Identifying the Inertial Cavitation Threshold and Skull Effects in a Vessel Phantom Using Focused Ultrasound and Microbubbles

Focused ultrasound (FUS) in combination with microbubbles has been shown capable of delivering large molecules to the brain parenchyma through opening of the blood-brain barrier (BBB). However, the mechanism behind the opening remains unknown. To investigate the pressure threshold for inertial cavitation of preformed microbubbles during sonication, passive cavitation detection in conjunction with B-mode imaging was used. A cerebral vessel was simulated by generating a cylindrical hole of 610 μm in diameter inside a polyacrylamide gel and saturating its volume with microbubbles. Definity microbubbles (Mean diameter range: 1.1-3.3 μm, Lantheus Medical Imaging, N. Billerica, MA, USA) were injected prior to sonication (frequency: 1.525 MHz; pulse length: 100 cycles; PRF: 10 Hz; sonication duration: 2 s) through an excised mouse skull. The acoustic emissions due to the cavitation response were passively detected using a cylindrically focused hydrophone, confocal with the FUS transducer and a linear-array transducer with the field of view perpendicular to the FUS beam. The broadband spectral response acquired at the passive cavitation detector (PCD) and the B-mode images identified the occurrence and location of the inertial cavitation, respectively. Findings indicated that the peak-rarefactional pressure threshold was approximately equal to 0.45 MPa, with or without the skull present. Mouse skulls did not affect the threshold of inertial cavitation but resulted in a lower inertial cavitation dose. The broadband response could be captured through the murine skull, so the same PCD set-up can be used in future in vivo applications. (E-mail: ek2191@columbia.edu)     

The threshold for thermally significant cavitation in dog's thigh muscle in vivo

In this study the threshold of thermally significant transient cavitation in vivo in dog's thigh muscle was investigated as a function of frequency from 0.246 MHz to 1.68 MHz. Cavitation, evidenced by strong emission of wide band noise monitored by a hydrophone, appeared to increase the energy absorption in tissue at the focal zone of a focused ultrasound beam as measured with an embedded thermocouple. This was indicated by a significant increase in the temperature, a loss of smooth temperature rise during the 1 s sound pulse and a significant reduction in the acoustic power transmitted through the thigh. This thermal phenomenon was associated with a strong emission of wide band noise which was monitored by a hydrophone. In addition, strong echoes appeared in ultrasound images during the pulses that caused the noise emission and the thermal effect. These echoes appeared preferentially at locations where there was acoustic heterogeneity. The measured cavitation pressure amplitude threshold was found to depend almost linearly on frequency with a slope of about 5.3 MPa MHz-1. (The extrapolated static pressure threshold was 0.6 MPa). When these measured levels are compared to those typical of clinical application, it appears that the transient cavitation can be avoided when perfusion independent high temperature hyperthermia is induced with focused and pulsed ultrasound fields. However, intensities required during scanned focused ultrasound hyperthermia, where sharply focused transducers are used to heat large tumors at low frequencies (1 MHz or below), could rise above the threshold. Thus, care should be taken when focused ultrasound systems are designed so that the maximum peak pressure is below the threshold in order to avoid unpredictable biological effects induced by transient cavitation. Finally it is unlikely that the present diagnostic ultrasound units which operate at higher frequencies and in pulsed mode could cause transient cavitation in vivo.     

