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Treatment of advanced heart failure with implantable LVADs is increasing, driven by profound unmet patient need despite potential serious complications: bleeding, infection, and thrombus. The experimental objective was to develop a sensitive imaging approach to assess early thrombus accumulation in LVADs under operational high flow and high shear rates. Methods: A monomeric bifunctional ligand with a fibrin-specific peptide, a short spacer, and 99mTc chelating amino acid sequence (F1A) was developed and compared to its tetrameric PEG analogue (F4A). Results:99mTc attenuation by LVAD titanium (1 mm) was 23%. 99mTc-F1A affinity to fibrin was Kd ~10 µM, whereas, the bound 99mTc-F4A probe was not displaced by F1A (120,000:1). Human plasma interfered with 99mTc-F1A binding to fibrin clot (p

A high r1 relaxivity manganese-gadolinium nanocolloid (αvβ3-MnOL-Gd NC) was developed and effectively detected atherosclerotic angiogenesis in rabbits fed cholesterol-rich diets for 12 months using a clinical MRI scanner (3T). 3D mapping of neovasculature signal intensity revealed the spatial coherence and intensity of plaque angiogenic expansion, which may, with other high risk MR bioindicators, help identify high-risk patients with moderate (40% to 60%) vascular stenosis. Microscopy confirmed the predominant media and plaque distribution of fluorescent αvβ3-MnOL-Gd NC, mirroring the MR data. An expected close spatial association of αvβ3-integrin neovasculature and macrophages was noted, particularly within plaque shoulder regions. Manganese oleate bioelimination occurred via the biliary system into feces. Gd-DOTA was eliminated through the bile-fecal and renal excretion routes. αvβ3-MnOL-Gd NC offers an effective vehicle for T1w neovascular imaging in atherosclerosis. From the clinical editor: Cerebrovascular accidents are a leading cause of mortality and morbidity worldwide. The acute formation of thrombus following atherosclerotic plaque rupture has been well recognized as the etiology of stroke. The authors studied microanatomical features of vulnerable atherosclerotic plaque in this article, in an attempt to identify those with high risk of rupture. Gadolinium-manganese hybrid nanocolloid (MnOL-Gd NC) was developed as a novel contrast agent for MRI. They show that this agent is effective in providing neovascular imaging.

Bone metastases occur in approximately 70% of metastatic breast cancer patients, often leading to skeletal injuries. Current treatments are mainly palliative and underscore the unmet clinical need for improved therapies. In this study, we provide preclinical evidence for an antimetastatic therapy based on targeting integrin β3 (β3), which is selectively induced on breast cancer cells in bone by the local bone microenvironment. In a preclinical model of breast cancer, β3 was strongly expressed on bone metastatic cancer cells, but not primary mammary tumors or visceral metastases. In tumor tissue from breast cancer patients, β3 was significantly elevated on bone metastases relative to primary tumors from the same patient (n = 42). Mechanistic investigations revealed that TGFβ signaling through SMAD2/SMAD3 was necessary for breast cancer induction of β3 within the bone. Using a micelle-based nanoparticle therapy that recognizes integrin αvβ3 (αvβ3-MPs of ∼12.5 nm), we demonstrated specific localization to breast cancer bone metastases in mice. Using this system for targeted delivery of the chemotherapeutic docetaxel, we showed that bone tumor burden could be reduced significantly with less bone destruction and less hepatotoxicity compared with equimolar doses of free docetaxel. Furthermore, mice treated with αvβ3-MP-docetaxel exhibited a significant decrease in bone-residing tumor cell proliferation compared with free docetaxel. Taken together, our results offer preclinical proof of concept for a method to enhance delivery of chemotherapeutics to breast cancer cells within the bone by exploiting their selective expression of integrin αvβ3 at that metastatic site. Cancer Res; 77(22); 6299–312. ©2017 AACR.

Eighty percent of lung cancers originate as subtle premalignant changes in the airway mucosal epithelial layer of bronchi and alveoli, which evolve and penetrate deeper into the parenchyma. Liquid-ventilation, with perfluorocarbons (PFC) was first demonstrated in rodents in 1966 then subsequently applied as lipid-encapsulated PFC emulsions to improve pulmonary function in neonatal infants suffering with respiratory distress syndrome in 1996. Subsequently, PFC nanoparticles (NP) were extensively studied as intravenous (IV) vascular-constrained nanotechnologies for diagnostic imaging and targeted drug delivery applications. Methods: This proof-of-concept study compared intratumoral localization of fluorescent paramagnetic (M) PFC NP in the Vx2 rabbit model using proton (1H) and fluorine (19F) magnetic resonance (MR) imaging (3T) following intratracheal (IT) or IV administration. MRI results were corroborated by fluorescence microscopy. Results: Dynamic 1H-MR and 19F-MR images (3T) obtained over 72 h demonstrated marked and progressive accumulation of M-PFC NP within primary lung Vx2 tumors during the first 12 h post IT administration. Marked 1H and 19F MR signal persisted for over 72 h. In contradistinction, IV M-PFC NP produced a modest transient signal during the initial 2 h post-injection that was consistent circumferential blood pool tumor enhancement. Fluorescence microscopy of excised tumors corroborated the MR results and revealed enormous intratumor NP deposition on day 3 after IT but not IV treatment. Rhodamine-phospholipid incorporated into the PFC nanoparticle surfactant was distributed widely within the tumor on day 3, which is consistent with a hemifusion-based contact drug delivery mechanism previously reported. Fluorescence microscopy also revealed similar high concentrations of M-PFC NP given IT for metastatic Vx2 lung tumors. Biodistribution studies in mice revealed that M-PFC NP given IV distributed into the reticuloendothelial organs, whereas, the same dosage given IT was basically not detected beyond the lung itself. PFC NP given IT did not impact rabbit behavior or impair respiratory function. PFC NP effects on cells in culture were negligible and when given IV or IT no changes in rabbit hematology nor serum clinical chemistry parameters were measured. Conclusion: IT delivery of PFC NP offered unique opportunity to locally deliver PFC NP in high concentrations into lung cancers with minimal extratumor systemic exposure.

