Validation of a combined ultrasound and bioluminescence imaging system with magnetic resonance imaging in orthotopic pancreatic murine tumors.

Preclinical mouse solid tumor models are widely used to evaluate efficacy of novel cancer therapeutics. Recent reports have highlighted the need for utilizing orthotopic implantation to represent clinical disease more accurately, however the deep tissue location of these tumors makes longitudinal assessment challenging without the use of imaging techniques. The purpose of this study was to evaluate the performance of a new multi-modality high-throughput in vivo imaging system that combines bioluminescence imaging (BLI) with robotic, hands-free ultrasound (US) for evaluating orthotopic mouse models. Long utilized in cancer research as independent modalities, we hypothesized that the combination of BLI and US would offer complementary advantages of detection sensitivity and quantification accuracy, while mitigating individual technological weaknesses. Bioluminescent pancreatic tumor cells were injected into the pancreas tail of C57BL/6 mice and imaged weekly with the combination system and magnetic resonance imaging (MRI) to serve as a gold standard. BLI photon flux was quantified to assess tumor activity and distribution, and US and MRI datasets were manually segmented for gross tumor volume. Robotic US and MRI demonstrated a strong agreement (R2 = 0.94) for tumor volume measurement. BLI showed a weak overall agreement with MRI (R2 = 0.21), however, it offered the greatest sensitivity to detecting the presence of tumors. We conclude that combining BLI with robotic US offers an efficient screening tool for orthotopic tumor models.

Nanoparticle Delivery of miR-122 Inhibits Colorectal Cancer Liver Metastasis.

Liver metastasis is a leading cause of cancer morbidity and mortality. Thus, there has been strong interest in the development of therapeutics that can effectively prevent liver metastasis. One potential strategy is to utilize molecules that have broad effects on the liver microenvironment, such as miR-122, a liver-specific miRNA that is a key regulator of diverse hepatic functions. Here we report the development of a nanoformulation miR-122 as a therapeutic agent for preventing liver metastasis. We engineered a galactose-targeted lipid calcium phosphate (Gal-LCP) nanoformulation of miR-122. This nanotherapeutic elicited no significant toxicity and delivered miR-122 into hepatocytes with specificity and high efficiency. Across multiple colorectal cancer liver metastasis models, treatment with Gal-LCP miR-122 treatment effectively prevented colorectal cancer liver metastasis and prolonged survival. Mechanistic studies revealed that delivery of miR-122 was associated with downregulation of key genes involved in metastatic and cancer inflammation pathways, including several proinflammatory factors, matrix metalloproteinases, and other extracellular matrix degradation enzymes. Moreover, Gal-LCP miR-122 treatment was associated with an increased CD8+/CD4+ T-cell ratio and decreased immunosuppressive cell infiltration, which makes the liver more conducive to antitumor immune response. Collectively, this work presents a strategy to improve cancer prevention and treatment with nanomedicine-based delivery of miRNA. SIGNIFICANCE: Highly specific and efficient delivery of miRNA to hepatocytes using nanomedicine has therapeutic potential for the prevention and treatment of colorectal cancer liver metastasis.

Assessing Polycystic Kidney Disease in Rodents: Comparison of Robotic 3D Ultrasound and Magnetic Resonance Imaging.

Polycystic kidney disease (PKD) is an inherited disorder characterized by renal cyst formation and enlargement of the kidney. PKD severity can be staged noninvasively by measuring total kidney volume (TKV), a promising biomarker that has recently received regulatory qualification. In preclinical mouse models, where the disease is studied and potential therapeutics are evaluated, the most popular noninvasive method of measuring TKV is magnetic resonance imaging (MRI). Although MRI provides excellent 3D resolution and contrast, these systems are expensive to operate, have long acquisition times, and, consequently, are not heavily used in preclinical PKD research. In this study, a new imaging instrument, based on robotic ultrasound (US), was evaluated as a complementary approach for assessing PKD in rodent models. The objective was to determine the extent to which TKV measurements on the robotic US scanner correlated with both in vivo and ex vivo reference standards (MRI and Vernier calipers, respectively). A cross-sectional study design was implemented that included both PKD-affected mice and healthy wild types, spanning sex and age for a wide range of kidney volumes. It was found that US-derived TKV measurements and kidney lengths were strongly associated with both in vivo MRI and ex vivo Vernier caliper measurements (R 2=0.94 and 0.90, respectively). In addition to measuring TKV, renal vascular density was assessed using acoustic angiography (AA), a novel contrast-enhanced US methodology. AA image intensity, indicative of volumetric vascularity, was seen to have a strong negative correlation with TKV (R 2=0.82), suggesting impaired renal vascular function in mice with larger kidneys. These studies demonstrate that robotic US can provide a rapid and accurate approach for noninvasively evaluating PKD in rodent models.

