Grace Hu

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.

Molecularly targeted nanocarriers deliver the cytolytic peptide melittin specifically to tumor cells in mice, reducing tumor growth

The in vivo application of cytolytic peptides for cancer therapeutics is hampered by toxicity, nonspecificity, and degradation. We previously developed a specific strategy to synthesize a nanoscale delivery vehicle for cytolytic peptides by incorporating the nonspecific amphipathic cytolytic peptide melittin into the outer lipid monolayer of a perfluorocarbon nanoparticle. Here, we have demonstrated that the favorable pharmacokinetics of this nanocarrier allows accumulation of melittin in murine tumors in vivo and a dramatic reduction in tumor growth without any apparent signs of toxicity. Furthermore, direct assays demonstrated that molecularly targeted nanocarriers selectively delivered melittin to multiple tumor targets, including endothelial and cancer cells, through a hemifusion mechanism. In cells, this hemifusion and transfer process did not disrupt the surface membrane but did trigger apoptosis and in animals caused regression of precancerous dysplastic lesions. Collectively, these data suggest that the ability to restrain the wide-spectrum lytic potential of a potent cytolytic peptide in a nanovehicle, combined with the flexibility of passive or active molecular targeting, represents an innovative molecular design for chemotherapy with broad-spectrum cytolytic peptides for the treatment of cancer at multiple stages.

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.

Imaging of Vx‐2 rabbit tumors with ανβ3‐integrin‐targeted 111In nanoparticles

Earlier tumor detection can improve 5‐year survival of patients, particularly among those presenting with cancers less than 1 cm in diameter. ανβ3‐Targeted 111In nanoparticles (NP) were developed and studied for detection of tumor angiogenesis. Studies were conducted in New Zealand white rabbits implanted 12 days earlier with Vx‐2 tumor. ανβ3‐Targeted 111In/NP bearing ∼10 111In/NP vs. ∼1 111In/NP nuclide payloads were compared to nontargeted radiolabeled control particles. In vivo competitive binding studies were used to assess ligand‐targeting specificity. ανβ3‐Integrin‐targeted NP with ∼10 111In/NP provided better (p < 0.05) tumor‐to‐muscle ratio contrast (6.3 ± 0.2) than ∼1 111In/NP (5.1 ± 0.1) or nontargeted particles with ∼10 111In/NP (3.7 ± 0.1) over the initial 2‐hr postinjection. At 18 hr, mean tumor activity in rabbits receiving ανβ3‐integrin‐targeted NP was 4‐fold higher than the nontargeted control. Specificity of the NP for the tumor neovasculature was supported by in vivo competition studies and by fluorescence microscopy of ανβ3‐targeted fluorescent‐labeled NP. Biodistribution studies revealed that the primary clearance organ in rabbits as a %ID/g tissue was the spleen. Circulatory half‐life (t1/2β) was estimated to be ∼5 hr using a 2‐compartment model. ανβ3‐Targeted 111In perfluorocarbon NP may provide a clinically useful tool for sensitively detecting angiogenesis in nascent tumors, particularly in combination with secondary high‐resolution imaging modalities, such as MRI.

Computed tomography in color: NanoK-enhanced spectral CT molecular imaging.

New multidetector cardiac computed tomography (MDCT) can image the heart within the span of a few beats, and as such, it is the favored noninvasive approach to assess coronary anatomy rapidly. However, MDCT has proven to be more useful for excluding coronary disease than for making positive diagnoses. The inability to detect unstable cardiac disease arises from the confounding attenuating effects of calcium deposits within atherosclerotic plaques, which obscure lumen anatomy, and from the insensitivity of CT X-rays to image low attenuating intraluminal thrombus adhered to a disrupted plaque cap, the absolute condition of ruptured plaque and unstable disease.[1–6] It is now well understood that the sensitive detection and quantification of small intravascular thrombus in coronary arteries with molecular imaging techniques could provide a direct metric to diagnose and risk stratify patients presenting with chest pain.[7,8]

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.

