Explorations of high-intensity therapeutic ultrasound and microbubble-mediated gene delivery in mouse liver.

Ultrasound (US) combined with microbubbles (MBs) is a promising technology for non-viral gene delivery. Significant enhancements of gene expression have been obtained in our previous studies. To optimize and prepare for application to larger animal models, the luciferase reporter gene transfer efficacy of lipid-based Definity MBs of various concentrations, pressure amplitudes and a novel unfocused high-intensity therapeutic US (HITU) system were explored. Luciferase expression exhibited a dependence on MB dose over the range of 0-25 vol%, and a strong dependence on acoustic peak negative pressure at over the range of 0-3.2 MPa. Gene expression reached an apparent plateau at MB concentration ≥2.5 vol% or at negative pressures >1.8 MPa. Maximum gene expression in treated animals was 700-fold greater than in negative controls. Pulse train US exposure protocols produced an upward trend of gene expression with increasing quiescent time. The hyperbolic correlation of gene expression and transaminase levels suggested that an optimum gene delivery effect can be achieved by maximizing acoustic cavitation-induced enhancement of DNA uptake and minimizing unproductive tissue damage. This study validated the new HITU system equipped with an unfocused transducer with a larger footprint capable of scanning large tissue areas to effectively enhance gene transfer efficiencies.

Ultrasound with microbubbles enhances gene expression of plasmid DNA in the liver via intraportal delivery.

Current ultrasound (US)-mediated gene delivery methods are inefficient due, in part, to a lack of US optimization. We systematically explored the use of microbubbles (MBs), US parameters and plasmid delivery routes to improve gene transfer into the mouse liver. Co-presentation of plasmid DNA (pDNA), 10% Optison MBs and pulsed 1-MHz US at a peak negative pressure of 4.3 MPa significantly increased luciferase gene expression with pDNA delivered by intrahepatic injection to the left liver lobe. Intraportal injection delivered pDNA and MBs to the whole liver; with insonation, all lobes expressed the transgene, thus increasing total gene expression. Gene expression was also dependent on acoustic pressure over the range of 0-4.3 MPa, with a peak effect at 3 MPa. An average of 85-fold enhancement in gene delivery was achieved. No enhancement was observed below 0.25 MPa. Increasing pulse length while decreasing pulse repetition frequency and exposure time to maintain a constant total energy during exposure did not further improve transfection efficiency, nor did extend the US exposure pre- or postinjection of pDNA. The results indicate that coupled with MBs, US can more efficiently and dose-dependently enhance gene expression from pDNA delivered via portal vein injection by an acoustic mechanism of inertial cavitation.

Hyperthermic teratogenicity, thermal dose and diagnostic ultrasound during pregnancy: implications of new standards on tissue heating.

Hyperthermia is a recognized teratogen in mammalian laboratory animals and is a suspected teratogen for humans. The purpose of this synopsis is to reanalyse existing data on hyperthermia-induced teratogenic effects in experimental mammalian systems in terms of a thermal dose (temperature:time) concept, and then to illustrate the utility of this concept to human situations involving potential thermal increments to post-implantation embryos and foetuses. For example, the threshold temperature elevation for hyperthermia-induced teratogenic effects in experimental mammals is estimated (but not rigorously tested) to be approximately 1.5 degrees C above core values for exposures of long duration, possibly with a thermal dose of approximately 5 min duration or more at 4 degrees C. This level of tissue temperature increment is within the capability of some modern diagnostic ultrasound (DUS) devices sold within the USA and abroad. Epidemiological studies have not indicated any hazard from the use of DUS, but such studies are limited in sensitivity and were conducted with DUS devices whose acoustic outputs were relatively low compared to those presently available. After a regulatory change that allowed for substantially increased acoustic outputs, modern DUS devices were mandated to provide the user with on-screen information (the Thermal Index, or 'TI') about ultrasound-induced temperature increments in the target tissue. The TI is generally accurate to within a factor of 2, but the factor may be as high as 6 in certain obstetric settings. Thus, informed use of and attention to the TI is strongly advised, with this admonition gaining increased emphasis if the present regulations regarding allowable acoustic outputs of DUS devices were to be further relaxed or eliminated.

A comparison of the hemolytic potential of Optison and Albunex in whole human blood in vitro: acoustic pressure, ultrasound frequency, donor and passive cavitation detection considerations.