Effect of Hydrostatic Pressure, Boundary Constraints and Viscosity on the Vaporization Threshold of Low-Boiling-Point Phase-Change Contrast Agents.

The vaporization of low-boiling-point phase-change contrast agents (PCCAs) using ultrasound has been explored in vitro and in vivo. However, it has been reported that the pressure required for activation is higher in vivo, even after attenuation is accounted for. In this study, the effect of boundary constraints, hydrostatic pressure and viscosity on PCCA vaporization pressure threshold are evaluated to explore possible mechanisms for variations in in vivo vaporization behavior. Vaporization was measured in microtubes of varying inner diameter and a pressurized chamber under different hydrostatic pressures using a range of ultrasound pressures. Furthermore, the activation threshold was evaluated in the kidneys of rats. The results confirm that the vaporization threshold is higher in vivo and reveal an increasing activation threshold inversely proportional to constraining tube size and inversely proportional to surrounding viscosity in constrained environments. Counterintuitively, increased hydrostatic pressure had no significant effect experimentally on the PCCA vaporization threshold, although it was confirmed that this result was supported by homogeneous nucleation theory for liquid perfluorocarbon vaporization. These factors suggest that constraints caused by the surrounding tissue and capillary walls, as well as increased viscosity in vivo, contribute to the increased vaporization threshold compared with in vitro experiments, although more work is required to confirm all relevant factors.

Ultrasound Measurement of Vascular Density to Evaluate Response to Anti-Angiogenic Therapy in Renal Cell Carcinoma.

BACKGROUND: Functional and molecular changes often precede gross anatomical changes, so early assessment of a tumor's functional and molecular response to therapy can help reduce a patient's exposure to the side effects of ineffective chemotherapeutics or other treatment strategies. OBJECTIVE: Our intent was to test the hypothesis that an ultrasound microvascular imaging approach might provide indications of response to therapy prior to assessment of tumor size. METHODS: Mice bearing clear-cell renal cell carcinoma xenograft tumors were treated with antiangiogenic and Notch inhibition therapies. An ultrasound measurement of microvascular density was used to serially track the tumor response to therapy. RESULTS: Data indicated that ultrasound-derived microvascular density can indicate response to therapy a week prior to changes in tumor volume and is strongly correlated with physiological characteristics of the tumors as measured by histology ([Formula: see text]). Furthermore, data demonstrated that ultrasound measurements of vascular density can determine response to therapy and classify between-treatment groups with high sensitivity and specificity. CONCLUSION/SIGNIFICANCE: Results suggests that future applications utilizing ultrasound imaging to monitor tumor response to therapy may be able to provide earlier insight into tumor behavior from metrics of microvascular density rather than anatomical tumor size measurements.

In Vivo Molecular Imaging Using Low-Boiling-Point Phase-Change Contrast Agents: A Proof of Concept Study.

Sub-micron phase-change contrast agents (PCCAs) have been proposed as a tool for ultrasound molecular imaging based on their potential to extravasate and target extravascular markers and also because of the potential to image these contrast agents with a high contrast-to-tissue ratio. We compare in vivo ultrasound molecular imaging with targeted low-boiling-point PCCAs and targeted microbubble contrast agents. Both agents were targeted to the intravascular (endothelial) integrin αvß3via a cyclic RGD peptide (cyclo-Arg-Gly-Asp-D-Tyr-Cys) mechanism and imaged in vivo in a rodent fibrosarcoma model, which exhibits angiogenic microvasculature. Signal intensity was measured using two different techniques, conventional contrast-specific imaging (amplitude/phase modulation) and a droplet vaporization imaging sequence, which detects the unique signature of vaporizing PCCAs. Data indicate that PCCA-specific imaging is more sensitive to small numbers of bound agents than conventional contrast imaging. However, data also revealed that contrast from targeted microbubbles was greater than that provided by PCCAs. Both control and targeted PCCAs were observed to be retained in tissue post-vaporization, which was expected for targeted agents but not expected for control agents. The exact mechanism underlying this observation remains unknown.

