This is a presentation of my final research project for my Introduction to Biomedical Engineering course.* Together with a partner, I conducted a thorough internet research of the newly emerging field of drug delivery using microbubbles activated by ultrasound technology. We presented to Biomedical Engineering faculty, graduates, and students at a formal poster session the advantages of microbubble technology as an alternative method to traditional chemotherapy, as well as its potential in thrombolysis and gene therapy. *Note: The bibliography for this project is not attached; it is a separate document also uploaded onto this website.

1. Christopher Plunkett
Christina Amaral
[15]

2. Abstract
Ultrasound, which originated as a medical imaging modality, has since
been discovered as a therapeutic tool to treat the diseases that it was
once only able to observe and diagnose. A recent application of
ultrasound lies with microbubble technology. Microbubbles are small
(on the order of micrometers), gas-filled spheres that act as contrast
agents by scattering an ultrasound signal. Current research involves
the loading of microbubbles with drugs and injecting them into the
body to be activated by localized ultrasound. Through a process called
cavitation, cell membranes are permeated and the microbubbles
rupture, releasing their payload into the intended target tissue.
Microbubble destruction via ultrasound has shown success clinically by
delivering chemotherapeutics and anti-cancer drugs to tumor sites in
animals and slowing tumor growth rate, and it holds promise in the
areas of gene therapy and thrombolysis. As a relatively new
technology, ultrasound mediated drug delivery still has limitations that
it must overcome before being used regularly to treat diseases in
humans.
[16]

3. French physicist
Curie discovers
piezoelectricity
French physicist Langevin
permitted by
government to create
device to detect
submerged enemy
submarines; basis for
sonar in WWII
Austrian physician
Dussik first to use US in
medical diagnosis for
brain tumors; called
“hyperphonography”
Radiologist Howry
creates B-mode
equipment for
cross-sectional
anatomical images
Swedish physicist
Hertz and
cardiologist Edler
become “fathers of
echocardiography”
“Sonic Boom”
2D echo and pulsed
Doppler introduced
First proposed use
of microbubbles as
a contrast agent
for ultrasound by
Gramiak and Shah
[2]
Real-time US
developed, smaller
probes and
increased image
resolution
Drug delivery using
microbubbles as a
vector proposed by
Miyazaki et al. [3]
Unger et al. first to
incorporate drugs
inside microbubble
gas core [3]
Note: Uncited events taken from source [1]. [17]
[18]

4. Ultrasound Design
• Ultrasound technology involves the use of 20kHz or
greater frequency pressure waves [3].
• In medical applications, waves are produced and
measured by piezoelectric materials. These
materials are crystals and two notable examples are
lead zirconate and lithium niobate. These materials
produce mechanical stress in response to a voltage
and vice versa [4].
• In ultrasound, piezoelectric materials are ideal
transducers. By oscillating the voltage in contact
with the material, a mechanical wave can be
produced. However, the reflected pressure waves
can mechanically stimulate the material causing a
voltage to be produced[4].
• Typically in medical imaging, a pulse is sent from the
pz material (Fig 2.) through a couplant (usually gel)
and into the body. Waves that contact a dense
object are reflected back to the transducer. The
resulting voltage is interpreted by a digital
receiver[4].
• In medical imaging, an ultrasound device records
the voltage produced by the piezoelectric material
and interpret that into an image. Drug delivery
however, is not as focused on what is reflected, but
rather what the waves can do inside the body.
Figure 1 shows a
transducer that would
be found in an
ultrasound device. It
is piezoelectric.
Figure 2 shows a typical transducer design.
Note the crystal and electrodes at the front
of the model.
[20]
[19]

