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Localized and targeted drug delivery can be achieved by the combined action of ultrasound and microbubbles on the tumor microenvironment, likely through sonoporation and other therapeutic mechanisms that are not well understood. Here, we present a perfusable in vitro model with a realistic 3D geometry to study the interactions between microbubbles and the vascular endothelium in the presence of ultrasound. Specifically, a three-dimensional, endothelial-cell-seeded in vitro microvascular model was perfused with cell culture medium and microbubbles while being sonicated by a single-element 1 MHz focused transducer. This setup mimics the in vivo scenario in which ultrasound induces a therapeutic effect in the tumor vasculature in the presence of flow. Fluorescence and bright-field microscopy were employed to assess the microbubble-vessel interactions and the extent of drug delivery and cell death both in real time during treatment as well as after treatment. Propidium iodide was used as the model drug while calcein AM was used to evaluate cell viability. There were two acoustic parameter sets chosen for this work: (1) acoustic pressure: 1.4 MPa, pulse length: 500 cycles, duty cycle: 5% and (2) acoustic pressure: 0.4 MPa, pulse length: 1000 cycles, duty cycle: 20%. Enhanced drug delivery and cell death were observed in both cases while the higher pressure setting had a more pronounced effect. By introducing physiological flow to the in vitro microvascular model and examining the PECAM-1 expression of the endothelial cells within it, we demonstrated that our model is a good mimic of the in vivo vasculature and is therefore a viable platform to provide mechanistic insights into ultrasound-mediated drug delivery. 35 31 10128 10138

Combining 4D echocardiography and tissue harmonic imaging (THI) may improve image quality and consequently diagnosis of heart disease. Beamforming considerations such as methodology for sampling the volume with ultrasound beams, beam shape, and the magnitude of the 2nd harmonic component generated in tissue are important for 4D cardiac THI. No studies have yet investigated harmonic generation produced by diagnostic matrix arrays for 4D cardiac THI. The objective of the present work is to evaluate harmonic generation in tissue by matrix arrays by considering different 4D beamforming approaches with a numerical model based on the KZK equation. We evaluated the spatial extent, level of the produced 2nd harmonic component in tissue, and the volume rate of 4D cardiac THI performed by different beamforming approaches. We used different source pressures (applied voltage to the elements) that resulted in peak MI=1.9 in tissue. Beamforming approaches with shallow foci produced the highest peak 2nd harmonic in tissue (1.2 MPa) yet lowest volume rate (1.7 Hz). Beamforming approaches with deep foci produced the highest 2nd harmonic at imaging depth of 150 mm (0.3 MPa). The volume rate of 4D cardiac THI performed with focused ultrasound, PWI, and DWI is 1.7-20, 81, and over 1275 Hz, respectively. 1834 1837

Despite advances in chemotherapeutic drug development, hepatocellular carcinoma (HCC) is still the third leading cause of cancer-related deaths worldwide with a < 30% 5-year survival rate. This poor prognosis can be attributed to the fact that HCC most commonly occurs in patients with pre-existing liver conditions, rendering many systemic options too aggressive. Patient survival rates could be improved by a more targeted approach. Ultrasound and microbubbles can provide a means for overcoming traditional barriers defining drug uptake. The goal of this work was to evaluate preclinical efficacy of image-guided drug delivery of doxorubicin with ultrasound and microbubbles. To this end, therapy settings were created on a Philips EpiQ and S5-1 phased array probe to provide focused sound for treatment. Sonovue was chosen as a clinically approved microbubble. A genetically engineered mouse model was bred and used as a physiologically relevant preclinical analog to human HCC. It was observed that ultrasound and microbubble therapy resulted in enhanced doxorubicin distribution as seen in fluorescent microscopy. Further, immediate vascular shutdown was observed in treated animals. The combination of these effects may be exploited to treat a challenging malignancy. 1527 1530

Recent advances in ultrafast contrast imaging have facilitated innovations, such as superresolution imaging and ultrafast contrast-enhanced Doppler imaging. Combining plane and diverging wave imaging (PWI/DWI) with tissue harmonic imaging (THI) may offer improvements in image quality in applications such as 3-D THI and harmonic color flow. However, no studies have reported simulations of the nonlinear acoustic fields produced by diagnostic arrays in either plane or diverging wave mode. The aim of this study is to model three typical diagnostic arrays that are used in clinical practice and research, Verasonics L11-4v linear array, C5-2v convex array, and P4-2v phased array with the Khokhlov-Zabolotskaya-Kuznetsov (KZK) equation. We have two specific objectives: first, to investigate whether there is increased bubble destruction due to the nature of the plane and diverging fields in contrast imaging and second, to investigate the feasibility of combining PWI/DWI and THI by quantifying the second harmonic generated by these fields. We showed in linear simulations that using such arrays for ultrafast contrast imaging produced pressures that are greater in the near field and lower in the far field than those of focused beams and thus may induce more near-field bubble destruction. In nonlinear simulations, the second harmonic produced by ultrafast THI was found to be 2-16 dB lower than that of focused beams for all arrays considered when operated at the same MI. This moderate difference of the second harmonic between PWI/DWI and focused ultrasound suggests that it is feasible to combine PWI/DWI and THI. 66 7 1158 1169

