Anne M. Neubauer

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.

19F magnetic resonance imaging for stem/progenitor cell tracking with multiple unique perfluorocarbon nanobeacons

MRI has been employed to elucidate the migratory behavior of stem/progenitor cells noninva‐sively in vivo with traditional proton (1H) imaging of iron oxide nanoparticle‐labeled cells. Alternatively, we demonstrate that fluorine (19F) MRI of cells labeled with different types of liquid perfluorocarbon (PFC) nanoparticles produces unique and sensitive cell markers distinct from any tissue background signal. To define the utility for cell tracking, mononuclear cells harvested from human umbilical cord blood were grown under proendothelial conditions and labeled with nanoparticles composed of two distinct PFC cores (perfluorooctylbromide and perfluoro‐15‐crown‐5 ether). The sensitivity for detecting and imaging labeled cells was defined on 11.7T (research) and 1.5T (clinical) scanners. Stem/progenitor cells (CD34+CD133+CD31+) readily internalized PFC nanoparticles without aid of adjunctive labeling techniques, and cells remained functional in vivo. PFC‐labeled cells exhibited distinct 19F signals and were readily detected after both local and intravenous injection. PFC nanoparticles provide an unequivocal and unique signature for stem/progenitor cells, enable spatial cell localization with 19F MRI, and permit quantification and detection of multiple fluorine signatures via 19F MR spectroscopy. This method should facilitate longitudinal investigation of cellular events in vivo for multiple cell types simultaneously.—Partlow, K. C., Chen, J., Brant, J. A., Neubauer, A. M., Meyerrose, T. E., Creer, M. H., Nolta, J. A., Caruthers, S. D., Lanza, G. M., Wickline, S. A. 19F magnetic resonance imaging for stem/progenitor cell tracking with multiple unique perfluorocarbon nanobeacons. FASEB J. 21, 1647–1654 (2007)

Molecular imaging and therapy of atherosclerosis with targeted nanoparticles.

Advances in bionanotechnology are poised to impact the field of cardiovascular diagnosis and therapy for decades to come. This review seeks to illustrate selected examples of newly developed diagnostic and therapeutic nanosystems that have been evaluated in experimental atherosclerosis, thrombosis, and vascular biology. We review a variety of nanotechnologies that are capable of detecting early cardiovascular pathology, as well as associated imaging approaches and conjunctive strategies for site‐targeted treatment with nanoparticle delivery systems. J. Magn. Reson. Imaging 2007.

In vitro demonstration using 19F magnetic resonance to augment molecular imaging with paramagnetic perfluorocarbon nanoparticles at 1.5 tesla

Objectives:This study explored the use of 19F spectroscopy and imaging with targeted perfluorocarbon nanoparticles for the simultaneous identification of multiple biosignatures at 1.5 T. Materials and Methods:Two nanoparticle emulsions with perfluoro-15-crown-5-ether (CE) or perfluorooctylbromide (PFOB) cores were targeted in vitro to fibrin clot phantoms (n = 12) in 4 progressive ratios using biotin–avidin interactions. The CE nanoparticles incorporated gadolinium. Fluorine images were acquired using steady-state gradient-echo techniques; spectra using volume-selective and nonselective sampling. Results:On conventional T1-weighted imaging, clots with CE nanoparticles enhanced as expected, with intensity decreasing monotonically with CE concentration. All clots were visualized using wide bandwidth fluorine imaging, while restricted bandwidth excitation permitted independent imaging of CE or PFOB nanoparticles. Furthermore, 19F imaging and spectroscopy allowed visual and quantitative confirmation of relative perfluorocarbon nanoparticle distributions. Conclusions:19F MRI/S molecular imaging of perfluorocarbon nanoparticles in vitro suggests that noninvasive phenotypic characterization of pathologic biosignatures is feasible at clinical field strengths.

Gadolinium-modulated 19F signals from perfluorocarbon nanoparticles as a new strategy for molecular imaging.

