Ultrasound-based cell sorting with microbubbles: A feasibility study.

The isolation and sorting of cells is an important process in research and hospital labs. Most large research and commercial labs incorporate fluorescently or magnetically labeled antibodies adherent to cell surface antigens for cell identification and separation. In this paper, a process is described that merges biochemical labeling with ultrasound-based separation. Instead of lasers and fluorophore tags, or magnets and magnetic particle tags, the technique uses ultrasound and microbubble tags. Streptavidin-labeled microbubbles were mixed with a human acute lymphoblastic leukemia cell line, CCL 119, conjugated with biotinylated anti-CD7 antibodies. Tagged cells were forced under ultrasound, and their displacement and velocity quantified. Differential displacement in a flow stream was quantified against erythrocytes, which showed almost no displacement under ultrasound. A model for the acoustic radiation force on the conjugated pairs compares favorably with observations. This technology may improve on current time-consuming and costly purification procedures.

Efficient microbubble- and ultrasound-mediated plasmid DNA delivery into a specific rat liver lobe via a targeted injection and acoustic exposure using a novel ultrasound system.

To develop efficient gene delivery in larger animals, based on a previous mouse study, we explored the luciferase reporter gene transfer in rats by establishing a novel unfocused ultrasound system with simultaneous targeted injection of a plasmid and microbubble mixture into a specific liver lobe through a portal vein branch. Luciferase expression was significantly enhanced over 0-30 vol % of the Definity microbubbles, with a plateau between 0.5 and 30 vol %. The increase of gene delivery efficiency also depended on the acoustic peak negative pressure, achieving over 100-fold enhancement at 2.5 MPa compared with plasmid only controls. Transient, modest liver damage following treatment was assessed by transaminase assays and histology, both of which correlated with gene expression induced by acoustic cavitation. In addition, pulse-train ultrasound exposures (i.e., with relatively long quiescent periods between groups of pulses to allow tissue refill with microbubbles) produced gene expression levels comparable to the standard US exposure but reduced the extent of liver damage. These results indicated that unfocused high intensity therapeutic ultrasound exposure with microbubbles is highly promising for safe and efficient gene delivery into the liver of rats or larger animals.

Blood vessel deformations on microsecond time scales by ultrasonic cavitation.

Transient interactions among ultrasound, microbubbles, and microvessels were studied using high-speed photomicrography. We observed liquid jets, vessel distention (motion outward against the surrounding tissue), and vessel invagination (motion inward toward the lumen). Contrary to current paradigms, liquid jets were directed away from the nearest vessel wall and invagination exceeded distention. These observations provide insight into the mechanics of bubble-vessel interactions, which appear to depend qualitatively upon the mechanical properties of biological tissues.

Treating Porcine Abscesses with Histotripsy: A Pilot Study.

Infected abscesses are walled-off collections of pus and bacteria. They are a common sequela of complications in the setting of surgery, trauma, systemic infections and other disease states. Current treatment is typically limited to antibiotics with long-term catheter drainage, or surgical washout when inaccessible to percutaneous drainage or unresponsive to initial care efforts. Antibiotic resistance is also a growing concern. Although bacteria can develop drug resistance, they remain susceptible to thermal and mechanical damage. In particular, short pulses of focused ultrasound (i.e., histotripsy) generate mechanical damage through localized cavitation, representing a potential new paradigm for treating abscesses non-invasively, without the need for long-term catheterization and antibiotics. In this pilot study, boiling and cavitation histotripsy treatments were applied to subcutaneous and intramuscular abscesses developed in a novel porcine model. Ultrasound imaging was used to evaluate abscess maturity for treatment monitoring and assessment of post-treatment outcomes. Disinfection was quantified by counting bacteria colonies from samples aspirated before and after treatment. Histopathological evaluation of the abscesses was performed to identify changes resulting from histotripsy treatment and potential collateral damage. Cavitation histotripsy was more successful in reducing the bacterial load while having a smaller treatment volume compared with boiling histotripsy. The results of this pilot study suggest focused ultrasound may lead to a technology for in situ treatment of acoustically accessible abscesses.

Blood vessel rupture by cavitation.

