Diagnostic and Therapeutic Applications with Microbubbles - HD

Introduction

Hi, I am Brian from the University of Michigan.
I'm professor of radiology and biomedical engineering,
and today I'll be discussing therapeutic
and diagnostic applications of microbubbles.
We'll be discussing some diagnostic
and therapeutic applications for microbubbles,
as they're used in ultrasound.

If you're at your local pub,
and I'll assume everybody here is over the age of 21.
If you fill your glass full of your favorite pale ale,
then what you'll notice when you look at the side
of the glass is that there's a series of gas bubbles
that will be floating up through the liquid,
but they're originating on the side of the glass.
And the reason that that happens is
that when you pour the liquid into the glass,
the glass surface does not completely wet,
and it traps a little bitty tiny pocket
of gas on the side of the glass.
And then because the beer
or your favorite beverage has
is carbonated, and it has a large amount of CO2,
carbon dioxide in it, then the gas diffuses into
that tiny pocket of gas
that's on the side of the glass and the bubble grows,
and then buoyancy causes it to pinch off,
and then it leaves a little bit of gas behind.
And that process continues to happen over and over again.
And if you have this type of beverage,
for instance, champagne in a champagne glass,
if the glass didn't have imperfections on the side of it,
you actually wouldn't get that beautiful
appearance for champagne.
This was the origin of one
of the first ultrasound contrast agents,
which gas was trapped in particles,
and that served to stabilize the gas pocket
because normally a gas bubble that spherical
has an internal pressure, gas pressure
that's greater than the outside pressure in the liquid.
And the gas naturally wants to diffuse out of the bubble.
They needed some sort of stabilization mechanism in order
to make the bubble stable.
And the pocket inside of a particle was one way
of being able to do that because at that point, that
flat surface that you see no longer has a curvature, and
therefore the gas wants to stay inside the pocket.
Subsequently, most of the microbubble contrast agents
that are available today are stabilized
spherical bubbles, but they have a shell
on the outside of them.
They have either a lipid or a protein
or some sort of material that's on the outside
of the gas bubble that will stabilize them against this
diffusion process.
And it becomes a very stable microbubble emulsion
and then it's injectable.
And the reason that it's injectable
that people don't suffer from the injection
of these microbubbles is that the microbubbles have a range
of sizes that are relatively small.
90, 95, 99%
of the gas bubbles are less than say, 10 microns in size.
And they're on the order of the size of red blood cell,
they'll circulate through the body
and they'll pass through the capillaries so
that there's no danger
of an embolization associated with them.

Microbubble Resonance and Properties

The other thing that's unique about that size distribution
of gas bubbles that you see is
that they have a natural mechanical resonance,
microbubbles will resonate at a frequency
related to their size.
And it turns out that microbubbles that are on the order
of about six microns in size
are gonna be resonant at about two megahertz.
And as the bubbles are smaller,
they actually have higher resonant frequency.
And it turns out completely fortuitous
that these bubbles in just the right size range to be able
to circulate through the body are also resonant in the
size range or the frequency range for diagnostic ultrasound.
It was a wonderful thing for this mechanical resonance
to exist at exactly the right frequency
and right bubble size.

Non-Linear Oscillation and Harmonic Imaging

This is what you see.
If you take a gas bubble
and you drive it with an ultrasound field,
normally you'd expect the oscillation of that bubble
to be just a simple sinusoid.
It would spend as much time
on a large size as it does at a small size.
The bubble is expanding and contracting
and changing its volume in response to the acoustic field.
What happens in gas bubbles that are being driven
by an ultrasound field if you drive them hard enough, is
that the oscillation actually goes non-linear.
And they collapse rapidly.
And these rapid collapses then generate a series
of higher order harmonics.
And those higher order harmonics are what we image
with diagnostic ultrasound
because it creates a unique signature that'll allow us
to detect the bubbles in the presence of the tissue
where the tissue can be suppressed
and the bubble signal can be imaged.
And although we're transmitting in say, two megahertz,
we actually get a series of harmonics,
and those harmonics can be quite large in amplitude such
that we'd be able to pick up four megahertz
and six megahertz in response to the
two megahertz drivers.
Although the tissue will in general be mostly sending
back two megahertz signal, the gas bubble will
amplify these higher order harmonics
and allow you to be able to detect the bubbles.