This project tested the hypothesis that a "second-generation" ultrasound (US) contrast agent (Optison), offering extended echogenicity over that of its "first-generation" predecessor (Albunex), would have the greater potential for sonolysis of human erythrocytes in vitro. Whole human blood, obtained from apparently healthy donors, was anticoagulated and subsequently exposed in vitro to US in the presence of one of each or neither of the two US contrast agents. The US exposures were for 30 s and involved frequency (1.0, 2.2 and 3.4 MHz) and amplitude (approximately 2.8 to 0.38 MPa P(-)) regimens; pulse duration (200 micros) and interpulse interval (20 ms) were held constant. The data supported the hypothesis, with an overall ratio of approximately 2.5 for relative extent of background-corrected US-induced hemolysis of the Optison/Albunex regimens. Passive cavitation detection analyses corroborated the results obtained with hemolysis.

Comparative sensitivity of human fetal and adult erythrocytes to hemolysis by pulsed 1 MHz ultrasound.

Human fetal and adult erythrocytes differ significantly in mean corpuscular volume (MCV), the fetal cells being larger than adult cells and diminishing in MCV as gestational age (GA) increases. Previous studies have shown that the sensitivity of erythrocytes from different species to lysis by mechanically applied shear stress increases as MCV increases. The tested hypotheses in the present project were: 1. fetal erythrocytes would be more sensitive to sonolysis than adult erythrocytes because of the former's larger size, and 2. erythrocyte sonolytic sensitivity would scale with MCV. Fetal and adult erythrocytes were resuspended to 40% hematocrit in oxygenated isotonic saline solution and 500 microL aliquots were exposed for 60 s to 200 micros bursts of 1-MHz ultrasound (US) (peak pressures: approximately 4.8 MPa positive, approximately 2.7 MPa negative; duty factor = 0.01), either with or without 3.6 volume % Albunex (ALX) present. Background-corrected hemolysis was indistinguishable from zero in sham-exposed fetal or adult erythrocyte suspensions. Without ALX, mean background-corrected US-induced hemolysis was significantly greater than zero for fetal and adult cells (0.42 +/- 0.15% vs. 0.62 +/- 0.15), but fetal cell lysis was not significantly greater than adult cell lysis. With ALX, US-induced hemolytic yields increased approximately 80-fold (fetal: 50.53 +/- 2.14; adult: 46.40 +/- 1.85%), and were significantly higher for fetal than for adult cells. There was also a statistically significant correlation between MCV and US-induced background-corrected hemolysis. Thus, the two hypotheses were supported.

Comparative sensitivity of human and bovine erythrocytes to sonolysis by 1-MHz ultrasound.

This project tested the hypothesis that human erythrocytes, being larger than bovine erythrocytes, would be the more sensitive to sonolysis induced by inertial cavitation. The rationale behind this hypothesis was an earlier demonstration that, among sized populations of erythrocytes, an inverse relation existed between erythrocyte volume and mechanically-induced shear forces in the surrounding medium; viz, the larger the cell, the less shear force required to rupture the cell's membrane. At low erythrocyte densities (i.e., approximately 5% hematocrit) the hypothesis was supported; at high cell densities (i.e., approximately 35% hematocrit) it was not supported. The data are consistent with an ultrasound (US)-induced symmetric implosion of affected gas nuclei as causing the effect at low cell densities; under such conditions there is ample spacing among cells for US-induced symmetric growth and collapse of gas nuclei and the concomitant production of radially-expanding shock waves (which lyse the cells); at high cell densities there is not sufficient spacing among cells for US-induced symmetric growth and collapse of bubbles and an alternative mechanism, possibly asymmetric bubble collapse, becomes operational.

Erosion of artificial endothelia in vitro by pulsed ultrasound: acoustic pressure, frequency, membrane orientation and microbubble contrast agent dependence.

The erosion of cells from fibroblast monolayers simulating the vascular endothelium by 20 micros pulses of ultrasound at 500 Hz PRF was studied in relation to the peak negative acoustic pressure (P-; 0.0-2.5 MPa), ultrasound (US) frequency (1.0, 2.1 or 3.5 MHz), orientation of the monolayer (i.e., simulating the sites of ultrasound entry/exit from a blood vessel) and the presence or absence of a microbubble contrast agent (3 Vol% Albunex). The a priori hypotheses were that erosion of the monolayers would: 1. arise due to insonation treatment, 2. arise as a consequence of cavitation activity and, thus, increase with increasing P- at constant frequency, and decrease with increasing frequency at constant P-, 3. be significantly increased by the presence of a microbubble contrast agent, and 4. have a weak dependence on monolayer orientation. The data support these hypotheses. Under the most severe exposure conditions used, most of the affected cells appeared to have been lysed; however, a substantial number of viable cells were dislodged from the monolayer surface.

