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A combination of ultrahigh-speed optical imaging (5 × 106 frames/s), B-mode ultrasound and passive cavitation detection was used to study the vaporization process and determine both the acoustic droplet vaporization (ADV) and inertial cavitation (IC) thresholds of phospholipid-coated perfluorobutane nanodroplets (PFB NDs, diameter = 237 ± 16 nm). PFB NDs have not previously been studied with ultrahigh-speed imaging and were observed to form individual microbubbles (1–10 μm) within two to three cycles and subsequently larger bubble clusters (10–50 μm). The ADV and IC thresholds did not statistically significantly differ and decreased with increasing pulse length (20–20,000 cycles), pulse repetition frequency (1–100 Hz), concentration (108–1010 NDs/mL), temperature (20°C–45°C) and decreasing frequency (1.5–0.5 MHz). Overall, the results indicate that at frequencies of 0.5, 1.0 and 1.5 MHz, PFB NDs can be vaporized at moderate peak negative pressures (

Lyso-thermosensitive liposomes (LTSLs) are specifically designed to release chemotherapy agents under conditions of mild hyperthermia. Preclinical studies have indicated that magnetic resonance (MR)-guided focused ultrasound (FUS) systems can generate well-controlled volumetric hyperthermia using real-time thermometry. However, high-throughput clinical translation of these approaches for drug delivery is challenging, not least because of the significant cost overhead of MR guidance and the much larger volumes that need to be heated clinically. Using an ultrasound-guided extracorporeal clinical FUS device (Chongqing HAIFU, JC200) with thermistors in a non-perfused ex vivo bovine liver tissue model with ribs, we present an optimised strategy for rapidly inducing (5-15 min) and sustaining (>30 min) mild hyperthermia (ΔT

Aims: Testing ultrasound-mediated cavitation for enhanced delivery of the therapeutic antibody cetuximab to tumors in a mouse model. Methods: Tumors with strong EGF receptor expression were grown bilaterally. Cetuximab was coadministered intravenously with cavitation nuclei, consisting of either the ultrasound contrast agent Sonovue or gas-stabilizing nanoscale SonoTran Particles. One of the two tumors was exposed to focused ultrasound. Passive acoustic mapping localized and monitored cavitation activity. Both tumors were then excised and cetuximab concentration was quantified. Results: Cavitation increased tumoral cetuximab concentration. When nucleated by Sonovue, a 2.1-fold increase (95% CI 1.3- to 3.4-fold) was measured, whereas SonoTran Particles gave a 3.6-fold increase (95% CI 2.3- to 5.8-fold). Conclusions: Ultrasound-mediated cavitation, especially when nucleated by nanoscale gas-entrapping particles, can noninvasively increase site-specific delivery of therapeutic antibodies to solid tumors.

Ultrasound-induced cavitation has been used as a tool of enhancing extravasation and tissue penetration of anticancer agents in tumours. Initiating cavitation in tissue however, requires high acoustic intensities that are neither safe nor easy to achieve with current clinical systems. The use of cavitation nuclei can however lower the acoustic intensities required to initiate cavitation and the resulting bio-effects in situ. Microbubbles, solid gas-trapping nanoparticles, and phase shift nanodroplets are some examples in a growing list of proposed cavitation nuclei. Besides the ability to lower the cavitation threshold, stability, long circulation times, biocompatibility and biodegradability, are some of the desirable characteristics that a clinically applicable cavitation agent should possess. In this study, we present a novel formulation of ultrasound-triggered phase transition sub-micrometer sized nanodroplets (~400 nm) stabilised with a biocompatible polymer, polydopamine (PDA). PDA offers some important benefits: (1) facile fabrication, as dopamine monomers are directly polymerised on the nanodroplets, (2) high polymer biocompatibility, and (3) ease of functionalisation with other molecules such as drugs or targeting species. We demonstrate that the acoustic intensities required to initiate inertial cavitation can all be achieved with existing clinical ultrasound systems. Cell viability and haemolysis studies show that nanodroplets are biocompatible. Our results demonstrate the great potential of PDA nanodroplets as an acoustically active nanodevice, which is highly valuable for biomedical applications including drug delivery and treatment monitoring.

