Rotating Tube Exposure Research Articles

Sonoporation of cultured cells in the rotating tube exposure system

Suspensions of Chinese hamster ovary cells were exposed to ultrasound in the presence of fluorescent dextran to determine the conditions needed for sonoporation with uptake of the large molecules. Albunex®, a gas-body–based ultrasound contrast agent, was added to enhance cavitation. Ultrasound was continuous wave at frequencies of 1.0, 1.68, 2.25, 3.3, 5.3, and 7.15 MHz. Sterile 4.5-mL exposure chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures. After exposure, cells were tested for sonoporation by counting fluorescent cells and for cell lysis by counting cells stained by trypan blue. Sonoporation was a sensitive bioeffects indicator that was detected at pressure amplitudes lower than were needed for transient cavitation or cavitation-induced cell lysis. For 10% Albunex, apparent thresholds for sonoporation, which were comparable to the levels required to perturb the gas bodies, were 0.084 MPa (spatial peak negative pressure amplitude) from 1.0–3.3 MHz and 0.27 MPa at 5.3 and 7.15 MHz. Sonoporation decreased slightly if the tube was not rotated. The effects increased for increasing Albunex concentration (with rotation). The plating efficiency of cells exposed to 0.2 MPa at 2.25 MHz and sorted by a flow cytometer was 19% (3.6% standard deviation [SD]) for fluorescent cells, compared to 67% (1% SD) for nonfluorescent exposed cells and 62% (6% SD) for sham-exposed cells. The reduced viability represents an important consideration for possible applications of sonoporation.

Transfection of a reporter plasmid into cultured cells by sonoporation in vitro

Cultured Chinese hamster ovary cells were exposed to 2.25-MHz ultrasound in sterile 4.5-mL polyethylene chambers and tested for cell lysis, sonoporation and DNA transfection. Ten percent of Albunex ®, a gas-body-based ultrasound contrast agent, was added to ensure cavitation nucleation, and the chambers were rotated at 60 rpm to promote cavitation activity during the 1-min exposures. Uptake of large fluorescent dextran molecules by some cells was observed for spatial peak pressure amplitudes as low as 0.1 MPa, which indicates transient permeabilization and resealing, i.e., sonoporation, of these cells during exposure. Significant lysis occurred for 0.2 MPa, and increased rapidly for exposures above the apparent cavitation threshold (using the H 2O 2 production test) of about 0.4 MPa spatial peak pressure amplitude. In the DNA transfection tests, 20 μg/mL luciferase reporter plasmid was added to the suspension during exposure, and cells were assayed for proliferation ability and luciferase gene expression 2 days after exposure. Cell proliferation was greatly reduced above the cavitation threshold. Luciferase production was significant for 0.20-MPa exposure, and reached 0.33 ng per 10 6 cells at 0.8-MPa exposure. The luciferase production was greater for cells exposed in medium supplemented with serum than for cells exposed in serum-free medium. Cells harvested for exposure either in the log phase or in the stationary phase of culture gave similar proliferation and transfection results. The effects essentially disappeared when the Albunex ® was omitted from the suspension and the tube was not rotated. Thus, sonoporation by ultrasonic cavitation in the rotating tube exposure system yields plasmid transfection with subsequent transient gene expression.

Induction of base damage in dna solutions by ultrasonic cavitation

Ultrasound can damage macromolecules by the mechanical (shearing) and sonochemical (free radical generating) action of ultrasonic cavitation. Attributing macromolecular damage to either direct mechanical stress or to indirect mechanisms involving free radicals or other sonochemicals is a challenging problem. DNA damage induced by ultrasound was evaluated by measuring the formation of purine and pyrimidine products using combined gas chromatography-mass spectrometry with selected ion monitoring. Samples of DNA were prepared in 10 mmol dm −3 phosphate buffered saline (pH 7.4) and saturated with a mixture of argon:oxygen (3:1). Continuous 2.17 MHz ultrasound exposures at 0.82 mPa spatial peak negative pressure amplitude were performed in a 60 rpm rotating tube exposure system. Hydrogen peroxide yields were measured after each exposure to quantify the cavitation activity and ranged up to 350 μmol dm −3 for 1-h exposures. Purine and pyrimidine products identified were those typically observed following exposure of DNA to hydroxyl radical-generating systems, such as ionizing radiation, hypoxanthine/xanthine oxidase, or hydrogen peroxide in the presence of transition metal ions. The yields of these products were directly correlated with cavitation activity as measured by residual hydrogen peroxide concentrations. The yields of DNA products increased in the following order: thymine glycol∼cytosine glycol>8-oxoAde>:FAPyAde-5-HMU∼5,6-diOHCyt>FAPyGua. Unexpectedly, 8-oxoguanine did not exhibit a dose-dependent increase above background levels, and this observation is inconsistent with processes involving metal ion-dependent formation of hydroxyl radicals from hydrogen peroxide. In addition, the product yields were far too large to result from the residual hydrogen peroxide. Thus, ultrasonic cavitation appears to have a mode of action distinct from either ionizing radiation or formation of hydroxyl radicals via Fenton-like reaction with transition metals.

