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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.

Molecular imaging contrast agents specifically detect the biochemical "signatures" of disease before anatomical manifestations are apparent. Sensitive and specific localization of fibrin both in vivo and in vitro has been demonstrated with the use of a ligand-directed liquid perfluorocarbon nanoparticle. Since the acoustic properties of perfluorocarbons are known to vary with temperature, it was hypothesized that temperature could be used to augment the magnitude of enhancement imparted by targeted nanoparticles. Accordingly, the acoustic backscatter of two different substrates, nitrocellulose membrane and human plasma clot, targeted by the nanoparticles was measured at temperatures ranging from 27 degrees to 47 degrees C in 5 degrees C increments. Classic avidin-biotin interactions were utilized to couple biotinylated nanoparticles to avidin-conjugated nitrocellulose membranes. Ultrasonic contrast enhancement of the nitrocellulose membrane at 25 MHz, measured by acoustic microscopy, increased from 2.0+/-0.3 dB at 27 degrees C to 3.7+/-0.4 at 47 degrees C. In a similar experiment, antifibrin nanoparticles bound to human plasma clots also exhibited temperature-dependent ultrasonic signal enhancement ranging from 13.9+/-1.5 dB at 27 degrees C to 18.1+/-1.5 dB at 47 degrees C. The increase in ultrasonic contrast enhancement measured was well described by a simple, acoustic transmission line model with temperature-dependent impedance. These results suggest that temperature-dependent changes in acoustic backscatter may be used to further differentiate tissues targeted with site-specific nanoparticles from surrounding normal soft tissues.

Complex molecular signaling heralds the early stages of pathologies such as angiogenesis, inflammation, unstable atherosclerotic plaques, and areas of remote thrombi. In previous studies, acoustic enhancement of blood clot morphology was demonstrated with the use of a nongaseous, fibrin-targeted acoustic nanoparticle emulsion delivered to areas of thrombosis both in vitro and in vivo. In this study, a system was designed and constructed that allows visualization of the evolution of acoustic contrast enhancement. To evaluate the system, two targets were examined: avidin-complexed nitrocellulose membrane and human plasma clots. The time evolution of enhancement was visualized in 10-min increments for 1 h. A monotonic increase was observed in ultrasonic reflection enhancement from specially treated nitrocellulose membranes for targeted emulsions containing perfluorooctylbromide (1.30+/-0.3 dB) and for perfluorooctane (2.64+/-0.5 dB) within the first 60 min of imaging. In comparison, the inherently nonechogenic plasma clots showed a substantial increase of 12.0+/-0.9 dB when targeted with a perfluoro-octane emulsion. This study demonstrates the concept of molecular imaging and provides the first quantifiable time-evolution report of the binding of a site-targeted ultrasonic contrast agent. Moreover, with the incorporation of specific drug treatments into the nanoparticulate contrast agent, ultrasonic molecular imaging may yield reliable detection and quantification of nascent pathologies and facilitate targeted drug therapy.

Molecular imaging permits tissues to be functionally characterized by identification of specific cell-surface receptors with targeted contrast agents. In our study, a ligand-targeted acoustic nanoparticle system was used to identify the angioplasty-induced expression of tissue factor by smooth muscle cells within the tunica media. Pig carotid arteries were overstretched bilaterally with balloon catheters, treated with a tissue factor-targeted or a control nanoparticle system, and imaged with intravascular ultrasound (20 MHz) before and after treatment. Carotid wall acoustic reflectivities were unaffected by overstretch injury. Tissue factor-targeted nanoemulsion bound and increased the echogenicity of smooth muscle cells expressing tissue factor within the tunica media. The targeted emulsion increased the arterial wall gray scale (99.4+/-14.5; P.05) relative to pretreatment (41.8+/-11.1, P0.05) and the control gray scale (pre-emulsion: 49.3+/-9.5; post-emulsion: 43.7+/-6.4; P.05). The area of acoustic enhancement appeared to coincide with expression of induced tissue factor in the tunica media confirmed by immunohistochemistry. We have demonstrated that this novel nanoemulsion can infiltrate into arterial walls after balloon injury and localize the expression of overstretch-induced tissue factor within pig carotid arteries. Molecular imaging and quantification of complex, biochemical change, such as tissue factor expression after angioplasty, may prove to be a prognostically important predictor of subsequent restenosis.

