Patrick M. Winter

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.

Novel MRI Contrast Agent for Molecular Imaging of Fibrin Implications for Detecting Vulnerable Plaques

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 Gd3+ 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.

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.

Targeted Antiproliferative Drug Delivery to Vascular Smooth Muscle Cells With a Magnetic Resonance Imaging Nanoparticle Contrast Agent Implications for Rational Therapy of Restenosis

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 T1-weighted MRI at 4.7T. MRI 19F 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.

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.

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.

Quantitative “magnetic resonance immunohistochemistry” with ligand‐targeted 19F nanoparticles

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 1H MRI. In this work, the perfluorocarbon content of the targeted nanoparticles is exploited for the purposes of 19F 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 19F 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. Magn Reson Med 52:1255–1262, 2004.

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.

Antiangiogenic synergism of integrin-targeted fumagillin nanoparticles and atorvastatin in atherosclerosis.

OBJECTIVESnStudies were performed to develop a prolonged antiangiogenesis therapy regimen based on theranostic alpha(nu)beta(3)-targeted nanoparticles.nnnBACKGROUNDnAntiangiogenesis therapy may normalize atherosclerotic plaque vasculature and promote plaque stabilization. alpha(nu)beta(3)-targeted paramagnetic nanoparticles can quantify atherosclerotic angiogenesis and incorporate fumagillin to elicit acute antiangiogenic effects.nnnMETHODSnIn the first experiment, hyperlipidemic rabbits received alpha(nu)beta(3)-targeted fumagillin nanoparticles (0, 30, or 90 microg/kg) with either a continued high fat diet or conversion to standard chow. The antiangiogenic response was followed for 4 weeks by cardiac magnetic resonance (CMR) molecular imaging with alpha(nu)beta(3)-targeted paramagnetic nanoparticles. In a second 8-week study, atherosclerotic rabbits received atorvastatin (0 or 44 mg/kg diet) alone or with alpha(nu)beta(3)-targeted fumagillin nanoparticles (only week 0 vs. weeks 0 and 4), and angiogenesis was monitored with CMR molecular imaging. Histology was performed to determine the location of bound nanoparticles and to correlate the level of CMR enhancement with the density of angiogenic vessels.nnnRESULTSnThe alpha(nu)beta(3)-targeted fumagillin nanoparticles reduced the neovascular signal by 50% to 75% at 1 week and maintained this effect for 3 weeks regardless of diet and drug dose. In the second study, atherosclerotic rabbits receiving statin alone had no antineovascular benefit over 8 weeks. The alpha(nu)beta(3)-targeted fumagillin nanoparticles decreased aortic angiogenesis for 3 weeks as in study 1, and readministration on week 4 reproduced the 3-week antineovascular response with no carry-over benefit. However, atorvastatin and 2 doses of alpha(nu)beta(3)-targeted fumagillin nanoparticles (0 and 4 weeks) achieved marked and sustainable antiangiogenesis. Microscopic studies corroborated the high correlation between CMR signal and neovessel counts and confirmed that the alpha(nu)beta(3)-targeted nanoparticles were constrained to the vasculature of the aortic adventia.nnnCONCLUSIONSnThe CMR molecular imaging with alpha(nu)beta(3)-targeted paramagnetic nanoparticles demonstrated that the acute antiangiogenic effects of alpha(nu)beta(3)-targeted fumagillin nanoparticles could be prolonged when combined with atorvastatin, representing a potential strategy to evaluate antiangiogenic treatment and plaque stability.