Ultrasound molecular imaging contrast agent binding to both E- and P-selectin in different species

Ultrasound molecular imaging is increasingly used in preclinical studies to measure the expression of vascular markers during inflammation process. In this context, a new ultrasound contrast agent functionalized with a recombinant P-selectin glycoprotein ligand-1 analogue (rPSGL-Ig) was developed (MBrPSGL-Ig). This agent was assayed in vitro and in vivo to evaluate its binding performance and potential to image expression of inflammatory markers E- and P-selectin. Performance of this newly developed agent was compared with that of antibody (MBAb) or sialyl Lewis X (MBsLe) containing microbubbles and with control microbubbles (MBC).

The targeted ultrasound contrast agents were prepared by coupling biotin-conjugated ligands onto streptavidin-functionalized microbubbles. First, in vitro experiments were performed to measure the adhesion efficiency of these microbubble constructs under static or flow conditions (114 sec), on cell monolayer (human umbilical vein endothelial cells and bEnd.5), or coatings of E- or P-selectin of various animal species, respectively. Second, molecular imaging studies were performed in a rat inflammatory model 24 hours after intramuscular injection of lipopolysaccharide in the hind limb. Finally, immunohistochemistry staining of rat inflamed muscle tissue was performed to assess expression of E- and P-selectin.

Microbubbles functionalized with rPSGL-Ig (MBrPSGL-Ig) displayed firm in vitro binding on the coating of both recombinant E- or P-selectin, with an efficiency similar to microbubbles comprising antibody specific for E-selectin (MBE) or P-selectin (MBP). In contrast, lower binding capacity was measured with MBsLe. At the surface of inflamed endothelial cells, MBrPSGL-Ig were able to interact specifically with E- and P-selectin. Binding specificity was demonstrated by performing blocking experiments with target-specific antibodies, resulting in an 80% to 95% decrease in binding. Ten minutes after microbubble injection, echo signal measured with MBrPSGL-Ig in the inflamed muscles was 20-fold higher compared with MBC. Moreover, the in vivo adhesion of MBrPSGL-Ig was 2- and 7-fold higher compared with P-selectin or E-selectin-specific microbubbles, respectively. Immunohistochemistry revealed a temporal coexpression of E- and P-selectin in the vascular bed of inflamed rat muscle 24 hours after lipopolysaccharide injection.

The molecular imaging study demonstrates that MBrPSGL-Ig provide imaging signal higher than those measured with antibody or sialyl Lewis X containing microbubbles. These results suggest that MBrPSGL-Ig is a powerful agent to image the expression of both E- and P-selectin in the context of an inflammatory process.