Sonoporation, the permeabilization of cell membranes following exposure to microbubbles and ultrasound, has considerable potential for therapeutic delivery. To date, engineering of microbubbles for these applications has focused primarily upon optimizing microbubble size and stability, or attachment of targeting species and/or drug molecules. In this work, it is demonstrated that the microbubble coating can also be tailored to directly influence cell permeabilization. Specifically, lipid exchange mechanisms between phospholipid microbubbles and cells can be exploited to significantly increase sonoporation efficiency in vitro. A theoretical analysis of the energy required for pore formation was carried out. From this, it was hypothesized that sonoporation could be promoted by the transfer of lipid molecules with appropriate carbon chain length and/or shape (cylindrical or conical). Spectral imaging with a hydration-sensitive membrane probe (C-Laurdan) was used to measure changes in the membrane lipid order of A-549 cancer cells following exposure to suspensions of different phospholipids. Two candidate lipids were identified, a short-chain-length phospholipid (1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)) and a medium-chain-length lysolipid (1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine (16:0 lyso-PC)). Microbubbles were prepared with matched concentrations, size distributions, and acoustic responses. Confocal microscopy was used to measure cell uptake of a model drug (propidium iodide) with and without ultrasound exposure (1 MHz, 250 kPa peak negative pressure, 1 kHz pulse repetition frequency, 10% duty cycle, 15 s exposure). Despite significantly decreasing the cell membrane lipid order, DLPC did not increase sonoporation. Microbubbles containing 16:0 lyso-PC, however, produced a ∼5-fold increase in sonoporation compared to control microbubbles. Importantly, the lyso-PC molecules were incorporated into the microbubble coating and did not affect cell permeability prior to ultrasound exposure. These findings indicate that microbubbles can be engineered to exploit lipid exchange between microbubble shells and cell membranes to enhance drug delivery, a new optimization route that may lead to enhanced therapeutic efficacy of ultrasound-mediated treatments.

Purpose To demonstrate the feasibility and safety of using focused ultrasound planning models to determine the treatment parameters needed to deliver volumetric mild hyperthermia for targeted drug delivery without real-time thermometry. Materials and Methods This study was part of the Targeted Doxorubicin, or TARDOX, phase I prospective trial of focused ultrasound-mediated, hyperthermia-triggered drug delivery to solid liver tumors ( ClinicalTrials.gov identifier NCT02181075). Ten participants (age range, 49-68 years; average age, 60 years; four women) were treated from March 2015 to March 2017 by using a clinically approved focused ultrasound system to release doxorubicin from lyso-thermosensitive liposomes. Ultrasonic heating of target tumors (treated volume: 11-73 cm3 [mean ± standard deviation, 50 cm3 ± 26]) was monitored in six participants by using a minimally invasive temperature sensor; four participants were treated without real-time thermometry. For all participants, CT images were used with a patient-specific hyperthermia model to define focused ultrasound treatment plans. Feasibility was assessed by comparing model-prescribed focused ultrasound powers to those implemented for treatment. Safety was assessed by evaluating MR images and biopsy specimens for evidence of thermal ablation and monitoring adverse events. Results The mean difference between predicted and implemented treatment powers was -0.1 W ± 17.7 (n = 10). No evidence of focused ultrasound-related adverse effects, including thermal ablation, was found. Conclusion In this 10-participant study, the authors confirmed the feasibility of using focused ultrasound-mediated hyperthermia planning models to define treatment parameters that safely enabled targeted, noninvasive drug delivery to liver tumors while monitored with B-mode guidance and without real-time thermometry. Published under a CC BY 4.0 license. Online supplemental material is available for this article. See also the editorial by Dickey and Levi-Polyachenko in this issue.

