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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.

Perfluorocarbon nanoparticles consisting essentially of liquid perfluoro-octyl bromide (PFOB) core surrounded by a lipid monolayer can serve as highly specific site-targeted contrast and therapeutic agents after binding to cellular biomarkers. Based on previous findings that ultrasound applied at 2 MHz and 1.9 mechanical index (MI) for a 5-min duration dramatically enhances the cellular interaction of targeted PFOB nanoparticles with melanoma cells in vitro without inducing apoptosis or other harmful effects to cells that are targeted, we sought to define mechanisms of interaction and the safety profile of ultrasound used in conjunction with liquid perfluorocarbon nanoparticles for targeted drug delivery, as compared with conventional microbubble ultrasound contrast agents under identical insonification conditions. Cell-culture inserts were used to grow a confluent monolayer of human umbilical vein endothelial cells. Definity in conjunction with continuous wave ultrasound (2.25 MHz for 1 and 5 min) increased the permeability of monolayer by four to six times above the normal, decreased transendothelial electrical resistance (a sign of reduced membrane integrity), and decreased cell viability by approximately 50%. Histological evaluation demonstrated extensive disruptions of cell monolayers. Nanoparticles (both nontargeted and targeted) elicited no changes in these different measures under similar insonification conditions and did not disrupt cell monolayers. We hypothesize that ultrasound facilitates drug transport from the perfluorocarbon nanoparticles not by cavitation-induced effects on cell membrane but rather by direct interaction with the nanoparticles that stimulate lipid exchange and drug delivery.

We describe characterization of digital signals using analogs of thermodynamic quantities: the topological entropy, Shannon entropy, thermodynamic energy, partition function, specific heat at constant volume, and an idealized version of Shannon entropy in the limit of digitizing with infinite dynamic range and sampling rate. We show that analysis based on these quantities is capable of detecting differences between digital signals that are undetectable by conventional methods of characterization based on peak-to-peak amplitude or signal energy. We report the results of applying thermodynamic quantities to a problem from nondestructive materials evaluation: detection of foreign objects (FO) embedded near the surface of thin graphite/epoxy laminates using backscattered waveforms obtained by C-scanning the laminate. The characterization problem was to distinguish waveforms acquired from the region containing the FO from those acquired outside. In all cases the thermodynamic analogs exhibit significant increases (up to 20-fold) in contrast and for certain types of FO materials permit detection when energy or amplitude methods fail altogether.

Anisotropy of ultrasonic scattering and attenuation in heart tissue depends on the specific orientation of myofibers with respect to angle of insonification. The present study was designed to delineate the effect of the angle of insonification with respect to the alignment of myofibers on measurements of integrated backscatter. A transmural cube of myocardium was cut from the anterior wall of the left ventricles from 5 normal canine hearts and their transmural scattering behavior was studied with the use of reflection acoustic microscopy at 50 MHz. A theoretical model for scattering based on the Born approximation (weak scattering) was employed to predict the relationship between backscatter and angle of insonification. Insonification of the basal face (basal view) demonstrated a wide transmural variation in integrated backscatter (/spl sim/15 dB), while insonification of the lateral face (lateral view) had much reduced variation (/spl sim/4 dB), despite an equivalent overall shift in transmural fiber angle of /spl sim/85/spl deg/ across the ventricular wall. Integrated backscatter was greatest in the midmyocardium when the basal face was viewed and least in the midmyocardium when the lateral face was viewed. The backscatter in the subepicardial and subendocardial regions was similar for both views. The maximum difference in backscatter from basal and lateral views at the midmyocardial level was approximately 18 dB, which represents a 64-fold change in the intensity of ultrasonic backscatter. The mathematical model developed for scattering based on the Born approximation (weak scattering) predicted the observed relationship between backscatter and angle of insonification. The rapid angular variation of integrated backscatter perpendicular to the fiber direction and the slow variation at parallel incidence observed experimentally were predicted by the model. This angular variation is due to the specific shape and elastic properties assumed for the predominant myocardial scatterer. There was a strong relationship between backscatter and fiber orientation, indicating that the view chosen for insonification of myocardium in clinical imaging may influence the estimation of scattering behavior. The mathematical model utilized here predicts the anisotropic behavior of scattering and suggests that the principal scattering structure in normal myocardium may be a stiff collagen shell surrounding a more compliant myocyte. This model might provide a valid approach for the study of material properties of the heart with the use of ultrasound. >

Paramagnetic and superparamagnetic metals are used as contrast materials for magnetic resonance (MR) based techniques. Lanthanide metal gadolinium (Gd) has been the most widely explored, predominant paramagnetic contrast agent until the discovery and association of the metal with nephrogenic systemic fibrosis (NSF), a rare but serious side effects in patients with renal or kidney problems. Manganese was one of the earliest reported examples of paramagnetic contrast material for MRI because of its efficient positive contrast enhancement. In this review, manganese based contrast agent approaches are discussed with a particular emphasis on their synthetic approaches. Both small molecules based typical blood pool contrast agents and more recently developed novel nanometer sized materials are reviewed focusing on a number of successful molecular imaging examples.