Control of Acoustic Cavitation for Efficient Sonoporation with Phase-Shift Nanoemulsions

Acoustic cavitation can be used to temporarily disrupt cell membranes for intracellular delivery of large biomolecules. Termed sonoporation, the ability of this technique for efficient intracellular delivery (i.e., >50% of initial cell population showing uptake) while maintaining cell viability (i.e., >50% of initial cell population viable) has proven to be very difficult. Here, we report that phase-shift nanoemulsions (PSNEs) function as inertial cavitation nuclei for improvement of sonoporation efficiency. The interplay between ultrasound frequency, resultant microbubble dynamics and sonoporation efficiency was investigated experimentally. Acoustic emissions from individual microbubbles nucleated from PSNEs were captured using a broadband passive cavitation detector during and after acoustic droplet vaporization with short pulses of ultrasound at 1, 2.5 and 5 MHz. Time domain features of the passive cavitation detector signals were analyzed to estimate the maximum size (R_max) of the microbubbles using the Rayleigh collapse model. These results were then applied to sonoporation experiments to test if uptake efficiency is dependent on maximum microbubble size before inertial collapse. Results indicated that at the acoustic droplet vaporization threshold, R_max was approximately 61.7 ± 5.2, 24.9 ± 2.8, and 12.4 ± 2.1 μm at 1, 2.5 and 5 MHz, respectively. Sonoporation efficiency increased at higher frequencies, with efficiencies of 39.5 ± 13.7%, 46.6 ± 3.28% and 66.8 ± 5.5% at 1, 2.5 and 5 MHz, respectively. Excessive cellular damage was seen at lower frequencies because of the erosive effects of highly energetic inertial cavitation. These results highlight the importance of acoustic cavitation control in determining the outcome of sonoporation experiments. In addition, PSNEs may serve as tailorable inertial cavitation nuclei for other therapeutic ultrasound applications.     

Cavitational mechanisms in ultrasound-accelerated fibrinolysis

The role of both inertial and stable cavitation was investigated during in vitro ultrasound-accelerated fibrinolysis by recombinant tissue plasminogen activator (rt-PA) in the presence and absence of Optison. A unique treatment configuration applied ultrasound, rt-PA and Optison to the interior of a plasma clot. Lysis efficacy was measured as clot weight reduction. Cavitational mechanisms were investigated by monitoring subharmonic and broadband noise. In the absence of Optison, 1.7 MHz pulsed ultrasound with 1.5 MPa peak-negative pressure applied for 30 min resulted in 45 +/- 19% lysis enhancement relative to rt-PA alone. Cavitation was not detected, indicating a role of noncavitational effects of ultrasound. The addition of Optison increased lysis enhancement to 88 +/- 25%. Inertial cavitation was present only at the start of the exposure, while low-amplitude subharmonic emissions persisted throughout. Additional protocols suggested a possible correlation between the increased lysis in the presence of Optison and the subharmonic emission, indicating a potentially important role of stable rather than inertial cavitation in microbubble-enhanced ultrasound-accelerated rt-PA-mediated thrombolysis.     

Non-linear Acoustic Emissions from Therapeutically Driven Contrast Agent Microbubbles

Non-linear emissions from microbubbles introduced to the vasculature for exposure to focused ultrasound are routinely monitored for assessment of therapy and avoidance of irreversible tissue damage. Yet the bubble-based mechanistic source for these emissions, under subresonant driving at typical therapeutic pressure amplitudes, may not be well understood. In the study described here, dual-perspective high-speed imaging at 210,000 frames per second (fps), and shadowgraphically at 10 Mfps, was used to observe cavitation from microbubbles flowing through a 500-µm polycarbonate capillary exposed to focused ultrasound of 692 kHz at therapeutically relevant pressure amplitudes. The acoustic emissions were simultaneously collected via a broadband calibrated needle hydrophone system. The observations indicate that periodic bubble-collapse shock waves can dominate the non-linear acoustic emissions, including subharmonics at higher driving amplitudes. Contributions to broadband emissions through variance in shock wave amplitude and emission timings are also identified. Possible implications for in vivo microbubble cavitation detection, mechanisms of therapy and the conventional classification of cavitation activity as stable or inertial are discussed.     

