**12. Medicinal application**

**Imaging the liver** – This clinical application of MBs in radiology is most promising. Some portion of MBs is taken up by the liver and spleen. The precise mechanism is hitherto uncertain, but the involvement of the reticulo-endothelial system is probable. In clinical trials, this liver phase generally lasts about 30 minutes when the licensed agent Levovist is used and a few hours with some agents. The liver is very well seen with MB-specific imaging models, such as harmonic imaging, during this phase. The primary practical importance is that numerous focal liver lesions, especially metastases and hepatocellular carcinoma, appear as defects and the use of MBs considerably increases their visibility [135-137].

**Imaging the heart** – MBs can enhance Doppler flow signals in cardiac ultrasonography, and this can be useful in several situations, such as detecting valvular stenoses. The left ventricular cavity is highlighted by MB contrast agents and thus the blood-tissue boundary becomes much clearer. This is useful in detecting abnormalities in wall motion in a given region, assessment of ejection fraction, and finding left ventricular thrombus. The evaluation of left ventricular function is crucial for managing of a number of heart diseases. Both resting and inducible ischaemia can be accurately diagnosed by real time perfusion imaging in stress echocardiog‐ raphy which can be further enhanced by applying intermittent high power pulses destroying the majority of the MBs in a scan plane and subsequently watch refilling: the rate at which it occurs is a measure of microcirculatory flow speed [138]. IDummy Text maging **the heart** – MBs can enhance Doppler flow signals in cardiac ultrasonography, and this can be useful in several situations, such as detecting valvular stenoses. The left ventricular cavity is highlighted by MB contrast agents and thus the blood-tissue boundary becomes much clearer. This is useful in detecting abnormalities in wall motion in a given region, assessment of ejection fraction, and finding left ventricular thrombus. The evaluation of left ventricular function is crucial for managing of a number of heart diseases. Both resting and inducible ischaemia can be accurately diagnosed by real time perfusion imaging in stress echocardiography which can be further enhanced by applying intermittent high power pulses destroying the majority of the MBs in a scan plane and subsequently watch refilling: the rate at which it occurs is a measure of microcirculatory flow speed[138].

Figure 16. The sonographs of the rabbit heart before and after the continual infusion of the pegylated micribubbles. (A) control - an image of the heart without MBs.

(A) control - an image of the heart without MBs. (B) Heard filled by MBs during 10 minutes of continual infusion

**ultrasound imaging** 

(B) Heard filled by MBs during 10 minutes of continual infusion

Right parasternal long-axis window, 4 - chamber view: LA = left atrium, LV = left ventricle, RA = right atrium, RV = right ventricle, IVS = intervetricular septum, LVW = left ventricular wall, RVW = right ventricular wall, P = pericardium - lung interface. (Adapted from [144]) **15. Therapeutic applications recent advances in molecular, multimodal and theranostic Figure 16.** The sonographs of the rabbit heart before and after the continual infusion of the pegylated micribub‐ bles.Right parasternal long-axis window, 4 - chamber view:LA = left atrium, LV = left ventricle, RA = right atrium, RV = right ventricle, IVS = intervetricular septum, LVW = left ventricular wall, RVW = right ventricular wall, P = pericardi‐ um - lung interface. (Adapted from [144])

#### targeting drug carriers. MBs can help in drug delivery in themselves (by acting as "cavitation nuclei") and as drug carriers for site-specific treatment. Due to these characteristics they can be employed as drug carriers for treatment of various diseases, such as inflammation, thrombus, lymph nodes, cancer, angiogenesis and atherosclerosis [136]. **16. Gene therapy 13. Therapeutic applications recent advances in molecular, multimodal and theranostic ultrasound imaging**

The use of MBs in gene therapy is most challenging. The delivery of genetic material to a selected site represents the main problem in this emerging area. Drug delivery can be potentiated by ultrasound which creates transient non-lethal

The importance of MBs usage in treatment is higher than their use as a diagnostic tool. At present, the different characteristic features of MBs (various sizes and the types of gases and shell materials) precondition them for use as

perforations in cell membranes facilitating penetration of large molecules and particles into the cells ("sonoporating"). During this process, high acoustic power is necessary, which is beyond that permitting for imaging. However, in the presence of MBs the amount of required power is substantially reduced because the MBs decrease the amount of energy needed for the process of cavitation, leading to MB collapse due to excessive oscillations induced by ultrasound pulses. Furthermore, cavitation of MBs in capillary beds leads to a capillary permeability increase, thus improving local access of The importance of MBs usage in treatment is higher than their use as a diagnostic tool. At present, the different characteristic features of MBs (various sizes and the types of gases and shell materials) precondition them for use as targeting drug carriers. MBs can help in drug delivery in themselves (by acting as "cavitation nuclei") and as drug carriers for site-specific treatment. Due to these characteristics they can be employed as drug carriers for treatment of various diseases, such as inflammation, thrombus, lymph nodes, cancer, angiogenesis and atherosclerosis [136].