To evaluate dynamic contrast-enhanced ultrasound (DCEUS) as a tool for measuring blood flow in the macro- and microcirculation of an ex-vivo machine-perfused pig liver and to confirm the ability of DCEUS to accurately detect induced flow rate changes so that it could then be used clinically for monitoring flow changes in liver tumors.Bolus injections of contrast agents in the hepatic artery (HA) and portal vein (PV) were administered to 3 machine-perfused pig livers. Flow changes were induced by the pump of the machine perfusion system. The induced flow rates were of clinical relevance (150 - 400 ml/min for HA and 400 - 1400 ml/min for PV). Quantification parameters from time-intensity curves [rise time (RT), mean transit time (MTT), area under the curve (AUC) and peak intensity (PI)] were extracted in order to evaluate whether the induced flow changes were reflected in these parameters.A linear relationship between the image intensity and the microbubble concentration was confirmed first, while time parameters (RT and MMT) were found to be independent of concentration. The induced flow changes which propagated from the larger vessels to the parenchyma were reflected in the quantification parameters. Specifically, RT, MTT and AUC correlated with flow rate changes.Machine-perfused pig liver is an excellent test bed for DCEUS quantification approaches for the study of the hepatic vascular networks. DCEUS quantification parameters (RT, MTT, and AUC) can measure relative flow changes of about 20 % and above in the liver vasculature. DCEUS quantification is a promising tool for real-time monitoring of the vascular network of tumors.ZIEL: Die Bewertung der dynamischen kontrastverstärkten Sonografie (DCEUS) als Methode zur Messung des Blutflusses in der Makro- und Mikrozirkulation einer Ex-Vivo maschinellen Perfusion einer Schweineleber. Bestätigung, dass DCEUS in der Lage ist, die induzierten Veränderungen der Flussrate exakt nachzuweisen und somit in der Klinik zur Überwachung von Flussveränderungen in Lebertumoren eingesetzt werden kann.Bolus-Injektionen der Kontrastmittel in die Leberarterie (HA) und die Pfortader (PV) wurden an 3 Schweinelebern mit maschineller Perfusion verabreicht. Die Flussänderungen wurden durch die Pumpe des maschinellen Perfusionssystems verursacht. Die induzierten Flussraten waren klinisch relevant (150 – 400 ml/min für HA und 400 – 1400 ml/min für PV). Die Quantifizierungsparameter aus den Zeit-Intensitätskurven [Anstiegszeit (RT), mittlere Laufzeit (MTT), Fläche unter der Kurve (AUC) und Spitzenintensität (PI)] wurden extrahiert, um zu bewerten, ob die induzierten Flussänderungen von diesen Parametern widergespiegelt wurden.Eine lineare Beziehung zwischen der Bildintensität und der Konzentration der Mikrobläschen wurde zuerst bestätigt, während die Zeitparameter (RT und MMT) unabhängig von der Konzentration gezeigt wurden. Die induzierten Flussänderungen, die sich von den größeren Gefäßen zum Parenchym verbreiten, werden durch die Quantifizierungsparameter reflektiert. Insbesondere die Parameter RT, MTT und AUC korrelierten mit Veränderungen der Flussrate.Die maschinelle Perfusion der Schweineleber ist ein hervorragendes Testsystem für den DCEUS-Quantifizierungsansatz bei der Untersuchung des Lebergefäßsystems. Die DCEUS-Quantifizierungsparameter (RT, MTT und AUC) können die relativen Flussänderungen in etwa 20 % und mehr im Gefäßsystem der Leber messen. Die DCEUS-Quantifizierung ist eine vielversprechende Methode für die Echtzeitüberwachung des Gefäßsystems von Tumoren.

High frame rate (ultrafast) ultrasound imaging has received a lot of attention recently. Ultrafast imaging facilitated innovations such as shear wave elastography, super-resolution imaging, and ultrafast contrast-enhanced Doppler imaging. It requires the use of large apertures to send plane or diverging waves to insonify the whole imaged area in one transmit event. Combining diverging wave imaging (DWI) with tissue harmonic imaging (THI) may offer improvements in image quality comparable to those obtained in conventional focused imaging. No studies have reported simulations of the nonlinear acoustic fields produced by diagnostic arrays in diverging wave mode. The aim of this study is to model convex arrays operating in diverging wave mode with the KZK equation. We considered 2 specific problems: (a) increased bubble destruction during ultrafast contrast imaging due to the nature of the diverging field and (b) whether the second harmonic generated in DWI is adequate for THI. In linear simulations, we showed that using convex arrays for ultrafast contrast imaging produced pressures that are greater in the nearfield and lower in the farfield than those of focused beams, and may induce more nearfield bubble destruction. In nonlinear simulations, the second harmonic produced by ultrafast THI was found to be 2-13 dB lower than that of focused beams when operated at the same maximum MI. This moderate difference of the second harmonic between DWI and focused ultrasound suggests it may be feasible to combine DWI and THI. 1 4

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