Pulse Sequences for Bubble Detection

The engineers that are out there in the world
were pretty smart guys,
and they took a page out of MRI
and started looking at pulse sequences, different ways
that you could transmit ultrasound in order
to preferentially detect gas bubbles.
If you have a gas bubble
and you send in a pulse of ultrasound,
you can send in one pulse that's at one phase,
and then you can send in another pulse
that's 180 degrees out of phase with the first.
And what happens then is this signal
that propagates in, it'll echo off
of linear scatters like tissue that are mostly linear.
There is some non-linear component to
tissue harmonics.
And then the scattered field that comes back
when you take the two signals from the linear scatterers
where one was at zero phase
and the other is 180 degrees out of phase.
With that, you sum them together
and you actually get nothing.
It nulls them out.
And this is a simple example of
how a pulse sequence might work.
Then what you do is for non-linear scatters,
you send the same type of pulse in,
and when you do, then the non-linearities, all
of the even harmonics that occur don't cancel one another.
The scattering that you get back from the gas bubbles
then sum together to actually give you a finite signal.
And that finite signal is what we detect
with diagnostic ultrasound.

Example of Contrast Agent Imaging

Here's an example from Dr. Morso.
And in this case,
what we're gonna do is we're gonna make an image
of just the ultrasound contrast agent that's percolating
through the tissue, and you actually are able
to see individual gas bubbles
that are circulating through the fluid.
That bright flash that you saw
is a higher amplitude pulse that used to
delete the bubbles.
You can cause the bubbles to dissolve
to get eliminated from the circulation.
And when you do, then you can watch the bubbles
refill into that imaging plane.
The other thing that you can do is you could take
that same type of sequence,
but instead of just updating the screen with each
frame, you can put in what's called a max hold.
And that max hold, basically, every time a pixel
appears bright,
or a brighter pixel will occur,
then it will keep that value.
It always is holding onto the highest pixel value
that is seen over the course of the sequence.
If I run this video, which is again,
bubble circulating through tissue,
and then you'll see a flash,
and then you'll see the brightest pixel being lit up at
every location, you can actually see the blood flow then
percolating through into the tissue.
This allows you to see this great sequence of
flow that occurs.
That gives you an idea of what you can do
with an ultrasound contrast agent.
And there is a lot
of work being done around the world.
In ultrasound contrast agent
with some clear clinical benefits associated with
the diagnosis of various conditions in the use
of contrast agents and contrast agents have been shown
to be a safe and effective way of being able to monitor
vascular conditions for a variety of conditions.

Acoustic Droplet Vaporization

The other thing that turns out a way
that you can make gas bubbles is
that you can start out
with a perfluorocarbon liquid.
And this perfluorocarbon liquid, then when it gets hit
by an ultrasound pulse, will cause that liquid
to transform from a liquid into a gas.
And that subsequent gas bubble then is available
for either an imaging process
or a therapeutic process in a process that we call
acoustic droplet vaporization.
Although you're starting out with a liquid, you hit it
with a pulse of ultrasound that liquid wants
to boil, it would normally boil at body temperature.
But because it's in the liquid state until it's triggered
by ultrasound, it stays in a liquid state.
For example, you can use a
perfluoropentane,
and that perfluorocarbon liquid then can be
formulated in droplets.
You can have a shell of albumin
or a protein shell around the outside.
And this pentane then will have a boiling point of 29 C
such that when you inject it into the body, it's ready
to boil, but then doesn't get triggered
until the ultrasound hits it.
You start out with particles that are on the order
of maybe two microns in diameter,
and then you trigger them to form gas bubbles.
And the threshold, the acoustic amplitude that's required
to trigger these is relatively low
and in the range of diagnostic ultrasound.
If you have a six megahertz ultrasound field,
you can trigger these droplets with just a few cycles, tens
of cycles, and that then will trigger the formation
of the gas bubbles.
And in this case, what happens is we gradually increase the
pressure amplitude of the acoustic field,
and then we reach a certain amplitude value.
And above that, then the droplets start to trigger.
And you see an increase in the echogenicity
from the gas bubbles appearing in the fluid.
The process that we're calling ADV, you start out
with this perfluorocarbon liquid, you hit it
with an ultrasound pulse, and then you form a gas bubble
where you start out with maybe a five micron
or four micron gas
or liquid particle.
And then you end up with a 40 to 50,
maybe 60 micron gas bubble in the end.