Transient poration and cell surface receptor removal from human lymphocytes in vitro by 1 MHz ultrasound.

The study objective was to gain insight into ultrasound-induced, sub-lytic cell surface modifications. Two primary hypotheses were tested by flow cytometric methods; viz., sonication will: 1. remove all or part of a specific cell surface marker in lymphocytes surviving insonation, and 2. induce transient pores in the cell membranes of some surviving cells. RPMI 1788 human lymphocytes were exposed in vitro to 1-MHz, continuous-wave ultrasound (approximately 8 W/cm2 ISP) for 30 s, which lysed approximately 50% of the cells. Insonation: 1. altered cell morphology, increasing the population of cells of reduced size but high structure (designated as population R2), many of which were nonviable, and diminishing the population of cells of large size and high structure (designated as population R1), most of which were viable, 2. diminished the fluorescence signal from the pan B lymphocyte marker CD19 in populations R1 and R2 to equivalent extents, and 3. increased by approximately 7-fold the number of transiently permeabilized cells in R1, as evidenced by simultaneous uptake of propidium iodide and fluorescein diacetate. The results indicate that ultrasound-induced CD19 removal from R1 cells can occur without accompanying gross membrane loss. The cell morphology/mortality shifts indicate that the ultrasound-induced morphological change is associated with lethal membrane poration, suggesting that the diminished CD19 fluorescence signal from insonated R2 cells arises partly by simultaneous loss of membrane fragments, CD19 and cytoplasm.

Sonolysis of Albunex-supplemented, 40% hematocrit human erythrocytes by pulsed 1-MHz ultrasound: pulse number, pulse duration and exposure vessel rotation dependence.

The hypotheses tested were that sonolysis of erythrocytes in the presence of a gas-based ultrasound contrast agent in vitro will be related quantitatively to the duration and number of ultrasound pulses applied using a constant pulse repetition period and, at least qualitatively, to the total exposure duration (i.e., the product of pulse number x pulse duration). An objective was to determine the influence of sample rotation during insonation on the amount of hemolysis produced under these conditions. Human erythrocytes, suspended to 40% hematocrit in autologous plasma containing 3.6% (V:V) Albunex, were exposed/sham-exposed to 1-100 pulses of 1-MHz ultrasound (6.2 MPa peak positive, 3.6 MPa peak negative acoustic pressures; I(SPTP) approximately 800 W/cm2) using a 1-s pulse repetition period. Pulse durations ranged from 20-20,000 micros; samples were either stationary or rotated (200 rpm) during insonation. Hemolysis was independent of vessel rotation treatment at all tested pulse durations and pulse numbers. Levels of hemolysis statistically greater than in sham-exposed samples were obtained with > or = 50 pulses of 20 micros duration, and > or = 1 pulse of 200, 2000 or 20,000 micros duration. Hemolysis increased with increasing pulse number and pulse duration. Approximately equivalent levels of hemolysis were produced by different pulse number x pulse duration combinations, yielding the same total exposure duration.

Sonochemicals increase the mutation frequency of V79 cells in vitro.

Phosphate buffered saline (PBS) was insonated or sham-insonated (1 MHz, 35 W/cm2, continuous wave, 30 min) in rotating (200 rpm) sterile polystyrene culture tubes. After treatment, the PBS was used immediately to suspend washed Chinese hamster V79 cells in vitro. Cells were incubated in the PBS at 37 degrees C for 15 min and then transferred to complete growth medium. Some insonation regimens also involved the inclusion of Albunex (ALX; an ultrasound microbubble contrast agent) to enhance ultrasound-induced inertial cavitation. Following exposure to the pretreated PBS and 6 d of subculture in complete medium, the cells were assayed for plating efficiencies and mutation frequencies (resistance to 6-thioguanine). X-rays (3 Gy) served as a positive control. Cells exposed to insonated PBS with or without ALX or x-rays had statistically significantly elevated mean mutation frequencies (4.37+/-0.97, 4.54+/-1.00, and 24.28+/-3.83 mutant colonies/10(6) viable cells, respectively) relative to corresponding control regimens (ultrasound sham, 2.44+/-0.56; x-ray sham, 2.96+/-0.88 mutant colonies/10(6) viable cells. The data supported the hypothesis that sonochemicals resulting from inertial cavitation have mutagenic potential.

Obstetric ultrasonography: a biophysical consideration of patient safety--the "rules" have changed.

