Anne H. Schmieder

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.

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.

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)

Minute dosages of ανβ3-targeted fumagillin nanoparticles impair Vx-2 tumor angiogenesis and development in rabbits

Fumagillin suppresses angiogenesis in cancer models and clinical trials, but it is associated with neurotoxicity at systemic doses. In this study, αvβ3‐targeted fumagillin nanoparticles were used to suppress the neovasculature and inhibit Vx‐2 adenocarcinoma development using minute drug doses. Tumor‐bearing rabbits were treated on days 6, 9, and 12 postimplantation with αvβ3‐targeted fumagillin nanoparticles (30 µg/kg), αvβ3‐targeted nanoparticles without drug, nontargeted fumagillin nanoparticles (30 µg/kg) or saline. On day 16, MRI was performed with αvβ3‐targeted paramagnetic nanoparticles to quantify tumor size and assess neovascularity. Tumor volume was reduced among rabbits receiving avβ3‐targeted fumagillin nanoparticles (470±120 mm3) compared with the three control groups: nontargeted fumagillin nanoparticles (1370±300 mm3, P<0.05), αvβ3‐targeted nanoparticles without drug (1080±180 mm3, P<0.05) and saline (980±80 mm3, P<0.05). MR molecular imaging of control rabbits (no fumagillin) revealed a predominant peripheral distribution of neovascularity representing 7.2% of the tumor rim volume, which decreased to 2.8% (P<0.05) with αvβ3‐targeted fumagillin nanoparticle treatment. Microscopically, the tumor parenchyma tended to show T‐cell infiltration after targeted fumagillin treatment, which was not appreciated in control animals. These results suggest that αvβ3‐targeted fumagillin nanoparticles could provide a safe and effective means to deliver MetAP2 inhibitors alone or in combination with cytotoxic or immunotherapy.—Winter, P. M., Schmieder, A. H., Caruthers, S. D., Keene, J. L., Zhang, H., Wickline, S. A., Lanza, G. M. Minute dosages of αvβ3‐targeted fumagillin nanoparticles impair Vx‐2 tumor angiogenesis and development in rabbits. FASEB J. 22, 2758–2767 (2008)

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.

Molecular MR Imaging of Neovascular Progression in the Vx2 Tumor with αvβ3-Targeted Paramagnetic Nanoparticles

PURPOSE To assess the dependence of neovascular molecular magnetic resonance (MR) imaging on relaxivity (r1) of αvβ3-targeted paramagnetic perfluorocarbon (PFC) nanoparticles and to delineate the temporal-spatial consistency of angiogenesis assessments for individual animals. MATERIALS AND METHODS Animal protocols were approved by the Washington University Animal Studies Committee. Proton longitudinal and transverse relaxation rates of αvβ3-targeted and nontargeted PFC nanoparticles incorporating gadolinium diethylenetrianime pentaacedic acid (Gd-DTPA) bisoleate (BOA) or gadolinium tetraazacyclododecane tetraacetic acid (Gd-DOTA) phosphatidylethanolamine (PE) into the surfactant were measured at 3.0 T. These paramagnetic nanoparticles were compared in 30 New Zealand White rabbits (four to six rabbits per group) 14 days after implantation of a Vx2 tumor. Subsequently, serial MR (3.0 T) neovascular maps were developed 8, 14, and 16 days after tumor implantation by using αvβ3-targeted Gd-DOTA-PE nanoparticles (n = 4) or nontargeted Gd-DOTA-PE nanoparticles (n = 4). Data were analyzed with analysis of variance and nonparametric statistics. RESULTS At 3.0 T, Gd-DTPA-BOA nanoparticles had an ionic r1 of 10.3 L · mmol(-1) · sec(-1) and a particulate r1 of 927000 L · mmol(-1) · sec(-1). Gd-DOTA-PE nanoparticles had an ionic r1 of 13.3 L · mmol(-1) · sec(-1) and a particulate r1 of 1 197000 L · mmol(-1) · sec(-1). Neovascular contrast enhancement in Vx2 tumors (at 14 days) was 5.4% ± 1.06 of the surface volume with αvβ3-targeted Gd-DOTA-PE nanoparticles and 3.0% ± 0.3 with αvβ3-targeted Gd-DTPA-BOA nanoparticles (P = .03). MR neovascular contrast maps of tumors 8, 14, and 16 days after implantation revealed temporally consistent and progressive surface enhancement (1.0% ± 0.3, 4.5% ± 0.9, and 9.3% ± 1.4, respectively; P = .0008), with similar time-dependent changes observed among individual animals. CONCLUSION Temporal-spatial patterns of angiogenesis for individual animals were followed to monitor longitudinal tumor progression. Neovasculature enhancement was dependent on the relaxivity of the targeted agent.

Simultaneous Dual Frequency

The combination of sensitive magnetic resonance techniques with a selective site-targeted nanoparticle contrast agent has a demonstrated utility for molecular imaging studies. By detecting a unique signature of the contrast agent, this approach can be employed to identify specific bio-molecular markers and observe cellular-level processes within a large and complex organism (e.g., in vivo rabbit). The objective of the present investigation was to design, fabricate and characterize a radio-frequency (RF) coil for the dual frequency (1H and 19F) simultaneous collection of both nuclei images in a 3T field, in order to facilitate studies of arthritic knee degradation in rabbits. The coil supports both transmit and receive modes. The supporting activities included: 1) establishing a technical database for calculating the required coil parameters, 2) selection of a favorable coil geometry, and 3) adaption of existing RF measurement techniques to the design, development and electrical evaluation of the coil. The coil is used in conjunction with a Philips Medical Systems clinical MRI scanner, requiring all RF simultaneous dual frequency (1H and 19F) coils to operate in both transmit and receive modes. A commercial version of SPICE (simulation program with integrated circuit emphasis) was used to estimate significant operational parameters prior to fabricating the imaging coil. Excellent images were obtained with the fabricated coil and no operational problems were observed that would limit the use of other coil geometries and field strengths.