Ultrasound-mediated cavitation thresholds of liquid perfluorocarbon droplets in vitro

This study was undertaken to measure the ultrasound (US)-mediated cavitation threshold of microdroplets as a function of its content and US parameters (frequency, amplitude and burst length). Albumin-coated droplets were prepared with perfluoropropane, perfluorohexane or perfluoromethylcyclohexane contents. The filtered suspensions were diluted to 1:1000 (v) and compared with Optison. The formulations were injected into an acoustically transparent vessel and sonicated with a single focused transducer. The frequencies employed were 0.74, 1.1, 2.18 and 3.3 MHz and the burst length and acoustic pressure were varied. The inertial cavitation threshold for each experiment was monitored through passive acoustic detection. The formation of droplet emulsion of the perfluorocarbon increased the natural boiling point of the perfluorocarbon. However, perfluorocarbon droplets having contents with higher molecular weights and boiling points did not have detectably higher inertial cavitation thresholds and, thus, the droplets do not need to be in a superheated state to be cavitated by US bursts. Therefore, higher boiling point perfluorocarbons should be investigated for this purpose and may prove to be useful for both imaging and therapy. The inertial cavitation threshold of perfluorocarbon droplets increases with frequency, and was approximately 0.7 MPa at 0.74 MHz and 1.75 MPa at 3.3 MHz. Optison, already in a gaseous state, has the lowest cavitation threshold of all formulations studied. Results show that, for the frequencies tested, there is no dependence between inertial cavitation threshold and burst lengths between 20 and 100 ms. As a conclusion, the inertial cavitation threshold of albumin-coated microdroplets of several perfluorocarbons was determined in vitro. The results indicate that the physical properties of these droplets are such that they may be useful for localized US therapies.     

Examination of inertial cavitation of Optison in producing sonoporation of chinese hamster ovary cells

The objective of this project was to elucidate the relationship between ultrasound contrast agents (UCAs) and sonoporation. Sonoporation is an ultrasound-induced, transient cell membrane permeability change that allows for the uptake of normally impermeable macromolecules. Specifically, this study will determine the role that inertial cavitation plays in eliciting sonoporation. The inertial cavitation thresholds of the UCA, Optison, are compared directly with the results of sonoporation to determine the involvement of inertial cavitation in sonoporation. Chinese hamster ovary (CHO) cells were exposed as a monolayer in a solution of Optison, 500,000 Da fluorescein isothiocyanate-dextran (FITC-dextran), and phosphate-buffered saline (PBS) to 30 s of pulsed ultrasound at 3.15-MHz center frequency, 5-cycle pulse duration and 10-Hz pulse repetition frequency. The peak rarefactional pressure (P(r)) was varied over a range from 120 kPa-3.5 MPa, and five independent replicates were performed at each pressure. As the P(r) was increased, from 120 kPa-3.5 MPa, the fraction of sonoporated cells among the total viable population increased from 0.63-10.21%, with the maximum occurring at 2.4 MPa. The inertial cavitation threshold for Optison at these exposure conditions has previously been shown to be in the range 0.77-0.83 MPa, at which sonoporation activity was found to be 50% of its maximum level. Furthermore, significant sonoporation activity was observed at pressure levels below the threshold for inertial cavitation of Optison. Above 2.4 MPa, a significant drop in sonoporation activity occurred, corresponding to pressures where >95% of the Optison was collapsing. These results demonstrate that sonoporation is not directly a result of inertial cavitation of the UCA, rather that the effect is related to linear and/or nonlinear oscillation of the UCA occurring at pressure levels below the inertial cavitation threshold.     

