Theragnostics for tumor and plaque angiogenesis with perfluorocarbon nanoemulsions.

Molecular imaging agents are extending the potential of noninvasive medical diagnosis from basic gross anatomical descriptions to complicated phenotypic characterizations based upon the recognition of unique cell-surface biochemical signatures. Although originally the purview of nuclear medicine, "molecular imaging" is now studied in conjunction with all clinically relevant imaging modalities. Of the myriad of particles that have emerged as prospective candidates for clinical translation, perfluorocarbon nanoparticles offer great potential for combining targeted imaging with drug delivery, much like the "magic bullet" envisioned by Paul Ehrlich 100 years ago. Perfluorocarbon nanoparticles, once studied in Phase III clinical trials as blood substitutes, have found new life for molecular imaging and drug delivery. The particles have been adapted for use with all clinically relevant modalities and for targeted drug delivery. In particular, their intravascular constraint due to particle size provides a distinct advantage for angiogenesis imaging and antiangiogenesis therapy. As perfluorocarbon nanoparticles have recently entered Phase I clinical study, this review provides a timely focus on the development of this platform technology and its application for angiogenesis-related pathologies.

Application of Renyi entropy for ultrasonic molecular imaging.

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.

Real-time calculation of a limiting form of the Renyi entropy applied to detection of subtle changes in scattering architecture.

Previously a new method for ultrasound signal characterization using entropy H(f) was reported, and it was demonstrated that in certain settings, further improvements in signal characterization could be obtained by generalizing to Renyi entropy-based signal characterization I(f)(r) with values of r near 2 (specifically r=1.99) [M. S. Hughes et al., J. Acoust. Soc. Am. 125, 3141-3145 (2009)]. It was speculated that further improvements in sensitivity might be realized at the limit r-->2. At that time, such investigation was not feasible due to excessive computational time required to calculate I(f)(r) near this limit. In this paper, an asymptotic expression for the limiting behavior of I(f)(r) as r-->2 is derived and used to present results analogous to those obtained with I(f)(1.99). Moreover, the limiting form I(f,infinity) is computable directly from the experimentally measured waveform f(t) by an algorithm that is suitable for real-time calculation and implementation.

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

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

Novel MRI contrast agent for molecular imaging of fibrin: implications for detecting vulnerable plaques.

BACKGROUND: Molecular imaging of thrombus within fissures of vulnerable atherosclerotic plaques requires sensitive detection of a robust thrombus-specific contrast agent. In this study, we report the development and characterization of a novel ligand-targeted paramagnetic molecular imaging agent with high avidity for fibrin and the potential to sensitively detect active vulnerable plaques. METHODS AND RESULTS: The nanoparticles were formulated with 2.5 to 50 mol% Gd-DTPA-BOA, which corresponds to >50 000 Gd(3+) atoms/particle. Paramagnetic nanoparticles were characterized in vitro and evaluated in vivo. In contradistinction to traditional blood-pool agents, T1 relaxation rate as a function of paramagnetic nanoparticle number was increased monotonically with Gd-DTPA concentration from 0.18 mL. s(-1). pmol(-1) (10% Gd-DTPA nanoparticles) to 0.54 mL. s(-1). pmol(-1) for the 40 mol% Gd-DTPA formulations. Fibrin clots targeted in vitro with paramagnetic nanoparticles presented a highly detectable, homogeneous T1-weighted contrast enhancement that improved with increasing gadolinium level (0, 2.5, and 20 mol% Gd). Higher-resolution scans and scanning electron microscopy revealed that the nanoparticles were present as a thin layer over the clot surface. In vivo contrast enhancement under open-circulation conditions was assessed in dogs. The contrast-to-noise ratio between the targeted clot (20 mol% Gd-DTPA nanoparticles) and blood was approximately 118+/-21, and that between the targeted clot and the control clot was 131+/-37. CONCLUSIONS: These results suggest that molecular imaging of fibrin-targeted paramagnetic nanoparticles can provide sensitive detection and localization of fibrin and may allow early, direct identification of vulnerable plaques, leading to early therapeutic decisions.

