Experimental observations and numerical modeling of lipid-shell microbubbles with
calcium-adhering moieties for minimally-invasive treatment of urinary stones
Yuri A. Pishchalnikov
, William Behnke-Parks
, Kazuki Maeda
, Tim Colonius
, Matthew Mellema
, Matthew
Hopcroft
, Alice Luong
, Scott Wiener
, Marshall L. Stoller
, Thomas Kenny
, and
Daniel J. Laser
Citation:
Proc. Mtgs. Acoust.
35
, 020008 (2018); doi: 10.1121/2.0000958
View online:
https://doi.org/10.1121/2.0000958
View Table of Contents:
https://asa.scitation.org/toc/pma/35/1
Published by the
Acoustical Society of America
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Experimental
observations
and
numerical
modeling
of
lipid-shell
microbubbles
with
calcium-adhering
moieties
for
minimally-invasive
treatment
of
urinary
stones
Yuri
A.
Pishchalnikov
R&D,
Applaud
Medical,
Inc.,
San
Francisco,
CA,
94107
;
yurapish@gmail.com
William
Behnke-Parks
Applaud
Medical,
Inc.,
San
Francisco
,
CA,
94107;
will.parks@applaudmedical.com
Kazuki
Maeda
Department
of
Mechanical
Engineering,
University
of
Washington,
Seattle
, WA
, 98105
;
maeda@uw.edu
Tim
Colonius
Division
of
Engineering
and
Applied
Science,
California
Institute
of
Technology,
Pasadena
, CA
, 91125
;
colonius@caltech.edu
Matthew
Mellema,
Matthew
Hopcroft
and
Alice
Luong
Applaud
Medical,
Inc.,
San
Francisco
,CA,
94107
;
matt.mellema@applaudmedical.com,
matt.hopcroft@applaudmedical.com,
alice.luong@applaudmedical.com
Scott
Wiener
and
Marshall
L.
Stoller
Department
of
Urology,
University
of
California,
San
Francisco
,
CA
, 94143
;
svwiener@gmail.com,
Marshall.Stoller@ucsf.edu
Thomas
Kenny
Department
of
Mechanical
Engineering,
Stanford
University,
Stanford
,
CA
, 94305
;
kenny@cdr
.stanford.edu
Daniel
J.
Laser
Applaud
Medical,
Inc.,
San
Francisco
,
CA
94107
;
daniel.laser@applaudmedical.com
A novel treatment modality incorporating calcium-adhering microbubbles has recently entered human clinical trials as a
new minimally-invasive approach to treat urinary stones. In this treatment method, lipid-shell gas-core microbubbles can
be introduced into the urinary tract through a catheter. Lipid moities with calcium-adherance properties incorporated into
the lipid shell facilitate binding to stones. The microbubbles can be excited by an extracorporeal source of quasi-collimated
ultrasound. Alternatively, the microbubbles can be excited by an intraluminal source, such as a fiber-optic laser. With
either excitation technique, calcium-adhering microbubbles can significantly increase rates of erosion, pitting, and
fragmentation of stones. We report here on new experiments using high-speed photography to characterize microbubble
expansion and collapse. The bubble geometry observed in the experiments was used as one of the initial shapes for the
numerical modeling. The modeling showed that the bubble dynamics strongly depends on bubble shape and stand-off
distance. For the experimentally observed shape of microbubbles, the numerical modeling showed that the collapse of the
microbubbles was associated with pressure increases of some two-to-three orders of magnitude compared to the excitation
source pressures. This in-vitro study provides key insights into the use of microbubbles with calcium-adhering moieties in
treatment of urinary stones.
© 2019 Acoustical Society of America. https://doi.org/10.1121/2.0000958
Proceedings of Meetings on Acoustics, Vol. 35, 020008 (2019)
Page 1
1. INTRODUCTION
Stone-adhering microbubbles
1
have recently entered human clinical trials as a medical device for min-
imally invasive approach to treat urinary stones. Gas-filled microbubbles are introduced through a catheter
and adhere to urinary stones with calcium-adhering moieties incorporated into encapsulating lipid shells.
1–3
The microbubbles can be excited either minimally invasively (e.g., with a laser coupled to an optical fiber
delivered through the ureter via a ureteroscope) or non-invasively with an extracorporeal source of ultra-
sound(Fig. 1).
1
With either excitation technique, recent studies suggest that the stone-adhering microbub-
bles can significantly increase the breakage of urinary stones.
2,3
To better understanding the mechanisms of
action of microbubbles in treatment of urinary stones, here we studied the dynamics of microbubbles at the
surface of urinary stones
in vitro
. This study is a continuation of the work presented at the previous 175
th
meeting of the Acoustical Society of America.
4
The microbubbles were driven with quasi-collimated ultra-
sound at low intensities and studied using a high-speed video microscopy. The observed bubble geometry
and the stand-off distance were used as input parameters for the numerical modeling of the collapsing bub-
bles. The modeling showed that the collapse of stone-adhering microbubbles can produce pressure spikes
with amplitudes significantly greater than the amplitude of the driving acoustic waves.