Nonlinear behaviors of contrast agents relevant to diagnostic and therapeutic applications

The nonlinear properties of an encapsulated microbubble of a contrast agent were studied theoretically and experimentally. A modified nonlinear differential equation (Herring equation) was used to describe the radial oscillation of the microbubble and solved numerically. It was found that the nonlinear resonance frequency, at which the peak radial oscillation amplitude occurs, was a decreasing function of the acoustic amplitude of a driving ultrasonic pulse. Optical images of the contrast agent microbubbles under various ultrasonic exposure conditions: 1. sham exposure; 2. 2-MHz spatial peak acoustic pressure = 200 kPa, I(SATA) = 260 mW/cm(2), duty cycle = 7.5%, repetition period = 0.0266 ms; 3. 0.5-MHz spatial peak acoustic pressure = 200 kPa, I(SATA) = 130 mW/cm(2), duty cycle = 7.5%, repetition period = 0.1067 ms; have also shown that the lower-frequency ultrasound (US) excitation (0.5 MHz) is more effective in disruption of the microbubbles due to acoustic inertial cavitation than the higher frequency US (2 MHz).     

Influence of Shell Composition on the Resonance Frequency of Microbubble Contrast Agents

The effect of variations in microbubble shell composition on microbubble resonance frequency is revealed through experiment. These variations are achieved by altering the mole fraction and molecular weight of functionalized polyethylene glycol (PEG) in the microbubble phospholipid monolayer shell and measuring the microbubble resonance frequency. The resonance frequency is measured via a chirp pulse and identified as the frequency at which the pressure amplitude loss of the ultrasound wave is the greatest as a result of passing through a population of microbubbles. For the shell compositions used herein, we find that PEG molecular weight has little to no influence on resonance frequency at an overall PEG mole fraction (0.01) corresponding to a mushroom regime and influences the resonance frequency markedly at overall PEG mole fractions (0.050–0.100) corresponding to a brush regime. Specifically, the measured resonance frequency was found to be 8.4, 4.9, 3.3 and 1.4 MHz at PEG molecular weights of 1000, 2000, 3000 and 5000 g/mol, respectively, at an overall PEG mole fraction of 0.075. At an overall PEG mole fraction of just 0.01, on the other hand, resonance frequency exhibited no systematic variation, with values ranging from 5.7 to 4.9 MHz. Experimental results were analyzed using the Sarkar bubble dynamics model. With the dilatational viscosity held constant (10^–8 N·s/m) and the elastic modulus used as a fitting parameter, model fits to the pressure amplitude loss data resulted in elastic modulus values of 2.2, 2.4, 1.6 and 1.8 N/m for PEG molecular weights of 1000, 2000, 3000 and 5000 g/mol, respectively, at an overall PEG mole fraction of 0.010 and 4.2, 1.4, 0.5 and 0.0 N/m, respectively, at an overall PEG mole fraction of 0.075. These results are consistent with theory, which predicts that the elastic modulus is constant in the mushroom regime and decreases with PEG molecular weight to the inverse 3/5 power in the brush regime. Additionally, these results are consistent with inertial cavitation studies, which revealed that increasing PEG molecular weight has little to no effect on inethe rtial cavitation threshold in the mushroom regime, but that increasing PEG molecular weight decreases inertial cavitation markedly in the brush regime. We conclude that the design and synthesis of microbubbles with a prescribed resonance frequency is attainable by tuning PEG composition and molecular weight.     

Correlation Between Brain Tissue Damage and Inertial Cavitation Dose Quantified Using Passive Cavitation Imaging

Focused ultrasound (FUS)-induced cavitation-mediated brain therapies have become emerging therapeutic modalities for neurologic diseases. Cavitation monitoring is essential to ensure the safety of all cavitation-mediated therapeutic techniques as inertial cavitation can be associated with tissue damage. The objective of this study was to reveal the correlation between the inertial cavitation dose, quantified by passive cavitation imaging (PCI), and brain tissue histologic-level damage induced by FUS in combination with microbubbles. An ultrasound image-guided FUS system consisting of a single-element FUS transducer (1.5 MHz) and a co-axially aligned 128-element linear ultrasound imaging array was used to perform FUS treatment of mice. Mice were sonicated by FUS with different peak negative pressures (0.5 MPa, 1.1 MPa, 4.0 MPa and 6.5 MPa) in the presence of systemically injected microbubbles. The acoustic emissions from the FUS-activated microbubbles were passively detected by the imaging array. The pre-beamformed channel data were acquired and processed offline using the frequency-domain delay, sum and integration algorithm to generate inertial cavitation maps. All the mice were sacrificed after the FUS treatment, and their brains were harvested and processed for hematoxylin and eosin staining. The obtained inertial cavitation maps revealed the dynamic changes of microbubble behaviors during FUS treatment at different pressure levels. It was found that the inertial cavitation dose quantified based on PCI had a linear correlation with the scale of histologic-level tissue damage. Findings from this study suggested that PCI can be used to predict histologic-level tissue damage associated with the FUS-induced cavitation.     