Effect of dietary aflatoxin concentration on the assessment of genetic variability of response in a randombred population of chickens.

The effect of graded levels of dietary aflatoxin on the assessment of genetic variability of body weight and gain and plasma protein response was tested utilizing the Athens-Canadian randombred population of chickens. Dietary aflatoxin was administered at levels of either 0, 1.25, 2.50 or 5.0 microg/g of diet ad libitum from 7 to 21 days of age to progeny from 58 sire families. Twenty-one-day body weights, gain and plasma protein concentration were used to assess the variation in response.-The administration of increasing levels of aflatoxin resulted in a dose-related decrease of gains and plasma protein concentrations. Plasma protein concentrations were significantly higher among males than females within the control group; however, this difference was reversed as the severity of the aflatoxin challenge increased. Heritability estimates for all responses increased as the level of aflatoxin administered increased. This change was most notable for total plasma protein concentration. Phenotypic correlations for plasma protein concentration and growth measurements tended to diminish with increasing levels of aflatoxin. A similar trend was noted for the genetic correlations; however, a moderate correlation between growth responses and plasma protein response was detected in the 5.0-microg/g aflatoxin treatment group. Genetic correlations were calculated for the same characters between the different levels of aflatoxin. Regardless of which aflatoxin challenges were compared, a very high genetic correlation for 21-day body weight and 7- to 21-day gain was estimated. This variation in growth potential in the toxic environment paralleled that observed in the control environment but at a lower plane. Genetic correlations for plasma protein response across aflatoxin levels diminished as the difference between the levels of aflatoxin administered increased. Plasma protein concentration in the control environment was positively correlated with plasma protein response in groups fed a low level of aflatoxin, but negatively correlated when an aflatoxin challenge of 2.5 microg/g or more was given, suggesting that selection for aflatoxin resistance using plasma protein response as a selection criterion should be made under an aflatoxin stress environment.

Comparison of the galactopoietic response to pituitary-derived and recombinant-derived variants of bovine growth hormone.

Two studies were designed to examine the differences in galactopoietic potency of molecular variants of pituitary- and recombinant-derived bovine GH (bGH). The recombinant bGH molecules included amino-terminal and position-127 amino acid substitutions which are representative of two of the four natural pituitary variants or of partially degraded bGH molecules. Amino-terminal variants of bGH included methionine (Met1), alanine (Ala1), serine (Ser1) or deletion of four amino acids (delta 1-4). The delta 1-4 variants were representative of degradation products previously isolated in pituitary bGH preparations. In the first study, 54 lactating Holstein cows received i.m. injections of a buffer solution (control), pituitary-derived bGH, or recombinant-derived [Met1,Leu127]-bGH, [Met1,Val127]-bGH, [Ala1,Leu127]-bGH, or [Ala1,Val127]-bGH. Cows received 25 mg bGH/day for 21 days. Substitution of the amino-terminal alanyl residue with methionine did not affect milk response. GH variants with Val127 elicited a greater milk response (8.5 kg/day) than Leu127 bGH variants (6.5 kg/day). The average milk response to the four recombinant bGH variants was 7.5 kg/day greater than controls compared with 4.4 kg/day for pituitary-derived bGH. In contrast, blood bGH concentrations were equivalent for pituitary and recombinant bGH treatments, approximately 20 micrograms/l more than control levels at 3 h after injection. Blood free fatty acid concentrations were increased, but insulin and glucose levels were unaffected by bGH treatment. In the second study, 54 lactating Holstein cows received i.m. injections of a buffer control solution or recombinant-derived [Met1,Leu127]-bGH, [Ser1,Leu127]-bGH, [Ser1,Val127]-bGH, [delta 1-4,Leu127]-bGH or [delta 1-4,Val127]-bGH. Cows received 25 mg bGH/day for 28 days. The milk response to full-length bGH variants was 6.6 kg/day greater than the response to the amino-terminal deletion variants (P less than 0.05). Substitution of valine for leucine did not affect milk response to either the deletion (delta 1-4) or full-length (Met1 or Ser1) bGH molecules. In conclusion, the lowered galactopoietic potency of pituitary bGH preparations was demonstrated, at least in part, to be due to the presence of amino-terminal amino acid deletions rather than differences in amino acid sequences of recombinant bGH. Ala1 bGH variants with valine at position 127 elicited a greater milk response than Leu127 variants.

