3.2.4. Multiple Targets

Besides single targeted UCA, few groups have synthesized and tested multi-targeted ones (Table 2). The idea is that addressing multiple targets at the same time improves MB binding affinity and avidity. This would reduce the amount of unbound bubbles, meaning a lower concentration would be needed to achieve similar results as with single-targeted MB, and the bubbles would persist for a longer time. Furthermore, in the case of a disease with heterogeneous expression of the individual markers, multi-targeted UCA could improve the accuracy of diagnosis.


**Table 2.** Summary of multi-targeted MB investigated for molecular US imaging.

First multi-targeted MB were functionalized using anti-ICAM-1 antibodies and sialyl Lewis X [133]. These dual-targeted MB had greater adhesion to inflammatory endothelial cells compared to their single targeted counterparts under shear flow. Similar results were reported for MB modified with anti-VCAM-1 and anti-P-selectin antibodies in flow chamber experiments [137]. Moreover, VEGFR2 and αvβ<sup>3</sup> integrin-targeted MB for angiogenesis imaging showed the benefits of dual-targeting in vivo [138]. Since dual-targeted MB comprise of two ligands it was expected that its binding affinity and avidity would be an average of the single-targeted MB counterparts. Interestingly, in all three studies, dual-targeted MB showed significantly higher binding affinity than their single targeted counterparts. Hence, the ligands on MB seem to synergistically increase the binding affinity.

The main drawback of dual-targeted UCA is the synthesis route. In the case of attaching two ligands to the MB, a ratio between the ligands has to be chosen. Controlling this ratio during synthesis can be difficult. This challenge can be overcome by using a single ligand that targets multiple markers, such as sialyl Lewis X that binds to P- and E-selectin. MB decorated with this natural ligand identified postischemic myocardium better than UCA targeted to just P- or E-selectin [128]. Moreover, even better results were achieved using P-selectin glycoprotein ligand-1 analog (PSGL-Ig) instead of sialyl Lewis X [80]. PSGL-Ig-targeted MB enabled the detection and quantification of inflammation in colitis induced mice [130] and swine [131,132]. Moreover, post-ischemic myocardium was detected in mice [84], rats [85], and even macaques [129]. Hence, inflammation detection using P- and E-selectin-targeted MB is possible and preferential over single-targeted UCA.

Furthermore, MB modified with VEGFR2, αvβ<sup>3</sup> integrin and P-selectin have been synthesized [139,140]. These triple-targeted UCA possess better binding to target cells than singleor even double-targeted MB (Figure 8). The in vivo experiments further confirmed these results. Interestingly, in vivo US intensity using triple-targeted UCA was higher than the cumulative intensity of all single-targeted MB [139]. Similar to dual-targeted MB, the ligands synergistically increased the efficiency of the binding. Moreover, these MB were used for monitoring antiangiogenic therapy in breast cancer-bearing mice [140]. Due to the high US signal produced by multi-targeted UCA, even small changes in angiogenesis could be detected. Therefore, an early response to the therapy can be assessed. However, no further studies using this triple-targeted MB have been reported so far.

**Figure 8.** In vitro microscopy images of control (MBC), single (MBS), double (MBD), and triple (MBT) targeted MB adhered on cells. Triple targeted MB adhere more on the cells than single or double targeted ones. Reproduced with permission from [139]. Copyright John Wiley and Sons, 2011.