We address the issue of health and safety in relation to exposure to diagnostic ultrasound, with particular emphasis given to obstetrics. In terms of fetal and maternal outcomes, the epidemiologic record of diagnostic ultrasound is exemplary but is primarily made on the basis of data derived from clinical devices whose outputs were relatively low compared with what is now allowable and available. The power outputs of clinical devices have been increasing over the past decade such that the potential for thermal and nonthermal insults is increased. For obstetric devices that use these higher outputs, the Food and Drug Administration now requires the presentation of 2 on-screen indexes, the thermal index and the mechanical index, in recognition of the 2 major mechanisms by which ultrasonography is known to affect cells and tissues. Greater responsibility for patient safety is now placed on the diagnostician; for the new indexes to be meaningful the diagnostician must be cognizant of the health and safety implications. The purpose of this article is to provide some guidance to the obstetrician in interpreting the indexes and to review the current status of ultrasonography biophysics in relation to the use of diagnostic ultrasound in obstetrics.

Comparative sensitivity of human erythrocytes and lymphocytes to sonolysis by 1-MHz ultrasound.

Many studies of ultrasonic hemolysis have used erythrocytes; other blood cells are less well studied. The hypothesis tested was that human lymphocytes, being large and relatively fragile, are more sensitive to sonolysis than are erythrocytes at equivalent cell concentrations. Human lymphocytes (RPMI 1788) grown in vitro and erythrocytes obtained by venipuncture were used at a nominal cell concentration of 2.5 x 10(6) cells/mL. Cells were contained in rotated (200 rpm) exposure vessels and were exposed/sham-exposed to 1-MHz continuous-wave ultrasound for 60 s. Cell lysis was determined by hemacytometer counts of aliquots taken before/after treatment. The hypothesis was supported; the mean levels of lysis in insonated lymphocyte and erythrocyte preparations were 89.8% +/- 0.6% and 78.9% +/- 3.1%, respectively. This difference was significant at p < 0.005.

Acoustic cavitation nuclei survive the apparent ultrasonic destruction of Albunex microspheres.

The hypothesis tested was that gas bodies capable of nucleating violent cavitation activity in vitro would survive the rapid disruption of Albunex microspheres by 1-MHz ultrasound. Human erythrocyte hemolysis was used as a proxy measure of cavitation. Fluid (5% human serum albumin [HSA]) with or without Albunex (ALX) was exposed or sham-exposed to 1-MHz ultrasound (P+ = 1.25 +/- 0.01 MPa, P- = 0.81 +/- 0.01 MPa; ISPTP approximately 35 W/cm2) for 60 s using 10-microseconds pulses and a duty factor of 0.5. An equal volume of whole human blood was then added to the fluid, followed by a second 60-s treatment. Insonation of cell suspensions prepared in previously sham-exposed HSA + ALX fluid produced about 4% hemolysis, a level significantly greater than in the controls. Insonation of cell suspensions prepared in previously insonated HSA + ALX fluid produced about 0.4% hemolysis; this also differed significantly from the controls. The data thus support the hypothesis.

Hemolysis of 40% hematocrit, Albunex-supplemented human erythrocytes by pulsed ultrasound: frequency, acoustic pressure and pulse length dependence.

The dependence of hemolysis produced by pulsed ultrasound on ultrasound frequency, acoustic pressure and pulse length was explored. Human erythrocytes (40% hematocrit; in Albunex-supplemented autologous plasma) were exposed (60 s) to 20 or 200 microns pulses of ultrasound at frequencies of 1.02, 2.24 or 3.46 MHz and at peak negative pressures [P-] ranging from 0.0 to approximately 3.0 MPa in 0.5 MPa increments. The duty factor was 0.01. At each frequency, hemolysis increased with increasing acoustic pressure and depended weakly on pulse duration. At relatively high acoustic pressures, hemolysis depended strongly on ultrasound frequency; at lower pressures, the frequency dependence was weaker. The potential clinical significance of ultrasonic hemolysis is discussed.

A test of the hypothesis that ELF magnetic fields affect calcium uptake in rat thymocytes in vitro.

The experiment's objective was to test the statistical hypothesis that a 60-min exposure of rat thymocytes in vitro to a unique combination of static and a.c. magnetic fields results in suppression of calcium influx triggered by concanavalin A (Con A). Con A (10 micrograms/ml) induced about a 50% increase in 45Ca2+ uptake relative to no Con A (control). Magnetic field exposures had no statistically significant effect on Con A-stimulated calcium uptake. The data did not support the hypothesis.

Effect of static pressure on acoustic transmittance of Albunex microbubble suspensions.