Molecular imaging of stretch-induced tissue factor expression in carotid arteries with intravascular ultrasound.

RATIONALE AND OBJECTIVES: Molecular imaging with targeted contrast agents enables tissues to be distinguished by detecting specific cell-surface receptors. In the present study, a ligand-targeted acoustic nanoparticle system is used to identify angioplasty-induced expression of tissue factor by smooth muscle cells within carotid arteries. METHODS: Pig carotid arteries were overstretched with balloon catheters, treated with tissue factor-targeted or a control nanoparticle system, and imaged with intravascular ultrasound before and after treatment. RESULTS: Tissue factor-targeted emulsions bound and increased the echogenicity and gray-scale levels of overstretched smooth muscle cells within the tunica media, versus no change in contralateral control arteries. Expression of stretch-induced tissue factor in carotid artery media was confirmed by immunohistochemistry. CONCLUSIONS: The potential for abnormal thrombogenicity of balloon-injured arteries, as reflected by smooth muscle expression of tissue factor, was imaged using a novel, targeted, nanoparticulate ultrasonic contrast agent.

In vivo molecular imaging of stretch-induced tissue factor in carotid arteries with ligand-targeted nanoparticles.

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, P<0.05) 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.

Plaque and structural characteristics of the descending thoracic aorta using transesophageal echocardiography.

The in vivo acoustic and structural characteristics of atherosclerosis in the descending thoracic aorta have not been well delineated. We prospectively evaluated the descending thoracic aorta of 147 patients (35 women and 112 men; age, 61 +/- 14 years) who underwent clinically indicated transesophageal echocardiography. Patients with suspected disease of the aorta were excluded. Thirty-eight patients (26%) had protruding plaques (men, 25%; women, 29%). Six patients had mobile intimal densities with the mobile area ranging up to 1 cm2. As expected, aortic lumen area was decreased (plaque-free, 3.53 cm2; plaque, 3.19 cm2; p less than 0.05) and wall area was increased (plaque-free, 1.51 cm2; plaque, 1.92 cm2; p less than 0.05) in the regions of the plaque. However, total arterial area was not increased (plaque-free, 5.04 cm2; plaque, 5.09 cm2; difference not significant) in a compensatory manner as observed in other arterial beds. Plaque gray scale was less than the gray scale of plaque-free wall (plaque-free, 141.2; plaque, 122.7; p less than 0.05) when compared at the same level of the descending thoracic aorta or with a second aortic plaque-free level (plaque-free, 150.4; plaque, 122.7; p less than 0.05). Standard deviation of gray scale level was similar between plaque and normal regions. Unsuspected protruding plaques in the descending thoracic aorta occurred in one quarter of the patients referred for routine transesophageal examination. Plaques tended to have lower echogenicity and were differentiated from plaque-free walls within patients. Plaque formation did not result in increased total arterial area. These data suggest that the degree or character of compensatory atherosclerotic remodeling in the highly elastic descending thoracic aorta may differ from other arterial beds.

Development of inherently echogenic liposomes as an ultrasonic contrast agent.

Ultrasonic contrast agents have been developed for improved assessment of blood flow and tissue perfusion. Many of these agents are not inherently acoustically reflective (echogenic), and nearly all are not suitable for tissue specific targeting. The purpose of this study was to develop acoustically reflective liposomes, which are suitable for antibody conjugation, without using gas or any other agent entrapment. Echogenic liposomes were prepared from phosphatidylcholine (PC), phophatidylethanolamine (PE), phosphatidylglycerol (PG), and cholesterol (CH), using a dehydration/rehydration method. The formulation was optimized for higher acoustic reflectivity by varying the lipid composition. Liposomes were imaged with a 20 MHz intravascular ultrasonic imaging catheter. Echogenicity levels were expressed using pixel gray scale. The presence of PE and PG at specific concentrations improved echogenicity due to their effects on liposomal morphology as confirmed by freeze-etch electron microscopy. The acoustic reflectivity of liposomes was retained when liposomes were treated with blood at room temperature and 37 degrees C under in vitro conditions. It was demonstrated that the liposomes were also acoustically reflective in vivo after they were injected into a miniswine model. We have developed echogenic liposomes that are stable and suitable for tissue specific targeting as a novel contrast agent. This new contrast agent can be used for ultrasonic image enhancement and/or treatment of targeted pathologic sites.