BACKGROUND Although angiotensin-converting enzyme (ACE) inhibitors have become a mainstay of treatment for chronic congestive heart failure (CHF), it is not known whether the cardiac remodeling effects are a secondary phenomenon, resulting from ACE inhibitors' hemodynamic actions of afterload reduction, or occur through an independent mechanism. METHODS AND RESULTS We used 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 and 6 normal (NL) hamsters were treated with captopril (2 g/L water ad libitum), and 10 CM hamsters and 10 NL hamsters were maintained untreated for 3 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 (IB). Treatment with captopril reduced left ventricular mass, calcium concentration, and IB in CM hearts without affecting myofiber size or collagen concentration. The IB from grossly normal regions of myocardium in NL hamsters, treated CM hamsters, and untreated CM hamsters was not significantly different. The IB from the microscopic regions of scar tissue in treated CM hamsters was significantly less (P = .0004) than that from scar tissue in untreated CM hamsters. CONCLUSIONS The reduced IB from treated scar tissue components reflects specific alterations in the material properties (elastic stiffness, density) of fibrous regions in CM hearts induced by captopril. This is the first report that defines specific cellular effects of ACE inhibitors on the material properties of isolated components of cardiac tissue in experimental cardiomyopathy. These alterations in material properties of scar tissue components represent a potential mechanism for the salutary actions of ACE inhibitors in heart failure.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by progressive weakness and wasting of skeletal and cardiac muscle; boys present with weakness by the age of 5 years and, if left untreated, are unable to walk without assistance by the age of 10 years. Therapy for DMD has been primarily palliative, with oral steroids emerging as a first-line approach even though this treatment has serious side-effects. Consequently, low-cost imaging technology suitable for improved diagnosis and treatment monitoring of DMD would be of great value, especially in remote and underserved areas. Previously, we reported use of the logarithm of the signal energy, log [E(f)], and a new method for ultrasound signal characterization using entropy, H(f), to monitor prednisolone treatment of skeletal muscle in a dystrophin-deficient mouse model. Three groups were studied: mdx mice treated with prednisolone, a control group of mdx mice treated with saline, and a control group of wild-type mice treated with saline. It was found that both log [E(f)] and H(f) were required to statistically differentiate the three groups. In the current study, we show that preprocessing of the raw ultrasound using optimal smoothing splines before computation of either log [E(f)] or a rapidly computable variant of Hf, denoted I(f,∞), permits delineation of all three groups by either metric alone. This opens the way to the ultimate goal of this study, which is identification and implementation of new diagnostically sensitive algorithms on the new generation of low-cost hand-held clinical ultrasonic imaging systems.

Paramagnetic and superparamagnetic metals are used as contrast materials for magnetic resonance (MR) based techniques. Lanthanide metal gadolinium (Gd) has been the most widely explored, predominant paramagnetic contrast agent until the discovery and association of the metal with nephrogenic systemic fibrosis (NSF), a rare but serious side effects in patients with renal or kidney problems. Manganese was one of the earliest reported examples of paramagnetic contrast material for MRI because of its efficient positive contrast enhancement. In this review, manganese based contrast agent approaches are discussed with a particular emphasis on their synthetic approaches. Both small molecules based typical blood pool contrast agents and more recently developed novel nanometer sized materials are reviewed focusing on a number of successful molecular imaging examples.

We have previously shown that high-frequency, high-resolution ultrasound can characterize the acoustic properties and composition of fatty plaques in cholesterol-fed rabbits. To determine whether quantitative ultrasound can delineate the regression of atherosclerotic lesions by detecting a change in their composition from fatty to fibrous types induced by alterations in dietary regimen, we fed six New Zealand White rabbits a 2% cholesterol diet for 3 months, followed by a standard diet for 3 additional months to promote the development of fibrous intimal lesions. Segments of aortas were excised, and backscattered radiofrequency data were acquired from 400 to 600 independent sites in each specimen with an acoustic microscope operated at 50 MHz. Control data were provided by measuring backscatter from adjacent portions of the aortas devoid of lesions. Histological and immunocytochemical analyses of the fibrous intimal lesions confirmed the presence of smooth muscle cells and abundant connective tissue with little appreciable lipid. Backscatter from normal aortic segments (-30.7 +/- 1.0 dB) was approximately 10-fold greater than that from fibrous lesions (-42.4 +/- 1.0 dB; P < .05). We previously reported that integrated backscatter from fatty lesions was -50.6 +/- 0.7 dB, or approximately 10-fold less than that from fibrous lesions (P < .05). Values for integrated backscatter from the media of each tissue type were approximately equal (-30.0 +/- 1.7 versus -30.7 +/- 1.6 versus -33.4 +/- 0.8 dB for normal versus fibrous versus fatty tissues, respectively; P = not significant).(ABSTRACT TRUNCATED AT 250 WORDS)