Cavitation is thought to be one mechanism for vessel rupture during shock wave lithotripsy treatment. However, just how cavitation induces vessel rupture remains unknown. In this work, a high-speed photomicrography system was set up to directly observe the dynamics of bubbles inside blood vessels in ex vivo rat mesenteries. Vascular rupture correlating to observed bubble dynamics were examined by imaging bubble extravasation and dye leakage. The high-speed images show that bubble expansion can cause vessel distention, and bubble collapse can lead to vessel invagination. Liquid jets were also observed to form. Our results suggest that all three mechanisms, vessel distention, invagination and liquid jets, can contribute to vessel rupture.

Displacement analysis of diagnostic ultrasound backscatter: a methodology for characterizing, modeling, and monitoring high intensity focused ultrasound therapy.

Accurate monitoring of high intensity focused ultrasound (HIFU) therapy is critical for widespread clinical use. Pulse-echo diagnostic ultrasound (DU) is known to exhibit temperature sensitivity through relative changes in time-of-flight between two sets of radio frequency (RF) backscatter measurements, one acquired before and one after therapy. These relative displacements, combined with knowledge of the exposure protocol, material properties, heat transfer, and measurement noise statistics, provide a natural framework for estimating the administered heating, and thereby therapy. The proposed method, termed displacement analysis, identifies the relative displacements using linearly independent displacement patterns, or modes, each induced by a particular time-varying heating applied during the exposure interval. These heating modes are themselves linearly independent. This relationship implies that a linear combination of displacement modes aligning the DU measurements is the response to an identical linear combination of heating modes, providing the heating estimate. Furthermore, the accuracy of coefficient estimates in this approximation is determined a priori, characterizing heating, thermal dose, and temperature estimates for any given protocol. Predicted performance is validated using simulations and experiments in alginate gel phantoms. Evidence for a spatially distributed interaction between temperature and time-of-flight changes is presented.

Inactivation of Planktonic Escherichia coli by Focused 1-MHz Ultrasound Pulses with Shocks: Efficacy and Kinetics Upon Volume Scale-Up.

This study addresses inactivation of E. coli in either 5- or 10-mL volumes, which were 50- to 100-fold greater than used in an earlier study (Brayman et al. 2017). Cells were treated with 1-MHz pulsed high-intensity focused ultrasound (10 cycles, 2-kHz repetition frequency, +65/-12.8 MPa focal pressures). The surviving fraction was assessed by coliform assay, and inactivation demonstrated curvilinear kinetics. The reduction of surviving fraction to 50% required 2.5 or 6 min in 5- or 10-mL samples, respectively. Exposure of 5 mL for 20 min reduced the surviving fraction to ∼1%; a similar exposure of 10-mL samples reduced the surviving fraction to ∼10%. Surviving cells from 5-min exposures appeared normal under light microscopy, with minimal debris; after 20 min, debris dominated. Transmission electron microscopy images of insonated samples showed some undamaged cells, a few damaged but largely intact cells and comminuted debris. Cellular damage associated with substantive but incomplete levels of inactivation can be variable, ranging from membrane holes tens of nanometers in diameter to nearly complete comminution.

Inactivation of Planktonic Escherichia coli by Focused 2-MHz Ultrasound.

This study was motivated by the desire to develop a non-invasive means to treat abscesses, and represents the first steps toward that goal. Non-thermal, high-intensity focused ultrasound (HIFU) was used to inactivate Escherichia coli (∼1 × 109 cells/mL) in suspension. Cells were treated in 96-well culture plate wells using 1.95-MHz ultrasound and incident focal acoustic pressures as high as 16 MPa peak positive and 9.9 MPa peak negative (free field measurements). The surviving fraction was assessed by coliform culture and the alamarBlue assay. No biologically significant heating was associated with ultrasound exposure. Bacterial inactivation kinetics were well described by a half-life model, with a half-time of 1.2 min. At the highest exposure levels, a 2log inactivation was typically achieved within 10 min. The free field-equivalent peak negative acoustic pressure threshold for inactivation was ∼7 MPa. At the highest acoustic pressures used, inactivation efficacy was insensitive to reciprocal changes in pulse length and pulse repetition frequency at constant duty factor. Although treated volumes were very small, proof of principle was provided by these experiments.