5. Ultrasound Wave Analysis
• Sound waves are variations in pressure over
time. Therefore the plot of an ultrasound
wave has an amplitude of pressure (Pa) and
a frequency over time (s) [4].
• Wave frequency (f) is equivalent to the
number of cycles per second of the wave.
Wave period (T), or the inverse of
frequency, is the number of seconds per
cycle.
• Amplitude of an ultrasound wave in terms
of stress (σ) is related to the strength of the
pulse. An assumed amplitude is shown in
our data table.
• In drug delivery, high frequency ultrasound
is necessary. Therefore, this poster will
focus on frequencies of 1 to 10MHz.
• A general equation for an oscillating wave is
a(t)=σsin(wt) where σ is the amplitude, w is
the product of frequency and 2π, and t is
the elapsed time [4]. A model wave is
plotted with an amplitude of 0.1MPa and a
frequency of 3.28MHz [5]. This simulates a
possible pulse used during microbubble
therapy.
Frequency (MHz) 3.28
Amplitude (MPa) 0.1
W (rad/s) 24001767.87
Sampling Frequency (MHz) 76400000
Sampling Period (s) 1.3089E-08
Assumptions
Assumptions on amplitude and frequency
taken from [5]
Time (s) Amplitude (Pa)
0 0
1.3089E-08 30901.69944
2.6178E-08 58778.52523
3.9267E-08 80901.69944
5.2356E-08 95105.65163
6.5445E-08 100000
7.8534E-08 95105.65163
9.1623E-08 80901.69944
1.04712E-07 58778.52523
1.17801E-07 30901.69944
1.3089E-07 -3.21574E-11
1.43979E-07 -30901.69944
1.57068E-07 -58778.52523
1.70157E-07 -80901.69944
1.83246E-07 -95105.65163
1.96335E-07 -100000
2.09424E-07 -95105.65163
2.22513E-07 -80901.69944
2.35602E-07 -58778.52523
2.48691E-07 -30901.69944
2.6178E-07 6.43149E-11
Data for One Cycle

6. Microbubble and the Outer Shell
• Microbubbles are small gas and lipid spheres that are measured in
micrometers (μm) [6].
• There are two components of a microbubble: the outer shell and
inner core. The outer shell can be made of rigid or flexible
materials depending on the design. The core is traditionally a
perflurocarbon (PFC) gas. Current FDA approved microbubbles
have a lipid coating [7].
• Outer shell design must balance stability with flexibility to achieve
optimal results. Rigid shells will reduce the echogenic effects of
the microbubble. However, thin shells can allow gas to diffuse out
of the bubble. In addition, the shell must be biocompatible [8].
• A thin lipid shell (Fig. 1) significantly reduces pressure within the
bubble and is biocompatible. However these shells will leak gas.
Therefore a water insoluble gas is essential in these types of shells
[8]. Figure 2: Optison enhanced image [GE]
Figure 1: Electron
microscope image
of a Lipid coated
Microbubble.
• Albumin shell coatings are more rigid than lipids and can
elicit an immune response. Despite this albumin shells
have been used in FDA approved products such as
OptisonTM (fig. 2) by GE [6].
• A thick shell such as a synthetic polymer will retain the
core and thus a water insoluble gas is not required.
However, to act as a contrast agent, the shell must be
destroyed by high frequency ultrasound [6].
Figure 3:
Comparison of
Lipid and Polymer
microbubbles [7]
[21]
[22]
[23]

7. The Core and Laplace Pressure
• Initially microbubble cores were filled with air.
However, bubble leaks caused excess air in the
bloodstream, leading to complications [3].
• Currently the inner portion of the microbubble
contains a gaseous PFC core. PFC’s have low
solubility, which results in longer circulation
periods, and are compatible with the human
body in small amounts. It is because the PFC is
highly echogenic (can easily return ultrasound
signals to a source), that it is ideal for use in
microbubbles as a contrast agent [7].
• Research is being conducted in alternatives to
PFC cores, including hybrid cores that change
phase based on acoustic energy. These phase
change contrast agents are promising but yet to
be FDA approved [7].
Phase change agents are prepared with a gaseous PFC
core. Low temperature and high pressure cause
condensation of the microbubble. Acoustic energy
from the ultrasound wave causes vaporization that
increases volume and echogenicity.
[7]

8. Clinical Relevance: Contrast Agents
• Ultrasound using microbubble contrast agents is an inexpensive, non-invasive, and fast method for
analyzing blood flow in the body.
• A dose of microbubbles ranges from 108 to 109 bubbles. An injection of this dose is placed into a
peripheral vein where it quickly spreads throughout the body [9].
• The wave scattering ability of a gas is substantially greater (by a factor of 10,000) than that of blood.
Therefore microbubbles containing a gas are excellent at scattering an ultrasound signal [9].
• Microbubbles are detectable by their own resonance frequency. The application of acoustic
pressure waves causes the bubble to oscillate at a unique frequency which the ultrasound device can
detect. Through signal processing, the ultrasound device can focus on the area surrounding this
frequency. This is beneficial in capillaries where few microbubbles can be present at one time [9].
Contrast
enhanced
ultrasound
images. Notice
the clear
distinction
between tissues.
[24] [25]