High-Speed Imaging of ADV

Along with some colleagues at Erasmus University
and University of Twente, we
took some high speed photographs of this process,
and that you can see
what the droplets look like when you trigger them
to form gas bubbles.
And here's a droplet,
and I'm gonna start the video, and then you'll see
that the droplet then is triggered to form a gas bubble.
Now there are about 128 frames
in this video, and they're at 12 million,
about 12 million frames per second.
The whole process here is occurring in only
10 microseconds.
It's a very rapid triggering
and then gas bubble formation that occurs as a consequence
of triggering these droplets with ultrasound.

Ultrasound Imaging of ADV

This is what it looks like in ultrasound imaging.
This is the ultrasound image,
and there are gonna be droplets
that are flowing in from the right hand side.
And then we focused ultrasound at this location here.
That's at a sufficiently high amplitude in order
to trigger the droplets.
And then you're gonna see contrast from the gas bubbles
that are flowing away from that site.
This is what it looks like
as you're triggering these gas bubbles.
And that creates a echogenicity that allows you
to see the conversion
of these liquid droplets into gas bubbles
on the ultrasound imaging.

Applications: Controlling Blood Flow

There are a variety of different applications
that you might think about for these type of gas bubbles
that are relatively large, but are being triggered on
the arterial side.
You can do an intravenous injection
of these liquid droplets,
and they're circulating then through the body.
And then on the arterial side, for instance, in the supply
of a tumor,
you might trigger these bubbles in order
to control the blood flow.
And you have specific sites next to the tumor,
and you would trigger these bubbles, they would flow in,
and then it would reduce the
blood flow at that location.
Here's an example, just in an externalized kidney,
in an animal model where the droplets are being infused,
they're in circulation.
And when I start the video, then you're gonna start,
you will see some interference appear on the screen,
which is low amplitude field.
And then I'll turn the amplitude up
and you can start to see a few bubbles
that are being formed in the renal artery,
which we're targeting down here.
And then we'll do another infusion of the droplets,
and they'll eventually make it around
to the arterial circulation.
There you can see they're being triggered.
And then you can see that we're generating enough gas
bubbles in the renal cortex
that we're actually shadowing out the kidney.
This allows you to control blood flow in a
given location.
This is just a demonstration.

Intravital Microscopy of Bubble Formation

The other thing that's interesting is to be able
to see exactly how these bubbles form in the vasculature.
If you do intravital microscopy in the rat cremaster
muscle, then you can see
bubbles that are being formed.
The ultrasound is gonna be focused
over in this area here.
And then we're gonna look at the variety of gas bubbles.
Then that will be formed inside the vasculature
of this muscle that's very thin,
and that you can actually see through it
in a microscope.
As we start the video, then the
bubbles are getting formed over here on the right hand side.
And then as we scan over, then we can see bubbles
that have collected in the vasculature.
We can see a few of those bubbles that are still
circulating slowly through these vessels.
And then you can see locations where the bubbles are
blocked in place.

Bubble Lodging and Theoretical Predictions

Interestingly enough, there was a theoretical
prediction that was done by colleagues
in biomedical engineering, Joe Bull and his group.
And they looked at what the conditions would be
for these bubbles to lodge in place.
And a bubble that might be flowing along through a vessel
and then comes to daughter vessels that are smaller,
what size bubble and what configuration
of bubble would it be that would lodge inside of
this bifurcation, this branch point that exists
inside of the vessel, the vascular structure.
And it turns out that we found
in this intravital microscopy, these dumbbell shaped
bubbles that were lodged very similar to this
diagram that you see here.
And the model predicts
that you should have a certain ratio, in other words,
a ratio between the length of the bubble in comparison
to the diameter bubble
that would lodge at a given location.
And that ratio turned out
to be from a theoretical prediction to be about two to one.
It's two times longer than it is in diameter.
And these blue dots
that you see on this curve here are actual measurements
that were taken in vivo in this
intravital microscopy imaging.
And you can see that these lie along a line that has a slope
of 2.11.
It's very close to two.
The aspect ratio
or the ratio of the bubble length to the hydraulic diameter,
the diameter of the gas bubble,
is about a ratio of two to one.
This looks at ways at which you can use
microbubbles to cause lodging
inside of tissue in order to control blood flow.