Inertial cavitation dose produced in ex vivo rabbit ear arteries with Optison by 1-MHz pulsed ultrasound.

Previous in vitro studies have shown that ultrasound-induced mechanical bioeffects with contrast agents present are highly correlated with inertial cavitation (IC) "dose" (Chen et al. 2003a, 2003c). The ex vivo experiments conducted here addressed the following hypotheses: 1. IC activity can be generated by insonating perfused rabbit ear blood vessel, and 2. the IC "dose" developed during insonation treatment can be reliably measured and will vary with varying acoustic parameters and Optison concentration. Ex vivo rabbit auricular arteries were perfused with Optison suspensions and then exposed to 1.1-MHz pulsed focused ultrasound. Experimental variables included peak negative acoustic pressure (0.2 MPa to 5.2 MPa), pulse-repetition frequency (5, 50 or 500 Hz), pulse length (50, 100, 500 or 1000 cycles), and Optison volume concentration (0, 0.2, 0.5 or 1%). Cavitation activity was quantified as IC dose, based on passive cavitation detection measurements. The results show that: 1. The IC pressure threshold decreases with higher concentrations of Optison, and 2. IC dose increases significantly with increasing acoustic pressure, Optison concentration, pulse length or with decreasing pulse-repetition frequency.

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.

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.

Ultrasound enhances gene delivery of human factor IX plasmid.

Delivery of plasmid DNA can be enhanced by treatment with ultrasound (US); acoustic cavitation appears to play an important role in the process. Ultrasound contrast agents (UCAs; stabilized microbubbles) nucleate acoustic cavitation, and lower the acoustic pressure threshold for inertial cavitation occurrence. Fifty micrograms of a liver-specific, high-expressing human factor IX plasmid, pBS-HCRHP-FIXIA, mixed with UCA or phosphate-buffered saline was delivered to mouse livers by intrahepatic injection, with simultaneous exposure to 1 MHz-pulsed US using various acoustic protocols. Variable pulse duration (PD) at constant treatment time, pulse repetition frequency, and an acoustic peak negative pressure amplitude of 1.8 MPa produced 2- to 13-fold enhancements in hFIX gene expression, but PD was not a strong determinant. In contrast, a dose-response relationship was demonstrated for the peak negative pressure (P-), with significant enhancement of gene transduction at P- >/= 2 MPa. Up to 63 ng/ml (approaching the therapeutic range for treating hemophilia patients) could be achieved by transducing one liver lobe at 4-MPa P-, corresponding to a 66- fold increment relative to treatment with naked DNA alone. Under the same conditions, mouse livers could also be transduced with a GFP plasmid. Histology showed transient liver damage caused by intrahepatic injection and US exposure at 4-MPa P-; however, the damage was repaired in a few days. We conclude that therapeutic US in combination with UCA has the potential to promote safe and efficient nonviral gene transfer of hFIX for the treatment of hemophilia.

Vascular effects induced by combined 1-MHz ultrasound and microbubble contrast agent treatments in vivo.

Previous in vivo studies have demonstrated that microvessel hemorrhages and alterations of endothelial permeability can be produced in tissues containing microbubble-based ultrasound contrast agents when those tissues are exposed to MHz-frequency pulsed ultrasound of sufficient pressure amplitudes. The general hypothesis guiding this research was that acoustic (viz., inertial) cavitation, rather than thermal insult, is the dominant mechanism by which such effects arise. We report the results of testing five specific hypotheses in an in vivo rabbit auricular blood vessel model: (1) acoustic cavitation nucleated by microbubble contrast agent can damage the endothelia of veins at relatively low spatial-peak temporal-average intensities, (2) such damage will be proportional to the peak negative pressure amplitude of the insonifying pulses, (3) damage will be confined largely to the intimal surface, with sparing of perivascular tissues, (4) greater damage will occur to the endothelial cells on the side of the vessel distal to the source transducer than on the proximal side and (5) ultrasound/contrast agent-induced endothelial damage can be inherently thrombogenic, or can aid sclerotherapeutic thrombogenesis through the application of otherwise subtherapeutic doses of thrombogenic drugs. Auricular vessels were exposed to 1-MHz focused ultrasound of variable peak pressure amplitude using low duty factor, fixed pulse parameters, with or without infusion of a shelled microbubble contrast agent. Extravasation of Evans blue dye and erythrocytes was assessed at the macroscopic level. Endothelial damage was assessed via scanning electron microscopy (SEM) image analysis. The hypotheses were supported by the data. We discuss potential therapeutic applications of vessel occlusion, e.g., occlusion of at-risk gastric varices.

