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

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

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

Gene Delivery with Ultrasound and Microbubbles 203

In vivo studies with plasmid DNA using the construct depicted in Figure 5 have shown high

We predict, however, that targeted constucts that would bind to cellular targets should be more effective for gene delivery. Since microbubbles are micron-sized structures, they are not expected to extravasate from the intravscular space. However, for the purposes of delivering genes to regions in close proximity to targeted tissues, they can be targeted to eptitopes expressed on endothelial cells. Because they can also be engulfed by phagocytic cells, such as immune cells, and targeted as intracellular passengers, the nanoparticles fused to microbubble surfaces as described below (Figure 8) can also be decorated with cell surface identifiers/targets, uptake enhancers, and even intracellular targets in order to provide additional selectivity. We have prepared targeted microbubbles to a variety of different targets. Depicted below is the design for a targeted microbubble to the integrin

The microbubble construct depicted above combines several features of nanotechnology. The parent microbubble is PEG'ylated to impart stealth properties in order to prevent reticuloendothelial system elimination and includes targeting ligands directed to epitopes expressed on endothelial cells. It could be monitored as an ultrasound contrast agent and then activated with higher energy ultrasound using low MI ultrasound for imaging to monitor delivery to the target site and high MI ultrasound energy for cavitation. Upon activation the nanoparticles containing DNA would be expected to extravasate from the vasculature into the interstitial tissues. The nanoparticles could be constructed to contain targeting ligands and/or cell penetration-enhancing agents to facilitate uptake by target cells. A challenge is to design linkers to attach the nanoparticles to the surface of the parent microbubbles. One approach would be to biotinylate the nanoparticles and to have avidin tethers attached to the parent microbubbles taking advantage of the avidin/biotin interaction. This approach could be utilized in proof-of-principle studies but would not be

Fig. 5. Depicts a PEG'ylated cationic microbubble binding DNA.

levels of gene expression in the zones of insonation.

αvβIII, Expressed on the endothelial surface in angiogenesis.


Table 1. Potential Compounds for Making Gaseous Cores of Microbubbles

Fig. 4. Depicts a microbubble for gene delivery. The gaseous core is coated by a monolayer of phospholipid containing cationic lipid imparting a net positive charge to the microbubble. DNA, as a polyanion, is adsorbed electrostatically to the exterior surface of the microbubble.

With respect to the microbubble membrane, the above design demonstrates how DNA is adsorbed to the surface via electrostatic adhesion with cationic lipids inserted in the membrane. However, note also that there is no steric protection of the DNA, making it susceptible to biodegradation/hydrolysis. As shown below in Figure 5, microbubbles can be designed to incorporate PEG'ylated lipid (e.g. 8-10 mole percent PEG'ylated lipid) and we have still found that cationic PEG'ylated microbubbles will still adsorb useful payloads of DNA.

Nitrogen 28 18071 -196

Oxygen 32 4865 -183

Sulfur Hexafluoride 146 5950 -64

Perfluoropropane 188 583 -36.7

Perfluorobutane 238 <500 -1.7

Perfluoropentane 288 >24 and <500 29

Perfluorohexane 338 24 56.6

Fig. 4. Depicts a microbubble for gene delivery. The gaseous core is coated by a monolayer of phospholipid containing cationic lipid imparting a net positive charge to the microbubble. DNA, as a polyanion, is adsorbed electrostatically to the exterior surface of the microbubble. With respect to the microbubble membrane, the above design demonstrates how DNA is adsorbed to the surface via electrostatic adhesion with cationic lipids inserted in the membrane. However, note also that there is no steric protection of the DNA, making it susceptible to biodegradation/hydrolysis. As shown below in Figure 5, microbubbles can be designed to incorporate PEG'ylated lipid (e.g. 8-10 mole percent PEG'ylated lipid) and we have still found that cationic PEG'ylated microbubbles will still adsorb useful payloads of DNA.

