3.2.3. Thrombosis

Thrombosis describes the formation of a blood clot in a vessel. In the clinics, Doppler US is a standard method for detecting deep venous thrombosis (DVT). However, the blood clot has to be big enough to produce visible circulation defects and the thrombus activity cannot be assessed. Moreover, the examination is user-dependent and hence the accuracy in detecting DVT varies [133]. MB targeting activated platelets have been proposed for thrombus detection and characterization. Acute thrombus should have a higher number of activated platelets than the chronic state. Hence, ligands, which only bind to activated platelets, have been proposed for targeting: lysine-glutamine-alanine-glycine-aspartate-valine (KQAGDV) [89,90], glycoprotein (GP) IIa/IIIb [93] and cyclic arginine-glycine-aspartate (RGD) [91,92]. All targeted MB formulations showed thrombus-specific US contrast in vitro and in vivo. Nonetheless, the exact concentration of activated platelets required to successfully identify a thrombus by targeted MB still needs to be elucidated, which is crucial for evaluating the potential of the method for early thrombus detection.

Furthermore, stimulus-responsive MB have been suggested for thrombus detection. The first group proposed MB decorated with aptamers [95,96]. When exposed to thrombin, the aptamers were detached and the bubble stiffness was reduced. The change in shell stiffness alters the harmonic signals generated by the bubble. For thrombus formation a critical concentration of 25 nmol is required [134,135]. At this concentration it was seen that all aptamers detach from the MB making the shell soft, while at lower concentrations (10 to 25 nmol) only a partial softness change was observed. Hence, these MB become active and give US contrast only when thrombin levels exceed the critical threshold. In vitro and in vivo experiments proved the concept. The stimulus-responsive MB increased the CEUS signal five-fold in presence of clots and even small lesions that were not visible by non-specific UCA were detected. Another group proposed MB with thrombin-sensitive activatable cell-penetrating peptides (ACPP) [97]. In this case, thrombin cleaved ACPP making the UCA positively charged. Due to Coulomb interaction, activated MB adhere to negatively charged surfaces like red blood cells, fibrin, and platelets. The concept of these UCA was confirmed in vitro using rabbit blood and an increase in the US signal was only seen if thrombin was present (Figure 7). Therefore, ACPP MB detect only acute blood clots. Both stimulus-responsive MB formulations can distinguish between acute and chronic thrombosis according to thrombin levels in the blood.

**Figure 7.** Molecular US imaging of thrombosis. Spatial maps of the molecular US signal color-coded and overlaid on B-mode images acquired at baseline after infusion of MB and washing with saline using targeted MB (left), non-targeted MB (middle), and targeted MB co-injected with hirudin (right). Targeted MB give specific signal only in the presence of thrombin. Reproduced with permission from [97]. Copyright American Chemical Society, 2017.
