*3.2. Intravascular Targeting (MB)*

Due to their size, MB do not leave the vasculature, so targets should be located within the vessels. This is the case for many angiogenesis, inflammation, and thrombosis markers, for which several ligands have been investigated (Table 1).


**Table 1.** Summary of intravascular targets investigated for molecular US imaging.


**Table 1.** *Cont.*

Due to the fast flow of MB in larger vessels, it is important that the ligands bind quickly to the target. For this purpose, a high kinetic association for binding is needed. Alternatively, an increase in binding can be achieved by using multiple ligands on one bubble (more about it in Section 3.2.4) or by applying acoustic radiation force pulses.

Acoustic radiation forces (primary and secondary) are the forces affecting MB localization and distribution. They cause movement of MB (primary radiation force) and interaction of the MB with each other (secondary radiation force) [98,99]. The primary radiation force is experienced by single particles resulting from the acoustic pressure field. It leads to the movement of the MB in the acoustic field and also allows to push a MB streamline towards the vessel wall bringing the bubbles closer to the target, which might increment targeting efficiency [99–101]. In this regard, Dayton et al. showed that during this process the flow of the MB concentrating close to the vessel wall was reduced compared to the MB floating in the streamline [100]. Secondary or Bjerknes force affects neighboring bubbles. It is produced by the scattered field of a resonating bubble. This leads to a reversible attraction and clustering of MB [99,100]. Moreover, the clustering of MB is affected by the distance between two bubbles [98]. It is hypothesized that bound bubbles can attract other bubbles thus increasing targeting efficacy and

the concentration of bound MB [100,101]. Following up, Zhao and colleagues showed in vitro that enhanced binding of targeted MB was observed when radiation force was applied [101]. In line with this, the in vivo study from Gessner et al. using a cyclic arginine–glycine–aspartic (RGD) peptide targeted to αvβ<sup>3</sup> demonstrated a significant increase in targeting efficacy and US signal intensity, when acoustic radiation force was applied [102]. In addition, Wang et al. programmed a conventional US imaging device with an acoustic radiation force inducing sequence for pushing the MB to the vessel wall by using low pressure and long duty cycles. The experiments successfully demonstrated higher MB binding to P-selectin in large blood vessels in vitro and in vivo, where good binding efficiency is usually difficult to achieve due to low contact between MB and the vessel wall at the high physiological flow rates [103]. Furthermore, with the introduced sequence the signal between molecularly adherent, non-specific adherent and free floating MB can be distinguished [104]. This method could be very helpful since all molecular US imaging methods reported above cannot distinguish between unspecific adherent stationary MB and target-bound ones, which increases the unspecific background signal.