Summary Background Previous preclinical research has shown that extracorporeal devices can be used to enhance the delivery and distribution of systemically administered anticancer drugs, resulting in increased intratumoural concentrations. We aimed to assess the safety and feasibility of targeted release and enhanced delivery of doxorubicin to solid tumours from thermosensitive liposomes triggered by mild hyperthermia, induced non-invasively by focused ultrasound. Methods We did an open-label, single-centre, phase 1 trial in a single UK hospital. Adult patients (aged ≥18 years) with unresectable and non-ablatable primary or secondary liver tumours of any histological subtype were considered for the study. Patients received a single intravenous infusion (50 mg/m 2 ) of lyso-thermosensitive liposomal doxorubicin (LTLD), followed by extracorporeal focused ultrasound exposure of a single target liver tumour. The trial had two parts: in part I, patients had a real-time thermometry device implanted intratumourally, whereas patients in part II proceeded without thermometry and we used a patient-specific model to predict optimal exposure parameters. We assessed tumour biopsies obtained before and after focused ultrasound exposure for doxorubicin concentration and distribution. The primary endpoint was at least a doubling of total intratumoural doxorubicin concentration in at least half of the patients treated, on an intention-to-treat basis. This study is registered with ClinicalTrials.gov, number NCT02181075, and is now closed to recruitment. Findings Between March 13, 2015, and March 27, 2017, ten patients were enrolled in the study (six patients in part I and four in part II), and received a dose of LTLD followed by focused ultrasound exposure. The treatment resulted in an average increase of 3·7 times in intratumoural biopsy doxorubicin concentrations, from an estimate of 2·34 μg/g (SD 0·93) immediately after drug infusion to 8·56 μg/g (5·69) after focused ultrasound. Increases of two to ten times were observed in seven (70%) of ten patients, satisfying the primary endpoint. Serious adverse events registered were expected grade 4 transient neutropenia in five patients and prolonged hospital stay due to unexpected grade 1 confusion in one patient. Grade 3–4 adverse events recorded were neutropenia (grade 3 in one patient and grade 4 in five patients), and grade 3 anaemia in one patient. No treatment-related deaths occurred. Interpretation The combined treatment of LTLD and non-invasive focused ultrasound hyperthermia in this study seemed to be clinically feasible, safe, and able to enhance intratumoural drug delivery, providing targeted chemo-ablative response in human liver tumours that were refractory to standard chemotherapy. Funding Oxford Biomedical Research Centre, National Institute for Health Research.

The efficient penetration of drugs into tumors is a major challenge that remains unmet. Reported herein is a strategy to promote extravasation and enhanced penetration using inertial cavitation initiated by focused ultrasound and cone-shaped gold nanoparticles that entrap gas nanobubbles. The cones are capable of initiating inertial cavitation under pressures and frequencies achievable with existing clinical ultrasound systems and of promoting extravasation and delivery of a model large therapeutic molecule in an in vitro tissue mimicking flow phantom, achieving penetration depths in excess of 2 mm. Ease of functionalization and intrinsic imaging capabilities provide gold with significant advantages as a material for biomedical applications. The cones show neither cytotoxicity in Michigan Cancer Foundation (MCF)-7 cells nor hemolytic activity in human blood at clinically relevant concentrations and are found to be colloidally stable for at least 5 d at 37 °C and several months at 4 °C.

The study of the effects of ultrasound-induced acoustic cavitation on biological structures is an active field in biomedical research. Of particular interest for therapeutic applications is the ability of oscillating microbubbles to promote both cellular and tissue membrane permeabilisation and to improve the distribution of therapeutic agents in tissue through extravasation and convective transport. The mechanisms that underpin the interaction between cavitating agents and tissues are, however, still poorly understood. One challenge is the practical difficulty involved in performing optical microscopy and acoustic emissions monitoring simultaneously in a biologically compatible environment. Here we present and characterise a microfluidic layered acoustic resonator (μLAR) developed for simultaneous ultrasound exposure, acoustic emissions monitoring, and microscopy of biological samples. The μLAR facilitates in vitro ultrasound experiments in which measurements of microbubble dynamics, microstreaming velocity fields, acoustic emissions, and cell-microbubble interactions can be performed simultaneously. The device and analyses presented provide a means of performing mechanistic in vitro studies that may benefit the design of predictable and effective cavitation-based ultrasound treatments.

Ultrasound and microbubbles are often used to enhance drug delivery and the suggested mechanisms are extravasation and sonoporation. Drug delivery schemes with ultrasound and microbubbles at both low and high acoustic amplitudes have been suggested. A diagnostic ultrasound scanner may play a double role as both an imaging and a therapy device. It was not possible to accurately measure microbubble response with an ultrasound scanner for a large range of acoustic pressures and microbubble concentrations until now, mainly because of signal saturation issues. A method for continuously adjusting the receive gain of a scanner and limiting signal saturation was developed to accurately measure backscattered echoes from microbubbles for mechanical indexes (MIs) up to 2.1. The intensity of backscattered echoes from microbubbles increased quarticly with MI without reaching any limit. The signal intensity from microbubbles was found to be linear with concentration at both low and high MIs. However, at very high concentrations, acoustic shadowing occurs which limits the delivered acoustic pressure in deeper areas. The contrastto- tissue ratio was also measured and found to stay constant with MI. These results can be used to better guide drug delivery approaches and to also develop imaging techniques for therapy procedures.