Stone
Ureter
Ultrasonic
Transducer
Figure 1: The concept of treating urinary stones using microbubbles with calcium-adhering moieties.
Gas-filled microbubbles are introduced into the urinary tract through a catheter and adhere to an urinary
stone (middle). The adhered microbubbles are excited with an extracorporeal source of quasi-collimated
ultrasound and erode the stone facilitating its passage through the urinary tract.
2. MATERIALS AND METHODS
A. LIPID-SHELL MICROBUBBLES AND URINARY STONES
Stone-adhering microbubbles (Applaud Medical, Inc.) were made of perfluoroalkane gas
(
C
4
F
10
)
encap-
sulated into lipid shells with calcium-adhering moieties.
1–3
The chemical composition of the moieties was
based on a synthetic pyrophosphate analog structure conferring adhering affinity for calcium constituents of
urinary stones.
These experiments were conducted with surgically retrieved calcium-oxalate-monohydrate urinary stones.
The stones were hydrated in deionized water and positioned in the test tank to study the dynamics of mi-
crobubbles with a high-speed video microscopy (Fig. 2).
Y.
A.
Pishchalnikov
et al.
Lipid-shell
microbubbles
with
calcium-adhering
moieties
for
treatment
of
urinary
stones
Proceedings
of
Meetings
on
Acoustics,
Vol.
35,
020008
(2019)
Page
2
B. HIGH-SPEED VIDEO MICROSCOPY
Bubble dynamics was captured using a high-speed (HS) camera Shimadzu Hyper Vision HPV-X2 (Shi-
madzu, Kyoto, Japan). The camera had a burst image sensor FTCMOS2 with ISO sensitivity of 16,000
and a monochrome 10-bit resolution. The camera recorded 400- by 250-pixel frames either in FP or HP
mode. The FP mode captured every pixel recording 128 frames at a rate up to five million frames per second
(Mfps). The HP mode captured every other pixel interpolating the images to 400- by 250-pixel frames and
recording 256 frames at a rate up to 10 Mfps. The physical size of sensor pixels was 32 by 32
μ
m.
The high-speed camera was used with a Nikon Eclipse TS100 microscope with a 4
×
objective (4
×
/0.13
PhL DL, WD 16.4, Nikon Plan Fluor), a 2.5
×
projection lens (Nikon CF PL2.5
×
), and a 34-cm exten-
sion tube (Thorlabs Inc., Newton, NJ, USA). The optical magnification was determined using a metallized
hemacytometer (Hausser Bright-Line, Hausser Scientific, Horsham, PA, USA) and was 1
μ
m per pixel.
Nikon Eclipse TS100 microscope had inverted configuration in which the objective was positioned at
the bottom. To use this configuration, a test tank had a transparent glass window at the bottom of the tank
(Fig. 2). The window was made of a microscope slide (75
×
25
×
1 mm, VistaVision, VWR International,
LLC, Radnor, PA) glued along its edges to the bottom of the tank. The tank was 3-D printed from a ther-
moplastic material—acrylonitrile butadiene styrene—and covered with a waterproof coating (Marine Grade
Epoxy 109 Medium, Tap Plastics, CA, USA). The test tank was filled with six liters of water (PURELAB
Chorus 1 for Life Science Applications, ELGA, Veolia Water Solutions and Technologies, UK) with an
electrical resistivity of 18.2 MOhm-cm and the ultrafiltration to particle size less than 0.05
μ
m. The water
remained in the tank for several days and was in equilibrium with atmospheric gases.
We used both continuous and flashlight illumination. The continuous lighting was provided by a fluores-
cence illumination system EXFO X-cite 120 (XE120, Photonic Solutions Inc., Mississauga, Ontario, CA).
This light source used a 120-W Metal Halide lamp coupled to a liquid lightguide. The end of the lightguide
was positioned at about 1 cm above the stone to backlit the stone (Fig. 2). The side lighting was provided
by a flashlamp WRF300 (Hadland Imaging LLC, Santa Cruz, CA). This spark-discharge lamp produced
a light pulse with the duration of about
10
μ
s. The spark light was delivered through a liquid lightguide
illuminating the side of the stone proximal to the incoming acoustic waves (Fig. 2).
Figure 2: Experimental setup. Left: general view with the HS-camera (top left), the spark-light source
(bottom left), and the water test tank positioned over the inverted microscope (center). Middle: view in
the test tank. Right: zoomed up view of an urinary stone positioned at the focus of the microscope. Back-
and side-illumination was provided by two liquid lightguides positioned at about 1 cm from the stone.
Y.
A.
Pishchalnikov
et al.
Lipid-shell
microbubbles
with
calcium-adhering
moieties
for
treatment
of
urinary
stones
Proceedings
of
Meetings
on
Acoustics,
Vol.
35,
020008
(2019)
Page
3
C. DRIVING ACOUSTIC WAVES
Driving acoustic waves were generated with a custom-made piezo-electric transducer (manufactured for
Applaud Medical by Sonic Concepts, Inc., Bothell, WA). The active element of the transducer was made
of a piezo-electric plate (72.3
×
30.3
×
3.18 mm) divided into eight elements and connected in pairs. Each
pair was driven by one of the four controllable power amplifiers (AP-400B, ENI, USA). The frequency and
duration of the acoustic bursts were computer controlled by a specially designed signal generator, allowing
us to not only reproduce the frequency modulation used in the clinic but also to study other driving regimes.