Complex molecular signaling heralds the early stages of pathologies such as angiogenesis, inflammation, and cellularresponses to mechanically damaged coronary arteries after balloon angioplasty. In previous studies, we have demonstratedacoustic enhancement of blood clot morphology with the use of a nongaseous, ligand-targeted acoustic nanoparticle emulsiondelivered to areas of thrombosis both in vitro and in vivo. In this paper, we characterize the early expression of tissue factorwhich contributes to subsequent arterial restenosis. Tissue factor is a 42kd glycoprotein responsible for blood coagulationbut also plays a well-described role in cancer metastasis, angiogenesis, and vascular restenosis. This study was designed todetermine whether the targeted contrast agent could localize tissue factor expressed within the wall of balloon-injuredarteries. Both carotid arteries of five pigs (20kg) were injured using an 8 x 20mm angioplasty balloon. The carotids weretreated in situ with a perfluorocarbon nanoparticle emulsion covalently complexed to either specific anti-tissue factorpolyclonal F(ab) fragments (treatment) or non-specific IgG F(ab) fragments (control). Intravascular ultrasound (30 MHz)images of the arteries were obtained before and after exposure to the emulsions. Tissue-factor targeted ultrasonic contrastagent acoustically enhanced the subintima and media at the site of balloon-induced injury compared with control contrastarteries (p

Time-domain–based integrated backscatter values obtained with the use of acoustic densitometry (AD) were compared with values determined from a spectral-based analysis of the radio-frequency (RF) signals with a modified Hewlett-Packard Sonos 1500 imaging system. Integrated backscatter images of five specimens of bovine tendon were acquired in the AD acquisition mode, and the corresponding signals related to the backscattered RF were digitized for each angle of insonification as the specimens were rotated in 10-degree increments. The integrated backscatter images were analyzed with the AD analysis package, and the corresponding values determined from the RF power spectra were obtained from the digitized ultrasonic signals. Good agreement was found between the two methods over the entire range of measured values. The mean anisotropy in the measured integrated backscatter (mean ± standard error) was found to be 27 ± 2 dB for time-domain–based analysis and 25 ± 2 dB for RF spectral-based analysis. (J Am Soc Echocardiogr 1997;10:511-7.)

Our overall goal is to develop clinically applicable tissue characterization methods, based on quantitative analyses of backscattered ultrasound, which can differentiate normal from diseased heart segments. In implementing these methods there is a need to compensate for the inherent anisotropic properties of the heart that are exhibited in echocardiographic images. [1–4] Furthermore, quantitative tissue characterization methods may be able to exploit the inherent anisotropy of the myocardium to achieve assessment of cardiac properties.[5–9] The specific aims of this investigation were to measure the spectral properties of backscattered ultrasound using a clinical imaging system and to determine effects of inherent tissue anisotropy on measured spectral properties of backscattered ultrasound.

Measurements of the frequency dependence of ultrasonic backscatter are presented for specific angles of insonification for regions of infarcted and noninfarcted human myocardium. A 5-MHz transducer was used to insonify cylindrical cores taken from 7 noninfarcted regions and 12 infarcted regions of the left ventricular free wall of 6 formalin-fixed human hearts explanted because of ischemic cardiomyopathy. The dependence of apparent (uncompensated for diffraction effects and attenuation) backscatter on frequency was approximated by a power-law dependence, magnitude of B(f)2 = afn. Under ideal conditions in a lossless medium, the effect of not compensating for the effects of diffraction and attenuation leads to the value of n to be 2.0 for Rayleigh scatterers while the frequency dependence of the fully compensated backscatter coefficient would be f4. The value of n was determined over the frequency range, 3-7 MHz. Both nonifarcted and infarcted myocardium exhibited anisotropy of the frequency dependence of backscatter, with maxima occurring at angles that were perpendicular to the predominant myofiber direction and minima when parallel to the fibers. Perpendicular insonification yielded results for n of 1.8 +/- 0.1 for noninfarcted myocardium and 1.2 +/- 0.1 for infarcted myocardium while parallel insonification yielded results of 0.4 +/- 0.1 for noninfarcted and 0.0 +/- 0.1 for infarcted myocardium. The functional form of the angle-dependent backscatter is similar for both noninfarcted and infarcted myocardium, although the frequency dependence is clearly different for both tissue states for all angles of insonification. The results of this study indicate that the anisotropy of the frequency dependence of backscatter may play a significant role in ultrasonic imaging and is an important consideration for ultrasonic tissue characterization in myocardium.