Delineation of the extracellular determinants of ultrasonic scattering from elastic arteries.

Elastic arteries consist of three primary components: elastin fibers, extracellular collagen matrix and smooth muscle cells. However, the relative contribution of elastin and collagen fibers to overall ultrasonic scattering from an intact arterial wall is poorly understood. To define the principal source of extracellular scattering from the medial layer of elastic arteries, canine ascending aortas (n = 10) were excised, fixed and sectioned for insonification. Subsequently, aortic specimens were restudied after treatment to dissolve all tissue components except extracellular collagen matrix (n = 5) and elastin fibers (n = 5). Histological staining revealed very few elastin fibers and sparse intact collagen in collagen-isolated and elastin-isolated tissues, respectively. Integrated backscatter, attenuation and backscatter coefficients differentiated these two treated tissues. The backscatter coefficient for elastin-isolated tissue demonstrated a fivefold increase over collagen-isolated tissue, suggesting that elastin fibers represent a primary scattering component within elastic arteries, and the collagen fibers may provide a secondary component of scattering.

High-frequency ultrasonic detection of thrombi with a targeted contrast system.

Site-targeted acoustic contrast agents used in conjunction with high-frequency intravascular ultrasound have the potential to localize and characterize intravascular pathology. The present study quantifies the utility of a novel, site-targeted ultrasonic contrast agent with high-frequency ultrasound (30 to 50 MHz) and demonstrates the feasibility of the new agent for augmenting detection of targeted pathology with intravascular ultrasonic catheters. High-frequency acoustic microscopy was used to image avidinconjugated nitrocellulose membranes after exposure to a control or biotinylated contrast agent. Increases (p < 0.05) in backscattered power of approximately 66 dB (4-fold) were found for the biotinylated, but not the control contrast agent. Intravascular ultrasonic images (30 MHz nominal center frequency) of plasma clots after exposure to the targeted contrast agent were brighter (p < 0.05) than in controls. These results demonstrate high-frequency acoustic enhancement with a novel targeted contrast agent and may extend the potential diagnostic spectrum of intravascular ultrasound.

Application of finite-element analysis with optimisation to assess the in vivo non-linear myocardial material properties using echocardiographic imaging.

An application of finite-element analysis with an optimisation technique to assess the myocardial material properties in diastasis in vivo is described. Using the data collected from an animal model, the three-dimensional geometry of the left ventricular chamber, at several times in diastole, was reconstructed. From the measurement of the ventricular chamber pressure during image acquisition, finite-element analysis was performed to predict the expansion during diastasis. Initially, by restricting the motion of the epicardial nodes and computing the reaction forces, an 'equivalent pericardial pressure' was determined and applied in subsequent analysis. The duration of diastasis was divided into three or four intervals and the analysis was performed at each interval to assess the material properties of the myocardium. Using such a step-wise linear approach, the non-linear material properties of the myocardium during passive expansion was determined. Our results demonstrated that the computed 'equivalent pericardial pressure' increased with and was smaller than the corresponding left ventricular chamber pressure. The passive myocardium exhibited a linear tangent modulus against chamber pressure relationship which is equivalent to an exponential stress/strain relationship, similar to those suggested by in vitro studies.

Experimental determination of phase velocity of perfluorocarbons: applications to targeted contrast agents.

Targeted acoustic contrast agents are designed to enhance the sensitivity and specificity of ultrasonic diagnoses. We have previously developed a ligand targeted ultrasonic contrast system that is a lipid-encapsulated, liquid-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. We have recently proposed a simple acoustic transmission line model that treats the emulsion particles as a thin layer over the targeted surface. In this model, the acoustic reflectivity of the sample increases for perfluorocarbons with smaller velocities of longitudinal sound or lower densities. In this study, we measure and report the velocity of longitudinal sound for 20 perfluorocarbons using a broadband phase spectroscopic approach for estimating phase velocities. Experimentally determined velocities ranged from 520+/-2 m/sec (perfluorohexane) to 705+/-5 m/s (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. Our 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 surface acoustic reflectivity.