Robust thrombus imaging is an unresolved clinical unmet need dating back to the mid 1970s. While early molecular imaging approaches began with nuclear SPECT imaging, contrast agents for virtually all biomedical imaging modalities have been demonstrated in vivo with unique strengths and common weaknesses. Two primary molecular imaging targets have been pursued for thrombus imaging: platelets and fibrin. Some common issues noted over 40 years ago persist today. Acute thrombus is readily imaged with all probes and modalities, but aged thrombus remains a challenge. Similarly, anti-coagulation continues to interfere with and often negate thrombus imaging efficacy, but heparin is clinically required in patients suspected of pulmonary embolism, deep venous thrombosis or coronary ruptured plaque prior to confirmatory diagnostic studies have been executed and interpreted. These fundamental issues can be overcome, but an innovative departure from the prior approaches will be needed.

Due to their small size, lower cost, short reproduction cycle, and genetic manipulation, rodents have been widely used to test the safety and efficacy for pharmaceutical development in human disease. In this report, MR cholangiography demonstrated an unexpected rapid (

Computed tomography (CT) is one of the most frequently pursued radiology technologies applied in the clinics today and in the preclinical field of biomedical imaging. Myriad advances have been made to make this technique more powerful with improved signal sensitivity, rapid image acquisition and faster reconstruction. Synergistic development of novel nanoparticles has been adopted to produce the next-generation CT contrasts agents for imaging specific biological markers. Nanometer-sized agents are anticipated to play a critical part in the prospect of medical diagnostics owing to their capabilities of targeting specific biological markers, extended blood circulation time and defined biological clearance. This review paper introduces the readers to the fundamental design principles of nanoparticulate CT contrast agents with a special emphasis on molecular imaging with noncrystalline high-metal-density nanobeacons. Copyright © 2014 John Wiley & Sons, Ltd.

Imaging has become a cornerstone for medical diagnosis and the guidance of patient management. A new field called image-guided drug delivery (IGDD) now combines the vast potential of the radiological sciences with the delivery of treatment and promises to fulfill the vision of personalized medicine. Whether imaging is used to deliver focused energy to drug-laden particles for enhanced, local drug release around tumors, or it is invoked in the context of nanoparticle-based agents to quantify distinctive biomarkers that could risk stratify patients for improved targeted drug delivery efficiency, the overarching goal of IGDD is to use imaging to maximize effective therapy in diseased tissues and to minimize systemic drug exposure in order to reduce toxicities. Over the last several years, innumerable reports and reviews covering the gamut of IGDD technologies have been published, but inadequate attention has been directed toward identifying and addressing the barriers limiting clinical translation. In this consensus opinion, the opportunities and challenges impacting the clinical realization of IGDD-based personalized medicine were discussed as a panel and recommendations were proffered to accelerate the field forward.

Purpose We sought to develop a unique sensor-reporter approach for functional kidney imaging that employs circulating perfluorocarbon nanoparticles and multinuclear 1H/19F MRI. Methods 19F spin density weighted and T1 weighted images were used to generate quantitative functional mappings of both healthy and ischemia-reperfusion (acute kidney injury) injured mouse kidneys. 1H blood-oxygenation-level-dependent (BOLD) MRI was also employed as a supplementary approach to facilitate the comprehensive analysis of renal circulation and its pathological changes in acute kidney injury. Results Heterogeneous blood volume distributions and intrarenal oxygenation gradients were confirmed in healthy kidneys by 19F MRI. In a mouse model of acute kidney injury, 19F MRI, in conjunction with blood-oxygenation-level-dependent MRI, sensitively delineated renal vascular damage and recovery. In the cortico-medullary junction region, we observed 25% lower 19F signal (P

X-ray contrast agents have heretofore been dominated by iodinated molecules imaged with computed tomography (CT) using traditional energy integrating detectors. Hardware and software developments in CT now present the option for dual-energy detection systems, which still retain the integrator detector approach. However, a new era of detectors called photon-counting detectors used for spectral CT has recently overcome many of the technical imaging barriers precluding clinical translation while offering a trade-off between higher image resolution or better signal to noise as well as lower radiation exposure. While these attributes are highly desirable, the unmet potential for Spectral CT is molecular imaging due to the energy discriminating properties of these systems. To date, contrast imaging is based on X-ray attenuation as seen routinely when high calcium content, such as in bone, appears white on the image. With spectral CT, individual metals, particularly gold, tantalum, bismuth, and ytterbium have emerged as the basis for new contrast media, which are referred to as K-edge contrast agents. These new agents could be used to localize and quantify important biomarkers to extend CT tissue characterization into the realm of molecular imaging. While many of the prototype attempts at such agents will likely not be translational to patients, an array of concepts, reviewed herein, could lead to clinical products in the not too distant future.

Photoacoustic imaging (PAI) represents a hybrid, nonionizing modality, which has been of particular interest because of its satisfactory spatial resolution and high soft tissue contrast. PAI has the potential to provide both functional and molecular imaging in vivo since optical absorption is sensitive to physiological parameters. In this review we summarize our effort to advance molecular PAI with colloidal gold nanobeacons (GNB). GNB represents a robust nanoparticle platform that entraps multiple copies of tiny gold nanoparticles (2–4 nm) within a larger colloidal particle encapsulated by biocompatible synthetic or natural amphilines. The utilization of numerous small gold particles greatly amplifies the signal without exceeding the renal elimination threshold size. With fibrin-targeted GNB, the robust detection of microthrombus formed over a ruptured atherosclerotic plaque has been achieved, which offers an important opportunity to recognize patients with moderate lumen stenosis but high risk of stroke. With the use of second-generation smaller GNBs, the potential to improve sentinel lymph node assessment and biopsy was advanced with respect to rapidity and sensitivity of detection in mice. Finally, for angiogenesis, an essential microanatomical biomarker of tumor and cardiovascular disease progression, integrin-targeted GNBs allowed visualization of numerous angiogenic sprouts and bridges that were otherwise undetectable from inherent blood signal alone, offering sensitive and specific discrimination and quantification of angiogenesis in vivo. Copyright © 2011 John Wiley & Sons, Ltd.