Vaporization Detection Imaging: A Technique for Imaging Low-Boiling-Point Phase-Change Contrast Agents with a High Depth of Penetration and Contrast-to-Tissue Ratio.

Phase-change contrast agents (PCCAs) possess advantages over microbubble contrast agents, such as the ability to extravasate and circulate longer in the vasculature that could enhance the diagnostic capabilities of contrast-enhanced ultrasound. PCCAs typically have a liquid perfluorocarbon (PFC) core that can be vaporized into echogenic microbubbles. Vaporization of submicron agents filled with liquid PFCs at body temperature usually requires therapeutic pressures higher than typically used for diagnostic imaging, but low-boiling-point PCCAs using decafluorobutane or octafluoropropane can be vaporized using pressures in the diagnostic imaging regime. Low-boiling-point PCCAs produce a unique acoustic signature that can be separated from tissue and bubble signals to make images with high contrast-to-tissue ratios. In this work, we explore the effect of pulse length and concentration on the vaporization signal of PCCAs and a new technique to capture and use the signals to make high contrast-to-tissue ratio images in vivo. The results indicate that using a short pulse may be ideal for imaging because it does not interact with created bubbles but still produces strong signals for making images. Furthermore, it was found that capturing PCCA vaporization signals produced higher contrast-to-tissue ratio values and better depth of penetration than imaging the bubbles generated by droplet activation using conventional contrast imaging techniques. The resolution of the vaporization signal images is poor because of the low frequency of the signals, but their high sensitivity may be used for applications such as molecular imaging, where the detection of small numbers of contrast agents is important.

A new preclinical ultrasound platform for widefield 3D imaging of rodents.

Noninvasive in vivo imaging technologies enable researchers and clinicians to detect the presence of disease and longitudinally study its progression. By revealing anatomical, functional, or molecular changes, imaging tools can provide a near real-time assessment of important biological events. At the preclinical research level, imaging plays an important role by allowing disease mechanisms and potential therapies to be evaluated noninvasively. Because functional and molecular changes often precede gross anatomical changes, there has been a significant amount of research exploring the ability of different imaging modalities to track these aspects of various diseases. Herein, we present a novel robotic preclinical contrast-enhanced ultrasound system and demonstrate its use in evaluating tumors in a rodent model. By leveraging recent advances in ultrasound, this system favorably compares with other modalities, as it can perform anatomical, functional, and molecular imaging and is cost-effective, portable, and high throughput, without using ionizing radiation. Furthermore, this system circumvents many of the limitations of conventional preclinical ultrasound systems, including a limited field-of-view, low throughput, and large user variability.

Ultrasound Molecular Imaging of VEGFR-2 in Clear-Cell Renal Cell Carcinoma Tracks Disease Response to Antiangiogenic and Notch-Inhibition Therapy.

