**6. Ultrasound imaging**

In the field of molecular imaging, ultrasound represents a unique imaging technique as it provides real-time images in a non-invasive way using relatively cheap and easily portable equipment; thus, ultrasonography is considered to be currently most widely used diagnostic imaging modality [19]. However, since the liquid phase material with low compressibility, such as blood, scatters the sound waves poorly, contrast agents were proposed to improve the ultrasound images, offering the following general advantages: a very small intravenous injection is needed for imaging; and they are composed of non-toxic, biodegradable materials [30]. There are several types of ultrasound contrast agents, such as liquid emulsions, liposomes, and gas-filled MBs [33]. MBs will be described in detail in this chapter. Since the MB-based contrast agents are similar in size and shape to erythrocytes and also rheologically behave similarly to the blood, they appear to be of utmost convenience for ultrasound imaging systems, providing bright contrast [30]. Moreover, due to their larger size (compared to lipid and polymer based nanocarriers), systematically delivered MBs do not extravasate; instead they remain in circulation until they dissolve or are actively cleared by the mononuclear phagocyte system (MPS) [21, 34, 35].

**Figure 7.** Pictures of MB obtained by various imaging techniques.


(Adapted from [48])

acoustic contrast, the intent is to produce larger MBs [26-29]. On the other hand, the MB size distribution should not exceed 10 μm of size in diameter; otherwise they get quickly filtered by the lungs and can pose a risk of embolus [22, 30]. Furthermore, the size distribution is also

In the field of molecular imaging, ultrasound represents a unique imaging technique as it provides real-time images in a non-invasive way using relatively cheap and easily portable equipment; thus, ultrasonography is considered to be currently most widely used diagnostic imaging modality [19]. However, since the liquid phase material with low compressibility, such as blood, scatters the sound waves poorly, contrast agents were proposed to improve the ultrasound images, offering the following general advantages: a very small intravenous injection is needed for imaging; and they are composed of non-toxic, biodegradable materials [30]. There are several types of ultrasound contrast agents, such as liquid emulsions, liposomes, and gas-filled MBs [33]. MBs will be described in detail in this chapter. Since the MB-based contrast agents are similar in size and shape to erythrocytes and also rheologically behave similarly to the blood, they appear to be of utmost convenience for ultrasound imaging systems, providing bright contrast [30]. Moreover, due to their larger size (compared to lipid and polymer based nanocarriers), systematically delivered MBs do not extravasate; instead they remain in circulation until they dissolve or are actively cleared by the mononuclear

believed to affect the drug delivery [22, 31, 32].

**6. Ultrasound imaging**

88 Advances in Bioengineering

phagocyte system (MPS) [21, 34, 35].

**Figure 7.** Pictures of MB obtained by various imaging techniques.

## **7. MBs in the ultrasound field – Different aspects**

When exposed to low acoustic pressures, MBs tend to oscillate in a symmetrical, linear way [36], which means that their expansion and compression is inversely proportional to the local ultrasound pressure [37]; the phenomenon is known as a stable cavitation or non-inertial cavitation [38] and comprises stable, low amplitude oscillations of MBs (Figure 8) [23]. Such stable oscillations create a liquid flow around the MBs, the so called microstreams, which can apply a shear stress on cell membranes resulting in a transient opening of the cell membranes, as the shear stress related to micro streaming is relatively high compared to the shear stress associated with blood flow [23, 36, 39]. With the driving pressure turning into violent or inertial cavitation [38, 40], MBs undergo rapid expansion and contraction, which result in a violent bubble destruction [21]. The collapse of MBs is accompanied by shock waves generated in the fluid near the MB; furthermore, jet formation may occur, which can both contribute to a cell membrane perforation and increased permeabilization of blood vessels, thus improving extravasation of circulating drugs into target cells and tissues [20, 23, 41-44]. This phenomenon is called sonoporation [45] and has been proved visualisation of the pores by scanning electron microscopy, showing the pores in the plasma membrane (Figure 9) [46, 47]. Despite initial belief that inertial cavitation of MBs was required to enhance uptake of low molecular weight drugs, there is now mounting evidence that these stable oscillations may also stimulate the intracellular delivery of macromolecular drugs and nanoparticles [36]. Two mechanisms have been postulated to contribute to the uptake of cell impermeable molecules: the formation of small pores and endocytosis [46, 49]. Whilst the involvement of endocytosis was shown to be more extensive for larger molecules, the pore formation is considered the main mechanism for low-weight molecules, such as dextrans [49]. Moreover, when studied in a single-cell model, it was reported that a direct contact is needed to induce pore formation by stable cavitation, as larger distances would hamper direct mechanical cavitating MB and cell membrane and decrease the influence of microstreaming on the cell membrane (Figure 10) [36, 50-52]. Additionally, MBs facilitate cavitation related phenomena, such as temperature rise and free radical formation [21, 42, 53]; it is assumed that the free radicals increase the cell membrane permeability for Ca2+ in primary endothelial cells and cardiomyocytes [54-56].

