Verification of protective effect of bubbles attached
to vascular endothelial cells on elastic wall from
cavitation under ultrasound exposure
Yoshiki Ito, Shunya Watanabe, Narumi Ogawa,
and Kohji Masuda
Grad. School of Bio-Applications and Systems Eng.,
Tokyo Univ. of Agriculture and Technology,
Koganei, Tokyo, Japan
ultrason@cc.tuat.ac.jp
Abstract—We investigated the viability of vascular
endothelial cells under ultrasound exposure in floating and
adherent cell conditions with the two types of lipid bubbles
which attach to the cells and do not attach to the cells. In the
floating and adherent conditions, continuous ultrasound was
irradiated to the suspension containing the cells and the bubbles
at a frequency of 3 MHz with a maximum sound pressure of 400
kPa-pp. Then, we acquired fluorescent images to derive the cell
viability in both conditions of the cells. Comparing the cell
viability on the adherent condition and the floating condition
using bubbles which do not attach to the cells, we confirmed that
higher cell viability in the adherent condition. In the adherent
condition, the cell viability with lipid bubbles attaching was
higher than that with floating bubbles, which indicates that the
cells were protected by the bubbles attached to the cell surface.
Keywords—Vascular endothelial cell, Viability, Lipid bubble,
Adherent condition, Floating condition
I. INTRODUCTION
Microbubbles have been utilized for physical drug
delivery to allow the uptake of larger molecules into cells,
where the advantage is the ease in determining the distribution
of bubbles functioning as contrast agent in blood flow. To
prevent the diffusion of bubbles after injection into the human
body and to reduce side effects such as relapse and metastasis
inhibitory effects, we have been researching a method to
control bubbles [1-4] using acoustic radiation force. Those
results showed the possibility of controlling bubbles in in
blood vessels by performing active induction in a bifurcated
path [5-7]. Considering an actual situation of the therapy, a
catheter should be used to make bubbles closer to the target
area. In those situations, the biological effect on vascular
endothelial cells due to cavitation that occurs upon the
destruction of bubbles under ultrasound exposure [8] should
be taken into account. In our previous study, we investigated
the effect on T-cells [9] and vascular endothelial cells [10] in
the presence of microbubbles, where cell damage increased
with respect to the sound pressure, exposure time, and
concentration of bubbles. However, those experiments were
conducted where the vascular endothelial cells were floating
in a medium, which is different from the in vivo conditions.
Therefore, in this study, we prepared a similar situation where
the vascular cells are fixed to compare the cell viability
between different presence conditions of the cells.
Meanwhile, using two types of bubbles, we investigate the
degree of cell damage caused by ultrasound irradiation with
the concentration of bubbles.
XXX-X-XXXX-XXXX-X/XX/$XX.00 ©20XX IEEE
Yoshitaka Miyamoto
National Center for Child Health and Development,
Setagaya, Tokyo, Japan
Daiki Omata, Ryo Suzuki
Faculty of Pharma-sciences, Teikyo University,
Itabashi, Tokyo, Japan
II. METHODS
A. Bubbles and cells
We used bovine-derived carotid epithelial HH cells (cells,
hereinafter) [10]. They were cultured in Eagle's minimal
essential medium containing 10% fetal calf serum at 37°C and
5% CO2 concentration. Also, we prepared lipid bubbles (LBs),
containing perfluoropropane (PFP, C3F8) gas and composed
of DSPC and DSPE-PEG2k [11,12]. The obtained LBs had an
average diameter of 100 nm and were encapsulated with
phosphate buffer solution in a liposome. Then, we prepared
modified LBs by conjugating cyclic-RGD peptides [13],
which covalently adhered to the vascular endothelial cells on
the LB surfaces. In the following description, the modified
LBs are denoted as “LBs (+)” and the original ones are
denoted as “LBs (−)”.