Contrast agent bubble and erythrocyte behavior in a 1.5-MHz standing ultrasound wave

Human erythrocytes and Optison contrast agent have been exposed to ultrasound, both alone and in combination, in a single-half-wavelength chamber driven at its resonance frequency (fo) of 1.5 MHz. Cell movements were recorded by video microscopy at speeds up to 500 frames/s. The hypothesis that cells near a standing wave pressure node might be stressed by the microbubble products of sonicated contrast agent was examined. In the absence of contrast agent, cells moved rapidly to form an aggregate in the standing wave pressure node plane. First subharmonic and second harmonic emissions were detected from cell-contrast agent suspensions immediately on exposure to a threshold peak pressure amplitude of 0.98 MPa. Emissions at 3fo/2 occurred at 1.47 MPa, whereas white noise and lower-order subharmonic emissions coincided with the appearance of visible bubbles at a threshold of approximately 1.96 MPa. Cells exposed together with contrast agent at a pressure of 0.98 MPa precessed very rapidly about the pressure node plane. This behavior was discussed in the context of a recent analysis predicting that, in contrast to the situation for lower-pressure amplitudes, subresonant size bubbles translate about pressure node plane if the driving pressure amplitude is sufficiently high. Many precessing erythrocytes were clearly spiculated and this morphology persisted after the cells had left the area of precession. Hemoglobin release was significant under conditions inducing precession with first subharmonic and first harmonic emissions. Protein release increased discontinuously near the pressure thresholds, where more complex categories of frequency emission were detected. The potential of this system, which induces erythrocyte morphology changes and some protein release at the first emission threshold, to provide some control on the membrane-permeabilizing stress experienced by cells in a cavitation field is discussed.     

Passive Cavitation Detection during Pulsed HIFU Exposures of Ex Vivo Tissues and In Vivo Mouse Pancreatic Tumors

Pulsed high-intensity focused ultrasound (pHIFU) has been shown to enhance vascular permeability, disrupt tumor barriers and enhance drug penetration into tumor tissue through acoustic cavitation. Monitoring of cavitation activity during pHIFU treatments and knowing the ultrasound pressure levels sufficient to reliably induce cavitation in a given tissue are therefore very important. Here, three metrics of cavitation activity induced by pHIFU and evaluated by confocal passive cavitation detection were introduced: cavitation probability, cavitation persistence and the level of the broadband acoustic emissions. These metrics were used to characterize cavitation activity in several ex vivo tissue types (bovine tongue and liver and porcine adipose tissue and kidney) and gel phantoms (polyacrylamide and agarose) at varying peak-rare factional focal pressures (1–12 MPa) during the following pHIFU protocol: frequency 1.1 MHz, pulse duration 1 ms and pulse repetition frequency 1 Hz. To evaluate the relevance of the measurements in ex vivo tissue, cavitation metrics were also investigated and compared in the ex vivo and in vivo murine pancreatic tumors that develop spontaneously in transgenic KrasLSL.G12 D/+; p53 R172 H/+; PdxCretg/+ (KPC) mice and closely re-capitulate human disease in their morphology. The cavitation threshold, defined at 50% cavitation probability, was found to vary broadly among the investigated tissues (within 2.5–10 MPa), depending mostly on the water-lipid ratio that characterizes the tissue composition. Cavitation persistence and the intensity of broadband emissions depended both on tissue structure and lipid concentration. Both the cavitation threshold and broadband noise emission level were similar between ex vivo and in vivo pancreatic tumor tissue. The largest difference between in vivo and ex vivo settings was found in the pattern of cavitation occurrence throughout pHIFU exposure: it was sporadic in vivo, but it decreased rapidly and stopped over the first few pulses ex vivo. Cavitation activity depended on the interplay between the destruction and circulation of cavitation nuclei, which are not only used up by HIFU treatment but also replenished or carried away by circulation in vivo. These findings are important for treatment planning and optimization in pHIFU-induced drug delivery, in particular for pancreatic tumors.     

