Todd A. Williams

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.

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.

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

OBJECTIVES Studies were performed to develop a prolonged antiangiogenesis therapy regimen based on theranostic alpha(nu)beta(3)-targeted nanoparticles. BACKGROUND Antiangiogenesis 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. METHODS In 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. RESULTS The 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. CONCLUSIONS The 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.

Three-dimensional MR mapping of angiogenesis with α5β1(ανβ3)-targeted theranostic nanoparticles in the MDA-MB-435 xenograft mouse model

Our objectives were 1) to characterize angiogenesis in the MDA‐MB‐435 xenograft mouse model with three‐dimensional (3D) MR molecular im aging using α5β1(RGD)‐ or irrelevant RGS‐targeted paramagnetic nanoparticles and 2) to use MR molecu lar imaging to assess the antiangiogenic effectiveness of α5β1(αvβ3)‐ vs. αvβ3‐targeted fumagillin (50 μg/kg) nanoparticles. Tumor‐bearing mice were imaged with MR before and after administration of either α5 β1(RGD) or irrelevant RGS‐paramagnetic nanopar ticles. In experiment 2, mice received saline or α5β1(αvβ3)‐ or αvβ3‐targeted fumagillin nanoparticles on days 7, 11, 15, and 19 posttumor implant. On day 22, MRI was performed using α5β1(αvβ3)‐targeted paramagnetic nanoparticles to monitor the antiangiogenic response. 3D reconstructions of α5β1(RGD)‐signal en hancement revealed a sparse, asymmetrical pattern of angiogenesis along the tumor periphery, which occupied <2.0% tumor surface area. α5β1‐targeted rhodamine nanoparticles colocalized with FITC‐lectin corroborated the peripheral neovascular signal. α5β1(αvβ3)‐fumagillin nanoparticles decreased neovas culature to negligible levels relative to control;αvβ3‐ targeted fumagillin nanoparticles were less effective (P>0.05). Reduction of angiogenesis in MDA‐MB‐435 tumors from low to negligible levels did not decrease tumor volume. MR molecular imaging may be useful for characterizing tumors with sparse neovasculature that are unlikely to have a reduced growth response to targeted antiangiogenic therapy.— Schmieder, A. H., Caruthers, S. D., Zhang, H., Williams, T. A., Robertson, J. D., Wickline, S. A., Lanza, G. M. Three‐dimensional MR mapping of angiogenesis with α5β1(αvβ3)‐targeted theranostic nanoparticles in the MDA‐MB‐435 xenograft mouse model. FASEB J. 22, 4179–4189 (2008)

Intramural Delivery of Rapamycin With αvβ3-Targeted Paramagnetic Nanoparticles Inhibits Stenosis After Balloon Injury

Background—Drug eluting stents prevent vascular restenosis but can delay endothelial healing. A rabbit femoral artery model of stenosis formation after vascular injury was used to study the effect of intramural delivery of &agr;v&bgr;3-integrin–targeted rapamycin nanoparticles on vascular stenosis and endothelial healing responses. Methods and Results—Femoral arteries of 48 atherosclerotic rabbits underwent balloon stretch injury and were locally treated with either (1) &agr;v&bgr;3-targeted rapamycin nanoparticles, (2) &agr;v&bgr;3-targeted nanoparticles without rapamycin, (3) nontargeted rapamycin nanoparticles, or (4) saline. Intramural binding of integrin-targeted paramagnetic nanoparticles was confirmed with MR molecular imaging (1.5 T). MR angiograms were indistinguishable between targeted and control arteries at baseline, but 2 weeks later they showed qualitatively less luminal plaque in the targeted rapamycin treated segments compared with contralateral control vessels. In a first cohort of 19 animals (38 vessel segments), microscopic morphometric analysis of the rapamycin-treated segments revealed a 52% decrease in the neointima/media ratio (P<0.05) compared to control. No differences (P>0.05) were observed among balloon injured vessel segments treated with &agr;v&bgr;3-targeted nanoparticles without rapamycin, nontargeted nanoparticles with rapamycin, or saline. In a second cohort of 29 animals, endothelial healing followed a parallel pattern over 4 weeks in the vessels treated with &agr;v&bgr;3-targeted rapamycin nanoparticles and the 3 control groups. Conclusions—Local intramural delivery of &agr;v&bgr;3-targeted rapamycin nanoparticles inhibited stenosis without delaying endothelial healing after balloon injury.

