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The contrast mechanisms used for photoacoustic tomography (PAT) and fluorescence imaging differ in subtle, but significant, ways. The design of contrast agents for each or both modalities requires an understanding of the spectral characteristics as well as intra- and intermolecular interactions that occur during formulation. We found that fluorescence quenching that occurs in the formulation of near-infrared (NIR) fluorescent dyes in nanoparticles results in enhanced contrast for PAT. The ability of the new PAT method to utilize strongly absorbing chromophores for signal generation allowed us to convert a highly fluorescent dye into an exceptionally high PA contrast material. Spectroscopic characterization of the developed NIR dye-loaded perfluorocarbon-based nanoparticles for combined fluorescence and PA imaging revealed distinct dye-dependent photophysical behavior. We demonstrate that the enhanced contrast allows detection of regional lymph nodes of rats in vivo with time-domain optical and photoacoustic imaging methods. The results further show that the use of fluorescence lifetime imaging, which is less dependent on fluorescence intensity, provides a strategic approach to bridge the disparate contrast reporting mechanisms of fluorescence and PA imaging methods.

The relaxivity and transmetallation of two lipophilic paramagnetic chelates incorporated onto perfluorocarbon nanoparticles, i.e., gadolinium-methoxy-tetraazacyclododecane-tetraacetic acid phosphatidylethanolamine (Gd-MeO-DOTA-PE) and gadolinium-methoxy-tetraazacyclododecane-tetraacetic acid triglycine phosphatidylethanolamine (Gd-MeO-DOTA-triglycine-PE (Gd-MeO-DOTA-triglycine-PE)), were compared to a prototypic gadolinium-diethylene-triamine-pentaacetic acid bis-oleate (Gd-DTPA-BOA) paramagnetic formulation. Nanoparticles with MeO-DOTA-based chelates demonstrated higher relaxivity (40% higher for Gd-MeO-DOTA-PE and 55% higher for Gd-MeO-DOTA-triglycine-PE) and less transmetallation than the original Gd-DTPA-BOA-based agent.

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.

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.

Although angiogenesis plays a critical role in the development of atherosclerosis, its participation in early valvular disease remains speculative. To delineate the presence of angiogenesis and its potential responsiveness to medical therapy in experimental valve disease, we used unique signaling ( 19 F), background-free molecular MR nanobeacons as simultaneous diagnostic and therapeutic (theranostic) tools to quantify valvular angiogenesis in 24 cholesterol-fed NZW rabbits (0.25% cholesterol, 6 mo.) and its response to nanoparticle-borne antiangiogenic therapy with an aminopeptidase-2 inhibitor, fumagillin. Perfluorocarbon nanobeacons (~250 nm) targeted to neovascular α v β 3 integrins and loaded with 0.2% fumagillin were given i.v. at 1 mL/kg for 4 weekly doses (n=8; 30 μg/kg fumagillin/dose). Controls included treatment with nontargeted drug-bearing nanoparticles (n=7) and saline (n=9). MR spectroscopy (470 MHz) for detection of 19 F signal from bound nanoparticles was performed after valve excision. Immunocytochemistry confirmed angiogenesis in all valves. In the targeted drug treatment group, both 50% fewer vessels and 50% lower 19 F signal were observed vs nontargeted or saline groups (p ). Across all animals, cholesterol levels exerted a strong linear effect on the extent of angiogenesis (r=0.52; p Figure 1: (Left) Least squares means of 19 F signal from the saline (S), targeted fumagillin (TF), and untargeted fumagillin (UF) treated groups, normalized to the saline control. Note 50% reduction of 19 F signal in the group treated with targeted fumagillin nanoparticles compared to the untreated and untargeted controls. (Right) LS means of vessel count on stained valve sections. Note 50% fewer vessels in targeted fumagillin group corresponding to reduction in 19 F signal. (* p

The authors have developed a nongaseous, ligand-targeted perfluorocarbon nanoparticle emulsion which can acoustically enhance the presence of molecular epitopes on tissue surfaces. In a two-part study, they first demonstrate how perfluorocarbons with varying phase velocities impact the acoustic reflectivity of plasma clots targeted with an anti-fibrin perfluorocarbon nanoparticle in vitro. The targeted perfluorocarbon contrast agents increased backscattered power from 18.2 to 23.4 dB with the greatest enhancement noted for formulations with low acoustic impedance. Porcine carotid arteries were injured by percutaneous balloon angioplasty and treated in situ with a tissue factor-targeted or control nanoparticulate immunoemulsion. Intravascular ultrasound images (30 MHz) of the arteries before and after application of contrast agent demonstrated the acoustic localization and enhancement of stretch-induced tissue factor expression within the carotid media, which was not apparent in the control vessels.

