Abstract
Targeted microbubble ultrasound contrast agents are being widely studied for potential clinical applications in molecular imaging and drug delivery. Rational design of microbubbles to improve targeting efficacy requires adequate understanding of microbubble adhesion dynamics in the vasculature. Local hemodynamics play a crucial role in microbubble adhesion to the vascular endothelium. Microbubble adhesion in vivo occurs at higher shear rates as compared to in vitro flow assays. The in vitro studies are typically conducted without red blood cells (RBCs) and do not account for the particulate nature of blood. The presence of RBCs at physiological hematocrit has been shown to enhance the adhesion of leukocytes, micro-particles, and platelets to the endothelium, but the role of RBC-microbubble interactions on microbubble adhesion dynamics is not fully understood. ‘Disturbed’ flow is commonly found in the arterial system at bifurcations and pathological sites like stenoses and aneurysms. The effect of disturbed flow on microbubble adhesion has also not been investigated. The central hypothesis of this dissertation is that red blood cells and locally disturbed hemodynamics play a critical role in microbubble adhesion to disease markers on the vascular endothelium.
The first part of this dissertation assessed the influence of red blood cells (RBCs) on microbubble targeting. The adhesion of biotin and P-selectin antibody-conjugated microbubbles to streptavidin (10 μg/ml) and murine P-selectin (μg/ml), respectively, was evaluated under dynamic flow conditions in a parallel-plate chamber. Fluorescent microscopy and ultrasound imaging was used for quantifying microbubble adhesion. At wall shear rates (WSR) greater than 100 s-1, the presence of RBCs (20% and 40% hematocrit) enhanced the adhesion efficiency of biotin microbubbles relative to control buffer (phosphate buffered saline). An order-of-magnitude improvement in adhesion efficiency was observed at 450 s-1 and 40% hematocrit. At lower WSR (≤ 100 s-1), RBCs did not significantly alter binding. For P-selectin targeting at high antibody coating concentration (1.5 μg/107 microbubbles) and at high WSR (450 and 600 s-1), the adherent microbubble ultrasound echo intensity was 40-50% greater when RBCs were present. In contrast, at low antibody concentration (0.075 μg/107 microbubbles), RBCs reduced P-selectin targeting by 35-50%. A two-fold increase in P-selectin targeting was observed in the presence of RBCs in an ex vivo carotid artery flow assay performed with high antibody coating concentration. The effect of RBCs on microbubble detachment from P-selectin substrate was examined using a flow detachment assay. At low antibody concentration, half-maximal detachment occurred at 600 s-1 and 3900 s-1 with RBCs and saline, respectively. At high antibody concentration, half-maximal detachment occurred at 3300 s-1 when RBCs were present, while with saline 60% of the microbubbles remained adherent even at WSR as high as 19200 s-1.
Thus, RBCs at physiological hematocrit enhanced microbubble targeting at high ligand densities. In contrast, RBCs decreased targeting at low ligand densities. Additional flow chamber studies were conducted to understand the mechanistic basis of these findings. At low streptavidin concentration (0.5 μg/ml), a large fraction of microbubble interactions with the substrate were transient when RBCs were present, lasting less than 5 s. This behavior was qualitatively different from the interactions seen at high substrate densities, where transient interactions were absent. Microbubble velocities were measured for a subset of the microbubbles that went on to adhere to the surface. The velocities were substantially higher when RBCs were present, with velocities as much as three times that found in saline. This indicates that microbubbles decelerate more before adhesion when RBCs are present. I performed total internal reflection fluorescent (TIRF) microscopy to obtain near-wall micro-particle trajectories. The trajectories exhibited punctate variation in intensity and deviations from the linear path when RBCs were present. These results suggest that the likely mechanistic basis of our findings lies in the hydrodynamic collisions of RBCs with microbubbles. These hydrodynamic interactions exert tangential (anti-adhesion) and normal (pro-adhesion) forces on the microbubbles. When receptor-ligand avidity is high, the normal forces aid the formation of sufficient number of bonds to overcome the additional tangential forces due to the RBCs. The microbubbles remain firmly bound to the surface and an overall increase in targeting is achieved. Conversely, at low ligand densities, due to the smaller number of bonds formed, the tangential forces imparted by flowing RBCs cannot be resisted; the bonds fail and microbubbles are sheared from the wall.
I hypothesized that localized microbubble adhesion would be promoted in vascular regions with disturbed flow. In the second part of the dissertation, I tested this hypothesis by analyzing microbubble adhesion in a model of disturbed flow implemented using a backward-step flow chamber. In this flow chamber a recirculation zone is established immediately downstream of the step. Microbubble adhesion was augmented in the vicinity of the recirculation zone, relative to the region of fully developed flow further downstream, under all tested flow conditions. For 1:2 expansion ratio (upstream channel height = 254 μm, downstream channel height = 508 μm), there was a 4-fold enhancement in adhesion near the recirculation zone at 700 s-1 and 2-fold enhancement at 350 s-1. With step expansion ratio of 1:3 (upstream channel height = 127 μm, downstream channel height = 381 μm), the adhesion was enhanced 8-fold. Computational fluid dynamics simulations were performed to compute near-wall velocities and wall shear rates. The presence of wall-directed normal velocity component along the flow path correlated strongly (r2 > 0.8) with the regions of elevated microbubble adhesion, indicating that convective transport towards the wall enhances adhesion.
Overall, this work establishes the critical role of the particulate nature of blood and locally disturbed hemodynamics in microbubble targeting. These factors should be taken into account while designing novel microbubble formulations to optimize targeting efficacy under physiological conditions.