Table 1. Potential Compounds for Making Gaseous Cores of Microbubbles

Aqueous Solubility (Ostwald's Coefficient)

Boiling Point °C

Compound Molecular Weight

Fig. 5. Depicts a PEG'ylated cationic microbubble binding DNA.

In vivo studies with plasmid DNA using the construct depicted in Figure 5 have shown high levels of gene expression in the zones of insonation.

We predict, however, that targeted constucts that would bind to cellular targets should be more effective for gene delivery. Since microbubbles are micron-sized structures, they are not expected to extravasate from the intravscular space. However, for the purposes of delivering genes to regions in close proximity to targeted tissues, they can be targeted to eptitopes expressed on endothelial cells. Because they can also be engulfed by phagocytic cells, such as immune cells, and targeted as intracellular passengers, the nanoparticles fused to microbubble surfaces as described below (Figure 8) can also be decorated with cell surface identifiers/targets, uptake enhancers, and even intracellular targets in order to provide additional selectivity. We have prepared targeted microbubbles to a variety of different targets. Depicted below is the design for a targeted microbubble to the integrin αvβIII, Expressed on the endothelial surface in angiogenesis.

The microbubble construct depicted above combines several features of nanotechnology. The parent microbubble is PEG'ylated to impart stealth properties in order to prevent reticuloendothelial system elimination and includes targeting ligands directed to epitopes expressed on endothelial cells. It could be monitored as an ultrasound contrast agent and then activated with higher energy ultrasound using low MI ultrasound for imaging to monitor delivery to the target site and high MI ultrasound energy for cavitation. Upon activation the nanoparticles containing DNA would be expected to extravasate from the vasculature into the interstitial tissues. The nanoparticles could be constructed to contain targeting ligands and/or cell penetration-enhancing agents to facilitate uptake by target cells. A challenge is to design linkers to attach the nanoparticles to the surface of the parent microbubbles. One approach would be to biotinylate the nanoparticles and to have avidin tethers attached to the parent microbubbles taking advantage of the avidin/biotin interaction. This approach could be utilized in proof-of-principle studies but would not be

Gene Delivery with Ultrasound and Microbubbles 205

clinically translatable. A maleimide labeled spacer might be affixed to the microbubble and the nanoparticles might be thiolated to covalently bind the nanoparticle to the surface of the microbubbles in a potentially biocompatible manner. Another approach would be to use electrostatic interaction between the nanoparticle and the surface of the microbubbles but this must be optimized to ensure that the DNA is bound until it reaches the target site. Note also that if the bubble has excess cationic charge this may adversely affect biodistribution. Note also that it is more difficult to bind low molecular weight genetic material such as si-

Fig. 8. Depicts a unique microbubble construct for gene delivery. The parent microbubble is PEG'ylated but the DNA (or siRNA) is condensed into nanoparticles that are bound to the surface of the microbubble. The nanoparticles have targeting ligands to bind to cell specific epitopes. Note also that the construct could be modified to comprise endothelial targeting moieties on the microbubble so that the microbubble could bind to endothelial epitopes.

Ultrasound is a commonly used modality for biomedical imaging, only exceeded by Xrays in overall worldwide use. For medical imaging the power or intensity of ultrasound that can be used is limited by regulatory guidelines. The intensity of the ultrasound can be described by the mechanical index (MI) which is related to the peak negative pressure of the ultrasound wave divided by the square root of the center frequency of the ultrasound. The FDA-approved mechanical index for most body imaging ultrasound (e.g. cardiac and abdominal) is limited to an MI < 1.9. For neurovascular imaging it is limited to MI < 1.0

Cavitation is a phenomenon in which ultrasound exposure at the resonance frequency of the microbubble will induce expansion and collapse of microbubbles (Apfel, Hallow). This can occur spontaneously at high acoustic pressure in the absence of exogenous microbubbles. Sufficiently high acoustic power is sufficient to create a microbubble nidus in situ and cause expansion and collapse, i.e. cavitation (Marmottant). Cavitation causes

RNA using merely electrostatic interaction.

**4. Ultrasound – parameters and bioeffects** 

and for ophthalmic ultrasound to MI < 0.8.