High Sensitivity : High-Resolution SPECT-CT/MR Molecular Imaging of Angiogenesis in the Vx2 Model

Objectives:The use of antiangiogenic therapy in conjunction with traditional chemotherapy is becoming increasingly in cancer management, but the optimal benefit of these targeted pharmaceuticals has been limited to a subset of the population treated. Improved imaging probes that permit sensitive detection and high-resolution characterization of tumor angiogenesis could improve patient risk-benefit stratification. The overarching objective of these experiments was to develop a dual modality &agr;&ngr;&bgr;3-targeted nanoparticle molecular imaging agent that affords sensitive nuclear detection in conjunction with high-resolution MR characterization of tumor angiogenesis. Materials and Methods:In part 1, New Zealand white rabbits (n = 21) bearing 14d Vx2 tumor received either &agr;&ngr;&bgr;3-targeted 99mTc nanoparticles at doses of 11, 22, or 44 MBq/kg, nontargeted 99mTc nanoparticles at 22 MBq/kg, or &agr;&ngr;&bgr;3-targeted 99mTc nanoparticles (22 MBq/kg) competitively inhibited with unlabeled &agr;&ngr;&bgr;3-nanoparticles. All animals were imaged dynamically over 2 hours with a planar camera using a pinhole collimator. In part 2, the effectiveness of &agr;&ngr;&bgr;3-targeted 99mTc nanoparticles in the Vx2 rabbit model was demonstrated using clinical SPECT-CT imaging techniques. Next, MR functionality was incorporated into &agr;&ngr;&bgr;3-targeted 99mTc nanoparticles by inclusion of lipophilic gadolinium chelates into the outer phospholipid layer, and the concept of high sensitivity – high-resolution detection and characterization of tumor angiogenesis was shown using sequential SPECT-CT and MR molecular imaging with 3D neovascular mapping. Results:&agr;&ngr;&bgr;3-Targeted 99mTc nanoparticles at 22 MBq/kg produced the highest tumor-to-muscle contrast ratio (8.56 ± 0.13, TMR) versus the 11MBq/kg (7.32 ± 0.12) and 44 MBq/kg (6.55 ± 0.07) doses, (P < 0.05). TMR of nontargeted particles at 22.2 MBq/kg (5.48 ± 0.09) was less (P < 0.05) than the equivalent dosage of &agr;&ngr;&bgr;3-targeted 99mTc nanoparticles. Competitively inhibition of 99mTc &agr;&ngr;&bgr;3-integrin-targeted nanoparticles at 22.2 MBq/kg reduced (P < 0.05) TMR (5.31 ± 0.06) to the nontargeted control contrast level. Multislice CT imaging could not distinguish the presence of Vx2 tumor implanted in the popliteal fossa from lymph nodes in the same fossa or in the contralateral leg. However, the use of 99mTc &agr;&ngr;&bgr;3-nanoparticles with SPECT-CT produced a clear neovasculature signal from the tumor that was absent in the nonimplanted hind leg. Using &agr;&ngr;&bgr;3-targeted 99mTc-gadolinium nanoparticles, the sensitive detection of the Vx2 tumor was extended to allow MR molecular imaging and 3D mapping of angiogenesis in the small tumor, revealing an asymmetrically distributed, patchy neovasculature along the periphery of the cancer. Conclusion:Dual modality molecular imaging with &agr;&ngr;&bgr;3-targeted 99mTc-gadolinium nanoparticles can afford highly sensitive and specific localization of tumor angiogenesis, which can be further characterized with high-resolution MR neovascular mapping, which may predict responsiveness to antiangiogenic therapy.

Detecting vascular biosignatures with a colloidal, radio-opaque polymeric nanoparticle.

A synthetic methodology for developing a polymeric nanoparticle for targeted computed tomographic (CT) imaging is revealed in this manuscript. The work describes a new class of soft type, vascularly constrained, stable colloidal radio-opaque metal-entrapped polymeric nanoparticle using organically soluble radio-opaque elements encapsulated by synthetic amphiphile. This agent offers several-fold CT signal enhancement in vitro and in vivo demonstrating detection sensitivity reaching to the low nanomolar particulate concentration range.

Nanoparticle pharmacokinetic profiling in vivo using magnetic resonance imaging.

Contrast agents targeted to molecular markers of disease are currently being developed with the goal of identifying disease early and evaluating treatment effectiveness using noninvasive imaging modalities such as MRI. Pharmacokinetic profiling of the binding of targeted contrast agents, while theoretically possible with MRI, has thus far only been demonstrated with more sensitive imaging techniques. Paramagnetic liquid perfluorocarbon nanoparticles were formulated to target αvβ3‐integrins associated with early atherosclerosis in cholesterol‐fed rabbits to produce a measurable signal increase on magnetic resonance images after binding. In this work, we combine quantitative information of the in vivo binding of this agent over time obtained by means of MRI with blood sampling to derive pharmacokinetic parameters using simultaneous and individual fitting of the data to a three compartment model. A doubling of tissue exposure (or area under the curve) is obtained with targeted as compared to control nanoparticles, and key parameter differences are discovered that may aid in development of models for targeted drug delivery. Magn Reson Med 60:1353–1361, 2008.