Recent advances in the design of fluorinated nanoparticles for molecular magnetic resonance imaging (MRI) have enabled specific detection of 19F nuclei, providing unique and quantifiable spectral signatures. However, a pressing need for signal enhancement exists because the total 19F in imaging voxels is often limited. By directly incorporating a relaxation agent, gadolinium (Gd), into the lipid monolayer that surrounds the perfluorocarbon (PFC), a marked augmentation of the 19F signal from 200‐nm nanoparticles was achieved. This design increases the magnetic relaxation rate of the 19F nuclei fourfold at 1.5 T and effects a 125% increase in signal—an effect that is maintained when they are targeted to human plasma clots. By varying the surface concentration of Gd, the relaxation effect can be quantitatively modulated to tailor particle properties. This novel strategy dramatically improves the sensitivity and range of 19F MRI/MRS and forms the basis for designing contrast agents capable of sensing their surface chemistry. Magn Reson Med 60:1066–1072, 2008.

Nanomedicine opportunities for cardiovascular disease with perfluorocarbon nanoparticles

Nanomedicine promises to enhance the ability of clinicians to address some of the serious challenges responsible for cardiovascular mortality, morbidity and numerous societal consequences. Targeted imaging and therapy applications with perfluorocarbon nanoparticles are relevant to a broad spectrum of cardiovascular diseases, ranging from asymptomatic atherosclerotic disease to acute myocardial infarction or stroke. As illustrated in this article, perfluorocarbon nanoparticles offer new tools to recognize and characterize pathology, to identify and segment high-risk patients and to treat chronic and acute disease.

Nanoparticle pharmacokinetic profiling in vivo using magnetic resonance imaging.

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

Fluorine Cardiovascular Magnetic Resonance Angiography In Vivo at 1.5 T with Perfluorocarbon Nanoparticle Contrast Agents

While the current gold standard for coronary imaging is X-ray angiography, evidence is accumulating that it may not be the most sensitive technique for detecting unstable plaque. Other imaging modalities, such as cardiovascular magnetic resonance (CMR), can be used for plaque characterization, but suffer from long scan and reconstruction times for determining regions of stenosis. We have developed an intravascular fluorinated contrast agent that can be used for angiography with cardiovascular magnetic resosnace at clinical field strengths (1.5 T). This liquid perfluorocarbon nanoparticle contains a high concentration of fluorine atoms that can be used to generate contrast on 19F MR images without any competing background signal from surrounding tissues. By using a perfluorocarbon with 20 equivalent fluorine molecules, custom-built RF coils, a modified clinical scanner, and an efficient steady-state free procession sequence, we demonstrate the use of this agent for angiography of small vessels in vitro, ex vivo, and in vivo. The surprisingly high signal generated with very short scan times and low doses of perfluorocarbon indicates that this technique may be useful in clinical settings when coupled with advanced imaging strategies.

Perfluorocarbon Nanoparticles for Molecular Imaging and Targeted Therapeutics

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.

Molecular MR Imaging with Paramagnetic Perfluorocarbon Nanoparticles

Targeted contrast agents, such as perfluorocarbon (PFC) nanoparticles, have been developed to allow conventional imaging modalities, including MRI, to detect and characterize specific pathological biomarkers of early disease rather than simply observe the anatomical manifestations occurring at very late stages. PFC nanoparticles are typically 200–300 nm in diameter and are encapsulated in a phospholipid shell, which provides an ideal surface for the incorporation of targeting ligands and/or imaging agents. Through chemical modification of the paramagnetic chelates incorporated on the particle surface, nanoparticle relaxivity as well as stability can be increased to improve the efficacy of MR molecular imaging. PFC nanoparticles can be targeted to a number of different biological epitopes, including fibrin, an abundant marker of ruptured atherosclerotic plaques; αvβ3-integrin, an endothelial biomarker of angiogenesis associated with atherosclerosis, tumor growth, and vascular injury; collagen III, a component of the extracellular matrix that is exposed after balloon angioplasty; and tissue factor, a vascular smooth muscle cell (VSMC) marker that is overexpressed following vascular injury. In addition to paramagnetic nanoparticles for 1H MRI, the PFC core has a high fluorine content that can be detected with 19F MRI, providing unambiguous and quantitative mapping of the contrast agent distribution. Another distinctive advantage of PFC nanoparticles for molecular imaging applications is their compatibility with several imaging modalities, including MRI, ultrasound, nuclear imaging, and CT.