The emerging field of "molecular imaging" refers to in vivo diagnosis of complex pathological processes by specific detection of unique molecular signatures of disease states either at the cellular, subcellular, or gene level. Identification of specific molecular epitopes causally associated with pathologies can be accomplished with the use of various novel targeted contrast agents, which permit conjunctive delivery and local deposition of therapeutic agents directly at the site of disease. Combining imaging with drug delivery provides verification of drug delivery and quantification of treatment, serving in effect as a new paradigm for rational drug dosing. Important potential clinical applications for targeted contrast agents include: (1) characterization of vulnerable plaques by detection of microscopic deposits of fibrin in plaque fissures prior to rupture. (2) early detection and treatment of solid tumors and metastases (colon, breast, prostate, etc.) by localization of angiogenesis. (3) detection of early atherosclerosis by assessment of epitopes related to macrophage trafficking (e.g., VCAM), and (4) diagnosis and treatment of inflammatory pathologies (infection, arthritis, myocarditis, transplant rejection, etc.) by delineation of epitopes that are markers for inflammatory cell recruitment (adhesion molecules, selectins, etc.) We provide a brief review of the field and discuss specific applications.

v 3-Integrin is a well-recognized biomarker of angiogenesis expressed on activated endothelial cells. It is a critical element in vascular restenosis where its upregulation by balloon overstretch injury promotes cell adhesion to the damaged extracellular matrix, induces extracellular metalloproteinase expression, and facilitates smooth muscle cell migration. We have conducted a series of in vivo studies in which v 3-integrin was detected by MRI (1.5T) as an early biomarker of nascent tumor growth (Vx-2 in rabbit and melanoma mice), early atherosclerosis in cholesterol-fed rabbits, and balloon overstretch-injury patterns in pigs. Paramagnetic nanoparticles covalentlycomplexed to a peptidomimetic v 3-integrin ligand were produced by microfluidization. Nontargeted paramagnetic nanoparticles and v 3-targeted nonparamagnetic nanoparticles were produced identically for control and competition studies. New Zealand White (NZW) rabbits implanted with nascent Vx-2 tumors and mice implanted with C-32 melanoma were treated systemically with v 3-integrin targeted paramagnetic nanoparticles that specifically bound and significantly enhanced (p 0.05) the MRI signal in the developing neovasculature after 2 hours (125% and 177%, respectively). Competitive blockage of v 3-integrin sites with v 3 targeted nonparamagnetic nanoparticles greatly ( 50%) diminished contrast signal enhancement (p 0.05) in both model systems. In cholesterol-fed NZW rabbits, black-blood MRI of the abdominal aorta v 3-targeted nanoparticles localized to angiogenic vessels in the expanding vaso-vasorum and produced enhanced signal (p 0.05) compared with nontargeted nanoparticles or control diet animals. In competition studies MRI enhancement was blunted, which confirmed the specificity of v 3-targeted nanoparticles. Furthermore, delayed enhancement (20 min) after injection of Gd-DTPA could not identify these early atherosclerotic changes. Finally, carotid arteries of pigs subjected to balloonoverstretch injury were treated locally with v 3 targeted, paramagnetic nanoparticles, saline or the nontargeted control. Contrast enhancement with v 3-targeted paramagnetic nanoparticles was dramatic and revealed asymmetrical patterns of balloon injury that were 50% greater than the actual balloon length and allowed the volume of vascular injury to be computed (955 234 mm2). No enhancement was discernible for the injured control vessels. v 3-Targeted paramagnetic nanoparticles provide a versatile molecular imaging platform useful for delineating vascular injury, diagnosing early atherosclerosis, and detecting nascent cancers with clinically relevant MRI systems (1.5 T). Early detection, effective therapy and sensitive longitudinal follow-up of these major disease processes with new molecular techniques could serve to noninvasively characterize and ameliorate their morbidity and mortality.

For 30 years, medical professionals have sought the ‘‘magic bullet’’ which would allow sensitive detection of disease and facilitate localized administration of potent chemotherapeutic agents without systemic toxicities. Ligand targeted ultrasonic contrast agents have the potential to improve diagnostic sensitivity, port drugs to specific pathologic tissues, and provide dosimetry of the delivered therapeutics. The authors have recently developed a ligand directed, lipid encapsulated perfluorocarbon emulsion particle (250‐nm diameter). These particles, when bound to biological surfaces, strikingly increase the acoustic reflectivity, although the particles have low inherent echogenicity. Contrast enhancement of the reflectivity of blood thrombi, fibrin clots, and nitrocellulose membranes has been tentatively explained by a transmission line model. Intravenous injection of the perfluorocarbon emulsion particles has localized and acoustically enhanced remote thrombi which were otherwise difficult to detect. The contrast agent has a 1‐h circulatory half‐life which greatly facilitates successful systemic targeting. The small particle size of these emulsions affords access to tiny capillary beds and vascular endothelial disruptions such as those following angioplasty. Collectively, these studies suggest perfluorocarbon contrast agents can target molecular epitopes within and proximate to the systemic circulation. These agents are creating new and exciting opportunities for ultrasound in medical diagnosis and therapy.