Metastatic clear-cell renal cell carcinoma (ccRCC) affects thousands of patients worldwide each year. Antiangiogenic therapy has been shown to have beneficial effects initially, but resistance is eventually developed. Therefore, it is important to accurately track the response of cancer to different therapeutics in order to appropriately adjust the therapy to maximize efficacy. Change in tumor volume is the current gold standard for determining efficacy of treatment. However, functional variations can occur much earlier than measurable volume changes. Contrast-enhanced ultrasound (CEUS) is an important tool for assessing tumor progression and response to therapy, since it can monitor functional changes in the physiology. In this study, we demonstrate how ultrasound molecular imaging (USMI) can accurately track the evolution of the disease and molecular response to treatment. Methods A cohort of NSG (NOD/scid/gamma) mice was injected with ccRCC cells and treated with either the VEGF inhibitor SU (Sunitinib malate, Selleckchem, TX, USA) or the Notch pathway inhibitor GSI (Gamma secretase inhibitor, PF-03084014, Pfizer, New York, NY, USA), or started on SU and later switched to GSI (Switch group). The therapies used in the study focus on disrupting angiogenesis and proper vessel development. SU inhibits signaling of vascular endothelial growth factor (VEGF), which is responsible for the sprouting of new vasculature, and GSI inhibits the Notch pathway, which is a key factor in the correct maturation of newly formed vasculature. Microbubble contrast agents targeted to VEGFR-2 (VEGF Receptor) were delivered as a bolus, and the bound agents were imaged in 3D after the free-flowing contrast was cleared from the body. Additionally, the tumors were harvested at the end of the study and stained for CD31. Results The results show that MI can detect changes in VEGFR-2 expression in the group treated with SU within a week of the start of treatment, while differences in volume only become apparent after the mice have been treated for three weeks. Furthermore, USMI can detect response to therapy in 92% of cases after 1 week of treatment, while the detection rate is only 40% for volume measurements. The amount of targeting for the GSI and Control groups was high throughout the duration of the study, while that of the SU and Switch groups remained low. However, the amount of targeting in the Switch group increased to levels similar to those of the Control group after the treatment was switched to GSI. CD31 staining indicates significantly lower levels of patent vasculature for the SU group compared to the Control and GSI groups. Therefore, the results parallel the expected physiological changes in the tumor, since GSI promotes angiogenesis through the VEGF pathway, while SU inhibits it. Conclusion This study demonstrates that MI can track disease progression and assess functional changes in tumors before changes in volume are apparent, and thus, CEUS can be a valuable tool for assessing response to therapy in disease. Future work is required to determine whether levels of VEGFR-2 targeting correlate with eventual survival outcomes.

In Vivo Assessment of the Potential for Renal Bio-Effects from the Vaporization of Perfluorocarbon Phase-Change Contrast Agents.

Low-boiling-point perfluorocarbon phase-change contrast agents (PCCAs) provide an alternative to microbubble contrast agents. Although parameter ranges related to in vivo bio-effects of microbubbles are fairly well characterized, few studies have been done to evaluate the potential of bio-effects related to PCCAs. To bridge this gap, we present an assessment of biological effects (e.g., hemorrhage) related to acoustically excited PCCAs in the rodent kidney. The presence or absence of bio-effects was observed after sonication with various perfluorocarbon core PCCAs (decafluorobutane, octafluoropropane or a 1:1 mixture) and as a function of activation pulse mechanical index (MI; minimum activation threshold, which was a moderate MI of 0.81-1.35 vs. a clinical maximum of 1.9). Bio-effects on renal tissue were assessed through hematology and histology including measurement of blood creatinine levels and the quantity of red blood cell (RBC) casts present in hematoxylin and eosin-stained kidney tissue sections after sonication. Short-term (24 h) and long-term (2 and 4 wk) analyses were performed after treatment. Results indicated that bio-effects from PCCA vaporization were not observed at lower mechanical indices. At higher mechanical indices, bio-effects were observed at 24 h, although these were not observable 2 wk after treatment.

Optimizing Sensitivity of Ultrasound Contrast-Enhanced Super-Resolution Imaging by Tailoring Size Distribution of Microbubble Contrast Agent.

Ultrasound contrast-enhanced super-resolution imaging has recently attracted attention because of its extraordinary ability to image vascular features much smaller than the ultrasound diffraction limit. This method requires sensitive detection of separable microbubble events despite a noisy tissue background to indicate the microvasculature, and any approach that could improve the sensitivity of the ultrasound system to individual microbubbles would be highly beneficial. In this study, we evaluated the effect of varying microbubble size on super-resolution imaging sensitivity. Microbubble preparations were size sorted into different mean diameters and then were imaged at equal concentrations. Commercially manufactured Definity and Optison were also imaged for comparison. Both in vitro experiments in phantom vessels and in vivo experiments imaging rat tumors revealed that the sensitivity of contrast-enhanced super-resolution imaging can be improved by using microbubbles with a larger diameter.