Albunex (ALX), an albumin-stabilized microbubble echo contrast agent, is sensitive to pressures similar to those produced by the heart. The tested hypothesis was that the acoustic transmittance of ALX suspensions will increase with increasing hydrostatic pressure (Ps). The test involved an acoustic setup analogous to a spectrophotometer. The acoustic transmittance of microbubble suspensions was strongly Ps dependent. Transmittance at 1 MHz was essentially zero at ambient pressure, increasing to approximately 50%, approximately 63%, and nearly 100% at Ps of 80, 120, and 400 mm Hg, respectively. The ultrasound pulses used to interrogate samples were without measurable effect on the acoustic transmittance of suspensions maintained at ambient pressure during experimental measurements. The data indicate that many of the microbubbles are destroyed at Ps comparable to those produced by the heart.

Re-evaluation of the concept that high cell concentrations "protect" cells in vitro from ultrasonically induced lysis.

Diminution of apparent ultrasonic cell lysis in vitro with increasing cell concentration/volume fraction has often been observed. A substantial fraction of the cells may be lysed by ultrasound at low cell concentrations, but only a few percent are lysed when the concentration is a fraction of that of whole blood. This suggests that sonolysis of cells in vitro is suppressed by high cell concentrations, and, therefore, that sonolysis of cells in vivo is unlikely. We present the results of a retrospective analysis of experimental data and a theoretical analysis; these indicate that while relative sonolytic yield declines with increasing cell concentration, the "absolute" extent of ultrasound-induced lysis remains large. We find evidence that in vitro sonolysis of cells is limited at high cell densities by the number of available microbubbles and/or the number of cells each bubble may encounter and lyse prior to bubble "inactivation." Theory indicates the latter arises in consequence to the cell concentration-dependent formation of cell aggregates around pulsating bubbles.

Hemolysis of albunex-supplemented, 40% hematocrit human erythrocytes in vitro by 1-MHz pulsed ultrasound: acoustic pressure and pulse length dependence.

The tested hypothesis was that ultrasound-induced hemolysis in blood supplemented with a microbubble contrast agent varies with ultrasound intensity and pulse duration. Human erythrocytes in autologous plasma containing 3.6% v:v Albunex microspheres were exposed to 1.07-MHz ultrasound pulses of 5 to 1000 mus at SPTP intensities of 0 to 1100 W/cm2. The dependence of hemolysis on the mechanical index (MI) value of the exposures was also examined. Ultrasound-induced hemolysis: (1) was evident at all pulse/intensity combinations; (2) increased generally with increasing pulse duration at constant intensity; and (3) increased with increasing MI at constant pulse duration. For pulses of 10 to 30 mus, ultrasound-induced hemolysis remained low (< or = 2%) at MI values < approximately 2 and increased sharply with further increase in MI; for 5-mus pulses, this abrupt increase in hemolysis was associated with a larger MI (approximately 3).

A review of in vitro bioeffects of inertial ultrasonic cavitation from a mechanistic perspective.

This selective review of the biological effects of ultrasound presents a synopsis of our current understanding of how cells insonated in vitro are affected by inertial cavitation from the standpoint of physical and chemical mechanisms. The focus of this review is on the physical and chemical mechanisms of action of inertial cavitation which appear to be effective in causing biological effects. There are several fundamental conditions which must be satisfied before cavitation-related bioeffects may arise. First, bubbles must be created and then brought into proximity to cells. Exposure methods are critical in this regard, and simple procedures such as rotation of a vessel containing the cells during exposure can drastically alter the results. Second, once association is achieved between bubbles and cells, the former must interact with the latter to produce a bioeffect. It is not certain that the inertial event is the prime mechanism by which cells are lysed; there is evidence that the turbulence associated with bubble translation may cause lysis. Additionally, there appear to be chemical and other physical mechanisms by which inertial cavitation may affect cells; these include the generation of biologically effective sonochemicals and the apparent emission of ultraviolet (UV) and soft X-rays. The evidence for inertial cavitation occurring within cells is critically reviewed.

A test of the hypothesis that a 60-Hz magnetic field affects ornithine decarboxylase activity in mouse L929 cells in vitro.

Four replicate experiments were performed to test the hypothesis that a 4-hr exposure of L929 cells in vitro to a 60 Hz, 10 microT magnetic field results in a large increase in ornithine decarboxylase (ODC) enzyme activity (1-2). A positive control yielded a highly statistically significant increase in ODC activity. However, magnetic field exposure had no statistically significant effect on extractable ODC activity of L929 cells relative to that of sham-exposed cells.