Download Cavitation Induced Cell Detachment and Membrane Permeabilization

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.
"
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2003 IEEE ULTRASONICS SYMPOSIUM-839
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2003 IEEE ULTRASONICS SYMPOSIUM-840