#### **13.1. Gene therapy**

Blue line: gass pushing homogenaous dispersion of MBs

102 Advances in Bioengineering

**12. Medicinal application**

Red line: homogenaous dispesion of MBs driving from Infucon cell into rabbit ear vein via canula

**a.** The use of Infucon device during the ultrasound scanning of the rabbit heart.

**b.** Integration of the Infucon device which is injecting the MB suspension via vena aricularis

**Imaging the liver** – This clinical application of MBs in radiology is most promising. Some portion of MBs is taken up by the liver and spleen. The precise mechanism is hitherto uncertain, but the involvement of the reticulo-endothelial system is probable. In clinical trials, this liver phase generally lasts about 30 minutes when the licensed agent Levovist is used and a few hours with some agents. The liver is very well seen with MB-specific imaging models, such as harmonic imaging, during this phase. The primary practical importance is that numerous focal liver lesions, especially metastases and hepatocellular carcinoma, appear as defects and the

**Figure 15.** Schematic and real view of the device used for i.v. infusion of MBs into the rabbit.

into the bloodstream of the rabbit. (Adapted from [144])

use of MBs considerably increases their visibility [135-137].

The use of MBs in gene therapy is most challenging. The delivery of genetic material to a selected site represents the main problem in this emerging area. Drug delivery can be poten‐ tiated by ultrasound which creates transient non-lethal perforations in cell membranes facilitating penetration of large molecules and particles into the cells ("sonoporating"). During this process, high acoustic power is necessary, which is beyond that permitting for imaging. However, in the presence of MBs the amount of required power is substantially reduced because the MBs decrease the amount of energy needed for the process of cavitation, leading to MB collapse due to excessive oscillations induced by ultrasound pulses. Furthermore, cavitation of MBs in capillary beds leads to a capillary permeability increase, thus improving local access of the released therapeutic drug [139]. The clinical use of virus-based vectors for gene therapy is limited. This is due to the fact that immune response can be induced by viral proteins in the target tissue and elicit a strong inflammatory activation of endothelial cells. However, it has been shown that when e.g. plasmids and antisense oligonucleotides are used in non-viral delivery vehicles, transfection efficiency is decreased and the expression of the gene product is transient. Commonly used MB preparation Levovist enhanced the beta-Gal gene transfection efficiency more than 6-fold and Optison were used for gene transfer to cardiomyocytes. The transfection efficiencies are significantly higher than that of no MB and it is similar to Levovist [97, 100, 114].

## **13.2. Blood-brain barrier**

The blood-brain barrier (BBB) is a specialized system of capillary endothelial cells preventing harmful substances in the blood stream from entering the brain. They also supply the brain with nutrients necessary for proper brain functioning. The BBB regulates transport through both tight junctions and metabolic barriers and is often a rate-limiting factor in determining permeation of therapeutic drugs into the brain. It is a significant obstacle affecting delivery of both small molecules and macromolecular agents. Despite the fact that a variety of drugs could be potentially used to treat brain diseases, no method exists that would allow non-invasivetargeted delivery through the BBB.

Ultrasound can be used to locally deliver a drug or gene to a specific region of interest in the brain. The benefit of the MB technique is that it can be used on a very small area of the BBB. MBs are used in combination with ultrasound exposure. The effects of ultrasound can be focused upon the vasculature to reduce the acoustic intensity required for BBB opening production. Several pathways of transcapillary passage after ultrasound sonication have been recognized, including transcytosis, passage through endothelial cell cytoplasmic openings holeyed by "nanoschrapnels" produced by explosion of MB, opening of tight junctions by oscillating MBs and free passage through injured endothelium [136, 140, 141].

#### **13.3. Thrombolysis and thrombus destruction**

The major cause of death in Western countries is thrombo-occlusive cardiovascular disease, with acute ST-elevation myocardial infarction (STEMI) being a serious medical condition leading to high morbidity and mortality. Two main treatment strategies have been developed during the past years. Their aim is to immediately restore coronary blood flow. In the first studies it was shown that fibrinolysis may be accelerated by ultrasound alone. Besides that, fibrinolysis can be even more increased by the addition of MBs which are destroyed by ultrasound induced high pressure. Three mechanisms leading to this increase have been proposed. The first is the occurrence of complete stress based on micro-jets formed after the destruction of the MBs by ultrasound. The second mechanism is the generation of reactive oxygen species in endothelial cells. The third proposed mechanism is a temperature increase, also occurring after the MBs collapse induced by ultrasound exposure. Whereas the full mechanism still remains to be elucidated, evidence indicates that the use of ultrasound alone and ultrasound combined with MBs can enhance clot dissolution by creating microholes in freshly formed thrombi [142, 143].