Inertial cavitation dose and hemolysis produced in vitro with or without Optison.

Gas-based contrast agents (CAs) increase ultrasound (US)-induced bioeffects, presumably via an inertial cavitation (IC) mechanism. The relationship between IC dose (ICD) (cumulated root mean squared [RMS] broadband noise amplitude; frequency domain) and 1.1-MHz US-induced hemolysis in whole human blood was explored with Optison; the hypothesis was that hemolysis would correlate with ICD. Four experimental series were conducted, with variable: 1. peak negative acoustic pressure (P-), 2. Optison concentration, 3. pulse duration and 4. total exposure duration and Optison concentration. P- thresholds for hemolysis and ICD were approximately 0.5 MPa. ICD and hemolysis were detected at Optison concentrations >/= 0.01 V%, and with pulse durations as low as four or two cycles, respectively. Hemolysis and ICD evolved as functions of time and Optison concentration; final hemolysis and ICD values depended on initial Optison concentration, but initial rates of change did not. Within series, hemolysis was significantly correlated with ICD; across series, the correlation was significant at p < 0.001.

The pulse length-dependence of inertial cavitation dose and hemolysis.

Gas-based ultrasound (US) contrast agents increase erythrocyte sonolysis, presumably via enhancing inertial cavitation (IC) activity. The amount of IC activity (IC "dose") and hemolysis generated by exposure to 1.15 MHz US were examined with different US pulse lengths, but with the same delivered acoustic energy, for Optison and Albunex. The hypotheses were that 1. at longer pulse lengths, IC would generate more bubbles that could nucleate additional IC activity; 2. if the interval between pulse pairs were short enough for the next pulse to hit derivative bubbles before their dissolution, more IC could be induced; and 3. hemolysis would be proportional to IC activity. Two types of studies were performed. In the first, bubble generation after each burst of IC activity was quantified using an active cavitation detector (ACD), for different pulse lengths (5, 10, 20, 30, 50, 100 or 200 cycles), but the same pressure level (3 MPa) and total "on" time (173.16 ms). Low concentrations of either Optison or Albunex were added into the tank with high-intensity and interrogating transducers orthogonal to each other. For pulse lengths > 100 cycles, and pulse repetition intervals < 5 ms, a "cascade" effect (explosive bubble generation) was observed. In the second, IC was measured by passive detection methods. IC dose and hemolysis were determined in whole blood samples at a pressure level (3 MPa) and interpulse interval (5 ms) that induced the "cascade" effect. Each blood sample was mixed with the same number of contrast microbubbles (Optison approximately 0.3 v/v % and Albunex approximately 0.5 v/v %), but exposed to different pulse lengths (5, 10, 20, 30, 50, 100 or 200 cycles). With Optison, up to 60% hemolysis was produced with long pulses (100 and 200 cycles), compared with < 10% with short pulses (5 and 10 cycles). Albunex generated considerably less IC activity and hemolysis. The r(2) value was 0.99 for the correlation between hemolysis and IC dose. High pulse-repetition frequency (PRF) (500 Hz) generated more hemolysis than the low PRF (200 Hz) at 3 MPa. All experimental results could be explained by the dissolution times of IC-generated bubbles.

Biological Effects of Ultrasound. The Perceived Safety of Diagnostic Ultrasound Within the Context of Ultrasound Biophysics: A Personal Perspective.

This brief review addresses the issue of health and safety from exposure to diagnostic ultrasound. The exemplary historical record of diagnostic ultrasound exposures is coupled with great patient benefit. However, the power outputs of clinical devices have been increasing over the past decade such that inertial cavitation seems reasonably likely to occur if appropriate gas nuclei are present. The use of microbubble contrast agents for certain diagnostic procedures ensures a well nucleated system. Under such conditions, the use of low signal output levels and short exam times will decrease the chance of cavitation related bioeffects.