Optimizing ultrasound molecular imaging of secreted frizzled related protein 2 expression in angiosarcoma.

Secreted frizzled related protein 2 (SFRP2) is a tumor endothelial marker expressed in angiosarcoma. Previously, we showed ultrasound molecular imaging with SFRP2-targeted contrast increased average video pixel intensity (VI) of angiosarcoma vessels by 2.2 ± 0.6 VI versus streptavidin contrast. We hypothesized that redesigning our contrast agents would increase imaging performance. Improved molecular imaging reagents were created by combining NeutrAvidin™-functionalized microbubbles with biotinylated SFRP2 or IgY control antibodies. When angiosarcoma tumors in nude mice reached 8 mm, time-intensity, antibody loading, and microbubble dose experiments optimized molecular imaging. 10 minutes after injection, the control-subtracted time-intensity curve (TIC) for SFRP2-targeted contrast reached a maximum, after subtracting the contribution of free-flowing contrast. SFRP2 antibody-targeted VI was greater when contrast was formulated with 10-fold molar excess of maleimide-activated NeutrAvidin™ versus 3-fold (4.5 ± 0.18 vs. 0.32 ± 0.15, VI ± SEM, 5 x 106 dose, p < 0.001). Tumor vasculature returned greater average video pixel intensity using 5 x 107 versus 5 x 106 microbubbles (21.2 ± 2.5 vs. 4.5 ± 0.18, p = 0.0011). Specificity for tumor vasculature was confirmed by low VI for SFRP2-targeted, and control contrast in peri-tumoral vasculature (3.2 ± 0.52 vs. 1.6 ± 0.71, p = 0.92). After optimization, average video pixel intensity of tumor vasculature was 14.2 ± 3.0 VI units higher with SFRP2-targeted contrast versus IgY-targeted control (22.1 ± 2.5 vs. 7.9 ± 1.6, p < 0.001). After log decompression, 14.2 ΔVI was equal to ~70% higher signal, in arbitray acoustic units (AU), for SFRP2 versus IgY. This provided ~18- fold higher acoustic signal enhancement than provided previously by 2.2 ΔVI. Basing our targeted contrast on NeutrAvidin™-functionalized microbubbles, using IgY antibodies for our control contrast, and optimizing our imaging protocol significantly increased the SFRP2-specific signal returned from angiosarcoma vasculature, and may provide new opportunities for targeted molecular imaging.

3-D Ultrasound Localization Microscopy for Identifying Microvascular Morphology Features of Tumor Angiogenesis at a Resolution Beyond the Diffraction Limit of Conventional Ultrasound.

Angiogenesis has been known as a hallmark of solid tumor cancers for decades, yet ultrasound has been limited in its ability to detect the microvascular changes associated with malignancy. Here, we demonstrate the potential of 'ultrasound localization microscopy' applied volumetrically in combination with quantitative analysis of microvascular morphology, as an approach to overcome this limitation. This pilot study demonstrates our ability to image complex microvascular patterns associated with tumor angiogenesis in-vivo at a resolution of tens of microns - substantially better than the diffraction limit of traditional clinical ultrasound, yet using an 8 MHz clinical ultrasound probe. Furthermore, it is observed that data from healthy and tumor-bearing tissue exhibit significant differences in microvascular pattern and density. Results suggests that with continued development of these novel technologies, ultrasound has the potential to detect biomarkers of cancer based on the microvascular 'fingerprint' of malignant angiogenesis rather than through imaging of blood flow dynamics or the tumor mass itself.

Enhancing Nanoparticle Accumulation and Retention in Desmoplastic Tumors via Vascular Disruption for Internal Radiation Therapy.

