LDC1267 Xie et al b reported that the combination of
Xie et al. (2009b) reported that the combination of diagnostic ultrasound impulses and cyclic RGD-bearing MBs targeted for GP IIb/IIIa resulted in the successful recovery of epicardial and microvascular blood flow in swine models with acute thrombotic occlusion. It is well known that the stability of RGD peptide is more robust in cyclic form than in linear form at neutral pH (pH 7) (Bogdanowich-Knipp et al. 1999). In addition, the affinity of RGD peptide for integrin receptors is higher in cyclic form rather than in linear form (Kato and Mrksich 2004). On the basis of these results, the targeting of lactadherin-bearing Sonazoid MBs to GP IIb/IIIa-expressing platelets might be lower than that of cyclic RGD-bearing MBs. This issue needs to be addressed further in in vivo studies.
For GP IIb/IIIa expressed on the surface of platelets, the conformational alterations triggered by agonists are essential in improving affinity to its ligands (Bennett, 2005, Ma et al., 2007). Although stimulating factors altering the conformation of GP IIb/IIIa were not added, the attachment of Sonazoid MBs to αIIbβ3 integrin was significantly augmented by the surface modification with lactadherin (Fig. 4). Additionally, pre-treatment with cyclic RGD LDC1267 or fibrinogen failed to decrease the number of lactadherin-bearing Sonazoid MBs attached to the αIIbβ3 integrin-coated plate (data not shown). These results might indicate that the solid-phase ELISA system using recombinant αIIbβ3 integrin does not necessarily reflect the GP IIb/IIIa expressed on the surface of platelets in vivo. Furthermore, pre-treatment with cyclic RGD also failed to decrease the number of lactadherin-bearing Sonazoid MBs attached to the activated platelets (data not shown). Therefore, the specificity of targeting of lactadherin-bearing Sonazoid MBs to GP IIb/IIIa should be clarified in further studies.
As illustrated in Figure 7b and 8a, VIs of clots after 10 min of incubation with MBs were comparable for Sonazoid and lactadherin-bearing Sonazoid MBs, whereas the increment in VI between 10 min and Destruction (ΔVI) was significantly higher in lactadherin-bearing Sonazoid than in Sonazoid MBs. These results are attributable to the lack of adjustment of the VI of clots before incubation with MBs (baseline). As a result, VIs at Destruction (similar to baseline) have a wide dispersion in both MBs (Fig. 7b). The correct adjustment of the VI of clots at baseline may facilitate the visual recognition of attached MBs and result in unnecessary of ΔVI images.
Considering the clinical translation of Sonazoid-based targeted MBs, attention should be paid to the alteration of plasma lactadherin level after injection of lactadherin-bearing Sonazoid MBs because lactadherin has been known to promote tumor growth in mice through its angiogenic potency (Neutzner et al., 2007, Silvestre et al., 2005). The mean plasma concentration of lactadherin in healthy human subjects was 3.5 ng/mL (Fig. 9), whereas the amount of lactadherin in 10 μL of Sonazoid MBs (1.2 × 107 MBs) was 1 μg. If the dosage of lactadherin-bearing Sonazoid MBs is estimated as equal to that of Sonazoid MBs for detecting a hepatic lesion (0.015 mL Sonazoid MBs/kg weight), the required dose of lactadherin is 1.5 μg/kg weight. Considering the body fluid volume, there may be a risk of this dose increasing the plasma lactadherin level. Therefore, further studies focused on reduction of the dosage of lactadherin-bearing Sonazoid MBs are required to clarify the safety and feasibility of clinical translation of lactadherin-bearing Sonazoid MBs.
Conclusions Our results suggest that lactadherin-bearing Sonazoid MBs have the potential to be GP IIb/IIIa-expressing platelet-targeted MBs. Further in vivo studies are required to clarify the feasibility of using lactadherin-bearing Sonazoid MBs as thrombus-targeted MBs.
Acknowledgments This study was supported by research grants from the Nakatani Foundation for Advancement of Measuring Technologies in Biomedical Engineering (to K.O.), the Fukuda Foundation for Medical Technology (to K.O.) and the Japan Agency for Medical Research and Development (AMED) under Grant No. JP16mk0101037 (to K.O.).