Ralph W. Fuhrhop

Molecular Imaging of Angiogenesis in Early-Stage Atherosclerosis With αvβ3-Integrin-Targeted Nanoparticles

Background—Angiogenesis is a critical feature of plaque development in atherosclerosis and might play a key role in both the initiation and later rupture of plaques that lead to myocardial infarction and stroke. The precursory molecular or cellular events that initiate plaque growth and that ultimately contribute to plaque instability, however, cannot be detected directly with any current diagnostic modality. Methods and Results—Atherosclerosis was induced in New Zealand White rabbits fed 1% cholesterol for ≈80 days. &agr;v&bgr;3-Integrin–targeted, paramagnetic nanoparticles were injected intravenously and provided specific detection of the neovasculature within 2 hours by routine magnetic resonance imaging (MRI) at a clinically relevant field strength (1.5 T). Increased angiogenesis was detected as a 47±5% enhancement in MRI signal averaged throughout the abdominal aortic wall among rabbits that received &agr;v&bgr;3-targeted, paramagnetic nanoparticles. Pretreatment of atherosclerotic rabbits with &agr;v&bgr;3-targeted, nonparamagnetic nanoparticles competitively blocked specific contrast enhancement of the &agr;v&bgr;3-targeted paramagnetic agent. MRI revealed a pattern of increased &agr;v&bgr;3-integrin distribution within the atherosclerotic wall that was spatially heterogeneous along both transverse and longitudinal planes of the abdominal aorta. Histology and immunohistochemistry confirmed marked proliferation of angiogenic vessels within the aortic adventitia, coincident with prominent, neointimal proliferation among cholesterol-fed, atherosclerotic rabbits in comparison with sparse incidence of neovasculature in the control animals. Conclusions—This molecular imaging approach might provide a method for defining the burden and evolution of atherosclerosis in susceptible individuals as well as responsiveness of individual patients to antiatherosclerotic therapies.

Targeted Antiproliferative Drug Delivery to Vascular Smooth Muscle Cells With a Magnetic Resonance Imaging Nanoparticle Contrast Agent Implications for Rational Therapy of Restenosis

Background—Restenosis is a serious complication of coronary angioplasty that involves the proliferation and migration of vascular smooth muscle cells (VSMCs) from the media to the intima, synthesis of extracellular matrix, and remodeling. We have previously demonstrated that tissue factor–targeted nanoparticles can penetrate and bind stretch-activated vascular smooth muscles in the media after balloon injury. In the present study, the concept of VSMC-targeted nanoparticles as a drug-delivery platform for the prevention of restenosis after angioplasty is studied. Methods and Results—Tissue factor–targeted nanoparticles containing doxorubicin or paclitaxel at 0, 0.2, or 2.0 mole% of the outer lipid layer were targeted for 30 minutes to VSMCs and significantly inhibited their proliferation in culture over the next 3 days. Targeting of the nanoparticles to VSMC surface epitopes significantly increased nanoparticle antiproliferative effectiveness, particularly for paclitaxel. In vitro dissolution studies revealed that nanoparticle drug release persisted over one week. Targeted antiproliferative results were dependent on the hydrophobic nature of the drug and noncovalent interactions with other surfactant components. Molecular imaging of nanoparticles adherent to the VSMC was demonstrated with high-resolution T1-weighted MRI at 4.7T. MRI 19F spectroscopy of the nanoparticle core provided a quantifiable approach for noninvasive dosimetry of targeted drug payloads. Conclusions—These data suggest that targeted paramagnetic nanoparticles may provide a novel, MRI-visualizable, and quantifiable drug delivery system for the prevention of restenosis after angioplasty.

Targeted nanoparticles for quantitative imaging of sparse molecular epitopes with MRI

Before molecular imaging with MRI can be applied clinically, certain problems, such as the potential sparseness of molecular epitopes on targeted cell surfaces, and the relative weakness of conventional targeted MR contrast agents, must be overcome. Accordingly, the conditions for diagnostic conspicuity that apply to any paramagnetic MRI contrast agent with known intrinsic relaxivity were examined in this study. A highly potent paramagnetic liquid perfluorocarbon nanoparticle contrast agent (∼250 nm diameter, >90000 Gd3+/particle) was imaged at 1.5 T and used to successfully predict a range of sparse concentrations in experimental phantoms with the use of standard MR signal models. Additionally, we cultured and targeted the smooth muscle cell (SMC) monolayers that express “tissue factor,” a glycoprotein of crucial significance to hemostasis and response to vascular injury, by conjugating an anti‐tissue factor antibody fragment to the nanoparticles to effect specific binding. Quantification of the signal from cell monolayers imaged at 1.5 T demonstrated, as predicted via modeling, that only picomolar concentrations of paramagnetic perfluorocarbon nanoparticles were required for the detection and quantification of tissue factor at clinical field strengths. Thus, for targeted paramagnetic agents carrying high payloads of gadolinium, it is possible to quantify molecular epitopes present in picomolar concentrations in single cells with routine MRI. Magn Reson Med 51:480–486, 2004.