Enhancing Tissue Heating with HIFU

The other thing that has been known for some time is
that if you have gas bubbles present in tissue
and you apply therapeutic ultrasound such as that used to
increase the temperature, like in HIFU,
that gas bubbles will enhance the amount
of heat deposition that you get
for the same acoustic parameters.
For example, if you take a HIFU exposure
that is five seconds in duration,
and you look at the size of the lesion
that you can produce the volume of a lesion
that you can produce, if you have no bubbles present,
this being no droplets, so
that the bubbles are not being produced
by the droplets, then you get a certain amount
of volume associated with the lesion that you produce.
But if you add droplets in circulation,
you can increase this by several fold,
maybe sevenfold increase in the size of the lesion
that you can produce, which would allow HIFU
to operate more efficiently
and be able to generate lesions faster.
The nice thing about using the droplets is
that the droplets only form gas bubbles
at the high amplitude portion of the field.
They only form these bubbles where the focus is.
Whereas if you have ultrasound contrast agent,
they'd be circulating throughout the
entire volume of tissue.
You'd have to be worried about
where else is it going to heat.
If you look in vivo, then these are MRI images
of the lesions produced by
circulating droplets and exposing to HIFU.
The lesions that you see here are the ones where
the droplets were present,
and the ones on this side are the ones that
where the droplets had been circulating for 30 minutes.
And you can see they're comparable in size.
The circulating droplets
or the droplets that were accumulated in the liver, which is
what is happening in this particular circumstance,
is triggered to form gas bubbles.
And those gas bubbles then increase the heating locally at
that location.
Over here are the size lesions
that you would produce if you didn't have any droplets.
We produce these lesions prior to the injection
of the droplets, and you can see the estimates then of the
lesion volume from these MRI images is about a 15
fold increase in volume.
Arguably then you might be able to go 15 times faster
with the droplets on board than you would be without.
That means that there's some interesting ways of being able
to accelerate the therapy process.
For example, these are images that are taken,
this is an optical image here.
And then these are MRI images of a sequence of spots
that are produced with the HIFU system,
and that HIFU system is then doing a spiral.
That spiral then has lots of gaps between these lesions.
And obviously there's a large gap
that exists within the spiral, but this is
before droplets were in place.
If you have droplets, then
this process completely fills in this volume.
For the same number of lesions that you produced here
and the time it took to produce these lesions,
you would completely treat that entire volume.

Drug Delivery with Microbubbles and ADV

The other thing that ultrasound contrast agents
has in its future is drug delivery.
And what I wanted to chat just a few minutes about is
what types of drug delivery mechanisms
might be involved for this acoustic droplet vaporization,
as a means of being able to do the drug delivery,
although there are a lot of other ways of being able
to use microbubbles to do this as well.
There are a few different things that you can do.
The drug delivery problem means
that you need some sort of payload.
There are a couple of different ways of doing that.
You can have a payload that is contained within an oil,
and you have an oil soluble drug,
and then you formulate that drug in a way
that produces a droplet so
that there's a perfluorocarbon component.
And then there is a drug component.
This drug component here, in this case, it's
just a fluorescent dye, can be seen on the inside
of the droplet, the overall droplet.
And then there's a perfluorocarbon component here,
which will be the triggerable component.
This is a perfluorocarbon oil water double emulsion.
The other is that you can do a two step emulsion
where the process will yield a drug that's contained
in an oil on the inside.
The perfluorocarbon now is on the outside,
and the drug oil component is in the core.
The nice thing about this is it puts this perfluorocarbon
here as a diffusion boundary, so
that the drug has a hard time diffusing out
through the perfluorocarbon to the outside.
Whereas up here, the drug was right on the outside,
so it was easier for it to diffuse through.
The other thing that you can do is that you can do this
with water-based emulsions as well.
Here is a picture of an emulsion
where these tiny particles, these nanoparticles
that you see on the inside here are actually water droplets,
and they're suspended on the inside of the perfluorocarbon.
You have a small amount of perfluorocarbon
that's sitting on the inside,
and then these water particles
that are containing the drug.
And you can tell that the drug is in there
'cause you can tag them and
show the fluorescence
of the drug on the inside of the particle.
Then these can be triggered by ultrasound.
If, for example, we
look back at high speed photography
for triggering these droplets with
the perfluorocarbon on the inside and then the droplet
or the oil to the outside,
then you see the triggering process.
These are triggerable droplets,
and then it would squeeze the drug out into the surrounding medium.
And the other thing you can do is trigger these
water-based double emulsions.
The thing that you'll notice here is
that this trigger is a lot slower.
Notice that the droplet does not expand,
or the gas bubble does not expand nearly as rapidly
as it did in the other case.
And that's because we're able to displace 90
plus percent of the perfluorocarbon on the
inside with the drug.
These are highly drug loaded.
The volume of drug
that you can actually contain within the particle
is really high.
These are just an example, a few examples of the ways that you'd be able to do this drug delivery.