Aggressive, desmoplastic tumors are notoriously difficult to treat because of their extensive stroma, high interstitial pressure, and resistant tumor microenvironment. We have developed a combination therapy that can significantly slow the growth of large, stroma-rich tumors by causing massive apoptosis in the tumor center while simultaneously increasing nanoparticle uptake through a treatment-induced increase in the accumulation and retention of nanoparticles in the tumor. The vascular disrupting agent Combretastatin A-4 Phosphate (CA4P) is able to increase the accumulation of radiation-containing nanoparticles for internal radiation therapy, and the retention of these delivered radioisotopes is maintained over several days. We use ultrasound to measure the effect of CA4P in live tumor-bearing mice, and we encapsulate the radio-theranostic isotope 177Lutetium as a therapeutic agent as well as a means to measure nanoparticle accumulation and retention in the tumor. This combination therapy induces prolonged apoptosis in the tumor, decreasing both the fibroblast and total cell density and allowing further tumor growth inhibition using a cisplatin-containing nanoparticle.

Optimizing Acoustic Activation of Phase Change Contrast Agents With the Activation Pressure Matching Method: A Review.

Submicrometer phase-change contrast agents (PCCAs) consist of a liquid perfluorocarbon (PFC) core that can be vaporized by ultrasound (acoustic droplet vaporization) to generate contrast with excellent spatial and temporal control. When these agents, commonly referred to as nanodroplets, are formulated with cores of low boiling-point PFCs such as decafluorobutane and octafluoropropane, they can be activated with low-mechanical-index (MI) imaging pulses for diagnostic applications. Since the utilization of minimum MI is often desirable to avoid unnecessary biological effects, enabling consistent activation of these agents in an acoustic field is a challenge because the energy that must be delivered to achieve the vaporization threshold increases with depth due to attenuation. A novel vaporization approach called activation pressure matching (APM) has been developed to deliver the same pressure throughout a field of view in order to produce uniform nanodroplet vaporization and to limit the amount of energy that is delivered. In this paper, we discuss the application of this method with a Verasonics V1 Research Ultrasound System to modulate the output pressure from an ATL L11-5 transducer. Vaporization-pulse spacing optimization can be used in addition to matching the activation pressure through depth, and we demonstrate the feasibility of this approach both in vivo and in vitro. The use of optimized vaporization parameters increases the amount of time a single bolus of nanodroplets can generate useful contrast and provides consistent image enhancement in vivo. Therefore, APM is a useful technique for maximizing the efficacy of PCCA while minimizing delivered acoustic energy.

High Resolution Ultrasound Superharmonic Perfusion Imaging: In Vivo Feasibility and Quantification of Dynamic Contrast-Enhanced Acoustic Angiography.

Mapping blood perfusion quantitatively allows localization of abnormal physiology and can improve understanding of disease progression. Dynamic contrast-enhanced ultrasound is a low-cost, real-time technique for imaging perfusion dynamics with microbubble contrast agents. Previously, we have demonstrated another contrast agent-specific ultrasound imaging technique, acoustic angiography, which forms static anatomical images of the superharmonic signal produced by microbubbles. In this work, we seek to determine whether acoustic angiography can be utilized for high resolution perfusion imaging in vivo by examining the effect of acquisition rate on superharmonic imaging at low flow rates and demonstrating the feasibility of dynamic contrast-enhanced superharmonic perfusion imaging for the first time. Results in the chorioallantoic membrane model indicate that frame rate and frame averaging do not affect the measured diameter of individual vessels observed, but that frame rate does influence the detection of vessels near and below the resolution limit. The highest number of resolvable vessels was observed at an intermediate frame rate of 3 Hz using a mechanically-steered prototype transducer. We also demonstrate the feasibility of quantitatively mapping perfusion rate in 2D in a mouse model with spatial resolution of ~100 µm. This type of imaging could provide non-invasive, high resolution quantification of microvascular function at penetration depths of several centimeters.

On the relationship between microbubble fragmentation, deflation and broadband superharmonic signal production.

