Dual targeting improves microbubble contrast agent adhesion to VCAM-1 and P-selectin under flow

Abstract

To improve ultrasound contrast agents targeted to the adhesion molecules P-selectin and VCAM-1 for the purpose of molecular imaging of atherosclerotic plaques, perfluorocarbon-filled phospholipid microbubble contrast agents were coupled by a polyethylene glycol-biotin-streptavidin bridge with mAb MVCAM.A(429), a sialyl Lewis(x) polymer (PAA-sLe(x)), or both (dual). Approximately three hundred thousand antibody molecules were coupled to the surface of each microbubble. Recombinant mouse P-selectin and/or VCAM-1 coated on flow chambers showed saturation of binding at approximately 15 ng/microl, resulting in 800 and 1200 molecules/microm(2) for P-selectin and VCAM-1, respectively. Dual substrates coated with equal concentrations of P-selectin and VCAM-1 had site densities between 50 and 60% of single substrates. When microbubbles were perfused through flow chambers at 5 x 10(6) microbubbles/ml (wall shear stress from 1.5 to 6 dyn/cm(2)) dual-targeted microbubbles adhered almost twice as efficiently as single-targeted microbubbles at 6 dyn/cm(2). The present study suggests that dual-targeted contrast agents may be useful for atherosclerotic plaque detection at physiologically relevant shear stresses.

Figure 1

Perfluorocarbon-filled microbubble targeted with MVCAM.A(429) against VCAM-1 (Y-shaped on bubble) and polymeric Sle^x (branch-shaped), which binds selectins. A biotin-streptavidin coupling system is used to graft biotinylated mAbs and carbohydrates on the PEG brush (black arms extending from microbubble surface) covering the phospholipid shell (thick, black circle) of ultrasound contrast agent microbubbles.

Figure 2

Site density (molecules/μm²) of VCAM-1 (A) and P-selectin (B) coated individually or together (dual substrates, C and D) on polystyrene flow chamber substrates. Results are measured in triplicate, ±SEM. Background radioactivity was subtracted from all data points.

Figure 3

Fluorescence Intensity and number of molecules/microbubble based on FACSCalibur data of microbubbles conjugated with Alexa 647-MVCAM.A(429). (A) Fluorescence histograms of microbubbles conjugated with fluorescent antibody at the indicated concentrations (μg/10⁷ microbubbles). Results from calibration beads were used to calculate the number of fluorescent molecules attached to the surface of microbubbles. (B) Table shows results of molecules/microbubble with respect to the fluorescent antibody concentration and microbubbles were incubated with. (C) MVCAM.A(429) site density per microbubble as a function of antibody concentration (μg/10⁷ microbubbles). Background fluorescence values were subtracted from results; n=4, MFI –mean fluorescence intensity.

Figure 4

MVCAM.A(429) site density as a function of MVCAM.A(429) concentration when microbubbles are incubated with the indicated concentrations (A) or as a function of PAA-sLe^x when microbubbles are incubated with a constant amount of MVCAM.A(429) (1 μg/10⁷ microbubbles, B).

Figure 5

Microbubble adhesion efficiency within a parallel plate flow chamber. Streptavidin microbubbles conjugated with 0.5 μg/10⁷ microbubbles PAA-sLe^x for 10 minutes at room temperature and, then, 1 μg/10⁷ microbubbles of MVCAM.A(429) for 10 minutes. Attachment efficiency (adherent microbubbles/all microbubbles passing field of view per 45 seconds) of dual, MVCAM.A(429)-targeted microbubbles and PAA-sLe^x microbubbles as a function of shear stress. “*”, p<0.05 from sLe^x microbubbles. “+”, p<0.05 from MVCAM.A(429) microbubbles. Error bars, SEM for n=8.