Strain variation in hematological response of broilers to dietary aflatoxin.

The response of clinical blood values to graded levels of dietary aflatoxin was studied in three populations of chickens. Male chicks of two commercial broiler strains, one color-sexed and the other feather-sexed, and a randombred strain were fed dietary aflatoxin at levels of 0, 1.25, 2.50 or 5.0 microgram/g of diet from 1 to 21 days of age. At the conclusion of the 21-day treatment period, all remaining birds were bled by cardiac puncture and packed cell volume (PCV), erythrocyte counts (RBC), hemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), total plasma protein, and total plasma cholesterol were determined. All strains of broilers were susceptible to aflatoxicosis as detected by total plasma protein and cholesterol. Anemia associated with aflatoxicosis was induced in the two commercial broiler lines, but not in the randombred line. The PCV, RBC, and Hb were diminished from control values in a dose-related fashion by graded levels of aflatoxin in the commercial broilers, but not in the randombred line. Decreases in MCV with no significant changes in MCHC were observed in all strains.

Genetic variability in growth response of chicks to cold brooding temperature.

Genetic variability in adaptation to brooding at a reduced temperature was examined by comparing 1 to 14-day body weight gains of progeny from 28 sire families of the Athens-Canadian randombred population brooded at 32.2 C and at 26.7 C. The 1 to 7-day gain of the chicks brooded at 26.7 C was significantly depressed, but 7 to 14-day gain was not significantly depressed by brooding at the reduced temperature. Heritability estimates averaged over the two trials were .31, .23, and .35 for the 1 to 7-, 7 to 14-, and 1 to 14-day weight gains of groups brooded at 32.2 C and .55, .59, and .67 for the gain of groups brooded at 26.7 C. The heritability estimates for the difference in weight gain of families brooded at the two temperatures was .78.

Variation with age in response of broilers to aflatoxin.

This study investigated the effects on growth and composition of the blood of dietary aflatoxin fed at levels of 0, 2.5, or 5.0 microgram/g of diet for a three week period beginning at 1, 7, 14, and 21 days of age in commercial broilers. Packed cell volume (PCV), erythrocyte counts, mean corpuscular volume, hemoglobin content and mean corpuscular hemoglobin concentration, body weight, and mortality were measured weekly. Plasma cholesterol and total plasma protein levels were determined weekly. Dietary aflatoxin at levels of 2.5 and 5.0 microgram of aflatoxin per gram of diet when fed to young chicks for three weeks, beginning at either one or seven days, depressed body weight and PCV in a dose related fashion. Body weight and PCV continued to be depressed by the 5.0 microgram/g diets in chicks treated from 2 to 5 weeks of age but not at the lower dosages. The feeding of aflatoxin at levels of up to 5.0 microgram aflatoxin per gram of diet from 3 to 6 weeks of age did not significantly depress body weight or PCV from control levels. Plasma cholesterol and total protein were found to be more sensitive to aflatoxin treatment at the later ages than body weight and other blood values. Plasma cholesterol was significantly depressed by the 5.0 microgram/g level of aflatoxin in all treatment periods, but the 2.5 mirogram/g level of aflatoxin did not significantly reduce cholesterol during the 3 to 6 week treatment period. Plasma proteins were found to be the most sensitive criteria for detecting broiler susceptibility to aflatoxin, being depressed by all levels of aflatoxin for all age groups.

Strain variation in 59Fe absorption during aflatoxicosis.

Strain variation of iron absorption in response to dietary aflatoxin was studied using two commercial broiler strains and a nonselected randombred strain. Aflatoxin depressed chick body weights and increased mortalities in each strain. Hematocrits were significantly reduced in the commercial broilers receiving the toxin, but not in the randombreds. Aflatoxin reduced iron absorption in each strain regardless of the presence of anemia.