High-grade atherosclerotic stenoses are reduced to zero or minimal residual stenosis grades by a single or a series of balloon angioplasties. Currently, stents are implanted to prevent immediate vascular recoil and elution of an anti-mitotic drug from the stent struts minimizes restenosis. An unwanted side-effect of this drug-elution is delayed re-endothelialization which requires the treatment with two anti-platelet drugs in many cases for a minimum of one year to prevent acute in-stent thrombosis. Advances in stent-design and drug-elution technology, now in its fourth generation, have not abated this issue. Nanotechnology-based local drug delivery has the potential to achieve restenosis prevention while not impeding endothelial healing. Molecularly targeted drugs can be aimed to specifically bind to epitopes in the injured media and adventitia. Thus, endothelial healing may progress unhindered. To prevent restenosis, this technology may be used with bare metal or biodegradable stents. In this article novel nanoparticulate agents will be compared regarding their potential to deliver drugs to molecular targets within the vascular wall. Potential molecular targets, targeting mechanisms, drug-delivery propensities, and biocompatibility will be reviewed.

Photoacoustic tomography (PAT) combines optical and acoustic imaging to generate high-resolution images of microvasculature. Inherent sensitivity to hemoglobin permits PAT to image blood vessels but precludes discriminating neovascular from maturing microvasculature. Α_vβ_3-Gold nanobeacons (α_vβ_3-GNBs) for neovascular molecular PAT were developed, characterized, and demonstrated in vivo using a mouse Matrigel-plug model of angiogenesis. PAT results were microscopically corroborated with fluorescent α_vβ_3-GNB localization and supporting immunohistology in Rag1^(tm1Mom) Tg(Tie-2-lacZ)182-Sato mice. Α_vβ_3-GNBs (154 nm) had 10-fold greater contrast than blood on an equivolume basis when imaged at 740 nm to 810 nm in blood. The lowest detectable concentration in buffer was 290 nM at 780 nm. Noninvasive PAT of angiogenesis using a 10-MHz ultrasound receiver with α_vβ_3-GNBs produced a 600% increase in signal in a Matrigel-plug mouse model relative to the inherent hemoglobin contrast pretreatment. In addition to increasing the contrast of neovessels detected at baseline, α_vβ_3-GNBs allowed visualization of numerous angiogenic sprouts and bridges that were undetectable before contrast injection. Competitive inhibition of α_vβ_3-GNBs with α_vβ_3-NBs (no gold particles) almost completely blocked contrast enhancement to pretreatment levels, similar to the signal from animals receiving saline only. Consistent with other studies, nontargeted GNBs passively accumulated in the tortuous neovascular but provided less than half of the contrast enhancement of the targeted agent. Microscopic studies revealed that the vascular constrained, rhodamine-labeled α_vβ_3-GNBs homed specifically to immature neovasculature (PECAM+, Tie-2−) along the immediate tumor periphery, but not to nearby mature microvasculature (PECAM+, Tie-2+). The combination of PAT and α_vβ_3-GNBs offered sensitive and specific discrimination and quantification of angiogenesis in vivo, which may be clinically applicable to a variety of highly prevalent diseases, including cancer and cardiovascular disease.—Pan, D., Pramanik, M., Senpan, A., Allen, J. S., Zhang, H., Wickline, S. A., Wang, L. V., Lanza, G. M. Molecular photoacoustic imaging of angiogenesis with integrin-targeted gold nanobeacons.

Aim: Receptor-specific small molecules and nanoparticles are widely used in molecular imaging of tumors. Although some studies have described the relative strengths and weaknesses of the two approaches, reports of a direct comparison and analysis of the two strategies are lacking. Herein, we compared the tumor-targeting characteristics of a small near-infrared fluorescent compound (cypate–peptide conjugate) and relatively large perfluorocarbon-based nanoparticles (250 nm diameter) for imaging ανβ3-integrin receptor expression in tumors. Materials & methods: Near-infrared fluorescent small molecules and nanoparticles were administered to living mice bearing subcutaneous or intradermal syngeneic tumors and imaged with whole-body and high-resolution optical imaging systems. Results: The nanoparticles, designed for vascular constraint, remained within the tumor vasculature while the small integrin-avid ligands diffused into the tissue to target integrin expression on tumor and endothelial cells. Targeted small-molecule and nanoparticle contrast agents preferentially accumulated in tumor tissue with tumor-to-muscle ratios of 8 and 7, respectively, compared with 3 for nontargeted nanoparticles. Conclusion: Fluorescent small molecular probes demonstrate greater overall early tumor contrast and rapid visualization of tumors, but the vascular-constrained nanoparticles are more selective for detecting cancer-induced angiogenesis. A combination of both imaging agents provides a strategy to image and quantify integrin expression in tumor tissue and tumor-induced neovascular systems.

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.