We demonstrate a novel approach to ultrasonic detection of molecularly targeted nanoparticle contrast agent accumulation in a xenographic mouse tumor model in vivo. This is accomplished by imaging the entire mouse tumor volume at 15-minute intervals following injection of a v β 3 -targeted perfluoro-carbon core nanoparticles. Radiofrequency (RF) data were stored for off-line analysis using signal energy and a limiting form of the Renyi entropy suitable for real-time calculation, I f, ∞ . The results indicate that I f, ∞ may be used to reliably detect accumulation of targeted nanoparticles within 15 minutes of injection without the need for image subtraction, whereas signal energy images showed less sensitivity to nanoparticle accumulation. The ability to detect nanoparticle accumulation without requiring image subtraction or fixing the transducer in place are necessary prerequisites for translation of this technology to the clinic.

In several investigations of molecular imaging of angiogenic neovasculature using a targeted contrast agent, Renyi entropy (I f (r)) and a limiting form of Renyi entropy (I f, ∞ ) exhibited significantly more sensitivity to subtle changes in scattering architecture than energy-based methods. Many of these studies required the fitting of a cubic spline to backscattered waveforms prior to calculation of entropy (either I f (r) or I f, ∞ ). In this study it is shown that the robustness of I f, ∞ may be improved by using a smoothing spline. Results are presented showing the impact of different smoothing parameters. In addition, if smoothing is preceded by lowpass filtering of the waveforms, further improvements may be obtained.

Previously, we reported new methods for ultrasound signal characterization using entropy, H(f); a generalized entropy, the Renyi entropy, I(f)(r); and a limiting form of Renyi entropy suitable for real-time calculation, I(f),(infinity). All of these quantities demonstrated significantly more sensitivity to subtle changes in scattering architecture than energy-based methods in certain settings. In this study, the real-time calculable limit of the Renyi entropy, I(f),(infinity), is applied for the imaging of angiogenic murine neovasculature in a breast cancer xenograft using a targeted contrast agent. It is shown that this approach may be used to reliably detect the accumulation of targeted nanoparticles at five minutes post-injection in this in vivo model.

BACKGROUND Noninvasive approaches to the evaluation of idiopathic cardiomyopathy are limited. Recent work from our laboratory has used quantitative ultrasound to define the three-dimensional structure of normal human myocardium and the myocardial remodeling associated with infarction. Our goal was to define the role of ultrasonic tissue characterization for detection of specific alterations in the three-dimensional transmural architecture of idiopathic dilated cardiomyopathy. METHODS AND RESULTS We measured frequency-dependent backscatter from 22 cylindrical biopsy specimens from nine explanted fixed hearts of patients who underwent heart transplantation for idiopathic cardiomyopathy, seven specimens from normal portions, and 12 specimens of infarcted tissue from six explanted fixed human hearts. Consecutive transmural levels from each specimen were insonified with a 5-MHz broadband transducer. The dependence of apparent (uncompensated for attenuation) backscatter, B(f), on frequency (f) was computed from radiofrequency (rf) data as: magnitude of B(f)2 = afn, where n is an index that reflects in part the size of the dominant scatterers in myocardial tissue. Myofiber diameter and percentage fibrosis were determined at each transmural level for each specimen. For cardiomyopathic tissue, the frequency dependence of backscatter (n) increased progressively from epicardial to endocardial (0.02 +/- 0.37 to 1.01 +/- 0.12, p less than 0.05) levels in conjunction with a progressive decrease in myofiber diameter (29.5 +/- 0.9 to 21.4 +/- 0.6 microns, p less than 0.0001). In contrast, in tissue from areas of infarction, the frequency dependence decreased progressively from epicardium to endocardium (0.91 +/- 0.20 to 0.23 +/- 0.21, p less than 0.05) in conjunction with a progressive increase in the percentage of fibrosis (23.5 +/- 9.4% to 54.5 +/- 4.9%, p less than 0.005). Normal tissue exhibited no significant transmural trend for frequency dependence, myofiber diameter, or percentage fibrosis. CONCLUSIONS These data indicate the presence of a heterogenous transmural distribution of scattering structures associated with human idiopathic cardiomyopathy and myocardial infarction that may be detected by ultrasonic tissue characterization. The divergence of these transmural trends for frequency dependence of backscatter reflects distinct mechanisms of structural heterogeneity for different pathological processes that comprise a transmural gradation of cell size and fibrosis for idiopathic cardiomyopathy and infarction, respectively.