Fibrin-targeted perfluorocarbon nanoparticles for targeted thrombolysis

BACKGROUND Reperfusion of the ischemic brain is the most effective therapy for acute stroke, restoring blood flow to threatened tissues. Thrombolytics, such as recombinant tissue plasminogen activator, administered within 3 h of symptom onset can improve neurologic outcome, although the potential for adverse hemorrhagic events limits its use to less than 3% of acute ischemic stroke patients. Targeting of clot-dissolving therapeutics has the potential to decrease the frequency of complications while simultaneously increasing treatment effectiveness, by concentrating the available drug at the desired site and permitting a lower systemic dose. OBJECTIVES We aimed to develop a fibrin-specific, liquid perfluorocarbon nanoparticle that is surface modified to deliver the plasminogen activator streptokinase. We also aimed to evaluate its effectiveness for targeted thrombolysis in vitro using quantitative acoustic microscopy. METHODS Human plasma clots were formed in vitro and targeted with streptokinase-loaded nanoparticles, control nanoparticles or a mixture of both. Depending on the treatment group, clots were then exposed to either phosphate-buffered saline (PBS), PBS with plasminogen or PBS with plasminogen and free streptokinase. Spatially registered ultrasound scans were performed at 15-min intervals for 1 h to quantify changes in clot morphology and backscatter. RESULTS Nanoparticles bound to the clot significantly increased the acoustic contrast of the targeted clot surface, permitting volumetric estimates. Profile plots of detected clot surfaces demonstrated that streptokinase-loaded, fibrin-targeted perfluoro-octylbromide nanoparticles in the presence of plasminogen induced rapid fibrinolysis (<60 min) without concurrent microbubble production and cavitation. Streptokinase-loaded or fibrin-targeted control nanoparticles insonified in PBS did not induce clot lysis. Morphologic changes in the treated group were accompanied by temporal and spatial changes in backscatter. Ultrasound exposure had no effect on the digestion process. Effective concentrations of targeted streptokinase were orders of magnitude lower than equivalently efficacious levels of free drug. Moreover, increasing competitive inhibition of fibrin-bound streptokinase nanoparticles reduced clot lysis in a monotonic fashion. As little as 1% surface targeting of streptokinase nanoparticles produced significant decreases in clot volumes (approximately 30%) in 1 h. CONCLUSION This new nanoparticle-based thrombolytic agent provides specific and rapid fibrinolysis in vitro and may have a clinical role in early reperfusion during acute ischemic stroke.

Conquering the Dark Side: Colloidal Iron Oxide Nanoparticles

Nanomedicine approaches to atherosclerotic disease will have significant impact on the practice and outcomes of cardiovascular medicine. Iron oxide nanoparticles have been extensively used for nontargeted and targeted imaging applications based upon highly sensitive T2* imaging properties, which typically result in negative contrast effects that can only be imaged 24 or more hours after systemic administration due to persistent blood pool interference. Although recent advances involving MR pulse sequences have converted these dark contrast voxels into bright ones, the marked delays in imaging from persistent magnetic background interference and prominent dipole blooming effects of the magnetic susceptibility remain barriers to overcome. We report a T1-weighted (T1w) theranostic colloidal iron oxide nanoparticle platform, CION, which is achieved by entrapping oleate-coated magnetite particles within a cross-linked phospholipid nanoemulsion. Contrary to expectations, this formulation decreased T2 effects thus allowing positive T1w contrast detection down to low nanomolar concentrations. CION, a vascular constrained nanoplatform administered in vivo permitted T1w molecular imaging 1 h after treatment without blood pool interference, although some T2 shortening effects on blood, induced by the superparamagnetic particles, persisted. Moreover, CION was shown to encapsulate antiangiogenic drugs, like fumagillin, and retained them under prolonged dissolution, suggesting significant theranostic functionality. Overall, CION is a platform technology, developed with generally recognized as safe components, that overcomes the temporal and spatial imaging challenges associated with current iron oxide nanoparticle T2 imaging agents and which has theranostic potential in vascular diseases for detecting unstable ruptured plaque or treating atherosclerotic angiogenesis.

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.