**2. Summary of gene delivery with microbubbles and ultrasound**

The basic outline of a microbubble is shown in Figure 1. Microbubbles are composed of gas with a stabilizing shell material oftentimes consisting of lipids, albumin, or biocompatible polymers. For biomedical application they range in size from several microns in diameter to several hundred nanometers in diameter. The original biomedical application was as ultrasound contrast agents for echocardiography. Two agents, Definity®, phospholipidcoated perfluoropropane microbubbles (Lantheus, Billerica, MA) and Optison®, albumincoated perfluoropropane microbubbles, are approved by the FDA in the US and are sold as contrast agents with approved claims for echocardiography.

Because of the large impedance mismatch between liquid and gas, when sound waves strike a microbubble, the waves are efficiently scattered back (microbubbles are excellent acoustic reflectors) and this is the basis for the use of microbubbles as ultrasound contrast agents. They are excellent reflectors of sound energy and hence are outstanding contrast agents for biomedical ultrasound imaging. Furthermore, the design of special ultrasound pulse sequences as 2nd harmonic imaging and phase inversion harmonic imaging has helped to increase ultrasound imaging by eliminating significant amounts of noise from tissue reflection.

Gene Delivery with Ultrasound and Microbubbles 201

microbubbles to enhance localized delivery via microbubble bursting and subsequent radiation force induced particle penetration through the membrane surface. Cavitation can be used to increase cell permeability and local delivery of materials such as DNA for gene

A number of preclinical studies have been performed for gene (described further below) and drug delivery, but phospholipid-coated microbubbles have entered human clinical trials for treatment of vascular thrombosis. In these studies microbubbles have been administered intravenously and been shown to permeate a thrombus. Ultrasound energy has then been

Fig. 3. Depicts and occlusive thrombus in an arterial blood vessel (upper left). Microbubbles are infused IV and permeate the clot (upper right). This can be seen on ultrasound imaging. High energy ultrasound is focused on the site of thrombus (lower left) and the microbubbles

Microbubbles are mainly composed of fluorinated gases. Air and nitrogen are relatively water-soluble and hence will diffuse into the blood and the bubbles will then rapidly shrink and eventually collapse from Laplace pressures. This is not to say that air and nitrogen cannot be used to make microbubbles for biomedical ultrasound applications, but that

As opposed to oxygen and nitrogen being relatively soluble in water, perfluorocarbons are virtually insoluble in the aqueous milieu. In fact perfluorocarbons are amphiphobic. The higher the molecular weight of the fluorinated compound the less water soluble and we would predict that microbubbles prepared from that gas should be correspondingly more stable in the blood stream (all else equal). Note that perfluoropentane has a boiling point of 29oC and that perfluorohexane volatilizes at 56.6oC and therefore will be a liquid at

stabilizing materials will need to be more robust to preserve the microbubbles.

biological temperature (presumably due to van der Waal's attractions).

cavitate, dissolving the clot and restoring blood flow.

**3. Details of microbubbles for gene delivery** 

applied to the site of the clot to cavitate the microbubbles and dissolve the thrombus.

therapy.

Fig. 1. Depicts a microbubble coated with a film of stabilizing material.

Fig. 2. Shows a contrast-enhanced echocardiogram image of a porcine heart after occlusion of the left anterior descending coronary artery (model of acute myocardial infarction). The area of decreased perfusion in the left ventricular wall is clearly seen on the post contrast images but was not detectable without contrast.

When ultrasound encounters the acoustic interface of a microbubble not only may the ultrasound be scattered, but also, because of their size and the fortuitous insonation frequencies used clinically, the microbubbles also can oscillate (stable cavitation). Depending upon the acoustic pressure, oscillating microbubbles may rupture (cavitate – described further in section 4, inertial cavitation). On the basis of cavitation it was discovered that microbubbles had therapeutic applications for gene and drug delivery and treatment of vascular thrombosis. Ultrasound effects on cell membranes may be two-fold; 1) ultrasound itself can enhance membrane permeability, thereby allowing more diffusion from the extracellular milieu (sonoporation); or, 2) can be used in conjunction with

Fig. 1. Depicts a microbubble coated with a film of stabilizing material.

Fig. 2. Shows a contrast-enhanced echocardiogram image of a porcine heart after occlusion of the left anterior descending coronary artery (model of acute myocardial infarction). The area of decreased perfusion in the left ventricular wall is clearly seen on the post contrast

When ultrasound encounters the acoustic interface of a microbubble not only may the ultrasound be scattered, but also, because of their size and the fortuitous insonation frequencies used clinically, the microbubbles also can oscillate (stable cavitation). Depending upon the acoustic pressure, oscillating microbubbles may rupture (cavitate – described further in section 4, inertial cavitation). On the basis of cavitation it was discovered that microbubbles had therapeutic applications for gene and drug delivery and treatment of vascular thrombosis. Ultrasound effects on cell membranes may be two-fold; 1) ultrasound itself can enhance membrane permeability, thereby allowing more diffusion from the extracellular milieu (sonoporation); or, 2) can be used in conjunction with

images but was not detectable without contrast.

microbubbles to enhance localized delivery via microbubble bursting and subsequent radiation force induced particle penetration through the membrane surface. Cavitation can be used to increase cell permeability and local delivery of materials such as DNA for gene therapy.

A number of preclinical studies have been performed for gene (described further below) and drug delivery, but phospholipid-coated microbubbles have entered human clinical trials for treatment of vascular thrombosis. In these studies microbubbles have been administered intravenously and been shown to permeate a thrombus. Ultrasound energy has then been applied to the site of the clot to cavitate the microbubbles and dissolve the thrombus.

Fig. 3. Depicts and occlusive thrombus in an arterial blood vessel (upper left). Microbubbles are infused IV and permeate the clot (upper right). This can be seen on ultrasound imaging. High energy ultrasound is focused on the site of thrombus (lower left) and the microbubbles cavitate, dissolving the clot and restoring blood flow.