This work demonstrates the utility of Shannon entropy imaging of myocardial anisotropy in mice. Conventional acoustic microscopy measurements of excised short-axis myocardial slices may be used to delineate this anisotropy ex vivo. This approach often requires processing of specular echoes, which can cause artifacts in images that obscure detailed structures near interfacial boundaries. Two different ultrasonic signal receivers were evaluated using the same raw data as input: log energy(log[E f ]) and Shannon entropy imaging( H S ), entropic imaging is least influenced by specular echoes and is able to resolve anisotropy near interfacial boundaries.

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.

Background and Objective: Augmentation of local drug delivery from targeted nanoparticles (NP) with nondestructive, noncavitational clinical ultrasound (US) offers the potential to enhance therapeutic efficacy in diseases such as cancer while limiting adverse systemic side effects. This lab has demonstrated augmentation of drug delivery from alphavbeta3-targeted perfluorocarbon NP and US in cancer cells in vitro (Crowder et al, 2005). The present study investigates the utility of conventional, clinical US for augmentation of drug delivery in vivo in a transgenic mouse cancer model (K14-HPV16) that develops squamous cell carcinomas.

Previously we reported on an entropy-based signal receiver and demonstrated that, in certain settings, it was more sensitive to subtle changes in scattering architecture than conventional energy-based signal characterization. In this paper we present results 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.

Duchenne muscular dystrophy is a severe wasting disease, involving replacement of necrotic muscle tissue by fibrous material and fatty infiltrates. One primary animal model of this human disease is the X chromosome-linked mdx strain of mice. The goals of the present work were to validate and quantify the capability of both energy and entropy metrics of radio-frequency ultrasonic backscatter to differentiate among normal, dystrophic, and steroid-treated skeletal muscle in the mdx model. Thirteen 12-month-old mice were blocked into three groups: 4 treated mdx-dystrophic that received daily subcutaneous steroid (prednisolone) treatment for 14 days, 4 positive-control mdx-dystrophic that received saline injections for 14 days, and 5 negative-control animals. Biceps muscle of each animal was imaged in vivo using a 40-MHz center frequency transducer in conjunction with a Vevo-660 ultrasound system. Radio-frequency data were acquired (1 GHz, 8 bits) corresponding to a sequence of transverse images, advancing the transducer from "shoulder" to "elbow" in 100-micron steps. Data were processed to generate both "integrated backscatter" (log energy), and "entropy" (information theoretic receiver, Hf) representations. Analyses of the integrated-backscatter values delineated both treated-and untreated-mdx biceps from normal controls (p

Stem/progenitor cells participate in many pathological and regenerative processes, which can be studied in vivo with molecular imaging approaches. Labeling cells with contrast agents for non-invasive imaging and tracking typically requires long exposure times or adjunctive methods, such as electroporation or transfection agents that can compromise cell viability. We have previously utilized perfluorocarbon (PFC) nanoparticles (~200 nm) for cell tracking, but this process entailed 12 hours of incubation to achieve efficient cellular labeling. We sought to develop an approach that reduces the prolonged labeling time by enhancing PFC nanoparticle-to-cell interactions using clinical levels of ultrasound energy. Stem/progenitor (CD34+ CD133+ CD31+ ) cells were derived from human umbilical cord mononuclear cells grown in fibronectin- coated OptiCelltrade cassettes. Ultrasound (US) was applied using a medical imaging system (Acuson Sequoia) and broadband (2-3 MHz) phased array transducer, which was coupled to cells in a heated (37degC) waterbath. The transducer was advanced mechanically to expose the entire surface to calibrated levels of US energy (MI: 1.9, frequency: 2 MHz). Estimated exposure time for individual cells was ~5 min. Results: Flow cytometry revealed US application greatly improved labeling when compared with cells not exposed to US (55plusmn9% vs. 17plusmn12% respectively, p

Background: Reperfusion of the ischemic brain is the most effective therapy for acute stroke, restoring blood flow to threatened tissues. Thrombolytics, such as recombinant tissue plasminogen activator, administered within 3 h of symptom onset can improve neurologic outcome, although the potential for adverse hemorrhagic events limits its use to less than 3% of acute ischemic stroke patients. Targeting of clot-dissolving therapeutics has the potential to decrease the frequency of complications while simultaneously increasing treatment effectiveness, by concentrating the available drug at the desired site and permitting a lower systemic dose. Objectives: We aimed to develop a fibrin-specific, liquid perfluorocarbon nanoparticle that is surface modified to deliver the plasminogen activator streptokinase. We also aimed to evaluate its effectiveness for targeted thrombolysis in vitro using quantitative acoustic microscopy. Methods: Human plasma clots were formed in vitro and targeted with streptokinase-loaded nanoparticles, control nanoparticles or a mixture of both. Depending on the treatment group, clots were then exposed to either phosphate-buffered saline (PBS), PBS with plasminogen or PBS with plasminogen and free streptokinase. Spatially registered ultrasound scans were performed at 15-min intervals for 1 h to quantify changes in clot morphology and backscatter. Results: Nanoparticles bound to the clot significantly increased the acoustic contrast of the targeted clot surface, permitting volumetric estimates. Profile plots of detected clot surfaces demonstrated that streptokinase-loaded, fibrin-targeted perfluoro-octylbromide nanoparticles in the presence of plasminogen induced rapid fibrinolysis (

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.