9. Cavitation and Drug Delivery
• Ultrasound alone induces cavitation—the formation,
oscillation, and destruction of gas-filled bubbles—due to
high and low pressures of the wave [2]. The presence of
microbubbles can lower the cavitation threshold and
increase cell permeability [2].
• Cavitation of microbubbles creates microstreams of
extracellular fluid that exert a shear stress on surrounding
cells and perforate them [soft matter]. The microbubbles
are also ruptured by the ultrasound wave and release their
payload into the open target site [6].
• Other methods of drug delivery include endocytosis of
microbubbles and fusion with the cell membrane [2].
Diagram of
the behavior
of
microbubble
s at different
points along
the
ultrasound
wave [3].
Ultrasound mediated destruction of a
microbubble with externally mounted
payload [6].
[3]
[6]

10. Microbubble Transport through a Capillary
Assumptions taken
from “Physics of
the Body” [10].
Length of Capillary (m) 1.00E-03
Viscosity of Blood (Pa*s) 3.50E-03
Velocity of Blood (m/s) 1.00E-03
Arterial end blood pressure in a capillary 4.00E+03
Assumptions
Volume Flow Rate (m3
/s) Change in Pressure(Pa) Venous End Pressure (Pa)
7.85E-16 112000 -1.08E+05
3.14E-15 28000 -2.40E+04
7.07E-15 12444.44444 -8.44E+03
1.25664E-14 7000 -3.00E+03
1.96E-14 4480 -4.80E+02
2.83E-14 3111.111111 8.89E+02
3.85E-14 2285.714286 1.71E+03
5.03E-14 1750 2.25E+03
6.36E-14 1382.716049 2.62E+03
7.85E-14 1120 2.88E+03
9.50E-14 925.6198347 3.07E+03
1.13E-13 777.7777778 3.22E+03
1.33E-13 662.7218935 3.34E+03
1.54E-13 571.4285714 3.43E+03

11. Drug Attachment
• Various approaches have been tested for the attachment of
drugs to microbubbles for targeted delivery in the body. Drugs
can be contained within the gas core, which reduces the risk of
premature release [3]; however, interactions between the
perfluorocarbon core and the drug create complications [6].
• Incorporation of drugs into microbubble
membranes uses noncovalent bonding and depends
upon charge. This technique is particularly useful
when implanting large, negatively charged DNA
molecules into a positively charged membrane [2].
• A favorable method is to incorporate drugs into the
outer shell of the microbubble for timely release
upon cavitation by ultrasound. This technique is
tailored for certain drug properties, such as
hydrophilicity and lipophilicity [2]. For instance, oil
introduced into lipid shells was used to dissolve and
successfully release chemotherapeutics in vitro [6].
[3]
[26]

12. “Smart” Microbubbles: Target Specificity
The process of loading microbubbles
with a specific payload, injecting them
into a vein in the general vicinity of a
target site, and locally applying
ultrasound leaves room for error. Upon
entering the bloodstream,
microbubbles are capable of traveling
through the entire systemic circulation
and settling in the incorrect site [2].
A solution to the circulation problem is
creating targeted microbubbles that have
ligands integrated in their shells. The ligands
are specific and bind to receptors on the
surface of target cells, for instance
endothelial cells or thrombi [2]. They are
attached to the microbubble shell by
covalent bonding or interaction of biotin and
avidin proteins. Types of ligands include
vitamins, antibodies, and peptides [6].
Studies show that once the
microbubble has successfully
attached to its target cell, shear
stress exerted by blood flow was
not enough to break the
connection; hence successful
payload uptake was observed [2].
Two types of ligand binding: (a) small hydrophilic ligands and (b) large protein ligands linked by biotin and
avidin [6].
[6]