Correlation between inertial cavitation dose and endothelial cell damage in vivo

Previous in vivo studies have demonstrated that vascular endothelial damage can result when vessels containing gas-based microbubble ultrasound contrast agent (UCA) are exposed to MHz-frequency pulsed ultrasound (US) of sufficient pressure amplitudes, presumably as a result of inertial cavitation (IC). The hypothesis guiding this research was that IC is the primary mechanism by which the vascular endothelium (VE) is damaged when a vessel is exposed to pulsed 1-MHz frequency US in the presence of circulating UCA. The expectation was that a correlation should exist between the magnitude and duration of IC activity and the degree of VE damage. Rabbit auricular vessels were exposed in vivo to 1.17-MHz focused US of variable peak rarefaction pressure amplitude (1, 3, 6.5 or 9 MPa), using low duty factors (0.04% or 0.4%), pulse lengths of 500 or 5000 cycles, with varying treatment durations and with or without infusion of a shelled microbubble contrast agent. A broadband passive cavitation detection system was used to measure IC activity in vivo within the targeted segment of the blood vessel. The magnitude of the detected IC activity was quantified using a previously reported measure of IC dose. Endothelial damage was assessed via scanning electron microscopy image analysis. The results supported the hypothesis and demonstrate that the magnitude of the measured IC dose correlates with the degree of VE damage when UCA is present. These results have implications for therapeutic US-induced vascular occlusion.     

Intravascular inertial cavitation activity detection and quantification in vivo with Optison

Inertial cavitation (IC) is an important mechanism by which ultrasound (US)-induced bioeffects can be produced. It has been reported that US-induced in vitro mechanical bioeffects with the presence of ultrasound contrast agents (UCAs) are highly correlated with quantified IC "dose" (ICD: cumulated root-mean-squared broadband noise amplitude in the frequency domain). The ICD has also been used to quantify IC activity in ex vivo perfused rabbit ear vessels. The in vivo experiments reported here using a rabbit ear vessel model were designed to: (1) detect and quantify IC activity in vivo within the constrained environment of rabbit auricular veins with the presence of Optison and (2) measure the temporal evolution of microbubble IC activity and the ICD generated during insonation treatment, as a function of acoustic parameters. Preselected regions-of-interest (ROI) in the rabbit ear vein were exposed to pulsed focused US (1.17 MHz, 1 Hz PRF). Experimental acoustic variables included peak rarefaction pressure amplitude ([PRPA]: 1.1, 3.0, 6.5 or 9.0 MPa) and pulse length (20, 100, 500 or 1000 cycles). ICD was quantified based on passive cavitation detection (PCD) measurements. The results show that: (1) after Optison injection, the time to onset of measurable microbubble IC activity was relatively consistent, approximately 20 s; (2) after reaching its peak value, the IC activity decayed exponentially and the half-life decay coefficient (t(1/2)) increased with increasing PRPA and pulse length; and (3) the normalized ICD generated by pulsed US exposure increased significantly with increasing PRPA and pulse length.     

