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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.

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.

Liquid perfluorocarbon nanoparticles serve as sensitive and specific targeted contrast and drug delivery vehicles by binding to specific cell surface markers. We hypothesized that application of acoustic energy at diagnostic power levels could promote nanoparticle-associated drug delivery by stimulating increased interaction between the nanoparticle's lipid layer and the targeted cell's plasma membrane. Ultrasound (mechanical index = 1.9) applied with a conventional ultrasound imaging system to nanoparticles targeted to alpha(v)beta3-integrins on C32 melanoma cancer cells in vitro produced no untoward effects. Within 5 min, lipid delivery from nanoparticles into cell cytoplasm was dramatically augmented. We also demonstrate the operation of a potential physical mechanism for this effect, the acoustic radiation force on the nanoparticles, which may contribute to the enhanced lipid delivery. Accordingly, we propose that local delivery of lipophilic substances (e.g., drugs) from targeted nanoparticles directly into cell cytoplasm can be augmented rapidly and safely with conventional ultrasound imaging devices through nondestructive mechanisms.

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 ( approximately 250 nm diameter, >90,000 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.

In an earlier studies we reported on the application of Renyi entropy, I f (r), r ≪ 2, for the detection of precancerous lesions, by detection subtle changes in backscattered waveforms f(t) that occured as targeted nanoparticles slowly accumulated near the lesion. In contrast, the signal energy, defined as the sum of squares of the over the same moving window, was unable to detect this change (as was conventional B-mode imaging). Although the computational effort to obtain the result precluded its clinical application with currently available equipment, the study raised the possibility of further sensitivity improvements by using values of r closer to the limiting value of 2, where I f (r) approaches infinity. The current study demonstrates that by extracting the asymptotic form of I f (r) as r → 2 there is no loss of sensitivity. However, the resulting algorithm is must faster than previous approaches and has an operation count that is suitable for implementation in a real-time imaging system.

Previous work has demonstrated that a signal receiver based on a limiting form of the Shannon entropy is, in certain settings, more sensitive to subtle changes in scattering architecture than conventional energy-based signal receivers [M. S. Hughes et al., J. Acoust. Soc. Am. 121, 3542–3557 (2007)]. In this paper new results are presented demonstrating further improvements in sensitivity using a signal receiver based on the Renyi entropy.

Targeted acoustic contrast agents are designed to enhance the sensitivity and specificity of ultrasonic diagnoses. The authors have previously developed a ligand targeted ultrasonic contrast system which is a liquid, lipid encapsulated perfluorocarbon emulsion. The emulsion particles are small (250 nm) and have inherently low echogenicity unless bound to a surface by a pretargeted ligand through avidin-biotin interactions. The authors have recently proposed a simple acoustic transmission line model that treats the emulsion particles as a thin layer over the targeted surface. The acoustic reflectivity of the contrast layer is related to the longitudinal velocity and density of the perfluorocarbon. Improvement of the contrast effect may be achieved by using perfluorocarbons with slower longitudinal velocities and/or lower densities. The authors report the longitudinal velocities as a function of frequency of 20 perfluorocarbons using a broadband phase spectroscopic approach for estimating phase velocities. Experimentally determined velocities ranged from 520/spl plusmn/2 m/sec (perfluorohexane) to 705/spl plusmn/5 m/sec (perfluorodecalin). No measurable dispersion was observed over the useful bandwidth of 2 to 22 MHz. Increasing carbon backbone chain length and fluorine substitution with halogens of greater atomic weight increased the measured speed of sound. The authors' experimental data were consistent (R=0.87) with a published empirical model that predicts velocity as a function of molecular structure. These data provide a rational basis for optimizing targeted perfluorocarbon-based contrast agents and offer further insight into the physical mechanisms responsible for the observed enhancement of the acoustic scattering.

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.

