**9. Multifunctional MBs for ultrasound imaging and theranostics**

The concept of therapeutic agents being encapsulated in or conjugated with MBs has been developed over the past few years. In addition to the synergistic effects of ultrasound and MBs to enhance the permeability of biological barriers such as cell membranes, small blood vessels, and the BBB, as discussed above, MBs can serve as protective drug carriers. Drugs can be preincorporated into carriers such as liposomes, micelles, or microspheres, and these structures can then be easily attached to lipid MBs, usually via avidin-biotin interactions (Figure 11) [66]. Such advanced MB complexes have extremely high drug loading capacities and the advantage of being able to encapsulate both hydrophilic and hydrophobic drugs. Encapsulating unstable agents protects them from degradation in blood, thus prolonging their half-lives in vivo, improving treatment efficacy, and lowering the required dose [67]. Another advantage is that the encapsulated agents can be released during the ultrasound-triggered MB destruction process. Chemotherapeutic drugs can thus be directly and specifically delivered to target tissues via ultrasound-mediated perforations, whereas the uptake of the drugs by non-target tissues is reduced. The encapsulated agents are conjugated close to the shell of MBs, increasing the opportunity for microstreams, shock waves, and microjets to drive them toward the tissues and enhance their uptake in the ultrasound-treated region [68]. Since MBs act as ultrasound contrast agents, the drug delivery process can also be concurrently monitored by detecting the drug-loaded MBs [69].

Another advantage is that the encapsulated agents can be released during the ultrasound-triggered MB destruction process. Chemotherapeutic drugs can thus be directly and specifically delivered to target tissues via ultrasound-mediated perforations, whereas the uptake of the drugs by non-target tissues is reduced. The encapsulated agents are conjugated close to the shell of MBs, Lipid-Based Nanoparticles and Microbubbles – Multifunctional Lipid-Based Biocompatible Particles for *in vivo*… http://dx.doi.org/10.5772/59870 93

The concept of therapeutic agents being encapsulated in or conjugated with MBs has been developed over the past few years. In addition to the synergistic effects of ultrasound and MBs to enhance the permeability of biological barriers such as cell membranes, small blood vessels, and the BBB, as discussed above, MBs can serve as protective drug carriers. Drugs can be pre-incorporated into carriers such as liposomes, micelles, or microspheres, and these structures can then be easily attached to lipid MBs, usually via avidin-biotin interactions (**Figure 11**) [66]. Such advanced MB complexes have extremely high drug loading capacities and the advantage of being able to encapsulate both hydrophilic and hydrophobic drugs. Encapsulating unstable agents protects them from degradation in blood, thus prolonging their half-lives in vivo, improving treatment efficacy, and lowering the required dose [67].

increasing the opportunity for microstreams, shock waves, and microjets to drive them toward the tissues and enhance their uptake in the ultrasound-treated region [68]. Since MBs act as ultrasound contrast agents, the drug delivery process can also be concurrently

**Multifunctional MBs for ultrasound imaging and theranostics** 

monitored by detecting the drug-loaded MBs [69].

*Figure 11. Targeted and multifunctional MBs.*  A) Structure of plain MB (upper – schematic picture; bottom – optical microscopy –Nomarski contrast) B) Functionalised MB with bound liposomes (upper - schematic picture showing various possibility of functionalization and modification; bottom – MB with surface bound liposomes – optical microscopy –Nomarski contrast) C) Transmission electron microscopy of the functionalised MB with bound liposomes. Black arrow - rupture of MB shell owing to vacuum in electron microscope; white arrows - liposomes bound onto the surface of MBs. (Adapted from [48]) A) Structure of plain MB (upper – schematic picture; bottom – optical microscopy –Nomarski contrast) B) Functionalised MB with bound liposomes (upper - schematic picture showing various possibility of functionaliza‐ tion and modification; bottom – MB with surface bound liposomes – optical microscopy –Nomarski contrast) C) Transmission electron microscopy of the functionalised MB with bound liposomes. Black arrow - rupture of MB shell owing to vacuum in electron microscope; white arrows - liposomes bound onto the surface of MBs. (Adapted from [48])

**Figure 11.** Targeted and multifunctional MBs.

#### **10. Targeted MBs**

**8. Commercial preparations**

92 Advances in Bioengineering

Definity® Lantheus Medical

drug-loaded MBs [69].

