An ultrasound contrast agent targeted to P-selectin detects activated platelets at supra-arterial shear flow conditions

Abstract

Objectives: To evaluate targeting of a microbubble contrast agent to platelets under high shear flow using the natural selectin ligand sialyl Lewis.

Materials and methods: Biotinylated polyacrylamide Sialyl Lewis or biotinylated carbohydrate-free polymer (used as a control) were attached to biotinylated microbubbles via a streptavidin linker. Activated human platelets were isolated and attached to fibrinogen-coated culture dishes. Fibrinogen-coated dishes without platelets or platelet dishes blocked by an anti-P-selectin antibody served as negative control substrates. Dishes coated by recombinant P-selectin served as a positive control substrate. Microbubble adhesion was assessed by microscopy in an inverted parallel plate flow chamber, with wall shear stress values of 40, 30, 20, 10, and 5 dynes/cm2. The ratio of binding and passing microbubbles was defined as capture efficiency.

Results: There was no significant difference between the groups regarding the number of microbubbles in the fluid flow at each shear rate. Sialyl Lewis-targeted microbubbles were binding and slowly rolling on the surface of activated platelets and P-selectin-coated dishes at all the flow conditions including 40 dynes/cm2. Capture efficiency of targeted microbubbles to activated platelets and recombinant P-selectin decreased with increasing shear flow: at 5 dynes/cm2, capture efficiency was 16.11% on activated platelets versus 21.83% on P-selectin, and, at 40 dynes/cm2, adhesion efficiency was still 3.4% in both groups. There was neither significant adhesion of Sialyl Lewis-targeted microbubbles to control substrates, nor adhesion of control microbubbles to activated platelets or to recombinant P-selectin.

Conclusions: Microbubble targeting using sialyl Lewis, a fast-binding ligand to P-selectin, is a promising strategy for the design of ultrasound contrast binding to activated platelets under high shear stress conditions.

Figure 1

Targeting scheme of sLe^a-targeted microbubbles in the flow chamber. MB had biotinylated PEG molecules on their shell and were coupled with biotinylated sialyl Lewis^a polyacrylamide or a biotinylated control carbohydrate-free polymer. Streptavidin served as a linker.

Figure 2

Flow chamber experiment at 10 dynes/cm². Dishes are covered with activated platelets that are expressing P-Selectin on their surface. Field of view is 420 × 350 μm: A: Control MB. The grey arrow indicates a non-specific MB adhesion that happened prior to the high shear flow application. B: sLe^a MB. Black arrows indicate adherent MB which are rolling along the flow path; white arrows indicate MB in the flow which were near the surface and appear as grey stripes.

Figure 3

Mean capture efficiency (± SEM) of sLe^a-MB on recombinant P-selectin and on activated platelets. 35mm culture dishes covered by P-selectin (n=4) or activated platelets (n=5) were perfused by a dispersion of sLe^a MB (5×10⁶ MB/ml PBS) at wall shear stress 40, 30, 20, 10, and 5 dynes/cm². * indicates a significant difference in capture efficiency at 5 dynes/cm², # indicates no significant difference at the other wall shear stress conditions. Capture efficiency was decreasing with increasing shear flow.