In this work, the acoustic bursts were generated with a frequency set of 400, 400, 433, and 433 kHz.
Acoustic waves were measured using a needle hydrophone with a frequency range of 50 kHz–1.9 MHz
(Y-104, Sonic Concepts, Inc., Bothell, WA). The sensitive element of the hydrophone was a ceramic crystal
with a diameter of 1.5 mm. The sensitive element was embedded at the rounded tip of the hydrophone
enclosed in a metal tube with a diameter of 3 mm. As the diameter of the tip was comparable with the
wavelength of acoustic waves (
∼
3
.
4
−
3
.
8
mm), the hydrophone sensitivity depended on hydrophone’s
orientations and was about 6 V/MPa for the normal angle of incidence of acoustic waves. The angular
response of the hydrophone was measured and taken into account in these measurements. The uncertainty
of pressure measurements was estimated to be on the order of 30
%
. Another uncertainty was related to the
scattering of acoustic waves by the irregular surface of the urinary stone. As the characterization of wave
scattering was beyond the scope of this report, the driving acoustic pressure was measured without the stone
by positioning the sensitive tip of the hydrophone at the focus of the microscope. The pressure at the stone,
however, could be greater due to reflection of pressure waves from the nearly rigid surface of the stone.
A typical trace of the hydrophone is shown in Fig. 3. The central frequency of the driving acoustic
bursts was 416.5 kHz with the duration of the beat envelope of
∼
30
μ
s. During the first four acoustic
cycles (
∼
90
−
100
μ
s), the driving pressure increased reaching a pressure amplitude of
∼
1
.
5
±
0
.
5
MPa.
The increase of the driving acoustic pressure was associated with the growth of bubbles to a larger size from
cycle to cycle. The collapse of the larger bubbles could produce daughter microbubbles that became visible
in the subsequent acoustic cycles. In this study, we focus on the dynamics of a single microbubble during
one acoustic cycle marked by a red rectangle starting at
∼
95
μ
s and lasting for a period of
2
.
4
μ
s (Fig. 3).
-2
-1
0
1
2
Pressure (MPa)
105
100
95
90
Time (μs)
Figure 3: The driving acoustic pressure measured with a hydrophone positioned at the focus of the
microscope. The red rectangle marks the acoustic period shown in HS-camera movies (Figs. 4 and 5).
D. NUMERICAL MODELING
The collapse of the gas bubble in the liquid was simulated using a compressible multi-component flow
solver.
5
In the solver, an anti-diffusion based interface sharpening technique
6
was used to suppress numer-
ical diffusion of the gas-liquid interface. We neglect viscosity, surface tension, and heat and mass transfer
across the gas-liquid interface. The bubble was assumed to remain axisymmetric. The stone was modeled
as an acoustically-rigid infinite plane wall. Both fluids were initially at rest, assuming that the bubble was
fully expanded before its collapse. The initial pressure of the liquid was set to
P
∞
= 1
.
55
MPa. The initial
pressure and density of the gas, the shape of the bubble, and its stand-off distance from the wall were varied.
Y.
A.
Pishchalnikov
et al.
Lipid-shell
microbubbles
with
calcium-adhering
moieties
for
treatment
of
urinary
stones
Proceedings
of
Meetings
on
Acoustics,
Vol.
35,
020008
(2019)
Page
4
3. RESULTS
A. HIGH-SPEED VIDEO MICROSCOPY OF BUBBLE DYNAMICS
Figures 4 and 5 show typical growth-collapse cycles of representative microbubbles at the surface
of an urinary stone. These sequences of images were recorded with the high-speed camera during one
acoustic cycle of the driving wave marked by the red rectangle in Fig. 3. Under these driving conditions,
microbubbles grew to several tens of micrometers. The bubble in Fig. 5 was smaller and collapsed earlier
than the bubble in Fig. 4. Among hundreds of microbubbles observed in this system over the course of
months of experiments, the vast majority exhibit a slightly non-spherical shape at their maximum expansion
similar to that seen in the
t
∼
1
μ
s frames in Figs. 4 and 5, with a cross-section well modeled as an ellipse.
Figure 4: High-speed imaging of the growth (top row) and collapse (bottom row) of a bubble at the sur-
face of an urinary stone during one acoustic cycle recorded in FP mode at 5 Mfps and 100-ns exposure.
Figure 5: High-speed imaging of the growth and collapse of a bubble recorded in HP mode at 10 Mfps
and 50-ns exposure. The bubble growth (top row) is shown at 0.2-
μ
s step skipping every other frame.
Y.
A.
Pishchalnikov
et al.
Lipid-shell
microbubbles
with
calcium-adhering
moieties
for
treatment
of
urinary
stones
Proceedings
of
Meetings
on
Acoustics,
Vol.
35,
020008
(2019)
Page
5