Lipid and protein-shelled microbubbles have been the mainstay of cavitation nucleation strategies for therapeutic applications for several decades. However, a growing number of therapeutic ultrasound applications require nuclei that (i) are significantly smaller in size, in order to overcome a particular biological barrier such as the leaky vasculature of tumours or the stratum corneum; (ii) offer greatly increased cavitation persistence, both during a single extended ultrasound pulse and in terms of extended circulation following intravenous administration; and (iii) have better resilience to sudden ambient pressure changes in order to enable direct injection without nuclei destruction into tissue targets via a needle and syringe. We will review a range of novel cavitation agents currently under development, including sub-micron gas-stabilizing solid particles and nanodroplets, and provide an overview of their known characteristics in terms of acoustic emissions, activation and cavitation thresholds, cavitation persistence, and circulation. Where possible, the relevant performance of these cavitation nucleation agents will be compared to microbubbles for applications ranging from drug delivery to nucleated tissue fractionation.

Sustained inertial cavitation seeded by microbubbles, sub-micron cavitation nuclei or even without any cavitation nucleation agent has been shown to enable increased delivery and penetration of drugs and vaccines across the skin, into solid tumours and across the blood-brain barrier. The safety and efficacy of these applications are typically monitored using single-element passive cavitation detectors, or more recently using multi-element arrays that enable real-time passive acoustic mapping of cavitation activity. We will provide an overview of the different types of cavitation nucleation agents which are available to use, the type and persistence of acoustic emissions that they are typically associated with, and, where possible, a direct comparison between the performance of different nucleation strategies to achieve the desired drug delivery bioeffect in the target of interest. We will then turn our attention to the presently available strategies for real-time monitoring of safety and efficacy, with specific reference to the benefits offered by the ability to achieve spatiotemporal monitoring of cavitation activity to correlate it with bioeffects in subsets of the overall region of interest.Sustained inertial cavitation seeded by microbubbles, sub-micron cavitation nuclei or even without any cavitation nucleation agent has been shown to enable increased delivery and penetration of drugs and vaccines across the skin, into solid tumours and across the blood-brain barrier. The safety and efficacy of these applications are typically monitored using single-element passive cavitation detectors, or more recently using multi-element arrays that enable real-time passive acoustic mapping of cavitation activity. We will provide an overview of the different types of cavitation nucleation agents which are available to use, the type and persistence of acoustic emissions that they are typically associated with, and, where possible, a direct comparison between the performance of different nucleation strategies to achieve the desired drug delivery bioeffect in the target of interest. We will then turn our attention to the presently available strategies for real-time monitoring of safety and efficacy, with sp...

Sonoporation is the transient and reversible cell membrane permeability change induced with ultrasound and microbubbles. It allows for the uptake of normally impermeable macromolecules and has been suggested for improving drug delivery. The exact sonoporation mechanism and the optimal ultrasound parameters are still under investigation. Ex vivo machine perfused porcine livers are an excellent platform for investigating the sonoporation parameters and specifically the interaction of ultrasound driven microbubbles with the capillaries. Our objective was to identify the ultrasound parameters that are capable of causing detectable perfusion changes in the sonoporation area. Three types of perfusion changes were considered: large mechanical damage void of perfusion, reduced perfusion due to capillary destruction, and unaltered perfusion.

New strategies are required to enhance the penetration and tumor-wide distribution of cancer therapeutics, including viruses, antibodies, and oligonucleotides. We have shown previously that increasing the density of a nanoparticle can enhance its ultrasound-mediated transport, particularly when exposed to microstreaming. The current study investigates how the physical characteristics and dynamics of different cavitation nucleation agents affect the cavitation-mediated transport of therapeutics not directly bound to the gas nuclei. SonoVue (Bracco, Milan, Italy) microbubble contrast agent (with 3 μm mean initial bubble diameter) and polymeric gas-entrapping nanoparticles (with 260 nm mean initial bubble diameter) were first compared in terms of inertial cavitation threshold and cavitation persistence. Under the same ultrasound exposure conditions (centre frequencies of either 0.5 MHz or 1.6 MHz, and peak negative pressures from 0.2 MPa to 3.5 MPa) their respective cavitation emissions were found to differ ...

