**11. Drug- and gene-loading strategies**

The main advantage of the use of MB-based particles as a delivery tool for drugs and genes lies in the dose reduction while maintaining its therapeutic efficacy; the method thus provides much less toxicity than conventional cytotoxic therapies and at the same time has more specificity, which has a great potential particularly in cancer treatment [84]. Generally, drugs and/or genes can be either (a) co-administered with MBs, (b) enclosed within the MB shell, (c) incorporated in the MB shell, (d) attached to the surface of the MBs (e) incorporated in liposomes or nanoparticles that are associated with the MB surface.

#### **11.1. Co-administration of drugs with MBs**

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

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 factthattargeting ligands 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

The main advantage of the use of MB-based particles as a delivery tool for drugs and genes lies in the dose reduction while maintaining its therapeutic efficacy; the method thus provides much less toxicity than conventional cytotoxic therapies and at the same time has more specificity, which has a great potential particularly in cancer treatment [84]. Generally, drugs

MB seems more attractive.

96 Advances in Bioengineering

applied in a prostate cancer clinical trial [101].

**11. Drug- and gene-loading strategies**

In this case, the MBs enhance the permeability of the endothelial wall in different manners; (1) by rupture of the endothelial wall, so that larger particles such as erythrocytes and polymer particles are able to extravasate [102]; (2) by MBs causing the temporary membrane opening when the low pressure is applied [103]; (3) by sonoporation, allowing for radionuclides or plasmid DNA to pass through [104, 105]. For more detailed description of the mechanisms concerned, please see the above part "Microbubbles in the Ultrasound Field".
