Theranostic Applications of Microbubble Sonography: An Overview

Introduction to Microbubbles in Theranostics

I am Art Fleischer, chief of Ultrasound at Vanderbilt University Medical Center.

I'm very excited to present some initial work that we've done using microbubbles for not only diagnosis but therapy.

This is a whole new area called Theranostics, and I'm going to show some very preliminary data.

This is a very exciting area for applications of ultrasound and in particular, application of microbubbles, not only in diagnosis but therapy.

Microbubbles are about a third to two thirds the size of a red blood cell.

The shell is made up of albumin, lipid, or polymer, and the center is a gas either perfluorocarbon, sulfur, hdi, or in our case of doing new work is oxygen in the center using oxygen as the gas.

The diameter at rest is somewhere between one and two microns.

And as you can see from this picture, these microbubbles can oscillate and distend up up to almost 50 microns, which is 10 times their original size.

The resonant frequencies are anywhere from two to 10 megahertz, and the non-linearity of the microbubble allow it to be imaged above the echoes arrived from tissue.

These persist for a few cycles through the lungs, and they circulate for about five minutes or so.

This is just a picture of a macro bubble that Dr. Robin and I encountered in Korea.

This is a picture of a microbubble in a field, and as you can see here, the microbubble and largest and then actually explodes right in front of our eyes.

And we could see this because this was in front of a camera that took 6 million frames per second courtesy of Dr. Caskey.

Theranostics: Diagnosis and Therapy with Microbubbles

Now theranostic, the word theranostic implies that the microbubbles are used not only for diagnosis, they go to, in this case, cancer, but they can also be used as a therapeutic means.

And this usually requires the microbubbles to be labeled, and they can be labeled with vegf two, which goes to tumor microvessels or selectin, which goes to an inflammatory site, or alpha two, beta three, which goes to areas of thrombosis.

Now, the microbubbles can be used for assessment of tumor response, but most exciting area is the use of microbubbles to enhance drug delivery.

Assessing Tumor Response in Hepatocellular Carcinoma

So we had the opportunity of evaluating patients with hepatocellular carcinoma that underwent an anti-angiogenesis new drug.

And this diagram shows a vessel with the VGF receptor hanging down into the lumen and the anti-VEGF antibody attaching to it.

Now, if there's no interaction with the anti-VEGF, VEGF promotes the production of new blood vessels to supply the tumor.

As we all know from Dr. Folkman's original work, the theory is that if you can destroy the tumor's ability to produce blood vessels, you greatly hamper its ability to grow and also metastasize.

Now, these are basically two patients that we examine that had both an example of a good responder and a poor responder.

This is an image, a 3D image of a hepatocellular carcinoma.

And as you can see, the tumor itself is nicely outlined and the vessels supplying it on color Doppler are shown in 3D after 15 days of treatment.

You can see the tumor here, which had not really grown in over the 15 days.

And visibly the vessels and vascularity of the tumor were unchanged.

Now, this is a side-by-side comparison of the microbubbles, and as you can see, we start with the breakage of microbubble and look at the perfusion on this image.

And this is the baseline image, and this is the same tumor right here at day 15.

Now, of course, it's difficult to visually quantitate this.

So we have the ability to do a region of interest and look at the profusion when we do that.

In the bolus sequence, you can see this is the pretreatment line, and at day 15, there's marked decrease in this line.

And if you do destruction, reperfusion this difference in the pretreatment and post-treatment holds true.

So here's a comparison of the volume, the contrast enhancement, micro vessels, density, which is correlated by the slope of the curve and the height of the curve and the blood velocity.

So this was considered a good responder.

This on the other hand, is a patient that did not respond.

Here's the tumor at the initial phase.

As you can see, it's relatively hypovascular.

And at day 15, in fact, it became more vascular.

And these changes were quantitated.

As you can see on this graph where there's a difference in the pre-treatment, which is here post-treatment.

Here, there's actually more blood flow in the post-treatment curve.

And I might add that Dr. Lasso in France is using ultrasound to determine tumor response and is very successful at that.

Targeted Microbubbles and Drug Delivery

Now, when we look at targeted microbubbles, here's the diagram showing the lipid shell and the ability of putting a ligand on that shell.

Of course, we can also put drug into the microbubble itself as we see in this diagram.

And I credit Dr. Andre Lich for doing this original work.