We describe characterization of backscatter from tumor tissue targeted with a nanoparticle-based ultrasound contrast agent in vivo using analogs of thermodynamic quantities. We apply these waveform characteristics to detection of tumor neovasculature in tumors implanted in athymic nude mice, which were imaged using a research ultrasound scanner over a 2-hour period after injection of alpha upsilon beta3-targeted perfluorocarbon nanoparticles. Images were constructed from backscattered ultrasound using two different approaches: fundamental B-mode imaging and a signal receiver based on a thermodynamic analog (H(C)). The study shows that the thermodynamic analog is capable of detecting differences in backscattered signals that are not apparent with the B-mode approach.

Thrombolytic agents, i.e. "clot-busters" such as tPA, have been shown to be effective therapeutics for acute ischemic stroke. However, systemic administration of such powerful agents may be complicated by increased risk of cerebral hemorrhage and expanding stroke. Targeting of clot-dissolving therapeutics has the potential to decrease the incidence of these complications while simultaneously increasing effectiveness of treatment, by concentrating the available drug at the desired site and permitting a lower overall systemic dose. We have previously demonstrated targeting of liquid perfluorocarbon nanoparticles to thrombi in vitro and in vivo, with concomitant enhancement of acoustic reflectivity from the targeted clot surfaces. In the current study, a traditional thrombolytic enzyme (streptokinase) was incorporated into fibrin-targeted nanoparticles and targeted to plasma clots in vitro to demonstrate use of the nanoparticles as both an acoustic contrast agent and an adjunctive targeted therapeutic delivery system. Human plasma clot samples targeted with streptokinase-loaded liquid perfluorocarbon nanoparticles and immersed in plasminogen/saline solution exhibited 7% loss of volume after 3 hrs exposure, as well as marked time-dependent variation in surface topography and acoustic reflectivity. Control samples exposed to either 1) targeted, streptokinase-loaded nanoparticles in the presence of saline only, or 2) targeted nanoparticles without streptokinase in the presence of plasminogen/saline solution exhibited no significant changes over time. These results suggest the potential utility of targeted nanoparticles for combined diagnosis and treatment of thrombus-initiated ischemic stroke.

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.

Perfluorocarbon (PFC) nanoparticles can serve as a very specific site-targeted contrast and therapeutic agent after binding to specific cellular biomarkers. Ultrasound has been used in conjunction with microbubbles to enhance the delivery of drugs and genes into the cell, mainly through the process of cavitation. We have demonstrated earlier that ultrasound at 2 MHz (1.9 MI) for 5 min can enhance the interaction of nanoparticles with targeted cells. In this study, we sought to establish that nanoparticles used in conjunction with ultrasound do not act as "cavitation" foci. Cell-culture inserts were seeded with human umbilical vein endothelial cells (HUVECs) and exposed to ultrasound in the presence of either Definity ® or PFC nanoparticles. Definity ® in conjunction with continuous wave ultrasound (2.25 MHz for 1 and 5 minutes) increased the permeability of monolayer by four to six times above the normal, decreased trans-endothelial electrical resistance (a sign of reduced membrane integrity), and decreased cell viability by ~50%. Histological evaluation demonstrated extensive disruptions of cell monolayers. Nanoparticles (both non-targeted and targeted) elicited no changes in the measured parameters under similar insonification conditions. Sonically enhanced delivery of drugs from liquid perfluorocarbon nanoparticles to targeted cells does not involve cavitation but increased lipid exchange with targeted cells.

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.