Effect of Magnetite Nanoparticle Agglomerates on Ultrasound Induced Inertial Cavitation

High intensity focused ultrasound (HIFU) induced inertial cavitation has been shown to improve release and cellular uptake of drugs. The effects of magnetite nanoparticle agglomerates (290 ± 10 nm diameter), silica coated magnetite nanoparticle agglomerates (320 ± 10 nm diameter) and silica particles (320 ± 10 nm diameter) suspended in MilliQ water on the degree of inertial cavitation due to HIFU were investigated. The HIFU transducer was operated at a frequency of 1.1 MHz, 1.67 kHz pulse repetition frequency, with applied duty cycles (DC) between 0% and 5% and different peak negative focal pressures (PNFPs) applied up to 7.2 MPa. The inertial cavitation dose (ICD: time averaged root-mean-squared broadband noise amplitude in the frequency domain) was measured in the presence and absence of nanoparticles when subjected to HIFU. Magnetite nanoparticle agglomerates caused a significant increase in the ICD above 2.7 MPa PNFP compared with MilliQ water, silica coated magnetite agglomerates and silica particles. With the dramatic increase in ICD on introduction of these magnetite agglomerates, this technique could provide a method of HIFU triggered drug delivery by enhancing inertial cavitation. The superparamagnetic properties of these particles offer the possibility of magnetic targeting to the site of disease.     

Ultrasound-induced cavitation enhances the delivery and therapeutic efficacy of an oncolytic virus in an in vitro model

We investigated whether ultrasound-induced cavitation at 0.5 MHz could improve the extravasation and distribution of a potent breast cancer-selective oncolytic adenovirus, AdEHE2F-Luc, to tumour regions that are remote from blood vessels. We developed a novel tumour-mimicking model consisting of a gel matrix containing human breast cancer cells traversed by a fluid channel simulating a tumour blood vessel, through which the virus and microbubbles could be made to flow. Ultrasonic pressures were chosen to maximize either broadband emissions, associated with inertial cavitation, or ultraharmonic emissions, associated with stable cavitation, while varying duty cycle to keep the total acoustic energy delivered constant for comparison across exposures. None of the exposure conditions tested affected cell viability in the absence of the adenovirus. When AdEHE2F-Luc was delivered via the vessel, inertial cavitation increased transgene expression in tumour cells by up to 200 times. This increase was not observed in the absence of Coxsackie and Adenovirus Receptor cell expression, discounting sonoporation as the mechanism of action. In the presence of inertial cavitation, AdEHE2F-Luc distribution was greatly improved in the matrix surrounding the vessel, particularly in the direction of the ultrasound beam; this enabled AdEHE2F-Luc to kill up to 80% of cancer cells within the ultrasound focal volume in the gel 24 hours after delivery, compared to 0% in the absence of cavitation.     

Effects of acoustic insonation parameters on ultrasound contrast agent destruction

Ultrasound contrast agents (UCAs) are used to enhance the acoustic backscattered intensity of blood and thereby assist the assessment of blood perfusion. Characterization of UCA destruction provides important information for the design of contrast-assisted perfusion imaging. High-speed optical observation of single microbubble destruction during acoustic insonation has been performed in previous studies. The results identified that pressure, center frequency and transmission phase have significant effects on the fragmentation threshold. We proposed an acoustic-based experiment method to demonstrate the relationship between different acoustic exposure conditions and the degree of UCA destruction. The method also provides a simple and convenient way to determine the microbubble destruction threshold. The experiments introduced three insonation parameters, including acoustic pressure (0 to 1 MPa), pulse frequency (1, 2.25, 5 and 7.5 MHz) and pulse length (1 to 10 cycles). The term of surviving percentage (SP) was proposed to represent the ratio of UCA backscattered power with and without acoustic insonation. The results showed that the SP decreased with decreasing pulse frequency, but with increasing transmission acoustic pressure and pulse length. In addition, there was an exponential relationship between SP and acoustic pressure, and thus the UCA destruction pressure threshold could be predicted from the fitted exponential curve. The results also show that the degree of UCA destruction was not related to mechanical index (MI). Potential applications of this method include UCA high-resolution destruction/replenishment imaging model, microbubble cavitation, sonoporation in drug delivery and gene therapy.     