Chemotherapeutic Drug Delivery Example

One is a chemotherapeutic.
In this case, if you look here, this is the case
for chlorambucil.
If you look at a set of VCaP cells
that are being grown, those cells
experience a growth inhibition
as a consequence of the exposure to the drug.
And in this case, it's about an 80% reduction in the
growth of these cells.
If the droplets contain chlorambucil,
then, and we don't trigger them, so there's no ultrasound
then, the drug does leak out slowly,
but you can trigger them and release the full contents
of the chlorambucil.
And you can see that we get as much effect here as we did
with just the drug alone.
That was not encapsulated at all.
The one thing to remember is that the droplets
are actually able to affect the cells as well.
This graphic that you see down here in the bottom,
because the droplets are heavy
and the cells are an adherent cell line,
we can have the droplets settle to the bottom
of the container, trigger them away from the cells,
and we get very little effect.
If you reverse this process
and allow the droplets to sit right on top of the cells,
then you'll actually experience a growth inhibition
that's on the same order as the actual drug delivery.

Thrombin for Clotting Control

In keeping with the notion of a control of blood flow.
You can also formulate these droplets to contain thrombin,
which is a thrombolytic agent
that will trigger a clotting cascade.
If you look at the time it takes
to clot the clot activation time, then
you can have a substantial reduction in the clot activation
by releasing the thrombin from the inside
of a certain formulation.
The T1, T3, T4, T5 that you see down
below are just different formulations of the droplets,
with the T3 formulation being particularly effective.

Tissue Engineering and Regenerative Medicine

Finally, I just wanna spend
just a few minutes talking about other ways of being able
to control drug delivery
and specifically within the idea of tissue
engineering and regenerative medicine.
One of the important things in regenerative medicine is
the ability to temporally
and spatially control when the
regenerative factors are released.
If you have a bone defect that you're wanting to
fix, then what you wanna be able
to do is recruit the vascular
structure into this region in order to be able to supply
nutrients into that region.
And then at some later point in time in the
central core of this defect,
then you wanna be able to trigger the bone formation so
you can then release certain factors that will grow bone.
The nice thing about ultrasound is it provides you an
opportunity to be able to temporally
and spatially control
when these factors would be released.
And where. We've developed a
couple of different strategies for being able to do this.
One is what we might call gene painting,
where we're using a heat activated expression of
vascular endothelial growth factor
and bone morphogenic proteins.
And we can do those, we can trigger those
at different points in time.
For example, if you have an implant, a tissue implant
that you wanted to grow bone within that implant,
then you could trigger the vascular component
and then recruit vascularity
and then trigger these bone morphogenic protein
factors to cause the bone to be generated.
The other thing is, and I'll talk about it a little bit
later, is the use of the ADV,
the acoustic droplet vaporization in order to
control what's happening inside
of these tissue constructs as well.
I don't wanna go into a huge amount of detail here,
but the idea is that this
heat shock protein mediated cascade that you see
has been developed so that it requires a certain
component to be present in order for the activation
to occur, and then to be able to release the factor,
different factors, for instance, the VEGF and the BMP-2.
Without this growth factor,
and in the absence of heating, you get basically no effect.
But in the combination of these two,
then you can get a release of the
or the production of the endothelial growth factor and
or the BMP-2, and you can control that spatially.
With the ultrasound, you can heat at specific locations.
This is a luciferase reporter, so that you can control
by the duration of time that you do the heating,
how much expression that you get,
you can spatially control it, which if you release a bunch
of graduate students to work on a project at University
of Michigan, you end up with S-blocks being made.
And then the nice thing is
that you can spatially control it both in, say,
the lateral direction where the
ultrasound is propagating into the material.
But because it's focused ultrasound,
you can control the depth within the tissue structure
that you'd be able to activate,
and you can produce different gradients, so
that you can have a gradient structure so
that there can be larger growth on one side
of an object than you have on the other.