The purpose of this study was to evaluate the suitability of a macromolecular MRI contrast agent (paramagnetic nanoparticles, PNs) for the characterization of tumor angiogenesis. Our aim was to estimate the permeability of PNs in developing tumor vasculature and compare it with that of a low molecular weight contrast agent (Gd-DTPA) using dynamic contrast-enhanced MRI (DCE). Male New Zealand white rabbits (n = 5) underwent DCE MRI 12–14 days after Vx-2 tumor fragments were implanted into the left hind limb. Each contrast agent (PNs followed by Gd-DTPA) was evaluated using a DCE protocol and transendothelial transfer coefficient (Ki) maps were calculated using a two-compartment model. Two regions of interest (ROIs) were located within the tumor core and hindlimb muscle and five ROIs were placed within the tumor rim. Comparisons were performed using repeated measures analysis of variance (ANOVA). The Ki values estimated using PNs were significantly lower than those obtained for Gd-DTPA (p = 0.018). When PNs and Gd-DTPA data were analyzed separately, significant differences were identified among tumor rim ROIs for PNs (p

Molecular imaging and targeted drug delivery play an important role toward personalized medicine, which is the future of patient management. Of late, nanoparticle-based molecular imaging has emerged as an interdisciplinary area, which shows promises to understand the components, processes, dynamics and therapies of a disease at a molecular level. The unprecedented potential of nanoplatforms for early detection, diagnosis and personalized treatment of diseases, have found application in every biomedical imaging modality. Biological and biophysical barriers are overcome by the integration of targeting ligands, imaging agents and therapeutics into the nanoplatform which allow for theranostic applications. In this article, we have discussed the opportunities and potential of targeted molecular imaging with various modalities putting a particular emphasis on perfluorocarbon nanoemulsion-based platform technology.

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 alpha(nu)beta3-targeted nanoparticle molecular imaging agent that affords sensitive nuclear detection in conjunction with high-resolution MR characterization of tumor angiogenesis.In part 1, New Zealand white rabbits (n = 21) bearing 14d Vx2 tumor received either alpha(nu)beta3-targeted 99mTc nanoparticles at doses of 11, 22, or 44 MBq/kg, nontargeted 99mTc nanoparticles at 22 MBq/kg, or alpha(nu)beta3-targeted 99mTc nanoparticles (22 MBq/kg) competitively inhibited with unlabeled alpha(nu)beta3-nanoparticles. All animals were imaged dynamically over 2 hours with a planar camera using a pinhole collimator. In part 2, the effectiveness of alpha(nu)beta3-targeted 99mTc nanoparticles in the Vx2 rabbit model was demonstrated using clinical SPECT-CT imaging techniques. Next, MR functionality was incorporated into alpha(nu)beta3-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.alpha(nu)beta3-Targeted 99mTc nanoparticles at 22 MBq/kg produced the highest tumor-to-muscle contrast ratio (8.56 +/- 0.13, TMR) versus the 11 MBq/kg (7.32 +/- 0.12) and 44 MBq/kg (6.55 +/- 0.07) doses, (P0.05). TMR of nontargeted particles at 22.2 MBq/kg (5.48 +/- 0.09) was less (P0.05) than the equivalent dosage of alpha(nu)beta3-targeted 99mTc nanoparticles. Competitively inhibition of 99mTc alpha(nu)beta3-integrin-targeted nanoparticles at 22.2 MBq/kg reduced (P0.05) 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 alpha(nu)beta3-nanoparticles with SPECT-CT produced a clear neovasculature signal from the tumor that was absent in the nonimplanted hind leg. Using alpha(nu)beta3-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.Dual modality molecular imaging with alpha(nu)beta3-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.

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. © 2008 Wiley-Liss, Inc.

A principal objective in cancer research and diagnosis is the noninvasive detection of small preclinical lesions, which afford opportunities for early intervention. Molecular imaging with targeted MRI contrast agents has emerged as a promising diagnostic approach offering high resolution depiction of pathological anatomy and detection of associated disease biomarkers (1,2). However, molecular MRI with contrast agents (lanthanide- or iron oxide-based) entails indirect assessment of the effect of targeted agents on the relaxation of surrounding water molecules (3), achieving only relative tumor contrast enhancement above the background proton signal. Additionally, intravenous injection of iron oxide compounds for molecular MRI require a delay of 24 h or more for blood clearance to avoid detection of an overwhelming blood pool signal (4). As lengthy delays pose a problem for efficient clinical imaging of patients, it would be preferable to use an agent with a unique MR signature that could be detected shortly after injection and was devoid of background signal or susceptibility artifact. Our laboratory has developed targeted liquid perfluorocarbon (PFC) nanoparticles for molecular imaging that are operational at both clinical (1.5 Tesla [T]) and research (11.7T) field strengths (5). Nanoparticles have been targeted to angiogenesis by incorporating high-affinity peptidomimetic binding ligands specific for αvβ3-integrin, which is highly up-regulated on activated malignant neovasculature endothelium (6). These 200-nm PFC particles contain very high concentrations of 19F (~100M), a high-signal nucleus for MRI. Because the low physiological concentration of 19F in biological tissues is essentially undetectable by MRI (7), PFC nanoparticles offer a unique MR signature with no inherent tissue background signal. To date, most cancer-related molecular MRI efforts have focused on detecting relatively large and readily apparent xenograft tumors, while studies on early detection of precancerous lesions remain lacking. Genetically engineered mouse models exhibiting well-defined multi-stage carcinogenesis offer opportunities for using molecular imaging to track stage-specific molecular events that drive lesion growth. The K14-HPV16 mouse is a model of squamous cell cancer, derived by incorporating human papilloma virus (HPV) transgenic segments E6 and E7 into the mouse genome under control of the K14 keratin promoter (8). At 5–8 months of age, dysplastic lesions in the ear epidermis are associated with expansive dermal papillation and a dense supporting network of neovasculature. In 18% of mice, these lesions contain foci of microinvasive cancer, which appear as grossly visible malignancies by 12 months of age (9). We propose that the early angiogenic substrate in this model might serve as an image-based biomarker of precancerous lesions with an appropriate targeted molecular contrast agent. In this work, we used a targeted MRI contrast agent with a unique 19F-based signature in conjunction with a novel application of 19F diffusion sensitive MRI techniques similar to those used by Neil and Ackerman to study the pseudo-diffusion of intravascular perfluorocarbon emulsions (10). We used these techniques to selectively suppress the unbound and flowing 19F nanoparticle signal in the blood pool. This strategy allowed for specific and rapid detection of the 19F signal derived only from targeted nanoparticles bound to lesion neovasculature. We demonstrated that 19F diffusion sensitive MRS directly reports nanoparticle binding to the up-regulated microvasculature of precancerous squamous lesions by comparing the decay of diffusion sensitive 19F signal in the epidermis of the ear in K14-HPV16 mice and controls.