Last but not least, acoustic radiation forces that may translate MBs in the direction of the propagating ultrasound wave should be taken into account [21, 36, 57, 58]. Not only can the acoustic radiation forces be applied to adhere to a specific target, but they can also help to push the MBs towards the cell surface and thus stimulate interaction with the cell membrane and promote drug delivery to specific cells [59-62]. Through acoustic radiation forces, MBs can squeeze through endothelium and tunnel through soft tissue, possibly increasing the deposi‐ tion of shell-loaded materials beyond the vasculature [21, 63, 64]. MBs was required to enhance uptake of low molecular weight drugs, there is now mounting evidence that these stable oscillations may also stimulate the intracellular delivery of macromolecular drugs and nanoparticles [36]. Two mechanisms have been postulated to contribute to the uptake of cell impermeable molecules: the formation of small pores and endocytosis [46, 49]. Whilst the involvement of endocytosis was shown to be more extensive for larger molecules, the pore formation is considered the main mechanism for low-weight molecules, such as dextrans [49]. Moreover, when studied in a single-cell model, it was reported that a direct contact is needed to induce pore formation by stable cavitation, as larger distances would hamper direct mechanical cavitating MB and cell membrane and decrease the influence of microstreaming on the cell membrane (**Figure 10**) [36, 50-52]. Additionally, MBs facilitate cavitation related phenomena, such as temperature rise and free radical formation [21, 42, 53]; it is assumed that the free radicals increase the cell membrane permeability for Ca2+ in primary endothelial cells and cardiomyocytes [54-56]. Last but not least, acoustic radiation forces that may translate MBs in the direction of the propagating ultrasound wave should be taken into account [21, 36, 57, 58]. Not only can the acoustic radiation forces be applied to adhere to a specific target, but they can also help to push the MBs towards the cell surface and thus stimulate interaction with the cell membrane and promote drug delivery tissues [20, 23, 41-44]. This phenomenon is called sonoporation [45] and has been proved visualisation of the pores by scanning electron microscopy, showing the pores in the plasma membrane (**Figure 9**) [46, 47]. Despite initial belief that inertial cavitation of MBs was required to enhance uptake of low molecular weight drugs, there is now mounting evidence that these stable oscillations may also stimulate the intracellular delivery of macromolecular drugs and nanoparticles [36]. Two mechanisms have been postulated to contribute to the uptake of cell impermeable molecules: the formation of small pores and endocytosis [46, 49]. Whilst the involvement of endocytosis was shown to be more extensive for larger molecules, the pore formation is considered the main

to specific cells [59-62]. Through acoustic radiation forces, MBs can squeeze through endothelium and tunnel through soft tissue,

mechanism for low-weight molecules, such as dextrans [49]. Moreover, when studied in a single-cell model, it was reported that a direct contact is needed to induce pore formation by stable cavitation, as larger distances would hamper direct mechanical cavitating MB and cell membrane and decrease the influence of microstreaming on the cell membrane (**Figure 10**) [36, 50-52]. Additionally, MBs facilitate cavitation related phenomena, such as temperature rise and free radical formation [21, 42, 53]; it is assumed that the

possibly increasing the deposition of shell-loaded materials beyond the vasculature [21, 63, 64].