The objective of this study was to assess the feasibility of image-based identification of nanoparticle targeted angiogenic neovasculature using backscattered ultrasound in several frequency ranges (7 to 15 and ~20- 35MHz). We employed a liquid-perfluorocarbon nanoparticle contrast agent to target the expression of αvβ3 in tumors implanted two different animal models. Ten K14-HPV16 transgenic mice were treated with either normal saline (n=5) or 0.3 mg/kg i.v. of αvβ3-targeted nanoparticles (n=5) and imaged dynamically for two hours using a research ultrasound imager (Vevo 660 30MHz probe) modified to store digitized RF waveforms. Data were analyzed for all mice at all times post-injection using conventional grayscale and C H (an information-theoretic quantity). These data demonstrate the ability and complementarity of information-theoretic receivers in conjunction with targeted nanoparticles to elucidate the presence of αvβ3-integrins in primordial neovasculature, particularly in acoustically unfavorable environments.

Molecular imaging agents are extending the potential of non-invasive medical diagnosis from basic gross anatomical descriptions to complicated phenotypic characterizations, based upon the recognition of unique cell-surface biochemical signatures. Originally the purview of nuclear medicine, molecular imaging is now a prominent feature of most clinically relevant imaging modalities, in particular magnetic resonance imaging and ultrasound. Nanoparticulate molecular imaging agents afford the opportunity not only for targeted diagnostic studies but also for image-monitored site-specific therapeutic delivery. Combining imaging with drug delivery permits verification and quantification of treatment and offers new clinical strategies to many diseases, e.g., cardiovascular, oncologic, and rheumatologic pathology. Ligand-targeted perfluorocarbon nanoparticles represent a novel platform technology in this emerging field, providing molecular imaging for all relevant imaging modalities and vascular-targeted drug delivery across a broad disease spectrum.

the potential for premature loss of ovarian function. A leading theory for these ovarian changes is that dechelation of gadolinium following Omniscan injection leads to heavy metal toxicity with subsequent microvascularity effects. Further studies evaluating subchronic toxicity are warranted, examining in greater depth the toxicity observed with Omniscan and evaluating other chelates with similar instability (Optimark).[2]

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.

Our laboratory previously has demonstrated the ability of liquid perfluoro-carbon (PFC) nanoparticles to serve as site-targeted contrast and therapeutic agents by specifically binding molecular markers. In this study, we sought to enhance the delivery of targeted PFC nanoparticles to cells expressing the integrin /spl alpha//sub v//spl beta//sub 3/ using clinical levels of ultrasound energy. Using an Acuson Sequoia imager, we demonstrate that ultrasound applied at 2MHz and 1.9 mechanical index (MI) for a five minute duration enhances the cellular interaction of targeted perfluorocarbon nanoparticles by melanoma cancer cells in vitro without any untoward effect from the ultrasound energy itself upon either the cells or the perfluorocarbon nanoparticles. Employing a custom designed specimen chamber, we have visualized a fundamental physical mechanism for this effect, that of acoustic radiation force (primary and secondary) on the particles, that may contribute to enhanced delivery. Accordingly, our study shows that enhancement of cellular interaction with targeted nanoparticles is feasible by noncavitational mechanisms. Ultrasonically-enhanced delivery of tracers or drugs to a wide variety of pathologic tissues may be useful for augmenting drug delivery after targeting, while limiting untoward effects on other tissues.