Atherosclerosis is a chronic, inflammatory disease in which ruptured plaques can lead to serious thrombotic events, including myocardial infarction or stroke. Often these cardiovascular events occur with no previous recognition of symptoms and only moderate stenosis. New diagnostic techniques are needed for earlier diagnosis and staging of atherosclerotic disease, so appropriate treatments, interventional procedures, or lifestyle changes can begin. Recent developments in nanotechnology could advance clinical imaging of molecular biomarkers, particularly for cardiovascular diagnosis.In this review, selected nanotechnologies under development for early detection of atherosclerotic disease and identification of vulnerable plaques are presented.The scope of this review encompasses molecular imaging of atherosclerosis using nanoparticle contrast agents. Nanoparticle approaches are grouped by their corresponding diagnostic imaging modality.Diagnostic imaging techniques employing nanoparticle contrast agents targeted to molecular signatures of atherosclerotic disease offer hope for improved non-invasive detection.

Fumagillin suppresses angiogenesis in cancer models and clinical trials, but it is associated with neurotoxicity at systemic doses. In this study, ανβ3-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 ανβ3-targeted fumagillin nanoparticles (30 μg/kg), ανβ3-targeted nanoparticles without drug, nontargeted fumagillin nanoparticles (30 μg/kg) or saline. On day 16, MRI was performed with ανβ3-targeted paramagnetic nanoparticles to quantify tumor size and assess neovascularity. Tumor volume was reduced among rabbits receiving ανβ3-targeted fumagillin nanoparticles (470±120 mm3) compared with the three control groups: nontargeted fumagillin nanoparticles (1370±300 mm3, P

Molecular imaging is a novel tool that has allowed noninvasive diagnostic imaging to transition from gross anatomical description to identification of specific tissue epitopes and observation of biological processes at the cellular level. Until recently, this technique was confined to the field of nuclear imaging; however, advances in nanotechnology have extended this research to include magnetic resonance (MR) imaging, positron emission tomography (PET), single photon emission computed tomography (SPECT), and ultrasound (US), among others. The application of nanotechnology to MR, SPECT, and US molecular imaging has generated several candidate contrast agents. We discuss the application of one multimodality platform, a targeted perfluorocarbon nanoparticle. Our results show that it is useful for noninvasive detection with all three imaging modalities and may additionally be used for local drug delivery.

Earlier tumor detection can improve 5-year survival of patients, particularly among those presenting with cancers less than 1 cm in diameter. alpha(nu)beta(3)-Targeted (111)In 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. alpha(nu)beta(3)-Targeted (111)In/NP bearing approximately 10 (111)In/NP vs. approximately 1 (111)In/NP nuclide payloads were compared to nontargeted radiolabeled control particles. In vivo competitive binding studies were used to assess ligand-targeting specificity. alpha(nu)beta(3)-Integrin-targeted NP with approximately 10 (111)In/NP provided better (p < 0.05) tumor-to-muscle ratio contrast (6.3 +/- 0.2) than approximately 1 (111)In/NP (5.1 +/- 0.1) or nontargeted particles with approximately 10 (111)In/NP (3.7 +/- 0.1) over the initial 2-hr postinjection. At 18 hr, mean tumor activity in rabbits receiving alpha(nu)beta(3)-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 alpha(nu)beta(3)-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 (t(1/2)beta) was estimated to be approximately 5 hr using a 2-compartment model. alpha(nu)beta(3)-Targeted (111)In 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.

As atherosclerosis remains one of the most prevalent causes of patient mortality, the ability to diagnose early signs of plaque rupture and thrombosis represents a significant clinical need. With recent advances in nanotechnology, it is now possible to image specific molecular processes noninvasively with MRI, using various types of nanoparticles as contrast agents. In the context of cardiovascular disease, it is possible to specifically deliver contrast agents to an epitope of interest for detecting vascular inflammatory processes, which serve as predecessors to atherosclerotic plaque development. Herein, we review various applications of nanotechnology in detecting atherosclerosis using MRI, with an emphasis on perfluorocarbon nanoparticles and fluorine imaging, along with theranostic prospects of nanotechnology in cardiovascular disease.

Objective— Angiogenic expansion of the vasa vasorum is a well-known feature of progressive atherosclerosis, suggesting that antiangiogenic therapies may stabilize or regress plaques. α ν β 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 α ν β 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 μg/kg or 30 μg/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 α ν β 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.

In this study, porcine carotid arteries were subjected to balloon overstretch injury followed by local delivery of paramagnetic nanoparticles targeted to alphavbeta3-integrin expressed by smooth muscle cells or collagen III within the extracellular matrix. Carotid T1-weighted angiography and vascular imaging was performed at 1.5T. While MR angiograms were indistinguishable between control and targeted vessel segments, alphavbeta3-integrin-and collagen Ill-targeted nanoparticles spatially delineated patterns and volumes of stretch injury. In conclusion, MR molecular imaging with alphavbeta3-integrin or collagen Ill-targeted nanoparticles enables the non-invasive, three-dimensional characterization of arterial pathology unanticipated from routine angiography.

