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Cavitation induced cell detachment and membrane permeabilization Bernhard Wolfrum, Robert Mettin, Thomas Kurz, and Werner Lauterborn Drittes Physikalisches Institut, Bürgerstr. 42-44, Universität Göttingen, D-37073 Göttingen, Germany Claus-Dieter Ohl Department of Applied Physics, Physics of Fluids, TU Twente, Postbus 217, 7500 AE Enschede, The Netherlands. Abstract— The effects of cavitation bubble dynamics on cells has been investigated with respect to cell damage, cell detachment and membrane permeabilization. High-speed images show the dynamics of pressure excited contrast agents and of lithotripter generated cavitation bubbles adjacent to cells. It is shown that cells are detached from a substrate by the action of cavitation bubbles. Furthermore, fluorescence labeling techniques indicate destruction and transient permeabilization of cell membranes after cavitation activity conterminous to the sites of cell detachment. experiment, contrast agent bubbles located adjacent to rat kidney fibroblast (NRK) cells in culture are excited by a spark-induced pressure wave. Images of the bubble dynamics and its effect on cells are captured with a high-speed camera connected to a table top microscope, as depicted in Fig. 1. The pressure created by the spark is measured with a fiber optical hydrophone (FOPH 300) at the point of observation. The pressure wave consists of a few short ns) with amplitudes at pulses (approximate width MPa followed by a low amplitude tensile around pulse ( MPa) lasting about a microsecond. I. I NTRODUCTION Ultrasound and shock waves are widely used in medical applications such as ultrasonic imaging techniques and lithotripsy. Some of these applications bear the risk of generating cavitation inside the human body. Particularly during shock wave lithotripsy, tissue damage has been observed as a side effect, which is probably caused by the action of cavitation bubbles [1], [2]. Furthermore, it was shown that shock waves and ultrasound may transiently enhance the permeability of cell membranes to facilitate drug delivery of usually non membrane-permeant molecules [3]–[9]. Drug delivery is a precondition for other applications including gene therapy, and is therefore subject to intensive research. In this work we concentrate on the interaction between cavitation bubbles and cells in culture and show that rapid bubble dynamics may lead to drug delivery and cell damage. II. M ATERIALS AND M ETHODS The interaction of cavitation bubbles with cells is investigated with two distinct setups. In the first 0-7803-7922-5/03/$17.00 (c) 2003 IEEE flash lamp lens fibre bundle filling (electrode liquid) objective with adjustable aperture fiber−optic hydrophone electrodes fiber electrode coating spark generator cells + contrast agent transient recorder objective (microscope) controlling computer trigger trigger images camera delay generator Fig. 1. General setup for the excitation of contrast agents and subsequent observation of bubble dynamics. In the second setup human epithelial uterus cancer cells (HeLa) adhering to the bottom of a culture flask are exposed to lithotripter generated shock waves 2003 IEEE ULTRASONICS SYMPOSIUM-837 and cavitation. High-speed imaging techniques are used to capture pictures of cells before, during and after bubble formation. Adhering and detached cells are subsequently analyzed to provide evidence for transient membrane permeabilization. The medium is supplemented with a non membrane-permeant fluorescent dye (FITC-Dextran, 20 kDa) prior to shock wave application. After shock wave exposure the cells are washed in phosphate buffered saline solution (PBS) to dispose of the dispensable fluorescent dye. Cells appearing green under the fluorescence microscope show molecular uptake facilitated by transient membrane permeabilization. The viability of cells was assessed using an acridine orange/ethidium bromide staining procedure. is subjected to the flow field induced by the bubble dynamics. The shear stress caused by the bubble induced flow is especially strong during collapse of the previously expanded bubbles. Figure 3 shows the collapse of an expanded contrast agent bubble as seen through a cover slide. The slide itself poses as a rigid boundary. During asymetrical collapse a jet evolves, III. C ONTRAST AGENT BUBBLE DYNAMICS In Fig. 2 we can see several contrast agent bubbles in the vicinity of a single NRK cell. After pressure 1 5 2 6 3 7 4 8 Fig. 2. Expanding contrast agent bubbles close to a cell after excitation by a pressure wave. A rectangular area of each frame around the cell has been contrast enhanced. The bubbles collapse first (frame 2) and subsequently rebound and expand due to a negative pressure pulse. The exposure and interframe times are 200 ns and 1 s, respectively. The frame width is m. wave application, the small bubbles first collapse as seen in frame 2 and subsequently expand due to the low amplitude tensile pulse. In the beginning of the rebound phase (frame 3) the center bubble has fragmented into three parts, which coalesce during later expansion (frame 4 and 5). The cell is squeezed in between the