International audience; Targeted drug delivery under image guidance is gaining more interest in the drug-delivery field. The use of microbubbles as contrast agents in diagnostic ultrasound provides new opportunities in noninvasive image-guided drug delivery. In the present study, the imaging and therapeutic properties of novel doxorubicin liposome-loaded microbubbles are evaluated. The results showed that at scanning settings (1.7 MHz and mechanical index 0.2), these microbubbles scatter sufficient signal for nonlinear ultrasound imaging and can thus be imaged in real time and be tracked in vivo. In vitro therapeutic evaluation showed that ultrasound at 1 MHz and pressures up to 600 kPa in combination with the doxorubicin liposomeloaded microbubbles induced 4-fold decrease of cell viability compared with treatment with free doxorubicin or doxorubicin liposome-loaded microbubbles alone. The therapeutic effectiveness is correlated to an ultrasound-triggered release of doxorubicin from the liposomes and an enhanced uptake of the free doxorubicin by glioblastoma cells. The results obtained demonstrate that the combination of ultrasound and the doxorubicin liposome-loaded microbubbles can provide a new method of noninvasive image-guided drug delivery.

Sub-micron cavitation nuclei with the ability to passively extravasate through the leaky tumor vasculature have been shown to enhance both extravasation and penetration of pharmaceuticals in tumors. The aim of the present work is to optimize the acoustic parameters and maximize drug delivery mediated by cavitational microstreaming. Either lipid-shell microbubbles, or gas-stabilizing polymeric submicron cups of mean diameter 480 nm are co-administered with a model drug through a flow channel formed in agarose gel. The effect of ultrasound frequency, pressure amplitude, pulse duration, duty cycle, and pulse repetition frequency on the delivery of the drug is first evaluated, using both a single-layer and a dual-layer flow phantom that better represents the leaky tumor vasculature and dense extracellular matrix. The optimal parameters at 0.5 MHz and 1.6 MHz are then selected and used to deliver an oncolytic virus, genetically modified to express the RFP reporter gene, thus allowing the quantification of the ...

For effective cancer therapy, therapeutic agents must extravasate from the blood stream into the tumour mass, then overcome the elevated intratumoural pressure and dense extracellular matrix to reach each and every cancer cell. Several recent studies have suggested that inertial cavitation in the absence or presence of artificial cavitation nuclei can significantly enhance the delivery, penetration, and distribution of small-molecule or nanoparticulate therapeutic agents into tumors. We first present a comparison of the reported enhancements in delivery achieved for a range of frequencies and therapeutic sizes without or with pre-seeding of cavitation, with particular emphasis on the potential role of cavitation persistence and spatial distribution. With microstreaming hypothesized to be the dominant transport mechanism for drug delivery, the likely benefit of using sub-micron cavitation nucleation agents capable of extravasating alongside the therapeutic, rather than microbubbles confined to the blood po...

The combination of focused ultrasound (FUS) with thermosensitive liposomes (TSL) is a promising method for drug delivery since it allows a localized release upon moderate heating with ultrasound. Besides thermal effects, FUS also induces mechanical stresses on drug nanocarriers. We propose in this study to examine the influence of both effects (thermal and mechanical) on drug release. For this, an in-vitro setup allowing liposomal drug delivery using FUS was first evaluated. Calcein was used as a model drug. FUS experiments were performed in water at 37°C using a 1 MHz transducer focused at 48 mm, at 1 kHz PRF and 40% duty cycle. The driving pressure and the insonation duration were varied from 1 to 2 MPa and from 0 to 30 min, respectively. Thermal heating using a water-bath was also performed with temperatures from 37 to 49°C. For TSL, the release reaches a plateau above 42°C (45%) after 10 min heating while no release is observed for non-thermosensitive liposomes (NTSL). Using FUS, a rapid calcein release is observed for pressures from 1 to 1.5 MPa (from 0% to 49%) for TSL. Above 1.5 MPa, the release increases slightly (59% at 2 MPa). For NTSL, a weak calcein release is measured for acoustic pressures higher than 1.5 MPa. This release is attributed to the mechanical stress generated by FUS which is sufficient to destabilize the liposomal membrane. Mechanical stress alone can enhance the calcein release by up to 17% for pressures higher than 1.75 MPa.

