 = 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. Magn Reson Med 50:411–416, 2003.

Molecular imaging and therapy of atherosclerosis with targeted nanoparticles.

Advances in bionanotechnology are poised to impact the field of cardiovascular diagnosis and therapy for decades to come. This review seeks to illustrate selected examples of newly developed diagnostic and therapeutic nanosystems that have been evaluated in experimental atherosclerosis, thrombosis, and vascular biology. We review a variety of nanotechnologies that are capable of detecting early cardiovascular pathology, as well as associated imaging approaches and conjunctive strategies for site‐targeted treatment with nanoparticle delivery systems. J. Magn. Reson. Imaging 2007.

Ligand-directed nanobialys as theranostic agent for drug delivery and manganese-based magnetic resonance imaging of vascular targets.

Although gadolinium has been the dominant paramagnetic metal for MR paramagnetic contrast agents, the recent association of this lanthanide with nephrogenic systemic fibrosis, an untreatable disease, has spawned renewed interest in alternative metals for MR molecular imaging. We have developed a self-assembled, manganese(III)-labeled nanobialys (1), a toroidal-shaped MR theranostic nanoparticle. In this report, Mn(III) nanobialys are characterized as MR molecular imaging agents for targeted detection of fibrin, a major biochemical feature of thrombus. A complementary ability of nanobialys to incorporate chemotherapeutic compounds with greater than 98% efficiency and to retain more than 80% of these drugs after infinite sink dissolution, point to the theranostic potential of this platform technology.

Applications of Nanotechnology to Atherosclerosis, Thrombosis, and Vascular Biology

The role of nanotechnology in cardiovascular diagnosis is expanding rapidly. The goal of this brief review is to illustrate selected examples of nanosystems that have been applied to the arenas of atherosclerosis, thrombosis, and vascular biology. The technologies for producing targeted nanosystems are multifarious and reflect end uses in many cases. The results to date indicate rapid growth of interest and capability in the field. The future of cardiovascular diagnosis already is being impacted by nanosystems that can both diagnose pathology and treat it with targeted delivery systems.