Influence of Acoustic Reflection on the Inertial Cavitation Dose in a Franz Diffusion Cell

The exposure of the skin to low-frequency (20–100?kHz) ultrasound is a well-established method for increasing its permeability to drugs. The mechanism underlying this permeability increase has been found to be inertial cavitation within the coupling fluid. This study investigated the influence of acoustic reflections on the inertial cavitation dose during low-frequency (20?kHz) exposure in an in vitro skin sonoporation setup. This investigation was conducted using a passive cavitation detector that monitored the broadband noise emission within a modified Franz diffusion cell. Two versions of this diffusion cell were employed. One version had acoustic conditions that were similar to those of a standard Franz diffusion cell surrounded by air, whereas the second was designed to greatly reduce the acoustic reflection by submerging the diffusion cell in a water bath. The temperature of the coupling fluid in both setups was controlled using a novel thermoelectric cooling system. At an ultrasound intensity of 13.6 W/cm², the median inertial cavitation dose when the acoustic reflections were suppressed, was found to be only about 15% lower than when reflections were not suppressed.     

Sonoluminescence, in water and in human blood plasma, generated using ultrasonic therapy equipment

Sonoluminescence in a liquid indicates both cavitation and free radical formation. This experimental study showed that it can be generated in both water and blood plasma using ultrasound at therapeutic intensities (less than 2W cm⁻², spatial average) and frequencies (0.75 and 1.5 MHz). Pulsing the ultrasound raised the intensity threshold for sonoluminescence. Recordings of acoustic emission from the liquid (for 0.75 MHz continuous insonation) showed that sonoluminescence was accompanied by the emission of ‘broad-band’ noise. The applicability of these results to tissue insonation, and the implications for ultrasound therapy, are discussed.     

An in vitro study of a phase-shift nanoemulsion: a potential nucleation agent for bubble-enhanced HIFU tumor ablation

Phase-shift nanoemulsions have the potential to nucleate bubbles and enhance high-intensity focused ultrasound (HIFU) cancer therapy. This emulsion consists of albumin-coated dodecafluoropentane (DDFP) droplets with a mean diameter of approximately 260 nm at 37°C. It is known that superheated perfluorocarbon droplets can be vaporized with microsecond long ultrasound pulses if the acoustic pressure exceeds a specific threshold. In addition, it is well documented that particles smaller than 400 nm can extravasate through leaky tumor vessels and accumulate in the tumor interstitial space. Thus, nanoemulsions may passively target solid tumors, thus localizing cavitation nuclei for bubble-enhanced HIFU-mediated heating. In this study, we investigate the acoustic droplet vaporization of a DDFP nanoemulsion in tissue-mimicking gels and demonstrate the ability to nucleate inertial cavitation (IC) and enhance HIFU-mediated heating. The nanoemulsion was dispersed throughout albumin-acrylamide gel phantoms and sonicated with microsecond-length HIFU pulses (f = 2 MHz). The pressure threshold needed to vaporize the nanoemulsion was measured as a function of degree of superheat, pulse length and nanoemulsion concentration. It was determined that the vaporization threshold was inversely proportional with degree of superheat and independent of pulse length and concentration within the range of values tested. It was also shown that the bubbles formed from vaporized nanoemulsions reduced the IC threshold in the gel phantoms. Finally, it was demonstrated that cavitation from vaporized nanoemulsions accelerated HIFU-mediated heating. The results from this study demonstrate that phase-shift nanoemulsions can be combined with HIFU to provide a high degree of spatial and temporal control of bubble-enhanced heating.