Temporality in Ultrasound-Induced Cell Lysis In Vitro.

To test the hypothesis that the full extent of in vitro cell lysis due to ultrasound becomes evident with time lapse following insonation, human erythrocytes (2% hematocrit) in autologous plasma were mixed with Albunex(R), a pulse echo contrast agent, and exposed to 1-MHz, continuous-wave ultrasound (US) (5 W/cm(2) SPTA intensity) for 60 seconds while in a rotating (200 rpm) dialysis membrane vessel. Exposed and sham-exposed samples were subsequently assayed for hemolysis colorimetrically, either immediately or after a delay of 3 hours. Hemolysis was dependent on the interval between US exposure and assay, with significantly greater lysis evident with delayed assay. There was also temporality in lytic yield with sample number, i.e., with time postpreparation of the blood sample, US-induced cell lysis decreased. The temporality of lytic yield was eliminated by maintenance at ice water temperatures, or by waiting about 1 hour before beginning treatments. The collective data indicate that the full extent of US-induced cell lysis is not evident upon assay immediately after insonation, and that with time postpreparation and preinsonation, erythrocytes may undergo changes in sensitivity to US. (ECHOCARDIOGRAPHY, Volume 13, January 1996)

Characteristic microvessel relaxation timescales associated with ultrasound-activated microbubbles.

Ultrasound-activated microbubbles were used as actuators to deform microvessels for quantifying microvessel relaxation timescales at megahertz frequencies. Venules containing ultrasound contrast microbubbles were insonified by short 1 MHz ultrasound pulses. Vessel wall forced-deformations were on the same microsecond timescale as microbubble oscillations. The subsequent relaxation of the vessel was recorded by high-speed photomicrography. The tissue was modeled as a simple Voigt solid. Relaxation time constants were measured to be on the order of ∼10 µs. The correlation coefficients between the model and 38 data sets were never lower than 0.85, suggesting this model is sufficient for modeling tissue relaxation at these frequencies. The results place a bound on potential numerical values for viscosity and elasticity of venules.

Preliminary observations on the spatial correlation between short-burst microbubble oscillations and vascular bioeffects.

The objective of this preliminary study was to examine the spatial correlation between microbubble (MB)-induced vessel wall displacements and resultant microvascular bioeffects. MBs were injected into venules in ex vivo rat mesenteries and insonated by a single short ultrasound pulse with a center frequency of 1 MHz and peak negative pressures spanning the range of 1.5-5.6 MPa. MB and vessel dynamics were observed under ultra-high speed photomicrography. The tissue was examined by histology or transmission electron microscopy for vascular bioeffects. Image registration allowed for spatial correlation of MB-induced vessel wall motion to corresponding vascular bioeffects, if any. In cases in which damage was observed, the vessel wall had been pulled inward by more than 50% of the its initial radius. The observed damage was characterized by the separation of the endothelium from the vessel wall. Although the study is limited to a small number of observations, analytic statistical results suggest that vessel invagination comprises a principal mechanism for bioeffects in venules by microbubbles.

Observations of translation and jetting of ultrasound-activated microbubbles in mesenteric microvessels.

High-speed photomicrography was used to study the translational dynamics of single microbubbles in microvessels of ex vivo rat mesenteries. The microbubbles were insonated by a single 2 µs ultrasound pulse with a center frequency of 1 MHz and peak negative pressures spanning the range of 0.8-4 MPa. The microvessel diameters ranged from 10-80 µm. The high-speed image sequences show evidence of ultrasound-activated microbubble translation away from the nearest vessel wall; no microbubble showed a net translation toward the nearest vessel wall. Microbubble maximum translation displacements exceeded 20 µm. Microjets with the direction of the jets identifiable were also observed; all microjets appear to have been directed away from the nearest vessel wall. These observations appear to be characteristic of a strong coupling between ultrasound-driven microbubbles and compliant microvessels. Although limited to mesenteric tissues, these observations provide an important step in understanding the physical interactions between microbubbles and microvessels.