The ability to enhance specifically molecular markers of pathology with ultrasound has been previously demonstrated by our group employing a nanoparticle contrast agent. One of the advantages of this agent is it's relative non-echogenicity in the blood pool that allows increased contrast-to-noise between the blood pool and the bound, site-targeted agent. We sought to define the contrast agent concentration and acoustic parameters necessary to detect contrast enhancement in the blood so that molecular contrast enhancement could be more precisely defined. This study addresses two potential mechanisms that have been proposed for backscatter from the nanoparticle contrast agent in the blood pool - concentration-related scattering and phase conversion from liquid to gaseous perfluorocarbon. The nanoparticles were produced by methods currently standard in our laboratory using perfluorooctyl bromide (PFOB: b.p. 142/spl deg/C) as the major component. Particle size was measured at 200/spl plusmn/30nm. Attenuation coefficient and backscatter of the agent were measured in whole porcine blood (hct 40%) and porcine plasma maintained at 37/spl deg/C. Specimens were insonified using a broadband, single element transducer (5 MHz, 2.54 cm diameter, 5.08 cm focal length). Acoustic pulses with usable bandwidth of 1.5 to 10 MHz, a repetition rate of 1kHz, and peak negative pressure of 3.9, 2.7, 1.5, and 0.8MPa (equivalent to M.I. of: 1.7, 1.2, 0.67, 0.36) were used to measure of attenuation coefficient and backscatter of nanoparticles at concentrations of 0.26, 0.51, 1.02, 2.04, 4.08/spl times/10/sup 14/ particles/mL while suspended in either whole porcine blood or porcine plasma. The attenuation coefficient was linear at all concentrations and power levels and shows no evidence of a resonant peak characteristic of liquid-to-gas phase conversion. The back-scatter coefficient in plasma increased with concentration. However, in blood, backscatter was only significantly different from baseline at 2.04/spl times/l0/sup 14/ particles/mL and above (8x the maximum anticipated dose). These data indicate that phase conversion of PFOB to gas is not the source of the contrast in molecular imaging with site targeted nanoparticles.

Detection of atherosclerosis in its early stages with MRI could lead to more effective treatment of the disease. However, the changes that occur in the vessels of animals given a high cholesterol diet to induce atherosclerosis are not visually apparent with conventional MRI techniques. This paper describes a semi-automated algorithm designed to quantify enhancement of the aortic wall after administration of targeted and non-targeted contrast agents in rabbits given a high cholesterol diet. The performance of the program was evaluated on multiple slices taken from eight rabbits. No lumenal narrowing or overall non-specific contrast enhancement was detected in cholesterol-fed animals. Enhancement after administration of a nanoparticle contrast agent specifically targeted to molecules associated with the initial development of atherosclerosis was higher in the cholesterol fed rabbits, providing an early indicator of disease.

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 ( approximately 250 nm diameter,90,000 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.

Previously we reported that useful ultrasound contrast enhancement could be obtained using a liquid emulsion contrast agent developed in our laboratory. Although the physical basis for this enhancement was not completely characterized, the effect was reproducible in vivo and in vitro. We now hypothesize that microbubble formation does not play a significant role in the acoustic behavior of these contrast agents. Otherwise, attenuation and backscatter would be significantly affected by changes in hydrostatic pressure and acoustic pressure, and would show evidence of scattering agent destruction as the duration of ultrasound exposure increased. Both low power (0.65 MPa) unipolar pulses and high-power (3 MPa) unipolar pulses were used to measure the attenuation coefficient of Optison and that of the emulsion. The coefficient was also measured using a high-power unipolar pulse preceded by 5 cycles of a 1 MHz sine-wave of the same amplitude, emitted at a high repetition frequency (5 kHz). Both specimens were held in a pressurized chamber in a water bath maintained at temperatures ranging from 37/spl deg/ to 50/spl deg/C. The emulsion nanoparticles were produced by methods standard in our laboratory using perfluorooctyl bromide (PFOB: boiling point 142/spl deg/C) as the major component. Particle size was measured at 276 nm. We varied ambient hydrostatic pressure (-50 to 200 mm Hg in 50 mm steps), duration of exposure to the acoustic field (2, 20, 40, 80 s), and peak positive and negative acoustic pressure (0.65 to 3.0 MPa).

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. α v β 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 α v β 3 -targeted, paramagnetic nanoparticles. Pretreatment of atherosclerotic rabbits with α v β 3 -targeted, nonparamagnetic nanoparticles competitively blocked specific contrast enhancement of the α v β 3 -targeted paramagnetic agent. MRI revealed a pattern of increased α v β 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.

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 T 1 -weighted MRI at 4.7T. MRI 19 F 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.