Further development of quantitative diagnostic procedures will enhance the selection and implementation of specific therapies that can reduce the damage to the heart muscle when applied to the victims of a heart attack. Ultrasonic imaging is one modality that provides real-time images of the beating heart that may be useful in forming diagnoses and evaluating therapies applied as well as providing insight into the underlying physiology. In characterizing the state of the cardiac muscle after a heart attack a clinician would like the ability to differentiate a segment of myocardial tissue with an old infarct (scarred tissue) from a region with acute ischemic injury where the muscle tissue may still be viable and could potentially be salvaged with the application of an appropriate therapy. Furthermore, once a specific therapy has been applied, it may be useful to monitor the reperfusion of the affected myocardial region. Our goal in ultrasonic tissue characterization is to provide an assessment of the state of the tissue based on quantitative analyses of the ultrasonic signal returned from a specific region of tissue. Implementation of quantitative ultrasonic tissue characterization procedures will complement conventional ultrasonic imaging which provides information regarding the dimensions and motion of the heart.

The clinical use of angiotensin-converting enzyme (ACE) inhibitors now represents the standard of care for the treatment of chronic congestive heart failure. We utilized ultrasonic tissue characterization to define potentially salutary effects of treatment with ACE inhibitors on the material properties of the heart and its potential influence on cardiac remodeling at the cellular level. Ten 1-month-old cardiomyopathic (CM) Syrian hamsters were treated with captopril (2 g/L water ad libitum), and 10 CM and 5 normal hamsters were maintained untreated for 9 months. Hearts were excised, and backscattered radiofrequency data were acquired from 1200 independent sites from each specimen with a high-resolution 50-MHz acoustic microscope for calculation of integrated backscatter. Treatment with captopril elicited relative decreases in left ventricular (LV) wet weight, LV calcium concentration and integrated backscatter (IB) in treated as compared with untreated cardiomyopathic hearts without affecting LV collagen concentration. The IB from hearts of treated cardiomyopathic hamsters was significantly less in both grossly normal regions of myocardium (P < 0.02) and scar tissue regions (P = 0.0005) as compared with IB from hearts of untreated hamsters. The reduced integrated backscatter from hearts of treated cardiomyopathic hamsters indicates direct alterations in the material properties of cardiomyopathic hearts after captopril therapy. The lower IB from scar tissue in the treated animals was associated with decreased scar tissue calcification, which represents a novel therapeutic effect of captopril. This is the first report that delineates direct effects of ACE inhibitors on the material properties of both scar tissue and grossly normal myocardium at the cellular level in experimental dilated cardiomyopathy. Quantitative ultrasonic tissue characterization may ultimately play a role in initiating and monitoring the effects ACE inhibitors on cardiac remodeling, calcium deposition and tissue stiffness in patients with dilated cardiomyopathy.

Methods and devices for enhanced ultrasound detection based upon changing temperature and ultrasound reflectivity of a temperature-dependent contrast agent bound to an ultrasound target are disclosed. The methods and devices can be used for enhanced imaging alone or in conjunction with drug delivery, with therapeutic approaches such as hyperthermia or cryotherapy or with other imaging modalities.

One of the exciting possible future clinical uses of ultrasound is site‐targeted drug delivery using focused acoustic energy. In an earlier study, it was shown that ultrasound exposure promoted lipid delivery from nanoparticles into melanoma cells in culture [Crowder et al., UMB 2005]. In order to extend the study to in vivo situations, a novel, combined ultrasound therapeutic and imaging system was developed. The system consisted of a therapeutic transducer (−6‐dB BW 1.1 to 2.3 MHz; natural focus 8 cm; f‐number 1) with a centrally located and coregistered imaging transducer connected to a commercial ultrasound imaging system. The system provided capability for arbitrary acoustic excitation, electronic axial steering, and mechanical translation of the therapy transducer as well as real‐time acquisition of images. The device was tested on bilateral murine tumor models (MDA435). Tumor images provided real‐time feedback for treatment planning. The animals were injected with perfluorooctyl bromide nanoparticl...

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.

✓ The authors report a retrospective study of 146 patients to assess the extent to which aneurysms, arteriovenous malformations, and pregnancy interact. The natural history of the lesions was modified if the women became pregnant. Clinical syndromes, diagnosis, neurosurgical and obstetrical management, and treatment are discussed.