Duchenne muscular dystrophy is a severe wasting disease, involving replacement of necrotic muscle tissue by fibrous material and fatty infiltrates. One primary animal model is the X chromosome-linked mdx strain of mice, which exhibit a clinical phenotype comparable to a mild expression of Duchenne muscular dystrophy in humans. The goals of the present work were to validate and quantify the capability of both energy and entropy metrics of radio-frequency ultrasonic backscatter to differentiate among normal, dystrophic, and steroid treated skeletal muscle in the mdx model

Lipidomics (the system-level analysis of lipids and their interacting moieties) is an emerging field in biomedical research that seeks to identify lipids as molecular targets for diagnosis and therapy. However, the delivery of lipids to pathological cells depends on the biochemical characteristics of the lipid including the overall charge, size of the polar head groups and length of fatty -acid side chains (Struck and Pagano, 1980). We have reported previously that focused ultrasound energy produced by clinical phase array imagers (2 MHz, 1.9 MI) can augment lipid delivery to cancer cells in vitro with the use of perfluorocarbon nanoparticles as non-cavitating carriers, which comprise a perfluorocarbon core and an outer lipid surfactant monolayer (Crowder et al, 2005). Accordingly, we now seek to establish the role of non-cavitational ultrasound in facilitating the delivery of lipids with a surrogate bulky polar head group. PFOB nanoparticles were synthesized by incorporating a bulky (1.4 nm) polar head group nanogold-DPPE (Nanoprobes, NY) at 0.172 mol% in the lipid monolayer. C-32 melanoma cells were grown on cell culture inserts and incubated with nanogold particles for 30 min followed by insonication with continuous wave ultrasound (2.25 MHz, 5 min, 0.6 MPa) and further incubation for 1 hour at 37 degC. The cells were then prefixed with glutaraldehyde, silver enhanced with HQ silver Enhancement Kit (Nanoprobes, NY) and fixed with sodium thiosulphate. After postfixation with osmium tetroxide the cells were treated with uranyl acetate, embedded in epoxy resin, thin-sectioned and stained with lead citrate. Sections 60 nm thick were examined with a Hitachi H-600 electron microscope. C-32 cells not exposed to ultrasound showed silver-enhanced gold particles partitioned only in the outer bilayer of the cell membrane and no evidence of intracellular nanogold. However, the cells exposed to ultrasound showed numerous nanogold-DPPE components inside the cell polarized to one end of intracellular vesicles. These observations are consistent with a mechanism that suggests ultrasound is capable of stimulating lipid internalization, sorting and intracellular trafficking. We surmise that non-cavitational ultrasound may facilitate the lipid uptake even under conditions not conducive to lipid transport by increasing the lipid diffusion coefficients. Therefore, adjunctive ultrasound could be used to increase the range of lipids delivered from perfluorocarbon nanoparticles to targeted cells, which might otherwise remain confined to the outer bilayer leaflet due to biophysical constraints

Liquid perfluorocarbon nanoparticle contrast agents can be used to target specific tissue types through incorporation of appropriate ligands into the nanoparticles' outer lipid monolayer. In this study we sought to characterize the specificity of targeting of perfluorooctyl bromide (PFOB) nanoparticles to tissue factor, a transmembrane glycoprotein expressed by smooth muscle cells as part of inflammatory response after vessel injury (e.g., angioplasty). Porcine smooth muscle cells constitutively expressing tissue factor on the cell surface were cultured on microporous membranes and targeted with PFOB nanoparticles by avidin-biotin binding techniques. Treatment groups included: 1) "targeted" cell samples exposed sequentially to biotinylated tissue factor antibody/avidin/biotinlyated nanoparticles, 2) "non-targeted" smooth muscle cells exposed to avidin/biotinlyated nanoparticles; 3) "control" smooth muscle cells left untreated. Ultrasonic imaging and quantification of surface reflectivity for each cell group was accomplished by scanning a 25MHz transducer in a grid over the membrane inserts and analyzing the reflected RF signal from the cell-covered surfaces. Total PFOB content for each sample (directly related to nanoparticle concentration) was determined independently by gas chromatography. Targeted cells exhibited significantly enhanced surface reflectivity (-22.6/spl plusmn/2.0 dB referenced to steel reflector, p

Advances in molecular biology and cellular biochemistry are providing new opportunities for diagnostic medical imaging. Molecular imaging provides an opportunity to "see" the earliest biochemical signatures of disease in vivo analogous to microscopic detection of epitopes with immunohistochemistry techniques. Previously the province of nuclear medicine, today numerous, highly active research programs can be found for all clinically relevant imaging modalities. In many instances, these emerging site-directed imaging agents also incorporate therapeutic agents for drug delivery, allowing noninvasive confirmation and quantification of the targeted therapeutic dose. We have developed a novel multi-modal site-targeted contrast agent for sensitive and specific imaging of molecular epitopes and local therapy, which illustrates the key features of these emerging platform technologies. Targeted nanoparticles are applicable to ultrasound, nuclear, CT, and magnetic resonance imaging and have been used to detect a variety of epitopes expressed on angiogenic vessels, thrombus, and within vascular walls. Moreover, this particular targeted agent can deposit a therapeutic payload through a unique mechanism termed "contact-facilitated delivery". The combined benefits of molecular imaging and therapeutic systems are merging and will potentially alter many current clinical paradigms in the next decade.

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.