Ultrasound contrast agents nucleate inertial cavitation in vitro

Some ultrasound contrast agents contain stable bodies of gas, and this study was undertaken to determine if these agents could provide nuclei for inertial cavitation. Inertial cavitation was detected and assessed by the measurement of the sonochemical hydrogen peroxide after exposure to 2.17-, 2.95- or 3.8-MHz ultrasound. A noncavitating system was obtained by removing cavitation nuclei from the rotating tube exposure chambers by vacuum degassing, and from the phosphate-buffered saline medium by filtering. Albunex ® added at 10 −2, 10 −3 or 10 −4 dilutions, or Levovist ® added at 2 mg mL −1, 0.2 mg mL −1 or 0.02 mg mL −1 all initiated significant H 2O 2 production for 2.17-MHz ultrasound at 0.41 MPa or higher spatial peak pressure amplitude for 5 min exposure gated at 0.25 s on and off with 60-rpm rotation. Not rotating the tube virtually eliminated H 2O 2 production. For 2.5-min continuous exposure, both agents initiated significant H 2O 2 production for 2.95-MHz exposure at 0.58 MPa or higher, but not for 3.8-MHz exposure up to 1.16 MPa. Bubble-based ultrasound contrast agents therefore appear to be able to provide nuclei for inertial cavitation in the rotating tube exposure system.

Frequency dependence of cavitation activity in a rotating tube exposure system compared to the mechanical index

Cavitation was produced by focused and unfocused ultrasonic beams in a 60-rpm rotating-tube exposure system. Hemolysis, free radical generation, and hydrogen peroxide were assayed after exposure to determine the dependence of cavitation activity on pressure-amplitude p and frequency f. For unfocused exposure, hemolysis rose rapidly from an apparent threshold pressure amplitude, to a maximum value and then declined gradually at higher amplitudes. Cavitation activity decreased with increasing ultrasonic frequency for a fixed pressure amplitude. Apparent thresholds for unfocused exposure were similar for hemolysis after 15 s and H2O2 production after 15 min. These thresholds increased roughly in proportion to increasing ultrasonic frequency. Cavitation activity was lower and thresholds were higher for focused exposures than for unfocused exposures. The approximate pf-1 = 0.20 MPa/MHz dependence of the experimental threshold of H2O2 production from 0.745 to 2.95 MHz differs from the pf-1/2 = 0.24 MPa/MHz1/2 dependence of the theoretical "mechanical index" threshold for inertial cavitation.

A comparison of hemolytic and sonochemical activity of ultrasonic cavitation in a rotating tube

Biological effects of in vitro ultrasonic exposure may result from mechanical and from sonochemical mechanisms related to ultrasonic cavitation. Mechanical cell lysis by 1.61 MHz ultrasonic cavitation was assessed in a rotating tube exposure system by hemolysis measurements. Free radical generation was assessed by the terephthalic acid dosimeter, calibrated by gamma-ray dosage. Sonochemical production was assessed by measuring residual hydrogen peroxide using the sensitive isoluminol method. Exposure conditions were similar for all tests, except longer durations were needed for the free radical and hydrogen peroxide tests. The sonochemical mechanisms were relatively more important for increasing intensity, and increasing temperature. Increasing rotation speed or bubbling with argon before exposure enhanced all cavitation activity. Stopping the rotation reduced all cavitation activity. Burst-mode ultrasound (10.5 μs bursts, 1:1 or 1:3 duty cycle) reduced cavitation activity, but gave relatively greater sonochemical activity under some conditions. These results indicate that the mechanical and sonochemical mechanisms can be separately favored to some extent by varying exposure conditions. The observed trends should be helpful for selecting exposure conditions favorable for studying bioeffects of the sonochemical mechanism.

