Morton W. Miller

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.

Biological effects of ultrasound

When ultrasound is absorbed by tissue, heat is produced which may be destructive or, if controlled, can be therapeutic. In addition, ultrasound produces biological effects which cannot be explained on the basis of heating alone. Under certain exposure conditions production of ultrasonic lesions in brain tissue can be explained on a purely thermal basis. Examples of nonthermal effects of ultrasound include changes in growth, mitotic index and the production of chromosomal aberrations in plant roots.

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.

Pulsed enhancement of acoustic cavitation: A postulated model

Abstract Iodine-131 labeled sodium iodide was used to demonstrate an iodine release reaction indicative of cavitation activity. Exposure of Na131I at varying pulsed regimes (1:1 duty cycle, 60 sec-60 μsec pulse duration) and intensities (10–30W/cm2) resulted in an increased efficiency of pulsed ultrasound to produce iodine release compared to continous wave exposures. A model based on the concurrent operation of two mechanisms has been proposed to explain this phenomenon.

Growth retardation in Chinese hamster V-79 cells exposed to 1 MHz ultrasound

Abstract Asynchronous Chinese hamster V-79 cells were exposed in suspension to continuous wave 1 MHz ultrasound for 1 min at an axial intensity of 2.5 W/cm 2 , then allowed to grow in monolayer culture. There was little if any growth, as determined by changes in the number of attached cells, for 24 hr after sonication, then a return to normal growth approximately 36 hr after sonication. The lack of growth during the first 24 hr appears to be due to an approximate balance between proliferation of viable cells and loss of non-viable cells into the medium.

Lysis and viability of cultured mammalian cells exposed to 1 MHz ultrasound

Abstract HeLa and CHO cells were exposed for 1 to 15 min to 1 MHz ultrasound at intensities up to 30 W/cm 2 . The threshold for cell lysis was approximately 1 W/cm 2 with the maximum effect at 10 W/cm 2 . Among the intact cells there was a decreased viability as determined both by trypan blue exclusion and by colony-forming ability; the intensity vs. response curve was similar to that fur cell lysis. Preliminary evidence also suggests that a decrease in proliferation rate and an increased incidence of giant cells occur for the remaining intact and viable cells.

Effect of a Stabilized Microbubble Contrast Agent on CW Ultrasound Induced Red Blood Cell Lysis In Vitro

Human red blood cells (RBCs) in vitro at various fractional hematocrits (HCTs) were exposed for 60–120 seconds in a dialysis tubing vessel to 1 MHz continuous wave ultrasound (0–5 W/cm2 SPTA intensity); exposure vessels were either rotated at 200 rpm or stationary. Some RBC suspensions also contained Albunex® (ALX; a commercially‐produced microbubble clinical ultrasound contrast agent) at final concentrations ranging from 0–41 μL/mL RBC suspension. Isonation was either by one transducer or by two opposing, continuously‐gated, balanced transducers. For the vessel rotation / no rotation experiments, ultrasound‐induced cell lysis was always increased with the ALX regimen relative to that of the no‐ALX regimen at HCTs up to about 10%; at higher HCTs (including whole blood), ultrasound‐induced hemol‐ysis essectially ceased to occur. The data are consistent with reports of the ineffectiveness of continuous wave ultrasound at 3–5 W/cm2 to lyse cells in vitro at physiological cell densities with or without echocontrast medium supplemantation.

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).

Effect of a Stabilized Microbubble Echo Contrast Agent on Hemolysis of Human Erythrocytes Exposed to High Intensity Pulsed Ultrasound

Microbubble contrast agents have been shown to enhance ultrasonic cell lysis in vitro when exposed to continuous‐wave ultrasound having spatial peak temporal average (SPTA) intensities of a few W/cm2. The response is strongly dependent upon the hematocrit (HCT) of the cell sample; detectable cell lysis essentially disappears as the HCT approaches 5%‐10%. This study was conducted to determine whether high intensity pulsedsound is an effective lytic agent in the presence of preexisting potential cavitation nuclei (Albunex® contrast agent). Human erythrocytes weresuspended in autologous plasma to HCTs ranging from 1%–40%. Suspensions were exposed or sham exposed for 60 seconds to focused, pulsed ultrasound. The pulse duration was 1 msec, and the pulse repetition frequency was 20 Hz. The pressure amplitudes, spatial peak pulse average (SPPA) intensity, and SPTA intensity were 4.7 MPa peak positive pressure, ‐2.7 MPa peak negative pressure, 420 W/cm2, and 8.5 /cm2, respectively. Samples were exposed to ultrasound in a dialysis membrane exposure vessel rotating at 200 rpm. When included in the erythrocyte samples, the Albunex concentration was 35 μL/mL suspension. Significant ultrasound‐induced hemolysis in the absence of Albunex was observed only at the lowest HCT value tested (1%). In the presence of Albunex significant cell lysis was observed at all tested HCT values. The relative fraction of cells lysed by the combination of ultrasound exposure and Albunex diminished with increasing HCT, but the number of cells lysed per sample was nearly constant over the range of 5%–40% HCT. The ultrasound exposure parameters used in this study differ substantially from those associated with diagnostic imaging equipment; it is not valid to infer from the present results that the use of Albunex in diagnostic applications will induce or enhance hemolysis in vivo.