Local drug delivery of doxorubicin holds promise to improve the therapeutic efficacy and to reduce toxicity profiles. Here, we investigated the release of doxorubicin from thermosensitive liposomes (Dox-TSL) into human glioblastoma (U-87MG) cells. Using Dox-TSL, experiments were carried out in a water bath and showed that 15 min incubation of TSL at 43°C induced the release of 80% doxorubicin loaded TSL compared to the release at 37°C. The cytotoxicity of a range of concentrations of Dox-TSL was also evaluated on U-87MG cells. At 37°C, no cytotoxicity was observed, whereas at 43°C the results showed that the cytotoxicity is dose dependent. At maximal dose of doxorubicin (30 μg/mL), the cell viability was less than 20%. Application of 15 min of HIFU at 1 MHz, 1.5 MPa and 50% duty cycle induced the release of 100% of doxorubicin from Dox-TSL. In the same experimental condition, the cell viability decreased to 40% and 20% at 12h and 48h, respectively, in comparison to that obtained during the incubation of cells with Dox-TSL alone without HIFU. In conclusion, a significant release of doxorubicin from temperature-sensitive liposomes can be achieved leading to an efficient treatment and cell death of tumor cells using HIFU.

Despite continuing advances in the sensitivity of diagnostic ultrasound systems, Doppler-based imaging techniques are unable to detect low velocity blood flow in the microcirculation. The main difficulty these techniques share is that blood is a weak reflector of ultrasound with received amplitude 40–60 dB smaller than that of tissue. As a result, Doppler-based techniques rely solely on the movement of red blood cells to differentiate blood flow from tissue. The removal of this tissue signal places a lower limit on the ability to detect low velocity blood flow (

Drug loaded temperature-sensitive liposomes (TSLs) release their payload with mild hyperthermia near their phase transition temperature (Tm = 43–45 °C). Such a release may improve therapeutic efficacy and reduce toxic side effects in cancer treatment. In the present work, two different approaches are considered where focused ultrasound is used to induce the required temperature elevation for the release of doxorubicin from TSLs: (a) primary heating due to thermo-viscous absorption of ultrasound in absorptive media (oil, glycerol) and (b) secondary heating in non-absorptive media (blood, cell medium) due to heat transfer from the surroundings. Fine-wire thermocouple readings where in close agreement with theoretical predictions of temperature elevation with the Bioheat equation. Pulsing schemes to elevate and maintain the temperature at the desired value were designed with the Bioheat equation and validated with experiments. Fluorescence spectroscopy was used to assess the release of free doxorubicin that ...

Localized drug delivery holds great promise in improving drug efficacy in cancer treatment. Newly developed temperature‐sensitive liposomes (TSLs) loaded with doxorubicin have been shown to release their payload with mild hyperthermia near their phase transition temperature (Tm = 43–45 °C). In the present work, high intensity focused ultrasound is used to induce the required temperature elevation for the release of the drug from TSLs. A theoretical model based on Pennes’ bioheat equation was initially used to calculate the conditions for temperature elevation in fluids and tissue phantoms under conditions for drug activation. Acoustic pressures of 1–2 MPa at the focus with varying duty cycle (typically 50%) at 1 MHz frequency were calculated. Measurements of temperature rise were found in good agreement with our theoretical predictions. Fluorescence measurements were used to assess the release of free doxorubicin that exhibits higher fluorescence intensity than the liposomal formulation. In vitro experime...

In current drug delivery approaches, microbubbles and drugs can be co‐administered intravenously while high intensity ultrasound is applied. The exact mechanisms of microbubble interactions with ultrasound the drug and the cells are not fully understood and sometimes, the optimal ultrasound parameters are simply a case of trial and error. A better understanding of microbubble response to long ultrasonic pulses is important for drug delivery. Two different in‐vitro setups to examine the response of microbubbles are presented: with the microbubbles suspended in a large enclosure and with the microbubbles flowing in a capillary. Various commercially available and drug delivery specific bubbles were considered. Acoustic streaming, which greatly influences the observed response from bubbles, was observed in many “typical” drug delivery conditions in the large enclosure setup. With the capillary setup, streaming was eliminated and accurate bubble responses were recorded. In the capillary setup and at high press...

Ultrasound contrast agents are used in cardiology for the assessment of myocardial perfusion and in radiology for the detection and characterization of tumors. One widely used approach of imaging contrast agents is to use a low Mechanical Index (MI) nonlinear imaging technique to avoid bubble destruction and image both the macro‐ and micro‐circulation in real‐time. Various pulsing schemes are employed for the detection of nonlinear echoes from contrast microbubbles. The objective of this paper is to evaluate the various pulsing schemes for low MI imaging of contrast microbubbles and better understand their similarities and differences. The pulsing schemes considered are pulse inversion, power modulation, and their combination. Emphasis is placed on identifying whether nonlinearity due to propagation in tissue may be discriminated from nonlinearity due to scattering from bubbles. Bubble destruction (use of high MI) and tissue motion were not considered in this work. The evaluation of the different pulsing ...