*3.1. Functionalization of Contrast Agents*

The easiest way to functionalize UCA is to have the targeting ligand incorporated in the shell layer of the bubble (Figure 4). This method favorably works for lipid-bound ligands such as phosphatidylserine [15] and phospholipid-heteropeptides binding to the vascular endothelial growth factor receptors 2 (VEGFR2) [16], because they can withstand harsh MB synthesis conditions.

**Figure 4.** Schematic representation of different functionalization methods of UCA.

Another way of functionalizing UCA is to incorporate a reactive moiety into the shell that can be coupled via a non-covalent or covalent bond to the targeting ligand. For non-covalent coupling, an avidin-biotin bond has been extensively used. In this case, biotin is incorporated into the shell layer and with the use of avidin as a "bridging" moiety biotinylated ligands are coupled. Since avidin has four possible binding sites multiple ligands can be attached. Additionally, it was shown that avidin itself can be incorporated into the bubble shell layer. The major drawback of using a biotin-avidin bond for UCA functionalization is the immunogenicity of avidin. Thus, avidin-biotin bubbles can only be used in preclinical research and not in the clinics.

Furthermore, two covalent binding methods (Figure 4) for UCA functionalization have been used. The first one is the carbodiimide coupling. In this case, the carboxyl group located on the shell coat reacts with an amine group present in the targeting ligand forming an amide bond. Due to the low yield of the reaction, an excess of ligand has to be used, which can become expensive [17]. Additionally, if the ligand contains multiple amine groups (as it is the case for most proteins) the coupling can occur at several sites leading to uncontrolled conjugation and a possible reduction in targeting affinity. Hence, the second method using maleimide-thiol coupling is preferred. Maleimide can be easily conjugated to polyethylene glycol (PEG)-lipids that can be used for the bubble synthesis, while the thiol group has to be attached to the ligand. In single-step "click" reaction maleimide and thiol groups form a thioether bond. This method has higher yield compared to carbodiimide coupling, thus fewer ligands are needed. Moreover, due to the single thiol group introduction to the targeting ligand the coupling to the UCA is better controlled. Though covalent bond avoids immunogenic materials like avidin, the unreacted chemical groups on the shell layer might as well trigger an immune response. Thus, it is important to have all chemical groups bonded.

In some cases, PEG was used as a spacer between the bubble and the targeting ligand (Figure 4). A long chain of PEG gives the targeting ligand more flexibility and mobility to interact with its receptors. It has been shown that ligand interaction to its receptor was increased with the increasing length of the spacer [18,19]. Moreover, the ligand could be shielded by introducing additional longer PEG chains without targeting moieties [20,21]. This prevented specific interactions with the target. Only when US irradiation was applied the longer PEG chains unfolded and exposed the ligand to its receptor. Then MB were able to bind to the target. Thus, the bimodal structure produces stimulus-responsive, targeted UCA. The advantage is reduced immunogenicity and low binding at not insonated sites.
