Monodispersity Increases Adhesion Efficiency and Specificity for Ultrasound-Targeted Microbubbles

Ultrasound molecular imaging with targeted microbubbles (MBs) can be used to noninvasively diagnose, monitor, and study the progression of different endothelial-associated diseases. Acoustic radiation force (Frad) can initiate and enhance MB adhesion at the target site. The goal of this study was to elucidate the effects of various MB parameters on Frad targeting. Monodisperse or polydisperse MBs with the immune-stealth cloaked (buried)-ligand architecture were conjugated with targeting RGD or nonspecific isotype control RAD peptides and then pumped through an αvβ3 integrin-coated microvessel phantom at a wall shear stress of 3.5 dyn/cm2. Targeting was assessed by measuring MB attachment for varying Frad time and frequency, as well as MB concentration and size distribution. We first confirmed that primary Frad is necessary to target the cloaked-ligand MBs. MB targeting increased monotonically with αvβ3 integrin density and Frad time. MB attachment and, to a lesser extent specificity, also increased when driven by Frad near resonance. MB targeting increased with MB concentration, although a shift in behavior was observed with increasing MB–MB interactions and aggregations forming from secondary Frad effects as MB concentration was increased. These secondary Frad effects reduced targeting specificity. Finally, after having validated our approach by testing different parameters with the appropriate controls, we then determined the effects of monodispersity on adhesion efficiency and specific targeting. We observed that both MB targeting efficiency and specificity were greatly enhanced for monodisperse vs polydisperse MBs. Analysis of videomicroscopy images indicated that secondary Frad effects may have disproportionally inhibited targeting of polydisperse MBs. In conclusion, our in vitro results indicate that monodisperse MBs driven near resonance and at a low concentration (∼106 MB/mL) can be used to maximize the adhesion efficiency (up to 88%) and specificity of RGD–MB targeting.