Imaging

**Table 1.** Specifications of commercial and self-made MBs.

The use of MBs in echocardiography was first reported in 1968 [65]. Since that time great progress has been done in preparation of MBs for human and veterinary use. The following three commercial MB agents Optison TM (GE Healthcare, WI, USA), Definity® (Lantheus Medical Imaging, MA, USA) and SonoVue® (Bracco, Milano, Italy) currently hold the license for clinical diagnostic applications (Table 1). The size of the commercial MBs in these agents

OptisonTM GE Healthcare Albumin C3F8 2.0 - 4.5 5-8x108 2.5-4.5

SonoVue® Bracco Phospholipid CF6 2.0-8.0 0.9-6x1010 3-6

The concept of therapeutic agents being encapsulated in or conjugated with MBs has been developed over the past few years. In addition to the synergistic effects of ultrasound and MBs to enhance the permeability of biological barriers such as cell membranes, small blood vessels, and the BBB, as discussed above, MBs can serve as protective drug carriers. Drugs can be preincorporated into carriers such as liposomes, micelles, or microspheres, and these structures can then be easily attached to lipid MBs, usually via avidin-biotin interactions (Figure 11) [66]. Such advanced MB complexes have extremely high drug loading capacities and the advantage of being able to encapsulate both hydrophilic and hydrophobic drugs. Encapsulating unstable agents protects them from degradation in blood, thus prolonging their half-lives in vivo, improving treatment efficacy, and lowering the required dose [67]. Another advantage is that the encapsulated agents can be released during the ultrasound-triggered MB destruction process. Chemotherapeutic drugs can thus be directly and specifically delivered to target tissues via ultrasound-mediated perforations, whereas the uptake of the drugs by non-target tissues is reduced. The encapsulated agents are conjugated close to the shell of MBs, increasing the opportunity for microstreams, shock waves, and microjets to drive them toward the tissues and enhance their uptake in the ultrasound-treated region [68]. Since MBs act as ultrasound contrast agents, the drug delivery process can also be concurrently monitored by detecting the

**9. Multifunctional MBs for ultrasound imaging and theranostics**

**(µm)**

Phospholipid C3F8 1.1 - 3.3 1.2x1010 2-10

**Concentration (MB/mL)**

**MB half life (min)**

is usually larger than 1 um with imaging durations being 5-10 min.

**MB type Manufacturer Shell material Gas Mean size**

Despite the possibility of employing MBs for targeted ultrasound imaging and drug and gene delivery using the local ultrasound insonation, attaching targeting ligands on the surface of MBs, which leads to their accumulation in the target region (Figure 12), makes the MBs even more specific so that the range of clinical use of MBs is vastly expanded [23]. Owing to their size, MBs are not supposed to leave the vasculature; thus their use is limited to thrombus, endothelial cells, atherosclerotic plaques, blood clots, and organs such as spleen and liver [23, 47]. Recently there have also appeared papers referring to the use of MB-enhanced ultrasound for facilitated brain delivery [70, 71]; although the exact mechanism is still unknown, the interaction between capillary walls and acoustically driven MBs was found to be one of the key factors which leads to the disruption of the blood-brain barrier – the main obstacle in the treatment of neurodegenerative diseases [70, 72]. Since the method is non-invasive, according to the authors, it could be the way for treatment of such diseases as Parkinson's disease or Alzheimer's disease.

**Figure 12.** Passive and active targeting of MBs.

There aredifferentwayshowtoattachthe targetingligands tothe shellofMBs that aregenerally distinguished by the method of preparation. The targeting ligand can be either co-incorporat‐ ed in the shell during the MB preparation, or attached to the surface of pre-formulated MBs by covalent or non-covalent methods [73, 74]. The simplest targeted MB design is based on the use of a phospholipid, phosphatidylserine, which is incorporated in the MB shell *during* prepara‐ tion. Due to the fact that phosphatidylserine is a membrane marker of apoptosis, it promotes the phagocytic uptake of cells and liposomes by Kupffer cells and macrophages [74-76]. While circulating in the bloodstream, MBs containing phosphatidylserine are actively targeting and captured by the Kupffer cells in the liver, macrophages in the spleen and active neutrophils in the areas of inflammation, which is the reason why phosphatidylserine-shell agents are used to detect the foci that lack active phagocytic capacity, e.g. tumour nods in the liver [73, 77-79]. In these agents, the shell is the ligand (phosphatidylserine); since the molecules of phopshati‐ dylserine are kept together by the hydrophobic, van der Waals, and other intermolecular interactions, the ligand is attached in a non-covalent manner [73].