Quantitative “magnetic resonance immunohistochemistry” with ligand‐targeted 19F nanoparticles

Unstable atherosclerotic plaques exhibit microdeposits of fibrin that may indicate the potential for a future rupture. However, current methods for evaluating the stage of an atherosclerotic lesion only involve characterizing the level of vessel stenosis, without delineating which lesions are beginning to rupture. Previous work has shown that fibrin‐targeted, liquid perfluorocarbon nanoparticles, which carry a high payload of gadolinium, have a high sensitivity and specificity for detecting fibrin with clinical 1H MRI. In this work, the perfluorocarbon content of the targeted nanoparticles is exploited for the purposes of 19F imaging and spectroscopy to demonstrate a method for quantifiable molecular imaging of fibrin in vitro at 4.7 T. Additionally, the quantity of bound nanoparticles formulated with different perfluorocarbon species was calculated using spectroscopy. Results indicate that the high degree of nanoparticle binding to fibrin clots and the lack of background 19F signal allow accurate quantification using spectroscopy at 4.7 T, as corroborated with proton relaxation rate measurements at 1.5 T and trace element (gadolinium) analysis. Finally, the extension of these techniques to a clinically relevant application, the evaluation of the fibrin burden within an ex vivo human carotid endarterectomy sample, demonstrates the potential use of these particles for uniquely identifying unstable atherosclerotic lesions in vivo. Magn Reson Med 52:1255–1262, 2004.

Noninvasive Photoacoustic and Fluorescence Sentinel Lymph Node Identification using Dye-Loaded Perfluorocarbon Nanoparticles

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.

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.

Improvements in the ultrasonic contrast of targeted perfluorocarbon nanoparticles using an acoustic transmission line model

Targeted acoustic contrast agents offer the potential for sensitive ultrasonic detection of pathologic tissues. We have previously reported the development of a ligand-targeted, lipid-encapsulated, liquid perfluorodichlorooctane ultrasonic contrast system with a small nominal particle size (approximately 250-nm diameter)Perfluorocarbon nanoparticles substantially increase reflectivity when bound to targeted surfaces, and we propose that this system can be approximated physically as a simple, thin layer, acoustic transmission line. In this study, we evaluate this model and compare the ultrasonic reflectivity of different perfluorocarbon formulations with widely varying acoustic impedances targeted to either nitrocellulose membranes or plasma thrombi in vitro. Five perfluorocarbons were investigated: perfluorohexane (PFH), perfluorooctane (PFO), perfluorooctyl bromide (PFOB), perfluorodichlorooctane (PFDCO), and perfluorodecalin (PFD). Ultrasonic reflection was measured by acoustic microscopy (17 to 35 MHz). Acoustic reflectivity was increased (P < 0.05) by all targeted perfluorocarbon formulations, and the magnitude of the contrast effect was inversely correlated with the perfluorocarbon acoustic impedance. PFH nanoparticles exhibited the greatest enhancement, and PFD nanoparticles showed the least. The acoustic transmission line model predicted well the relative differences in acoustic reflectivity and frequency dependence among the perfluorocarbon formulations. For future clinical applications, PFO nanoparticles may provide the best combination of acoustic enhancement, in vivo physical stability, and safety.