center and the topmost bubble and Fig. 3. Contrast agent bubble collapsing aspherically onto a glass surface after pressure wave excitation. The first frame is taken before pressure excitation. The second frame starts after expansion of the contrast agent due to a negative pressure pulse. The exposure and interframe times after frame 1 are 200 ns and 400 ns, respectively. which penetrates the bubble and is directed towards the boundary. This leads to the toroidal shape after collapse as seen in frame 4 of Fig. 3. During rebound the remaining fragments of the bubble torus round up due to the surface tension. Other factors such as neighboring bubbles and the propagation direction of the pressure wave also influence bubble collapse and jet formation [10]. Although single cells do not seem to significantly affect collapse behavior, they are still subjected to a strong shear flow during a jet-like bubble collapse in their vicinity. IV. C AVITATION INDUCED CELL DETACHMENT The effects of bubble induced shear flow on cells becomes evident during the collapse of lithotripter generated bubbles in the vicinity of cells adhering to a substrate. Figure 4 shows the detachment of cells caused by the action of a cavitation bubble after shock wave application. The bubbles are generated in the tension phase, which is following the focused lithotripter shock wave. The previously described collapse behavior of expanded contrast agents can be transferred similarly to the collapse of the larger lithotripter induced bubbles. 2003 IEEE ULTRASONICS SYMPOSIUM-838 −500 ms 17 µs 516 ms 30 s Fig. 4. Cell detachment caused by a lithotripter induced cavitation bubble. Time data is given with respect to shock wave impact on the substrate and exposure times are 2 s. The frame size is m . Fig. 5. The image shows cells after shock wave exposure under a fluorescence microscope. Cells displaying green color have taken up FITC-Dextran from the extracellular medium. Red color reveals dead cells because of the intercalation of ethidium bromide into the cells DNA and RNA. The frame size is m . A jet evolves, which is directed towards the boundary (i.e. the substrate supporting the cell layer). The jet induces a shear flow at the boundary, which causes cell detachment. As a result, roughly circular regions appear in a confluent cell layer, where the supporting substrate is cleared of cells as shown in Fig. 5. ! VI. C ONCLUSION In this work the interactions of cavitation bubbles with cells were investigated. High-speed images show that contrast agent bubbles with an initial diameter of a few microns may expand to more than 50 m after MPa application of small tensile pressures of amplitude and s duration. After expansion the bubbles collapse evolving a jet toward the cell substrate and exert a strong shear flow on neighboring cells. Furthermore, lithotripter generated bubbles have been shown to detach adherent cells from a substrate using the same mechanism. Cells lining the border to vacated regions on the substrate reveal molecular uptake of non membrane-permeant molecules indicating transient membrane permeabilization. " V. M EMBRANE PERMEABILIZATION AND MOLECULAR DELIVERY Shear flow does not only cause cell detachment but may also lead to transient and permanent membrane permeabilization. This is visible in Fig. 5, where the green color of cells reveals the uptake of FITCDextran potentiated by transient membrane permeabilization. As can be seen, the molecular uptake is concentrated at the ring lining the border to a vacated region. This strongly suggests that the membrane permeabilization is also caused by the bubble induced shear flow. The rapid breaking of cell-cell adhesion bonds may also contribute to the poration of cells. Applying multiple shock waves, the cavitation clouds generated by a lithotripter can be used to clear cells from the whole bottom of a culture flask. After such % of the cells are a cell detachment procedure permanently damaged, while % of the surviving cells reveal molecular uptake of FITC-Dextran. " R EFERENCES [1] M. Delius, R. Denk, C. Berding, H.-G. Liebich, M. Jordan, and W. Brendel, “Biological effects of shock waves: Cavitation by shock waves in piglet liver,” Ultrasound in Med. & Biol., vol. 16, no. 5, pp. 467–472, 1990. [2] A. J. Coleman and J. E. Saunders, “A review of the physical properties and biological effects of the high amplitude acoustic fields used in extracorporeal lithotripsy,” Ultrasonics, vol. 31, no. 2, pp. 75–89, 1993. 2003 IEEE ULTRASONICS SYMPOSIUM-839 [3] S. Gambihler, M. Delius, and J. W. Ellwart, “Permeabilization of the plasma membrane of l1210 mouse leukemia cells using lithotripter shock waves,” J. Membrane Biol., vol. 141, pp. 267–275, 1994. [4] S. Bao, B. D. Thrall, R. A. Gies, and D. L. 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Miller, “Gene transfection and drug delivery,” Ultrasound in Med. & Biol., vol. 26, no. Sup. 1, pp. 59–62, 2000. [10] B. Wolfrum, R. Mettin, T. Kurz, and W. Lauterborn, “Observations of pressure-wave-excited contrast agent bubbles in the vicinity of cells,” Appl. Phys. Lett., vol. 81, no. 26, pp. 5060–5062, 2002. 2003 IEEE ULTRASONICS SYMPOSIUM-840