Neovascularization is a critical component in the progression of malignant melanoma. The objective of this study was to determine whether alpha(nu)beta(3)-targeted paramagnetic nanoparticles can detect and characterize sparse alpha(nu)beta integrin expression on neovasculature induced by nascent melanoma xenografts ( approximately 30 mm(3)) at 1.5T. Athymic nude mice bearing human melanoma tumors were intravenously injected with alpha(v)beta(3)-integrin-targeted paramagnetic nanoparticles, nontargeted paramagnetic nanoparticles, or alpha(v)beta(3)-targeted-nonparamagnetic nanoparticles 2 hr before they were injected with alpha(v)beta(3)-integrin-targeted paramagnetic nanoparticles (i.e., in vivo competitive blockade) and imaged with MRI. Contrast enhancement of neovascularity in animals that received alpha(nu)beta(3)-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 alpha(nu)beta(3)-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.

Molecular imaging agents are extending the potential of noninvasive medical diagnosis from basic gross anatomic descriptions to complicated phenotypic characterizations based on the recognition of unique cell surface biochemical signatures. Although originally the purview of nuclear medicine, molecular imaging is now a prominent feature of most clinically relevant imaging modalities, in particular magnetic resonance (MR) imaging. MR nanoparticulate agents afford the opportunity not only for targeted diagnostic studies but also for image-monitored site-specific therapeutic delivery, much like the "magic bullet" envisioned by Paul Erhlich 100 years ago. Combining high-resolution MR molecular imaging with drug delivery will facilitate verification and quantification of treatment (ie, rational targeted therapy) and will offer new clinical approaches to many diseases.

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 r(1) relaxivities (1.5 T) for Gd-DTPA-PE (33.7 (s*mM)(-1) and 2.48 x 10(6) (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 R(1) values at 1.5 T (deltaR(1) = 0.77 +/- 0.02 1/s) relative to Gd-DTPA-BOA (deltaR(1) = 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.

The detection of regional lymph node metastases is important in cancer staging as it influences the prognosis of the patient and the strategy for treatment. Sentinel lymph node biopsy (SLNB) has emerged as the standard of care for axillary staging of clinically node-negative breast cancer.1,2 The SLN hypothesis states that the pathological status of the axilla can be accurately predicted by determining the status of the first (i.e., sentinel) lymph node(s) that drains from the primary tumor. Besides the presence of metastasis or micrometastasis detected in the SLN after excision and histological examination, the total number of involved regional lymph nodes is important in staging the disease, with the number predicting overall survival with an inverse relationship. Typically, the conventional SLNB procedure consists of injecting radioactive tracers and/or MB dye to mark the lymphatic system and guide the surgeon to the sentinel node. The radioactive tracer is injected a few hours prior to the surgery, while MB, which spreads relatively quickly through lymph vessels, is injected in the operating room. A few minutes following MB injection, a surgical incision is made in the area indicated by a handheld Geiger counter. The surgeon interrogates the axilla and identifies nodes that have been stained blue or nodes that are detected as radioactive with the Geiger counter. These nodes are then removed for histological examination to determine the presence of tumor metastases.

Expanded and aberrant bronchial vascularity, a prominent feature of the chronic asthmatic airway, might explain persistent airway wall edema and sustained leukocyte recruitment. Since it is well established that there are causal relationships between exposure to house dust mite (HDM) and the development of asthma, determining the effects of HDM in rats, mammals with a bronchial vasculature similar to humans, provides an opportunity to study the effects of bronchial angiogenesis on airway function directly. We studied rats exposed bi-weekly to HDM (Der p 1; 50 μg/challenge by intranasal aspiration, 1, 2, 3 weeks) and measured the time course of appearance of increased blood vessels within the airway wall. Results demonstrated that within 3 weeks of HDM exposure, the number of vessels counted within airway walls of bronchial airways (0.5–3 mm perimeter) increased significantly. These vascular changes were accompanied by increased airway responsiveness to methacholine. A shorter exposure regimen (2 weeks of bi-weekly exposure) was insufficient to cause a significant increase in functional vessels or reactivity. Yet, 19F/1H MR imaging at 3T following αvβ3-targeted perfluorocarbon nanoparticle infusion revealed a significant increase in 19F signal in rat airways after 2 weeks of bi-weekly HDM, suggesting earlier activation of the process of neovascularization. Although many antigen-induced mouse models exist, mice lack a bronchial vasculature and consequently lack the requisite human parallels to study bronchial edema. Overall, our results provide an important new model to study the impact of bronchial angiogenesis on chronic inflammation and airways hyperreactivity.

Duchenne muscular dystrophy in boys progresses rapidly to severe impairment of muscle function and death in the second or third decade of life. Current supportive therapy with corticosteroids results in a modest increase in strength as a consequence of a general reduction in inflammation, albeit with potential untoward long-term side effects and ultimate failure of the agent to maintain strength. Here, we demonstrate that alternative approaches that rescue defective autophagy in mdx mice, a model of Duchenne muscular dystrophy, with the use of rapamycin-loaded nanoparticles induce a reproducible increase in both skeletal muscle strength and cardiac contractile performance that is not achievable with conventional oral rapamycin, even in pharmacological doses. This increase in physical performance occurs in both young and adult mice, and, surprisingly, even in aged wild-type mice, which sets the stage for consideration of systemic therapies to facilitate improved cell function by autophagic disposal of toxic byproducts of cell death and regeneration.—Bibee, K. P., Cheng, Y.-J., Ching, J. K., Marsh, J. N., Li, A. J., Keeling, R. M., Connolly, A. M., Golumbek, P. T., Myerson, J. W., Hu, G., Chen, J., Shannon, W. D., Lanza, G. M., Weihl, C. C., Wickline, S. A. Rapamycin nanoparticles target defective autophagy in muscular dystrophy to enhance both strength and cardiac function.

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 Gd 3+ 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.

