**4. Ultrasound – parameters and bioeffects**

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 and for ophthalmic ultrasound to MI < 0.8.

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

Gene Delivery with Ultrasound and Microbubbles 207

Fig. 10. Videomicroscopy images of a single bubble in response to a single ultrasonic wave.

Fig. 11. A drug carrying-microbubble cavitating in response to ultrasound.

cavitation and can be used to increase efficacy from gene delivery.

microbubble-mediated gene delivery to treat the heart.

Another major mechanism in ultrasound that may be useful for drug and gene delivery is the mechanical force of microbubbles. Ultrasound exerts a radiation (pushing) force that can move microparticles and improve cellular delivery of biomaterials. Figure 12 below shows avidin-coated microbubbles flowing in blood over a biotinylated plate. The microbubbles flow and do not appear to bind but after application of relatively low MI ultrasound the microbubbles are pushed by the acoustic waves and bind to the surface of the plate. The same process can be used to improve cellular uptake of biomaterials such as DNA. The (pushing) radiation force of ultrasound occurs at lower energies than necessary for

An acoustic transducer that generally uses a piezoelectric material to convert electrical energy into ultrasound waves creates ultrasound waves. The transducer design varies depending upon the biomedical application. Figure 13 (below) shows the design of a three dimensional transducer for imaging and treating the heart. This transducer can be used to image the heart and visualize microbubble as they enter the myocardial circulation. Ultrasound energy can be applied to the heart to cavitate the microbubbles or for the radiation force to improve myocardial delivery. The transducer is currently being used in pre-clinical studies for microbubble enhanced sonothrombolysis to treat myocardial infarction and planned for use in clinical studies. The same transducer design could potentially be used in clinical studies for

Despite its ease of use, unwanted bioeffects can be experienced using ultrasound at high acoustic outputs. High levels of ultrasound energy with cavitation may cause cell damage, cell death and apoptosis (Miller). Ultrasound may also heat tissues and cause coagulative

acoustic streaming and local shock waves that may radiate on the order of microns or larger depending upon the acoustic intensity and other factors (Mehier-Humbert). Cavitation can be used to increase cell permeability (i.e. sonoporation)(Deng), open the blood brain barrier or destroy tissues (i.e. sonoablation)(Conger, Feril). The acoustic pressure can be controlled to create the desired effects (Forsberg). Within the ranges of allowable acoustic pressures, in the absence of exogenous microbubbles, biomedical ultrasound imaging does not generally cause violent cavitation and ultrasound imaging is generally quite safe, particularly compared to other technologies such as X-ray imaging that uses ionizing radiation.

As shown below in Figure 9, microbubbles lower the threshold of ultrasound energy necessary for cavitation to occur. The effect is frequency dependent with a greater proportional effect at 1 MHz than at 10 MHz. Note that at 1 MHz, cavitation occurs at MI < 0.5 within the allowable ultrasound power limits for biomedical imaging. Note that microbubbles in the size range of 0.5 to 2.0 microns are quite effective in lowering the thresh-hold of energy for cavitation. Stable microbubbles for gene delivery can certainly be made in this size range. If necessary, for therapeutic applications, higher levels of ultrasound energy can be employed. Higher levels of ultrasound energy are used therapeutically for hyperthermia and focused ultrasound surgery (Brujan).

Fig. 9. Microbubbles Lower Thresh-hold of Ultrasound Energy for Cavitation (Apfel).

Figure 10, below, shows images from ultra-high speed videomicroscopy of a single bubble in response to a single high MI pulse of ultrasound energy. The microbubble expands, collapses, and fragments. The daughter fragments then undergo one additional cycle of expansion, collapse and disappear. In this process genetic materials might be released from a genecarrying microbubble. The cavitation process will also create acoustic jets and streaming which might be used for delivering the genetic material to the target tissue or cells.