Previously we reported that useful ultrasound contrast enhancement could be obtained using a liquid emulsion contrast agent developed in our laboratory. The physical basis for this enhancement was unknown at the time of our report, although the effect was reproducible, in vivo and in vitro. We hypothesize that microbubble formation does not play a significant role in the acoustic behavior of these contrast agents, otherwise attenuation and backscatter would be. significantly affected by changes in hydrostatic pressure, acoustic pressure, and show evidence of scattering agent destruction as the duration of ultrasound exposure increased. Measurements of attenuation coefficient and backscatter of Optison and the emulsion were made in a water bath maintained at temperatures ranging from 37/spl deg/ to 50/spl deg/C. The emulsion nanoparticles were produced by methods standard in our laboratory using perfluorooctyl bromide (PFOB: boiling point 142/spl deg/C) or perfluorooctane (PFO: boiling point 99/spl deg/C) as the major component. Particle size was measured at 435 and 375 /spl plusmn/ 30 nm respectively. We varied ambient hydrostatic pressure (-50 to 200 mm Hg in 50 mm steps), duration of exposure to insonifying acoustic field (2, 20, 40, 80 s), peak positive and negative acoustic pressure (0.038 to 3.0 MPa).

Improved echocardiographic endocardial border delineation aids in the assessment of ventricular function and wall motion abnormalities. Microbubble-based contrast agents provide excellent echogenicity but a short-lived effect. In contrast, liquid perfluorocarbon (PFC) nanoparticles exhibit long circulatory times and are stable to insonification but have poor echogenicity. Ideally, a contrast agent should demonstrate long circulatory persistence, resistance to ultrasonic destruction, while displaying significant echogenicity. This study demonstrates the effectiveness of biocompatible, liquid PFC nanoparticles as a blood pool contrast agent. When imaged with power Doppler harmonic imaging (PDHI), this agent exhibited marked blood pool enhancement lasting over 1 hour. Furthermore, the contrast enhancement was achieved at doses of 0.7% blood volume. In order to investigate the influence of particle size on blood pool contrast effect, liquid PFC nanoparticles were formulated with diameters of 232 and 465 nm. The nanoparticles were then administered to canines (N=10), with 7 canines receiving the 232 nm nanoparticles and the remainder receiving the 465 nm nanoparticles at a dose of 0.5 ml/kg. PDHI revealed marked blood pool enhancement in all three canines receiving the 465 nm nanoparticles but minimal contrast effect in the canines receiving the 232 nm nanoparticles. This data suggests that PFC nanoparticles in conjunction with PDHI may serve as an important adjunct for the assessment of ventricular function and wall motion abnormalities.

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.

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.

The relaxivity and transmetallation of two lipophilic paramagnetic chelates incorporated onto perfluorocarbon nanoparticles, i.e., gadolinium-methoxy-tetraazacyclododecane-tetraacetic acid phosphatidylethanolamine (Gd-MeODOTA-PE) and gadolinium-methoxy-tetraazacyclododecane-tetraacetic acid triglycine phosphatidylethanolamine (Gd-MeO-DOTA-triglycine-PE (Gd-MeO-DOTA-triglycine-PE)), were compared to a prototypic gadoliniumdiethylene-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. r 2005 Published by Elsevier B.V.

The reaction of o-aminobenzenethiol with carbonyl sulfide in the presence of triethylamine afforded an alternate route for the synthesis of 2-benzothiazolinone (1) in 97–98% yield. The reaction of ammonium thiocarbamate (2) with 2-chlorocyclohexanone furnished the novel 4,5,6,7-tetrahydro-2-benzothiazolinone (3). 3-Ethoxy-2H-1,4-benzothiazin-2-one (7) was prepared by the reaction of o-aminobenzenethiol with diethyl oxalate. Possible pathways and supporting nmr, ir and mass spectra are discussed.

The reaction of o-aminobenzenethiol with carbonyl sulfide in the presence of triethylamine afforded an alternate route for the synthesis of 2-benzothiazolinone (1) in 97–98% yield. The reaction of ammonium thiocarbamate (2) with 2-chlorocyclohexanone furnished the novel 4,5,6,7-tetrahydro-2-benzothiazolinone (3). 3-Ethoxy-2H-1,4-benzothiazin-2-one (7) was prepared by the reaction of o-aminobenzenethiol with diethyl oxalate. Possible pathways and supporting nmr, ir and mass spectra are discussed.