^{1}{\rm H}

The primary objective of this study was to utilize MR molecular imaging to compare the 3‐dimensional spatial distribution of Robo4 and αVβ3‐integrin as biosignatures of angiogenesis, in a rapidly growing, syngeneic tumor. B16‐F10 melanoma‐bearing mice were imaged with magnetic resonance (MR; 3.0 T) 11 d postimplantation before and after intravenous administration of either Robo4‐ or αVβ3‐targeted paramagnetic nanoparticles. The percentage of MR signal‐enhanced voxels throughout the tumor volume was low and increased in animals receiving αVβ3‐ and Robo4‐targeted nanoparticles. Neovascular signal enhancement was predominantly associated with the tumor periphery (i.e., outer 50% of volume). Microscopic examination of tumors coexposed to the Robo4‐ and αVβ3‐targeted nanoparticles corroborated the MR angiogenesis mapping results and further revealed that Robo4 expression generally colocalized with αVβ3‐integrin. Robo4‐ and αVβ3‐targeted nanoparticles were compared to irrelevant or nontargeted control groups in all modalities. These results suggest that αVβ3‐integrin and Robo4 are useful biomarkers for noninvasive MR molecular imaging in syngeneic mouse tumors, but αVβ3‐integrin expression was more detectable by MR at 3.0 T than Robo4. Noninvasive, neovascular assessments of the MR signal of Robo4, particularly combined with αVβ3‐integrin expression, may help define tumor character prior to and following cancer therapy.—Boles, K. S., Schmieder, A. H., Koch, A. W., Carano, R. A. D., Wu, Y., Caruthers, S. D., Tong, R. K., Stawicki, S., Hu, G., Scott, M. J., Zhang, H., Reynolds, B. A., Wickline, S. A., and Lanza, G. M. MR angiogenesis imaging with Robo4‐ vs. αVβ3‐targeted nanoparticles in a B16/F10 mouse melanoma model. FASEB J. 24, 4262–4270 (2010). www.fasebj.org

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Stroke is the third-leading cause of death and most common etiology for long-term adult disability. Of the approximate 700 000 individuals experiencing new or recurrent stroke annually, less than 5% of these individuals receive thrombolytic therapy.1–3 Intravenous recombinant tissue plasminogen activator (rt-PA) is an approved pharmacological treatment for acute ischemic stroke, but the window of opportunity is considered to be 3 hours from the onset of symptoms following a diagnostic imaging study to rule out hemorrhage. At the thrombolytic dosages approved, the incidence of intracranial hemorrhage in Europe and North America has been 6.4%.4 More recently, this time-window guideline has been liberalized to 3 to 6 hours, primarily because very few patients present for treatment within 3 hours.5 However, this change could bring a potential increase in the complication rate. The potential impact and value of a nanomedicine approach to this problem becomes apparent after examination of the alternatives that have been explored. Neuroprotective approaches to stroke treatment have generated considerable interest because of the low effective use rate of thrombolytic therapies and, if successful, would extend the therapeutic window. However, hundreds of compounds and thousands of articles have been written on neuroprotecting agents to be administered in the advent of a cerebral vascular accident, but none has achieved regulatory-approved status.6 The evidence from PET studies has revealed that early reperfusion, either spontaneous or chemically initiated, primarily determines the final size of brain infarction; these hypoperfused regions account for 70% of the final infarct volume. Reperfusion of the ischemic brain is the most effective therapy for acute stroke, restoring nutrition and oxygen-rich blood flow to threatened tissues. Endogenous enzymes such as rt-PA and urokinase control the conversion of plasminogen to plasmin and the ultimate digestion of fibrin. A number of circulating proteins neutralize plasmin; the best …

^{19}{\rm F}

Although angiogenesis and osteogenesis are critically linked, the importance of angiogenesis for stress fracture healing is unknown. In this study, mechanical loading was used to create a non-displaced stress fracture in the adult rat forelimb. Fumagillin, an anti-angiogenic agent, was used as the water soluble analogue TNP-470 (25mg/kg) as well as incorporated into lipid-encapsulated α(v)β(3) integrin targeted nanoparticles (0.25mg/kg). In the first experiment, TNP-470 was administered daily for 5 days following mechanical loading, and changes in gene expression, vascularity, and woven bone formation were quantified. Although no changes in vascularity were detected 3 days after loading, treatment-related downregulation of angiogenic (Pecam1) and osteogenic (Bsp, Osx) genes was observed at this early time point. On day 7, microCT imaging of loaded limbs revealed diminished woven bone formation in treated limbs compared to vehicle treated limbs. In the second experiment, α(v)β(3) integrin targeted fumagillin nanoparticles were administered as before, albeit with a 100-fold lower dose, and changes in vascularity and woven bone formation were determined. There were no treatment-related changes in vessel count or volume 3 days after loading, although fewer angiogenic (CD105 positive) blood vessels were present in treated limbs compared to vehicle treated limbs. This result manifested on day 7 as a reduction in total vascularity, as measured by histology (vessel count) and microCT (vessel volume). Similar to the first experiment, treated limbs had diminished woven bone formation on day 7 compared to vehicle treated limbs. These results indicate that angiogenesis is required for stress fracture healing, and may have implications for inducing rapid repair of stress fractures.