Figure 11 depicts a gene carrying microbubble in response to cavitation. The stabilizing wall material of the microbubble fragments and the genetic material is ejected with the cavitation ballistically, thereby extravasating from the vasculature to the target tissue.

acoustic streaming and local shock waves that may radiate on the order of microns or larger depending upon the acoustic intensity and other factors (Mehier-Humbert). Cavitation can be used to increase cell permeability (i.e. sonoporation)(Deng), open the blood brain barrier or destroy tissues (i.e. sonoablation)(Conger, Feril). The acoustic pressure can be controlled to create the desired effects (Forsberg). Within the ranges of allowable acoustic pressures, in the absence of exogenous microbubbles, biomedical ultrasound imaging does not generally cause violent cavitation and ultrasound imaging is generally quite safe, particularly compared to other technologies such as X-ray imaging

As shown below in Figure 9, microbubbles lower the threshold of ultrasound energy necessary for cavitation to occur. The effect is frequency dependent with a greater proportional effect at 1 MHz than at 10 MHz. Note that at 1 MHz, cavitation occurs at MI < 0.5 within the allowable ultrasound power limits for biomedical imaging. Note that microbubbles in the size range of 0.5 to 2.0 microns are quite effective in lowering the thresh-hold of energy for cavitation. Stable microbubbles for gene delivery can certainly be made in this size range. If necessary, for therapeutic applications, higher levels of ultrasound energy can be employed. Higher levels of ultrasound energy are used

therapeutically for hyperthermia and focused ultrasound surgery (Brujan).

Fig. 9. Microbubbles Lower Thresh-hold of Ultrasound Energy for Cavitation (Apfel).

might be used for delivering the genetic material to the target tissue or cells.

ballistically, thereby extravasating from the vasculature to the target tissue.

Figure 10, below, shows images from ultra-high speed videomicroscopy of a single bubble in response to a single high MI pulse of ultrasound energy. The microbubble expands, collapses, and fragments. The daughter fragments then undergo one additional cycle of expansion, collapse and disappear. In this process genetic materials might be released from a genecarrying microbubble. The cavitation process will also create acoustic jets and streaming which

Figure 11 depicts a gene carrying microbubble in response to cavitation. The stabilizing wall material of the microbubble fragments and the genetic material is ejected with the cavitation

that uses ionizing radiation.

Fig. 10. Videomicroscopy images of a single bubble in response to a single ultrasonic wave.

Fig. 11. A drug carrying-microbubble cavitating in response to ultrasound.

Another major mechanism in ultrasound that may be useful for drug and gene delivery is the mechanical force of microbubbles. Ultrasound exerts a radiation (pushing) force that can move microparticles and improve cellular delivery of biomaterials. Figure 12 below shows avidin-coated microbubbles flowing in blood over a biotinylated plate. The microbubbles flow and do not appear to bind but after application of relatively low MI ultrasound the microbubbles are pushed by the acoustic waves and bind to the surface of the plate. The same process can be used to improve cellular uptake of biomaterials such as DNA. The (pushing) radiation force of ultrasound occurs at lower energies than necessary for cavitation and can be used to increase efficacy from gene delivery.

An acoustic transducer that generally uses a piezoelectric material to convert electrical energy into ultrasound waves creates ultrasound waves. The transducer design varies depending upon the biomedical application. Figure 13 (below) shows the design of a three dimensional transducer for imaging and treating the heart. This transducer can be used to image the heart and visualize microbubble as they enter the myocardial circulation. Ultrasound energy can be applied to the heart to cavitate the microbubbles or for the radiation force to improve myocardial delivery. The transducer is currently being used in pre-clinical studies for microbubble enhanced sonothrombolysis to treat myocardial infarction and planned for use in clinical studies. The same transducer design could potentially be used in clinical studies for microbubble-mediated gene delivery to treat the heart.