Acoustic angiography imaging of microbubble contrast agents uses the superharmonic energy produced from excited microbubbles and enables high-contrast, high-resolution imaging. However, the exact mechanism by which broadband harmonic energy is produced is not fully understood. To elucidate the role of microbubble shell fragmentation in superharmonic signal production, simultaneous optical and acoustic measurements were performed on individual microbubbles at transmit frequencies from 1.75 to 3.75 MHz and pressures near the shell fragmentation threshold for microbubbles of varying diameter. High-amplitude, broadband superharmonic signals were produced with shell fragmentation, whereas weaker signals (approximately 25% of peak amplitude) were observed in the presence of shrinking bubbles. Furthermore, when populations of stationary microbubbles were imaged with a dual-frequency ultrasound imaging system, a sharper decline in image intensity with respect to frame number was observed for 1-µm bubbles than for 4-µm bubbles. Finally, in a study of two rodents, increasing frame rate from 4 to 7 Hz resulted in decreases in mean steady-state image intensity of 27% at 1000 kPa and 29% at 1300 kPa. Although the existence of superharmonic signals when bubbles shrink has the potential to prolong the imaging efficacy of microbubbles, parameters such as frame rate and peak pressure must be balanced with expected re-perfusion rate to maintain adequate contrast during in vivo imaging.

Contrast-enhanced ultrasound imaging and in vivo circulatory kinetics with low-boiling-point nanoscale phase-change perfluorocarbon agents.

Many studies have explored phase-change contrast agents (PCCAs) that can be vaporized by an ultrasonic pulse to form microbubbles for ultrasound imaging and therapy. However, few investigations have been published on the utility and characteristics of PCCAs as contrast agents in vivo. In this study, we examine the properties of low-boiling-point nanoscale PCCAs evaluated in vivo and compare data with those for conventional microbubbles with respect to contrast generation and circulation properties. To do this, we develop a custom pulse sequence to vaporize and image PCCAs using the Verasonics research platform and a clinical array transducer. Results indicate that droplets can produce contrast enhancement similar to that of microbubbles (7.29 to 18.24 dB over baseline, depending on formulation) and can be designed to circulate for as much as 3.3 times longer than microbubbles. This study also reports for the first time the ability to capture contrast washout kinetics of the target organ as a measure of vascular perfusion.

Acoustic characterization of contrast-to-tissue ratio and axial resolution for dual-frequency contrast-specific acoustic angiography imaging.

Recently, dual-frequency transducers have enabled high-spatial-resolution and high-contrast imaging of vasculature with minimal tissue artifacts by transmitting at a low frequency and receiving broadband superharmonic echoes scattered by microbubble contrast agents. In this work, we examine the imaging parameters for optimizing contrast-to-tissue ratio (CTR) for dual-frequency imaging and the relationship with spatial resolution. Confocal piston transducers are used in a water bath setup to measure the SNR, CTR, and axial resolution for ultrasound imaging of nonlinear scattering of microbubble contrast agents when transmitting at a lower frequency (1.5 to 8 MHz) and receiving at a higher frequency (7.5 to 25 MHz). Parameters varied include the frequency and peak negative pressure of transmitted waves, center frequency of the receiving transducer, microbubble concentration, and microbubble size. CTR is maximized at the lowest transmission frequencies but would be acceptable for imaging in the 1.5 to 3.5 MHz range. At these frequencies, CTR is optimized when a receiving transducer with a center frequency of 10 MHz is used, with the maximum CTR of 25.5 dB occurring when transmitting at 1.5 MHz with a peak negative pressure of 1600 kPa and receiving with a center frequency of 10 MHz. Axial resolution is influenced more heavily by the receiving center frequency, with a weak decrease in measured pulse lengths associated with increasing transmit frequency. A microbubble population containing predominately 4-µm-diameter bubbles yielded the greatest CTR, followed by 1- and then 2-µm bubbles. Varying concentration showed little effect over the tested parameters. CTR dependence on transmit frequency and peak pressure were confirmed through in vivo imaging in two rodents. These findings may lead to improved imaging of vascular remodeling in superficial or luminal cancers such as those of the breast, prostate, and colon.

Genome Sequence of Streptomyces olindensis DAUFPE 5622, Producer of the Antitumoral Anthracycline Cosmomycin D.

Streptomyces olindensis DAUFPE 5622, which was isolated from a Brazilian soil sample, produces the antitumor anthracycline cosmomycin D. The genome sequence is 9.4 Mb in length, with a G+C content of 71%. Thirty-four putative secondary metabolite biosynthetic gene clusters were identified, including the cosmomycin D cluster.