Targeted contrast agents are expanding the detectability and diagnosis of pathology from a strict anatomic to biochemical basis. Moreover, these new agents, in their various forms, offer the potential for site-specific drug and gene delivery, ie, the ″magic bullet” first postulated by Paul Erhlich 100 years ago. The ability to direct drugs to the molecular signatures of disease, to confirm noninvasively their presence at the site-of-interest, and to quantify the adequacy of local drug concentration at the time of treatment, ie, rational targeted drug delivery, offers exciting new clinical paradigms in the near future. Copyright © 2001 by W.B. Saunders Company Progress in Cardiovascular Diseases, Vol. 44, No. 1, (July/August) 2001: pp 13-31

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).

Myocardial edema is associated with impaired ventricular compliance and diastolic dysfunction. To determine the sensitivity of high-frequency ultrasound to myocardial edema, we employed a model in which edema was induced by immersion of heart tissue in isotonic saline. Formation of edema after rapid-freezing and thawing of myocardium was also evaluated. Rat hearts were arrested at end-diastole and insonified fresh within 15 minutes of excision (n=5) or after being frozen for 24 hours and thawed (n=4). A high-frequency, acoustic microscope was employed to perform ultrasonic measurements of attenuation, backscatter, speed of sound, and tissue thickness at baseline and hourly for 4 hours while immersed in solution. Parametric images of these ultrasonic indices were constructed to monitor changes in microscopic material properties due to edema and to allow identification of localized regions of interest. Fresh tissue demonstrated a greater propensity for the development of edema than frozen tissue. Integrated backscatter increased in both tissues while the magnitude and slope of attenuation decreased as edema evolved. We conclude that high-frequency ultrasound sensitively detects myocardial edema and may prove useful for monitoring and treatment of cardiac edematous disease states.

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 highly ordered and complex arrangement of cardiac myofibers produces an angle dependence or anisotropy of ultrasonic scattering. The extent to which the magnitude of anisotropy may depend on the particular view used for insonification of the heart has not been determined. The transmural dependence of integrated backscatter (IB) on myofiber angle in vitro in sections of myocardial tissue is measured from two orthogonal directions. Ultrasonic anisotropy in vivo is characterized in volunteer patients with a method known as lateral gain compensation, which permits compensation for anisotropy in clinical echocardiography images. >

Specific ultrasonic tissue characterization parameters were correlated with the three-dimensional architecture and material properties (density, compressibility, size, and orientation) of aortic elastic elements at the microscopic level. The medial layer of 10 samples of normal canine aorta were insonified in vitro utilizing acoustic microscopy from 30 to 44 MHz. The following quantitative indexes exhibited substantial anisotropic elastic behavior in radial (R), circumferential (C), and longitudinal (L) directions: backscatter coefficient (R:0.9/spl plusmn/0.2; C:0.008/spl plusmn/0.0008; L:0.0077/spl plusmn/0.0008 sr/sup -1/ cm/sup -1/); frequency dependence of backscatter (R:3.3; C:1.4; L:1.5); attenuation coefficients (R:105/spl plusmn/22; L:135/spl plusmn/13; C:131/spl plusmn/14 dB/cm). Thus, the ultrasonic indexes are anisotropic: equivalent in the C and L directions, but markedly different in the R direction. These data are indicative of an aortic microstructure that interacts with ultrasonic waves as thin sheet-like elastic layers instead of independent elastin fibers. This specific sheet-like organization of elastin microfibers may function to limit shear injury to concentric aortic lamellae and prevent aortic dissection. The marked anisotropic behavior of normal aortas suggests that ultrasound may be useful for nondestructive characterization of vascular integrity.

Measurements of ultrasonic quasi-longitudinal velocity were made in the muscle fiber plane of excised human and canine cardiac tissue. The measurements were carried out in 2 degrees increments through the full 360 degrees relative to the myofibers. In addition, for each tissue specimen, measurements were made at a number of adjacent planes across the left ventricular wall in order to assess the transmural dependence of velocity. The data indicate a 1.0% to 1.5% anisotropy of velocity, with maximum velocity when insonifying along the myofibers and minimum velocity when insonifying perpendicular to the myofibers. The anisotropy of velocity is 90 degrees out of phase with the anisotropy of integrated backscatter, which was also measured. Both velocity and integrated backscatter were found to depend on transmural position. Each exhibited a progressive angular shift of maximal value from the epicardium to the endocardium. This shift in the position of peak in the backscatter and velocity graphs reflects the underlying shift in myofiber orientation. >

The progressive increase in stiffening of the myocardium associated with the aging process and abetted by comorbid conditions such as diabetes may be linked to an excessive number of collagen cross links within the myocardial extra-cellular matrix. The time-evolution of the crosslinking of proteins was monitored with high frequency ultrasound (30-50 MHz). Rats hearts (n=5 each group) were arrested at end-diastole and then insonified fresh within a few minutes of excision in a phosphate buffered solution. The solution was then replaced with a fixative and the tissue was insonified at 30-minute intervals thereafter for 24 hours. A parametric image of the magnitude and frequency dependence of ultrasonic attenuation in heart tissue was constructed at each time point to yield a time-lapse visualization of the process of protein crosslinking.