Nevertheless,thoughpreparedbystraightforwardshear-mixinginonesimplestep,thismethod is convenient only for stable ligands, which are able to survive the extreme conditions during the MB preparations [23]. Particularly some proteins and enzymes might be denatured in the harsh conditions and might lose binding affinity [73]. Therefore a different approach has been applied, involving attachment of ligands after MB preparation: non-covalent (strept)avidinbiotin interaction and covalent binding [23, 73, 80, 81]. Considering the fact that the (strept)avi‐ din-biotin interaction has the strongest effective dissociation constant, it has been the most widely used technique in biotechnology [82]. Biotin residues might be attached in one single step to antibodies, carbohydrates and many other targeting ligands, including the MB shell components [73, 83]. Biotinylated antibodies are abundant and can be easily linked to prema‐ nufactured biotinylated bubbles via a streptavidin linker [84]. Nonetheless, this coupling scheme is useful only in preclinical research: streptavidin as a foreign protein is a possible immunogen which might lead to undesired immune response after multiple injections [85].

Another attractive non-covalent strategy is to use the interaction of a metallochelating lipid complex, such as nickel-nitrilotriacetyl lipid, with a multiplicity of histidine residues (His-tag) of a protein [48]. The method offers an easy way to attach a genetically engineered protein to the MB surface with a His-tag, a commonly applied insert in recombinant protein preparation. The advantage of metallochelating complex over protein-ligand complexes, e.g. (streptavidin – biotin, glutathion – glutathion transferase) consists in its specific binding and very low immunogenicity, if any [86]. However, except for studies using metallochelating liposomes [86, 87], very little research has been devoted to this issue in terms of MBs; therefore there still appears to be a high potential for the His-tag – MB applications in the foreseeable future.

**Figure 12.** Passive and active targeting of MBs.

94 Advances in Bioengineering

There aredifferentwayshowtoattachthe targetingligands tothe shellofMBs that aregenerally distinguished by the method of preparation. The targeting ligand can be either co-incorporat‐ ed in the shell during the MB preparation, or attached to the surface of pre-formulated MBs by covalent or non-covalent methods [73, 74]. The simplest targeted MB design is based on the use of a phospholipid, phosphatidylserine, which is incorporated in the MB shell *during* prepara‐ tion. Due to the fact that phosphatidylserine is a membrane marker of apoptosis, it promotes the phagocytic uptake of cells and liposomes by Kupffer cells and macrophages [74-76]. While circulating in the bloodstream, MBs containing phosphatidylserine are actively targeting and captured by the Kupffer cells in the liver, macrophages in the spleen and active neutrophils in the areas of inflammation, which is the reason why phosphatidylserine-shell agents are used to detect the foci that lack active phagocytic capacity, e.g. tumour nods in the liver [73, 77-79]. In these agents, the shell is the ligand (phosphatidylserine); since the molecules of phopshati‐ dylserine are kept together by the hydrophobic, van der Waals, and other intermolecular

Nevertheless,thoughpreparedbystraightforwardshear-mixinginonesimplestep,thismethod is convenient only for stable ligands, which are able to survive the extreme conditions during the MB preparations [23]. Particularly some proteins and enzymes might be denatured in the harsh conditions and might lose binding affinity [73]. Therefore a different approach has been applied, involving attachment of ligands after MB preparation: non-covalent (strept)avidinbiotin interaction and covalent binding [23, 73, 80, 81]. Considering the fact that the (strept)avi‐ din-biotin interaction has the strongest effective dissociation constant, it has been the most widely used technique in biotechnology [82]. Biotin residues might be attached in one single step to antibodies, carbohydrates and many other targeting ligands, including the MB shell components [73, 83]. Biotinylated antibodies are abundant and can be easily linked to prema‐ nufactured biotinylated bubbles via a streptavidin linker [84]. Nonetheless, this coupling scheme is useful only in preclinical research: streptavidin as a foreign protein is a possible immunogen which might lead to undesired immune response after multiple injections [85].

Another attractive non-covalent strategy is to use the interaction of a metallochelating lipid complex, such as nickel-nitrilotriacetyl lipid, with a multiplicity of histidine residues (His-tag) of a protein [48]. The method offers an easy way to attach a genetically engineered protein to the MB surface with a His-tag, a commonly applied insert in recombinant protein preparation.

interactions, the ligand is attached in a non-covalent manner [73].