tissues [20, 23, 41-44]. This phenomenon is called sonoporation [45] and has been proved visualisation of the pores by scanning electron microscopy, showing the pores in the plasma membrane (**Figure 9**) [46, 47]. Despite initial belief that inertial cavitation of

production of fragmets ‐ nanoshrapnels

*Figure 8. Effect of acoustic pressure on MB oscillation and explosion.*  Inertial Cavitation of Lipid-Coated MBs: Ultrasound causes lipid-coated MBs to expand and contract. If the pressure is below a critical value, called the inertial cavitation threshold, then the bubble undergoes sustained oscillations – stable cavitation. If the pressure is above the inertial cavitation threshold, then inertial forces govern the collapse; this is typically associated with the bubble imploding and breaking into small fragments – nanoshrapnels. The inertial cavitation threshold pressure depends on the material properties of the lipid coating (e.g., area expansion modulus and surface tension), which are set by the monolayer composition (lipid chain length, PEG coating, nanoparticle bound to the MB surface) Inertial Cavitation of Lipid-Coated MBs: Ultrasound causes lipid-coated MBs to expand and contract. If the pressure is below a critical value, called the inertial cavitation threshold, then the bubble undergoes sustained oscillations – stable cavitation. If the pressure is above the inertial cavitation threshold, then inertial forces govern the collapse; this is typi‐ cally associated with the bubble imploding and breaking into small fragments – nanoshrapnels. The inertial cavitation threshold pressure depends on the material properties of the lipid coating (e.g., area expansion modulus and surface tension), which are set by the monolayer composition (lipid chain length, PEG coating, nanoparticle bound to the MB surface) production of fragmets ‐ nanoshrapnels

critical value, called the inertial cavitation threshold, then the bubble undergoes sustained oscillations – stable cavitation. If the pressure is above the inertial cavitation threshold, then inertial forces govern the collapse; this is typically associated with the bubble imploding and breaking into small fragments – nanoshrapnels. The inertial cavitation threshold pressure depends on the material properties of the lipid coating (e.g., area expansion modulus and surface tension), which are set by the monolayer composition (lipid

**Figure 8.** Effect of acoustic pressure on MB oscillation and explosion. *Figure 8. Effect of acoustic pressure on MB oscillation and explosion.*  Inertial Cavitation of Lipid-Coated MBs: Ultrasound causes lipid-coated MBs to expand and contract. If the pressure is below a

chain length, PEG coating, nanoparticle bound to the MB surface)

*Figure 9. MBs assisted sonoporation of cells.* GSEM micrographs of cells after sonoporation and fixation. The cells were insonated with 2.25 MHz and 570 kPa peak negative pressure pulses in the presence of MBs. (a) MAT B III cells; (b) red blood cells. Adapted from [45]. Arrows indicate pores in the cell membrane. (http://amazingbubbleworld.blogspot.cz/2012/05/classifications-of-cavitation.html) GSEM micrographs of cells after sonoporation and fixation. The cells were insonated with 2.25 MHz and 570 kPa peak negative pressure pulses in the presence of MBs. (a) MAT B III cells; (b) red blood cells. Adapted from [45]. Arrows indicate pores in the cell membrane. (http://amazingbubbleworld.blogspot.cz/2012/05/classifications-of-cavita‐ tion.html)

**Figure 9.** MBs assisted sonoporation of cells.

Lipid-Based Nanoparticles and Microbubbles – Multifunctional Lipid-Based Biocompatible Particles for *in vivo*… http://dx.doi.org/10.5772/59870 91