Testing Gene Activation

There have been some tests of this, again,
using the luciferase reporter to indicate the
activation by the heating process.
And in this case, you can see that
the luciferase was activated, the exposure
with ultrasound would occur,
but in the absence of the rapamycin as the
triggering mechanism,
then you don't get the activation.
It's nice you're able to control then the combination
of having heating, which might occur if you had fever,
but if you have ultrasound,
you can control when the actual heating occurs.
And only when the rapamycin is on board do you actually get
the activation of the gene sequence.

ADV in Tissue Constructs

And finally, it turns out
that the acoustic droplet vaporization may have some
impact as well
because it then will allow you
to change the structure of the implant.
These are gel implants then that you can trigger
a vascular response by adding ADV
and releasing different growth factors.
You can have a vascular response.
But the other thing that happens is
that you can change the porosity that exists
because now you've gone from a liquid state to a gas state,
you've generated pores on the inside of the
tissue construct, which will allow infiltration
of the vascular structures more easily.
This is just an example of the spatial control
that you have over the activation.
And this is an example, a fluorescent example of the
droplet with the drug on the inside of the droplet.
You can control then and release these growth factors.
The growth factor then,
this is the cumulative release.
This is a measure of the growth factor.
If you just introduce it
into the construct,
and then you look at what the accumulation
of the growth factor is over time,
then you can see that the growth factor,
once it's introduced, then accumulates within the
construct.
You can also formulate it as an emulsion
with the droplet, and then you can trigger it
and the release follows very much
that same progression.
If you release it at this point in time, then you get
a gradual accumulation
of this growth factor within the construct.
The nice thing is that you can look here,
the growth factor is gradually leaving the droplets
and accumulating in the outside
in the tissue construct.
But if you go over in time,
you can actually trigger it
and you can have a delayed trigger.
This is the temporal control that you'd like to have.
Even though the droplets were present starting at day
one, and there's some gradual leakage of the
material on the inside of
the droplet out into the medium, you only get
a substantial release where the slope
of this is similar to the slope
that you see over here when you actually trigger it
subsequently.
And as I was saying before,
because you're transitioning from a liquid droplet into a
gas state, then you actually change the porosity.
This is brightfield microscopy.
These are the particles that are on the inside of the
tissue construct.
And then once you trigger them with ultrasound,
then you can see the porosity has changed.
You can see the structures of the bubbles on the inside.
And then this is a fluorescent image of the fibrinogen
that is normally impregnated into these materials.
And the interesting thing is when you form these bubbles
on the inside, it concentrates apparently the fibrinogen
and causes the construct to actually become stiffer.
Not only are you controlling the porosity,
you're actually controlling the stiffness on the inside
of the construct.

Cell Viability in Constructs

And finally, it wouldn't do any good to have these kinds
of constructs and perform the vaporization of these
droplets on the inside if the cells didn't survive.
These are assays of fibrin and actin,
and then staining of the nuclei
of the cells using different types of dyes.
And you can see that the
cells are viable on the inside of the construct
without ultrasound
and then when ultrasound is applied as well.
It does appear that you can actually control the
material properties
and keep the cells viable in these constructs.

Summary

In summary, microbubbles have a wide range
of potential uses both in diagnostic
and therapeutic ultrasound.
They have unique non-linear properties
that create contrast signatures that allow us
to detect bubbles in the presence of tissue.
And bubbles can be formulated directly as gas bubbles
or formed from triggered evaporation
of the liquid perfluorocarbons.
For example, using this acoustic droplet vaporization,
microbubbles can then be used to control blood flow,
microbubbles either
as ultrasound contrast agents or bubbles coming from
this acoustic droplet
vaporization can enhance tissue heating
and drug delivery.
And there are many new applications,
including the new applications
that were presented here in tissue engineering.
Thank you and I hope you enjoyed this presentation.