RATIONALE AND OBJECTIVES Molecular imaging with targeted contrast agents enables tissues to be distinguished by detecting specific cell-surface receptors. In the present study, a ligand-targeted acoustic nanoparticle system is used to identify angioplasty-induced expression of tissue factor by smooth muscle cells within carotid arteries. METHODS Pig carotid arteries were overstretched with balloon catheters, treated with tissue factor-targeted or a control nanoparticle system, and imaged with intravascular ultrasound before and after treatment. RESULTS Tissue factor-targeted emulsions bound and increased the echogenicity and gray-scale levels of overstretched smooth muscle cells within the tunica media, versus no change in contralateral control arteries. Expression of stretch-induced tissue factor in carotid artery media was confirmed by immunohistochemistry. CONCLUSIONS The potential for abnormal thrombogenicity of balloon-injured arteries, as reflected by smooth muscle expression of tissue factor, was imaged using a novel, targeted, nanoparticulate ultrasonic contrast agent.

In this study, the sensitivity of a novel fibrin-targeted contrast agent for fibrin detection was defined in vitro on human thrombus. The contrast agent was a lipid-encapsulated perfluorocarbon nanoparticle with numerous Gd-DTPA complexes incorporated into the outer surface. After binding to fibrin clots, scanning electron microscopy of treated clots revealed dense accumulation of nanoparticles on the clot surfaces. Fibrin clots with sizes ranging from 0.5‐7.0 mm were imaged at 4.7 T with or without treatment with the targeted contrast agent. Regardless of sizes, untreated clots were not detectable by T1-weighted MRI, while targeted contrast agent dramatically improved the detectability of all clots. Decreases in T1 and T2 relaxation times (20 ‐ 40%) were measured relative to the surrounding media and the control clots. These results suggest the potential for sensitive and specific detection of microthrombi that form on the intimal surfaces of unstable atherosclerotic plaque. Magn Reson Med 44:867‐ 872, 2000. © 2000 Wiley-Liss, Inc.

Site-directed contrast enhancement of angiogenic vessels in vivo was demonstrated using antibody targeting of an MRI contrast agent to the αvβ3 integrin, a molecular marker characteristic of angiogenic endothelium. The agent was tested in a rabbit corneal micropocket model, in which neovasculature is induced in the cornea using basic fibroblast growth factor. The targeted contrast agent consists of Gd-perfluorocarbon nanoparticles linked to αvβ3 integrin antibody DM101. The animal group receiving the targeted contrast agent displayed a 25% increase in the average MR signal intensity after 90 min. Control groups in which the nanoparticles are either used alone, linked to an isotype-matched antibody, or linked to DM101 and administered following receptor blocking did not display MR contrast enhancement at similar dose levels. These findings indicate that the antibody-targeted agent enhances MR signal intensity in the capillary bed in a corneal micropocket model of angiogenesis, and is selectively retained within the angiogenic region via specific interaction with the αvβ3 epitope. Magn Reson Med 44:433–439, 2000. © 2000 Wiley-Liss, Inc.

Bicuspid pulmonary valves and pulmonary artery aneurysms are two rare entities, reported in association, and usually attributed to hemodynamic alterations caused by the bicuspid pulmonary valve. We present magnetic resonance images of a patient with a bicuspid pulmonary valve and pulmonary artery aneurysm, and propose an alternative mechanism for this association, based on recent embryologic studies that link anomalies of the semilunar valves and great vessels with derangement of the cardiac neural crest cell development.

Background Endothelial activation is one of the necessary initial steps in the formation of atherosclerotic plaque that facilitates immune cell recruitment and retention. To image and quantify early markers of endothelial inflammation, we recently developed a multiplexing peptide-based targeting system for post-formulation functionalization of perfluorocarbon (19F) nanoparticles (PFC NP) that is intrinsically quantifiable at a binding site based on magnetic resonance spectroscopy. The approach is now demonstrated in vivo to detect and quantify VCAM-1 in ApoE-/- mouse aorta. Methods

This study delineates the role of angiotensin II type I (AT1) receptor in the remodeling of Syrian cardiomyopathic hamsters. Twelve cardiomyopathic (T0-2) hamsters received L-158,809 treatment ad libitum in their drinking water (27 µg/ml) and 9 cardiomyopathic and 9 normal F1-B hamsters received tap water from 1 to 4 months of age. Although pharmacologically effective with regard to complete suppression of the blood pressure response to angiotensin II infusion, L-158,809 did not diminish the progression or severity of cardiomyopathy. Heart weight/100 g body weight and left ventricular wall thickness adjusted for body weight of both L-158,809 and cardiomyopathic control hamsters did not differ and exceeded those of F1-B controls (p < 0.05). Myocardial material properties (e.g., stiffness and density) of cardiomyopathic hamsters treated with L-158,809 were not affected. Thus, the progression of fibrosis, calcification, and necrosis in T0-2 cardiomyopathic hamsters was not sensitive to AT1 receptor blockade.

Herein, we review the use of non-nephrotoxic perfluorocarbon nanoparticles (PFC NPs) for noninvasive detection and therapy of kidney diseases, and we provide a synopsis of other related literature pertinent to their anticipated clinical application. Recent reports indicate that PFC NPs allow for quantitative mapping of kidney perfusion and oxygenation after ischemia-reperfusion injury with the use of a novel multinuclear (1)H/(19)F magnetic resonance imaging approach. Furthermore, when conjugated with targeting ligands, the functionalized PFC NPs offer unique and quantitative capabilities for imaging inflammation in the kidney of atherosclerotic ApoE-null mice. In addition, PFC NPs can facilitate drug delivery for treatment of inflammation, thrombosis, and angiogenesis in selected conditions that are comorbidities for kidney failure. The excellent safety profile of PFC NPs with respect to kidney injury positions these nanomedicine approaches as promising diagnostic and therapeutic candidates for treating and following acute and chronic kidney diseases.