Last but not least, acoustic radiation forces that may translate MBs in the direction of the propagating ultrasound wave should be taken into account [21, 36, 57, 58]. Not only can the acoustic radiation forces be applied to adhere to a specific target, but they can also help to push the MBs towards the cell surface and thus stimulate interaction with the cell membrane and promote drug delivery to specific cells [59-62]. Through acoustic radiation forces, MBs can squeeze through endothelium and tunnel through soft tissue, possibly increasing the deposi‐

tissues [20, 23, 41-44]. This phenomenon is called sonoporation [45] and has been proved visualisation of the pores by scanning electron microscopy, showing the pores in the plasma membrane (**Figure 9**) [46, 47]. Despite initial belief that inertial cavitation of MBs was required to enhance uptake of low molecular weight drugs, there is now mounting evidence that these stable oscillations may also stimulate the intracellular delivery of macromolecular drugs and nanoparticles [36]. Two mechanisms have been postulated to contribute to the uptake of cell impermeable molecules: the formation of small pores and endocytosis [46, 49]. Whilst the involvement of endocytosis was shown to be more extensive for larger molecules, the pore formation is considered the main mechanism for low-weight molecules, such as dextrans [49]. Moreover, when studied in a single-cell model, it was reported that a direct contact is needed to induce pore formation by stable cavitation, as larger distances would hamper direct mechanical cavitating MB and cell membrane and decrease the influence of microstreaming on the cell membrane (**Figure 10**) [36, 50-52]. Additionally, MBs facilitate cavitation related phenomena, such as temperature rise and free radical formation [21, 42, 53]; it is assumed that the free radicals increase the cell membrane permeability for Ca2+ in primary endothelial cells and cardiomyocytes [54-56].

Last but not least, acoustic radiation forces that may translate MBs in the direction of the propagating ultrasound wave should be taken into account [21, 36, 57, 58]. Not only can the acoustic radiation forces be applied to adhere to a specific target, but they can also help to push the MBs towards the cell surface and thus stimulate interaction with the cell membrane and promote drug delivery to specific cells [59-62]. Through acoustic radiation forces, MBs can squeeze through endothelium and tunnel through soft tissue,

Last but not least, acoustic radiation forces that may translate MBs in the direction of the propagating ultrasound wave should be taken into account [21, 36, 57, 58]. Not only can the acoustic radiation forces be applied to adhere to a specific target, but they can also help to push the MBs towards the cell surface and thus stimulate interaction with the cell membrane and promote drug delivery to specific cells [59-62]. Through acoustic radiation forces, MBs can squeeze through endothelium and tunnel through soft tissue,

Inertial Cavitation of Lipid-Coated MBs: Ultrasound causes lipid-coated MBs to expand and contract. If the pressure is below a critical value, called the inertial cavitation threshold, then the bubble undergoes sustained oscillations – stable cavitation. If the pressure is above the inertial cavitation threshold, then inertial forces govern the collapse; this is typically associated with the bubble imploding and breaking into small fragments – nanoshrapnels. The inertial cavitation threshold pressure depends on the material properties of the lipid coating (e.g., area expansion modulus and surface tension), which are set by the monolayer composition (lipid

Inertial Cavitation of Lipid-Coated MBs: Ultrasound causes lipid-coated MBs to expand and contract. If the pressure is below a critical value, called the inertial cavitation threshold, then the bubble undergoes sustained oscillations – stable cavitation. If the pressure is above the inertial cavitation threshold, then inertial forces govern the collapse; this is typi‐ cally associated with the bubble imploding and breaking into small fragments – nanoshrapnels. The inertial cavitation threshold pressure depends on the material properties of the lipid coating (e.g., area expansion modulus and surface tension), which are set by the monolayer composition (lipid chain length, PEG coating, nanoparticle bound to the MB