The Effects of the Microbubble Suspension SH U 454 (Echovist®) on Ultrasound‐Induced Cell Lysis In a Rotating Tube Exposure System

Human erythrocytes resuspended at different hematocrits in autologous plasma at 37 degrees C were exposed to the therapeutic intensities of continuous-wave 0.75-MHz ultrasound in vitro in a rotating tube exposure apparatus designed to maximize the destructive effects of cavitational activity. Provided that large numbers of additional gas bubbles had not been introduced during the various preparative and manipulatory procedures, the addition of Echovist at final concentrations comparable with those currently being used for clinical investigations resulted in a statistically significant increase in the amount of cell lysis in vitro in those samples having hematocrits less than 2%. The amount of cell lysis produced at any given ultrasound intensity decreased with increasing hematocrit in both the controls and the cell suspensions containing Echovist until it was virtually zero in both cases at hematocrits of 5.5% or greater. The addition of Echovist to samples that already contained large numbers of stabilized gas bubbles and/or had hematocrits greater than 5.5% produced no detectable cell lysis even at ultrasonic intensities as high as 3 W/cm 2 spatial average, temporal average (SATA). It is therefore unlikely that Echovist would cause appreciable amounts of cell lysis when the gas bubbles were being exposed to ultrasound under the conditions used for clinical investigations in vivo.

Ultrasonic cavitation indirectly induces single strand breaks in DNA of viable cells in vitro by the action of residual hydrogen peroxide

Direct exposure of cells to vigorous ultrasonic cavitation results predominantly in mechanical cell lysis, but latent effects due to production of toxic sonochemicals can also be present. Phosphate buffered saline (PBS) was exposed to 1.61 MHz ultrasonic cavitation at 20°C in a rotating tube exposure system to build up sonochemical products. Single strand DNA breaks (SSBs) were then induced by treating Chinese hamster ovary (CHO) cells with the cavitated PBS for 30 min on ice. The SSBs resided in viable cells, as evidenced by their ability to repair the breaks when warmed. This indirect effect could be explained by the action of cavitation-generated hydrogen peroxide that had built up (e.g., to 16 μM after 30 min exposure) in the PBS. Dissolution of argon gas in the PBS before exposure enhanced the SSB effect and the H202 production. Addition of catalase to the cavitated PBS before cell treatment eliminated the H 2O 2 and the SSB effect. Tests with hydrogen peroxide showed that 16 μM H 2O 2 treatment for 30 min on ice was as effective as a 1 Gy dose of 60Co gamma rays in producing single strand breaks. The SSB effect of H 2O 2 and gamma rays was reduced by addition of the radical scavenger cysteamine to the cells before treatment, but cysteamine did not reduce the SSB effect of direct exposure to ultrasonic cavitation. These results help to clarify the potential for genetic effects from ultrasonic cavitation.

Bubble cycling as the explanation of the promotion of ultrasonic cavitation in a rotating tube exposure system.

The cavitation-promotion effect of rotating a sample tube during ultrasound exposure is not yet fully understood. Cavitation-induced hemolysis was observed in 0.5% hematocrit suspensions of erythrocytes exposed for 5 min at 37 degrees C. For 1.61 MHz unfocused exposure an apparent threshold of 4 W/cm2 spatial-peak intensity was obtained for hemolysis in a 72 rpm rotating exposure chamber which was made of dialysis tubing. If the tube was not rotated, then no hemolysis was observed up to 16 W/cm2. Creation of standing waves in the sample by using a thick-walled culture tube, or by placing a styrofoam reflector behind the dialysis tube did not change the apparent threshold. Focused 1.45 MHz exposure gave the same apparent threshold in a rotating dialysis tube, but this increased to about 128 W/cm2 for the nonrotating case. When the focused ultrasound was turned on for half a rotation and off for half, results were similar to those for a nonrotating CW case, while on-off cycles synchronized to a quarter rotation gave results similar to the rotating CW case. A plausible explanation of the rotation effect, given these results, is that rotation brings bubbles on the rear wall, where the beam exits the tube, back around to the front again, where the bubbles may then re-nucleate the suspension, and thereby promote cavitation bioeffects in the rotating tube.