B. Ultrasound exposure with adherent condition of cells
In this procedure, we prepared a channel with a rectangular
cross-section having a width of 2.0 mm, a depth of 2.0 mm,
and a length of 80 mm, made of PDMS. At the bottom of the
channel, a basement membrane of collagen film was coated.
A solution of Cell matrix Type I-C (Nitta Gelatin) was filled
in the flow channel. Allowing to stand for 1 h after the
collagen fibers were precipitated, the supernatant was
removed and dried for 30 min. Figure 1 shows the procedure
for ultrasound exposure to the cells and LBs in the channel.
After the cells were seeded into the channel, the cells were
cultured for 24 hours in an incubator at a constant temperature
of 37°C with a CO2 concentration of 5% to achieve the
adherent condition. The LBs suspension was injected into the
channel, as shown in Fig. 1 (a). Next, the channel was placed
in a water tank filled with degassed water to expose
ultrasound, as shown in Fig. 1 (b). The ultrasound transducer
had a concave ceramic disc with a central frequency of 3 MHz
to irradiate continuous wave ultrasound with a maximum
sound pressure of 400 kPa-pp. After the ultrasound exposure
for 60 s, the channel was reversed to the original position to
be put in a CO2 incubator to culture the cells for 1 hour.
Fig. 1. Ultrasound exposure in the adherent condition of the cells.
C. Ultrasound exposure with floating condition of cells
Figure 2 shows the procedure to expose ultrasound to the
cells with floating conditions contained with the LBs. We used
the same experimental setup as described in the previous
section. The concentrations of the cells and the LBs were also
similar. In Fig. 2 (a), the cell suspension and the LBs were
confined in the channel. Next, the channel was exposed by
ultrasound in water, as shown in Fig. 2 (b). Then, the channel
was put in a CO2 incubator for 4 hours to allow the floating
cells to precipitate at the bottom of the vessel.
Fig. 2. Ultrasound exposure in the floating condition of the cells.
III. RESULTS
Figures 3 shows the cell survival rate versus applied
maximum sound pressure with LBs concentrations of 0.3
mg/mL. The error bars in Fig. 3 indicate the standard deviation
of the retention area obtained in 4 attempts. Although there
was no significant difference in cell damage when the
maximum sound pressure was lower than 300 kPa-pp, the
following tendencies were observed when the maximum
sound pressure was 400 kPa-pp: 1) the cell viability with LBs
(+) was higher than that with LBs (–) in the adherent condition
of the cells and 2) the viability of the cells in the adherent
condition was higher than that in the floating condition using
LBs (–).
Fig. 3. Comparison of cell survival rate between the adherent and the
floating conditions of the cells versus maximum sound pressure with the LBs
concentration of 0.3 mg/mL.
IV. CONCLUSION
We investigated the cell viability under ultrasound
exposure in floating and adherent cell conditions with various
concentrations of the two types of lipid bubbles; LBs (+)
which attach to the cells and LBs (–) which do not attach to
the cells. In the floating and adherent conditions, continuous
ultrasound was irradiated to the suspension containing the
cells and the LBs, before and after the precipitation of the cells
in a channel. Then, we acquired fluorescent images to derive
the cell viability in both conditions of the cells. In the adherent
condition, the cell viability with LBs (+) was higher than that
with LBs (–), which indicates that the cells were protected by
the LBs attached to the cell surface. In the floating condition,
we confirmed the decrease of the cell viability according to
the sound pressure and LBs concentration, as well as our
preceding research. The experiment with LBs (+) was not
possible due to the buoyancy of the cells, however, upon
comparing the adherent condition and the floating condition
using LBs (–), higher cell viability in the adherent condition
was confirmed. In the case of adherent cell condition using
LBs (+), cell damage was not confirmed through this study.
We are going to utilize the benefits for future development of
ultrasound therapy.
ACKNOWLEDGMENT
This research was supported by a grant from the Japan
Society for the Promotion of Science (JSPS) through
KAKENHI and the Uehara Memorial Foundation.
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