Acoustic characterization in whole blood and plasma of site-targeted nanoparticle ultrasound contrast agent for molecular imaging

The ability to enhance specific molecular markers of pathology with ultrasound has been previously demonstrated by our group employing a nanoparticle contrast agent [Lanza et al., Invest. Radiol. 35, 227-234 (2000); Ultrasound Med. Biol. 23, 863-870 (1997)]. One of the advantages of this agent is very low echogenicity in the blood pool that allows increased contrast between the blood pool and the bound, site-targeted agent. We measured acoustic backscatter and attenuation coefficient as a function of the contrast agent concentration, ambient pressure, peak acoustic pressure, and as an effect of duty cycle and wave form shape. Measurements were performed while the nanoparticles were suspended in either whole porcine blood or plasma. The nanoparticles were only detectable when insonified within plasma devoid of red blood cells and were shown to exhibit backscatter levels more than 30 dB below the backscatter from whole blood. Attenuation of nanoparticles in whole porcine blood was not measurably different from that of whole blood alone over a range of concentrations up to eight times the maximum in vivo dose. The resulting data provide upper bounds on blood pool attenuation coefficient and backscatter and will be needed to more precisely define levels of molecular contrast enhancement that may be obtained in vivo.

Properties of an entropy-based signal receiver with an application to ultrasonic molecular imaging

Qualitative and quantitative properties of the finite part, H(f), of the Shannon entropy of a continuous waveform f(t) in the continuum limit are derived in order to illuminate its use for waveform characterization. Simple upper and lower bounds on H(f), based on features of f(t), are defined. Quantitative criteria for a priori estimation of the average-case variation of H(f) and log E(f), where E(f) is the signal energy of f(t) are also derived. These provide relative sensitivity estimates that could be used to prospectively choose optimal imaging strategies in real-time ultrasonic imaging machines, where system bandwidth is often pushed to its limits. To demonstrate the utility of these sensitivity relations for this application, a study designed to assess the feasibility of identification of angiogenic neovasculature targeted with perfluorocarbon nanoparticles that specifically bind to alpha(v)beta3-integrin expression in tumors was performed. The outcome of this study agrees with the prospective sensitivity estimates that were used for the two receivers. Moreover, these data demonstrate the ability of entropy-based signal receivers when used in conjunction with targeted nanoparticles to elucidate the presence of alpha(v)beta3 integrins in primordial neovasculature, particularly in acoustically unfavorable environments.

Blood Contrast Enhancement with a Novel, Non-Gaseous Nanoparticle Contrast Agent

Modern ultrasound contrast agents primarily comprise microbubble formulations that circulate in the intravascular compartment and are designed to enhance acoustic signals reflected from the blood pool. A variety of shell materials have been utilized to stabilize gas bubbles of the order of 1–10 microns in diameter. Reflectivity from microbubbles is enhanced by resonance and non-linear physical effects. However, the overall efficacy of bubbles as contrast agents must be considered in light of their marked instability to insonification pressures, marked attenuation artifacts, “blooming” effects, and their short circulatory half-life. Low molecular weight gaseous perfluorocarbon formulations have been utilized in vivo because they may offer advantages in formulation and reflectivity. In contrast, higher molecular weight perfluorocarbon emulsions that are liquid at body temperature have been formulated as nongaseous nanoparticle preparations (diameters 100– 300 nanometers), originally for use as blood substitutes. Unfortunately they exhibit low inherent echogenicity and are poor blood pool contrast agents under conditions of conventional 2-D echocardiography or harmonic imaging, or when imaged with color flow or spectral Doppler. Nevertheless, these nanoparticle formulations are chemically inert, manifest long circulatory half-lives, are not destroyed by ultrasonic imaging, and they possess low acoustic attenuation. Such features might still render them of interest as blood pool contrast agents if properly formulated and imaged. Recently, a new ultrasonic imaging modality, Power Doppler Harmonic Imaging (PDHI), has been introduced (4). This technique color-encodes changes in acoustic signal amplitude and motion of ultrasonic scatterers between insonifying pulses. PDHI has been used in a number of clinical studies to assess coronary artery bypass graft patency, tumor blood flow, and myocardial perfusion. In view of the exquisite sensitivity of Doppler for detecting the presence of small scatterers with limited scattering cross-sections as compared to microbubbles (e.g., red blood cells), and the enhanced ability of PDHI to register backscatter power, we hypothesized that certain liquid perfluorocarbon nanoparticle emulsions (5) might be more efficiently detected with this new imaging modality. Furthermore, although we have demonstrated previously that the liquid nanoparticle emulsions do not manifest any appreciable resonance behavior at clinically relevant imaging frequencies, they have performed well as targeted imaging agents in vitro and in vivo over a very broad range of frequencies (5–50 MHz)(6–8). Thus we anticipated that the PDHI method might permit imaging of these nanoparticles in the blood pool without reliance on any intrinsic resonance behavior.