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.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.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.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.

Angiogenesis is an important constituent of many inflammatory pulmonary diseases, which has been unappreciated until recently. Early neovascular expansion in the lungs in preclinical models and patients is very difficult to assess noninvasively, particularly quantitatively. The present study demonstrated that (19)F/(1)H MR molecular imaging with αvβ3-targeted perfluorocarbon nanoparticles can be used to directly measure neovascularity in a rat left pulmonary artery ligation (LPAL) model, which was employed to create pulmonary ischemia and induce angiogenesis. In rats 3 days after LPAL, simultaneous (19)F/(1)H MR imaging at 3T revealed a marked (19)F signal in animals 2 h following αvβ3-targeted perfluorocarbon nanoparticles [(19)F signal (normalized to background) = 0.80 ± 0.2] that was greater (p = 0.007) than the non-targeted (0.30 ± 0.04) and the sham-operated (0.07 ± 0.09) control groups. Almost no (19)F signal was found in control right lung with any treatment. Competitive blockade of the integrin-targeted particles greatly decreased the (19)F signal (p = 0.002) and was equivalent to the non-targeted control group. Fluorescent and light microscopy illustrated heavy decorating of vessel walls in and around large bronchi and large pulmonary vessels. Focal segmental regions of neovessel expansion were also noted in the lung periphery. Our results demonstrate that (19)F/(1)H MR molecular imaging with αvβ3-targeted perfluorocarbon nanoparticles provides a means to assess the extent of systemic neovascularization in the lung.

Background: Bone metastases occur in 70% of metastatic breast cancer patients and are a leading cause of morbidity. Current therapies are often palliative, in part due to a lack of specificity for tumor targets within the bone. Integrin αvβ3 is overexpressed on neo-angiogenic blood vessels, tumor-promoting macrophages, osteoclasts, and more aggressive breast cancer cells, making it an attractive therapeutic target. Objective: The goal of this study was to use Sn2 lipase-labile docetaxel-prodrug nanoparticle, targeted against activated integrin αvβ3, to attenuate breast cancer metastases. Methods: A novel phospholipid-based micelle (∼12.5 nm) was functionalized with a peptidomimetic for activated integrin αvβ3, and designed to carry either rhodamine for fluorescent labeling or Sn2 lipase-labile prodrug of docetaxel (DTX-PD) for drug delivery. For microscopic localization studies, fluorescently labeled micelles were prepared with or without integrin αvβ3-targeting. C57BL/6 female mice received MMTV-PyMT breast cancer cell line (luciferase-labeled) via intracardiac (IC) injection to achieve tumor metastasis in all major organs. On day 8 post-IC injection, micelle preparations were administered i.v. and circulated within C57BL/6 mice for 3 hours prior to sacrifice and tissue collection. For drug efficacy studies, Sn2 lipase-labile docetaxel-prodrug was incorporated into αvβ3-micelles (αvβ3-DTX-PD). C57BL/6 female mice IC injected with MMTV-PyMT cells (luciferase-labeled) were treated with αvβ3-DTX-PD, or molar equivalent dose of free-DTX, or saline. Beginning on day 4 post-IC injection, mice were treated 3 times, once every 3 days (1.85mg/kg DTX per treatment). On day 12 post-IC injection, metastatic burden in the major organs was analyzed via ex vivo bioluminescent imaging. Results: Fluorescent histological analysis of the tibiofemoral bone region showed significant colocalization of αvβ3-micelles with breast cancer bone metastases, as compared with non-targeted micelles (6.5-fold increase, p Conclusion: These findings suggest that the unique elevated expression of integrin αvβ3 within breast cancer bone metastases could be exploited with αvβ3-DTX-PD micelles for effective therapy. Citation Format: Michael H. Ross, Alison K. Esser, Anne H. Schmieder, Grace Cui, Xiaoxia Yang, Xinming Su, Dipanjan Pan, Gregory M. Lanza, Katherine N. Weilbaecher. Integrin αvβ3-targeted lipase-labile docetaxel-prodrug micelles preferentially treat breast cancer bone metastases. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2198.

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.

Herein, initial results are presented as obtained in a hypercholesterol rabbit model with the simultaneous 19F/1H balanced UTE-SSFP technique and using ανβ3-targeted PFOB nanoparticles to establish the feasibility of high sensitivity MR molecular imaging of Gd-free, fluorine-based, clinically-relevant contrast agents.

Summary Mathematical modeling studies have suggested that nonspherical, disc-shaped nanoparticles may have optimal intravascular flow and homing characteristics. In this study, we report the development of a fibrin-specific high-relaxivity bialy-shaped polymeric nanoparticle using porphyrin-chelated manganese. We anticipate that this agent would be highly effective for molecular imaging of microthrombi in ruptured atherosclerotic plaques. Background Detection of microthrombi within fissures of vulnerable atherosclerotic plaques requires a sensitive molecular imaging contrast agent. Moreover, recent reports based on mathematical modeling suggest that nonspherical, disc-shaped nanoparticles could have improved intravascular flow characteristics, which may improve liganddirected targeting. In light of the concern surrounding the use of gadolinium in patients with severe renal disease, the goal of this research was to develop a nonspherical fibrin-targeted manganese-based molecular imaging agent.

Summary Duchene Muscular Dystrophy (DMD) is a lethal disease caused by ubiquitous lack of dystrophin, but the interaction with regional cardiac mechanical forces that may facilitate eventual expression of abnormal contractile function is unknown. Diffusion tensor MRI (DTI) was used to evaluate function in Langendorff perfused hearts in the mdx mouse model of DMD. Abnormal calcium kinetics (by optical mapping) and sheet mechanics (by DTMRI) occurred more prominently at the mid-upper ventricle, suggesting that regional mechanics influence the development of heart failure. Background