Despite its ease of use, unwanted bioeffects can be experienced using ultrasound at high acoustic outputs. High levels of ultrasound energy with cavitation may cause cell damage, cell death and apoptosis (Miller). Ultrasound may also heat tissues and cause coagulative

Gene Delivery with Ultrasound and Microbubbles 209

was added to the microbubble suspension and mixed for 2 hours at 4°C. The mixture was separated into 2 distinct layers. The upper layer consisted of microbubbles with attached virus; the bottom layer, which contained unattached virus, was discarded. The concentration of microbubbles with attached AdCMV-β-Gal was 1.2x109 bubbles/mL; the mean diameter was 3.5 um. The viral titer of these microbubbles was determined. Rats were anesthetized, and MBs were administered IV. Echocardiography was performed at 1.3 MHz with a mechanical index of 1.5. Images were ECG-triggered to deliver a burst of 3 frames of ultrasound every 4 to 6 cardiac cycles. The hearts of all 6 rats in the experimental group showed blue staining with 5 bromo-4-chloro-3-indolyl-β-D-galactopyranoside. None of the control rats showed myocardial staining, which confirmed that the destruction of the microbubbles containing the virus was

Initiated in 2001, researchers began using gas-filled, albumin-shelled MBs (Optison) and non-viral carriers to increase nascent serum HDL cholesterol in mice and Sprague-Dawley rats. Initial feasibility studies were performed using reporter genes, 5-bromo-4-chloro-3 indolyl-β-D-galactopyranoside and GFP. Subsequent studies now use ApoA-I DNA plasmids in combination with Optison. In a series of rat studies performed with partial support from an NIH SBIR award (44HL095238-01), the results revealed that an average peak response of HDL-C elevation occurred within 24 hours of treatment and 88% of the responses occurred within 48 hours. The control animal group included ultrasound only, apoA-I DNA only, and microbubble only rats. These data demonstrated a rapid incorporation of the plasmid into the cell and efficient ApoA-I protein production that was

Summarized data revealed an average increase of 16.8% on day one post treatment. Due to wide normal variation in individual rat HDL cholesterol values, all data is plotted as a percentage from an individual animal's baseline. When examining the overall response to treatment in mg/dL, the average peak response to treatment was 14.0% (p-value <0.0001) as seen in Figure 1 (below). This response was observed uniformly across different animals

Ongoing studies include additional design of experiments for the following: (1) optimization of sonoporation utility for raising HDL cholesterol, (2) efficient energy delivery algorithms, (3) modes and methods of delivery, and (4) mechanistic analyses of the intra-

Ongoing studies have been performed with lipid coated MBs binding plasmid DNA with genes to treat diabetes. In vivo studies in rats have shown long-term normalization of blood

Encouraging work has been performed in models of hypercholesterolemia showing the potential to perform gene therapy for H1Alpha to improve the capacity of the liver to produce HDL as treatment for atherosclerosis. Promising work continues to progress in the use of MBs binding genes to treat hemophilia. Cationic MBs binding DNA (similar to Figure 7) have been tested with ultrasound and compared to DefinityR MBs which do not bind DNA. Expression of Factor IX transgenes is more robust with the cationic MBs binding DNA than with MBs not binding DNA. In both the HI1Alpha/HDL and hemophilia treatment applications the target organ is the liver. The goal in both of these programs and in many others is to generate sustained transgene expression in the liver (Guo). Catheter mediated administration may prove attractive to maximize efficiency of delivery to hepatocytes and catheters can be

Ultrasound can be targeted to any organ for which it is possible to create an acoustic window. Most tissues are readily accessible to ultrasound. Ultrasound can be applied across

glucose levels in diabetic rats. Studies are planned to test this system in primates.

deployed for delivery to other organs such as the kidneys, heart and blood vessels.

responsible for the observed β-galactosidase expression in the rat myocardium.

and ranged from 40mg/dL HDL-C baseline to 100mg/dL HDL-C baseline.

later incorporated into serum HDL-C.

cellular plasmid location.

necrosis at high temperatures (Ter Haar). We emphasize, however, that ultrasound power levels within the limits for diagnostic ultrasound are generally safe. Ultrasound power levels can be optimized for gene therapy to maximize gene expression while minimizing unwanted bioeffects (Chen, Rahim).

Fig. 12. Drawing of three-dimensional transducer for imaging and treating the heart.