The goal of myocardial ultrasonic tissue characterization is to complement two-dimensional and Doppler echocardiography by providing information (such as assessment of regional viability based on localized values of backscatter) beyond that derived from an assessment of myocardial dimensions and motion. Quantitative backscatter imaging can be subdivided into three broad areas: (1) direct applications, in which specific pathologies are identified and monitored, (2) indirect applications, in which quantitative techniques designed for use in tissue characterization serve to expand the role of echocardiography, and (3) contributions to the understanding of cardiac structure and function.

The authors' goal is to apply the techniques of quantitative ultrasonic tissue characterization to intravascular ultrasound. They have developed a system to acquire radio frequency ultrasonic signals from a modified commercially available intravascular ultrasound scanner. This system incorporates a novel method of acoustic signal calibration. Experiments have been completed demonstrating the ability to make repeatable measurements of frequency-averaged backscattered power. In order to test and evaluate this system, intravascular ultrasound was used to characterize the enhancement of backscattered power from clot specifically targeted with an acoustic contrast agent recently developed in the authors' laboratory. The agent has the unique physical attribute of low inherent echogenicity in suspension, while attaining high acoustic reflectivity when bound to a biologic surface or to itself. Clots from canine plasma were formed in vitro on nitrocellulose membranes. Clots were incubated with either a clot-specific (n=2) or a clot-nonspecific (n=2) contrast emulsion. Specimens were imaged with a 30 MHz intravascular ultrasonic catheter. Radio frequency signals backscattered from the clots were acquired, gated, and their power spectra obtained by FFT. Frequency-averaged power backscattered from the clots exposed to clot-specific contrast exceeded that from clots exposed to clot-nonspecific contrast by 5.6/spl plusmn/0.7 dB (/spl plusmn/SD,p

Ventricular remodeling after myocardial infarction elicits structural and functional changes that can compromise global cardiac function. To determine whether age influences this process, we characterized remodeling after myocardial infarction in Fischer 344 rats that undergo natural age-related changes in heart structure and function that parallel those observed in human hearts. Young (3 month, n=7) and old (18 month, n=7) rats underwent myocardial infarction by coronary occlusion. Sham-infarct procedures were performed in both groups as controls. Twelve weeks after infarction, hearts were excised, fixed, and sliced into 2 mm thick cross-sections. Ultrasonic RF signals were acquired with a 50 MHz transducer and integrated backscatter was calculated from 30 to 50 MHz. Heart slices were then stained and their images digitized to permit histology with computer assisted planimetry. Infarct sizes were similar in young and old rats (12.1/spl plusmn/2.4% and 11.6/spl plusmn/5.4%; p=NS). Integrated backscatter from the infarct scar tissue in both the young and old rats were similar, (-41.4/spl plusmn/1.2 dB vs. -42.7/spl plusmn/1.1 dB, p=NS). Integrated backscatter from the remodeled but non-infarcted viable tissue was -48.4/spl plusmn/0.8 dB and -45.4/spl plusmn/1.0 dB in young and old rats, respectively, both statistically different from integrated backscatter from scar tissue (p

Progressive collagen cross-linking is a feature of many cardiovascular conditions (e.g., aging, diabetes, remodeling) and may contribute to impaired ventricular relaxation. However, it is not known whether cross-linking affects anisotropic tissue material properties more in one direction than another. We hypothesized that biological tissue comprising a highly ordered collagen matrix (porcine tendon) would manifest a directional dependence of the physical effects of cross-links that could be quantitatively delineated with ultrasound. Fresh porcine flexor tendons were sliced in 2-mm planes parallel (n=7) and perpendicular (n=7) to the tissue fiber axis for attenuation measurements (1-7 MHz). Tendons subjected to cross-linking treatment were immersed in 10% formalin, and control tendons in normal saline solution. Attenuation measurements were made immediately after immersion for both tendon groups at numerous intervals for 1 hr. The attenuation coefficient of the samples insonified perpendicular to the collagen fibers increased after formalin fixation over 1-hr period, while attenuation for parallel measurements decreased at selected frequencies during the first 20 min and subsequently increased modestly by 1 hr. Although the molecular mechanism responsible for the unexpected decrease in attenuation in the parallel direction as compared with the increase in the perpendicular direction is not clear, it may reflect rapid alterations in directionally dependent viscous and/or elastic components that contribute to material properties of well-ordered biological tissues.