Basically the nuclear glue, if you want to call it that, is a strep avidan biotin complex where one can get an anti antibody to vegf and hook it onto the microbubble and it attaches to the vegf site.

And this is basically a 3D picture of what's going on.

Now, this is an animation courtesy of Dr. Caskey.

As you can see, this is a vessel with endothelial gaps right here and the ana the VEGF receptor in projecting into the lumen.

And when we introduce our labeled microbubbles, some of them of course stick to the VGF receptor.

Others do not.

Those are free.

And if we examine this after a time, one minute in this case, we can determine which microbubbles are in fact bound to the receptor in this case, vegf.

And this is a picture from the literature using optical imaging of the little microbubble, those red dots attached to the vessels.

So basically to recapitulate how we do this, we inject the microbubbles.

And then after a good period of time, about three minutes, there is a high mechanical index applied, and the microbubbles that are in fact attached will remain.

This is a picture of a mouse tumor.

And these are es at one millimeter.

This is a mouse tumor that's implanted on a hind limb.

This is a example of the VEGF targeted microbubbles.

And as you can see, there's a very high green dot signal in this tumor that has very high vg GF.

And when you look at the differences between the two tumors, one with high VEGF versus one with low vegf, and you do vascular staining, the red, you can see visually and quantitate the amount of VEGF receptors in this tumor.

This is a nice diagram showing why certain medications are ineffective.

Either they get through the gap junction and they encounter a very acidic fluid environment, or they're basically destroyed by enzymatic reaction before they get to this site.

So it's obvious that a microbubble could determine how effectively drug is being produced.

I'm sorry, how effectively drug is distributed into the interstitial microbubbles, as you can see here, are various sizes, and their shell, as you can see, is very thin.

This is a diagram showing some of the concepts that we're using.

Oxygen-Filled Microbubbles for Tumor Hypoxia

In Theranostics, we have a microbubble and we're infusing oxygen into the center of it, which produces the gas.

In this case, it's a combination of oxygen and per fluorocarbon.

When the microbubble gets to the tumor, we can apply a high mechanical index.

And basically this helps the tumor become less hypoxic and therefore more susceptible to radiation therapy.

So these new microbubbles, as you can see, help the treatment of cancers, and it does that by decreasing the hypoxic areas inside of a tumor.

And it originally is shown that it improves oxygen within the tumor very significantly.

In fact, threefold increase in our initial work.

This is a photograph of a similar bubble with the oxygen inside of the center of the bubble.

Of course, other things can be put inside of a bubble, get to the target and then released.

And this is a diagram showing a viral component that can be delivered into tissues using this mechanism.

This is a picture of what the microbubble does at very high mechanical indexes, and this is Dr. Kakis work showing that the microbubble will oscillate, will increase in size, and then when it explodes, it produces these jets and the jets of fluid go through the endothelial gap.

Applications in Brain Tumors and Blood-Brain Barrier

And we've examined this mechanism in treating brain tumors.

As we all know, the blood brain barrier is very difficult to penetrate with drug, and we feel that the microbubbles will help the drug distribution into brain tumors.

This is a picture of a mouse who has a brain tumor, and as you, we can see from day three to day four with the ultrasound, the optical image shows much less activity inside the tumor.

And the similar type of principle has been reported by the group in Bri Brain Australia in patients with alt in mice with an analog of Alzheimer's disease.

And they've shown an a significant improvement in memory in mice treated with the microbubbles, which allow antibodies to cross the blood-brain barrier and attach to amyloid deposits.

This seems to be a very promising area for the use of microbubbles.

Future Applications of Microbubbles

So future applications include preselection of patients for certain specific treatments.

This is done by, has been shown by Beth McCarville, who's at St. Jude's Children's Hospital in Memphis, Tennessee in patients with refractory metastatic tumors.

There's a trial ongoing in Norway in patients with pancreatic cancers to use the microbubbles to improve drug delivery in China.

They're combining microbubbles with haifu to develop better ways of drug delivery.

And our institution, we're looking at drug delivery with microbubbles that get past the blood brain barrier.

This is a very small study that we hope to enlarge in mice with brain tumors.

Conclusion

I want to give special recognition to my associates, Charles Caskey at our Imaging Institute, and Andre Ach, who is now at Jefferson, worked for many years at Vanderbilt.

I'm very excited about the Theranostic applications of Microbubbles, and I hope this gives you an initial glimpse into their application.