Inertial Cavitation of Lipid-Coated MBs: Ultrasound causes lipid-coated MBs to expand and contract. If the pressure is below a critical value, called the inertial cavitation threshold, then the bubble undergoes sustained oscillations – stable cavitation. If the pressure is above the inertial cavitation threshold, then inertial forces govern the collapse; this is typically associated with the bubble imploding and breaking into small fragments – nanoshrapnels. The inertial cavitation threshold pressure depends on the material properties of the lipid coating (e.g., area expansion modulus and surface tension), which are set by the monolayer composition (lipid

production of fragmets ‐ nanoshrapnels

production of fragmets ‐ nanoshrapnels

GSEM micrographs of cells after sonoporation and fixation. The cells were insonated with 2.25 MHz and 570 kPa peak negative pressure pulses in the presence of MBs. (a) MAT B III cells; (b) red blood cells. Adapted from [45]. Arrows indicate pores in the cell

GSEM micrographs of cells after sonoporation and fixation. The cells were insonated with 2.25 MHz and 570 kPa peak negative pressure pulses in the presence of MBs. (a) MAT B III cells; (b) red blood cells. Adapted from [45]. Arrows indicate pores in the cell

GSEM micrographs of cells after sonoporation and fixation. The cells were insonated with 2.25 MHz and 570 kPa peak negative pressure pulses in the presence of MBs. (a) MAT B III cells; (b) red blood cells. Adapted from [45]. Arrows indicate pores in the cell membrane. (http://amazingbubbleworld.blogspot.cz/2012/05/classifications-of-cavita‐

membrane. (http://amazingbubbleworld.blogspot.cz/2012/05/classifications-of-cavitation.html)

membrane. (http://amazingbubbleworld.blogspot.cz/2012/05/classifications-of-cavitation.html)

tissues [20, 23, 41-44]. This phenomenon is called sonoporation [45] and has been proved visualisation of the pores by scanning electron microscopy, showing the pores in the plasma membrane (**Figure 9**) [46, 47]. Despite initial belief that inertial cavitation of MBs was required to enhance uptake of low molecular weight drugs, there is now mounting evidence that these stable oscillations may also stimulate the intracellular delivery of macromolecular drugs and nanoparticles [36]. Two mechanisms have been postulated to contribute to the uptake of cell impermeable molecules: the formation of small pores and endocytosis [46, 49]. Whilst the involvement of endocytosis was shown to be more extensive for larger molecules, the pore formation is considered the main mechanism for low-weight molecules, such as dextrans [49]. Moreover, when studied in a single-cell model, it was reported that a direct contact is needed to induce pore formation by stable cavitation, as larger distances would hamper direct mechanical cavitating MB and cell membrane and decrease the influence of microstreaming on the cell membrane (**Figure 10**) [36, 50-52]. Additionally, MBs facilitate cavitation related phenomena, such as temperature rise and free radical formation [21, 42, 53]; it is assumed that the free radicals increase the cell membrane permeability for Ca2+ in primary endothelial cells and cardiomyocytes [54-56].

tion of shell-loaded materials beyond the vasculature [21, 63, 64].

*Figure 8. Effect of acoustic pressure on MB oscillation and explosion.* 

chain length, PEG coating, nanoparticle bound to the MB surface)

*Figure 8. Effect of acoustic pressure on MB oscillation and explosion.* 

**Figure 8.** Effect of acoustic pressure on MB oscillation and explosion.

chain length, PEG coating, nanoparticle bound to the MB surface)

*Figure 9. MBs assisted sonoporation of cells.*

*Figure 9. MBs assisted sonoporation of cells.*

**Figure 9.** MBs assisted sonoporation of cells.

surface)

90 Advances in Bioengineering

tion.html)

possibly increasing the deposition of shell-loaded materials beyond the vasculature [21, 63, 64].

possibly increasing the deposition of shell-loaded materials beyond the vasculature [21, 63, 64].

**Figure 10.** Various aspect of haw MBs can effect endothelial cell in capillary vessels.