13. Clinical Relevance: Cancer Treatment via Drug
Delivery
• Selective drug delivery is a preferred model for
chemotherapy as it specifically targets tumor cells
and reduces unnecessary cell destruction from the
drugs. Microbubbles are ideal vectors as they can
contain a drug payload until treated with ultrasound.
• Initial animal tests show significant decrease in tumor
size using ultrasound activated microbubbles (Fig 1).
Tumor area decreased to less than half its original
area after treatment with this method. Traditional
chemotherapy using Genexol PM (GEN) showed
tumor reduction at a slower rate than the ultrasound
drug infused microbubble combination (Fig 2) [12].
• A control group of empty microbubbles treated with
ultrasound did not respond to the treatment (Fig 2.)
Thus it is the drug microbubbles that cause cell
death.
• After the first round of treatment, tumor growth
reemerged most likely because of drug resistant
tumor cells. Future focus should be placed on
refining current drug options to ensure full tumor
destruction [12].
Figure 1: Ovarian tumor growth. “US”
signifies area of ultrasound treatment
[12].
Figure 2: A.
Ovarian tumor
size (note
empty droplet
control) B.
Breast cancer
tumor size
[12].
[13]
[13]

14. Thrombolysis, or clot lysis, is the process of breaking down a blood clot by
degrading fibrinogen and fibrin, therefore freeing an occluded vessel [2]. Cavitation
of microbubbles due to ultrasound creates a shear stress that disrupts the clot and
enhances thrombolysis. Targeted microbubbles bearing thrombolytic agents are
especially powerful in their ability to deliver the clot-dissolving drugs directly to the
target site [6]. Cavitation induced by low frequency, high power ultrasound also
greatly improved thrombolysis [2].
The use of microbubbles to aid
thrombolysis is a promising technique,
for it can prevent cardiovascular
diseases caused by blood clots.
However, this concept is still in the
early stages of experimentation and
has not yet been applied clinically due
to potential dangers, such as how the
microbubbles will interact with living
cells [2].
Microbubble
with ligands
that bind to
receptors on
thrombus
and release
thrombolytic
agents to
break up the
clot [2].[2]
[27]

15. A Look to the Future: Gene Therapy
• Unlike viral vectors, microbubbles are generally
non-immunogenic. Genes contained in the gas
core or outer shell typically remain undigested by
the body and have a better chance of being
delivered to the target site [2].
• Microbubbles show sufficient transfection (gene
uptake). Though dependent on the duration and
frequency of the ultrasound wave (Fig 2),
expression of a target gene was observed when
using microbubbles in mice [13].
• Studies have incorporated DNA vectors into the
albumin- and lipid-based shells of microbubbles
for gene transfection [6]. DNA can be efficiently
bound to microbubbles in concentrations up to
100µg DNA per 1E9 microbubbles [13] .
Cancer treatment is one application of microbubble
gene therapy. Herpes simplex virus-1 thymidine
kinase (TK), which can destroy target cells, can be
delivered via microbubbles to a tumor. Current data
(Fig. 1) indicates that, with microbubble delivery,
there is a significant slowdown of tumor growth [13].
Figure 1(right):
Triangles represent
control data while
squares represent
microbubbles
treated with TK [13]
Figure 2(left): Table displaying various
gene therapy related studies involving
microbubble vectors. All outcomes show
significant gene uptake and tissue
response to the gene therapy [2].
[13]
[2]

16. Ethical Issues Concerning Microbubbles
• Mechanical stress from microstreams upon cavitation of microbubbles can lead to cell
injury or death. Hemolysis, the destruction of red blood cells, is one example.
Cavitation increases the formation of free radicals, highly reactive molecules that can
cause cell mutation [2].
• The use of microbubbles with ultrasound is tested on non-human subjects before it can
be applied clinically. Experiments are performed on animals such as rats, mice, and
rabbits [6]. Whether it is a painful experience for the animal or if painkillers are used is
an issue often voiced among animal rights activists, and it raises the question of
whether animals should suffer for human benefit [14].
• Large doses of microbubbles, on the order of 109 microbubbles/mL, were needed for
successful drug uptake in animal studies; this amount can be harmful to humans.
Ligands attached to the microbubble surface have the potential to bind to areas other
than the target site, and drug payloads can be washed out by systemic circulation
before reaching their destination. The small circulation times of microbubbles present
another challenge. Sonovue, for example, has a half-life of only 6 minutes, and multiple
circulation trials are necessary for successful uptake [6]. [28]