**Figure 13.** Schematic presentation of metallochelating MBs, confocal and electron microscopy of metallochelating MBs and coupling of proteins with HisTag anchor.

The rGFP-HisTag molecule bound onto the surface of metallochelating liposomes was directly detected by TEM (**A,B**). Fluorescence of rGFP was used to prove binding of this protein onto the MB (**C**). The binding did not affect the native conformation of rGFP as demonstrated by the preserved fluorescence characteristics of the protein. Homogenous population of highly fluorescent rGFP MB was detected by flow cytometry (**G**). Nonspecific binding of rGFP on both DPPC liposomes as well as MB lacking DOGS-NTA-Ni was not observed by flow cytometry and confocal microscopy. Confocal microscopy confirmed co-localisation of bound rGFP-HisTag (green fluorescence) with the lipids forming MB monolayer. The monolayer was marked with LisR-PE (red fluorescence) (**D, E, F**). Confocal microscopy revealed that a small fraction of MB had also attached liposomes or residual lipid structures of collapsed MB on their surface (as seen in **D, E, F**). This is in a good agreement with the electron microscopy data (**D**) (Adapted from [48]).

Concerning the covalent coupling methods, the techniques are basically the same as the ones developed for the attachment of ligands to liposomes [88]. As the covalent coupling does not require foreign proteins or His-tags (Figure 13), the chances of undesired immune response to the ligand are low [84]. The simple covalent coupling with the formation of peptide bond between the carboxyl-carrying MBs and protein aminogroups is achieved by adding carbo‐ diimide chemistry [89]. First, a carboxyl group is activated with carbodiimide in the presence of *N-*hydroxysulfosuccinimide, forming active ester [84]. The resulting ester then reacts with the protein aminogroup, forming an amide bond. This method is unfortunately unsuitable for clinical settings as well, as to achieve the desired number of targeting molecules per bubble, large quantities of expensive antibodies must be added to the reaction mixture, not to mention that only very few of them are actually coupled by the peptide bonding [73]. An alternative approach of covalent coupling scheme for antibody attachment to MB surface seems to be thiolmaleimide chemistry, developed for targeted liposome applications [90]. In this case, malei‐ mide on the shell is coupled with a thiol group on the ligand, forming thiol-ether [74]. This technique is convenient with respect to a possible oriented coupling; if a ligand has a single thiol, then a single point attachment to the MB shell should retain affinity of the ligand to this target [84]. Overall, selective oriented coupling with of thiol-protein with a maleimide-carrying MB seems more attractive.

Despite the possibility of attaching the ligand directly to the MB surface, it might seem more suitable to connect the ligand to lipid via an extended spacer arm, such as poly(ethylene glycol) known as PEG. Up to 30 nm long [91], flexible extended PEG spacer arm separates the ligand andtheanchorfromeachother[92], sothatthe spacerarmactsasa"fishing line" [93],improving the chancesofreachingits target andMBadhesionandretentiononthe receptor-coatedsurfaces [73, 92, 94]. On the other hand, similar to the design of long-circulating liposomes, PEG chains, or PEG chain derivatives, can be incorporated into the shell of MBs in order to form a steric barrier against coalescence and adsorption of macromolecules, such as antibodies [47, 95]. In recent papers, the protective role by the PEG chains was shown when the targeting ligand was attached to shorter PEG chains that were shielded by a PEG overbrush layer (buried-ligand architecture), significantly increasing the MB in vivo stability, prolonging their circulation lifetime andreducingligand-mediatedimmunogenicity;itwas alsoreportedthatthe extentofPEG protection to depended on the overbrush length [48, 96-99]. PEG overbrush architecture (PEG - polyethyleneimine with a high degree of substitution) was lately used for complexing and stabilizing the plasmid as well; as a result, the transfection was significantly enhanced [100]. One ofthe possible reasons for enhanced drug and gene delivery when PEG overbrush strategy is appliedis the factthattargetingligands aregenerallyaffixedto the free ends ofthePEGchains to direct the MBs to a given target [97], so that the bubbles usually bear thousands of ligands on their surface.Thus cooperativebindingofmultiple ligands toa targetmightincrease the affinity of particles to the target site, boosting the transfection efficacy. A combination of targeted MB agent BR55 and peptide ligand attached to the bubble shell via a PEG spacer are has even been applied in a prostate cancer clinical trial [101].
