Designing Microbubbles for Multi-Color Ultrasound Molecular Imaging

Current ultrasound molecular imaging (USMI) techniques using targeted microbubbles (tMBs) are limited to a single ligand-receptor pair per imaging scan. With the advent of the buried-ligand architecture, “cloaked” ligand-receptor binding and tMB adhesion can be activated by primary acoustic radiation force (Frad) pulses tuned to microbubble resonance, enabling multicolor USMI. This approach permits the selective activation of two or more different tMB reporters, each binding to its cognate receptor when driven by a select Frad pulse tuned its distinct resonance frequency (f0). We previously demonstrated tuning microbubble f0 and Frad responsiveness by engineering size distribution and shell composition. The goal of this study was to demonstrate frequency-selective tMB adhesion to receptor-bearing microvessel tubes in vitro, based on tMB diameter.

Size-isolated BLA tMBs of 1 and 5 μm diameter were synthesized with f0 equal to 7 and 4 MHz, respectively (within frequency limits of our ultrasound scan probe). The 1-μm tMBs were conjugated with IELLQAR peptide for P-selectin targeting (inflammation), while the 5-μm tMBs were conjugated with cyclo-RGD peptide for αvβ3 integrin targeting (angiogenesis). The MB gas volume fraction () was used to unify size and concentration into a single dose parameter. Frequency-selective tMB binding was quantified using fluorescent microscopy. Specific targeting was evaluated by comparing RGD- or IELLQAR-MB attachment to control RAD- or nonligand-bearing MBs, respectively. The results confirmed specific frequency-selective targeting of the two tMBs species to their cognate receptors when activated by Frad pulses at their respective f0, both individually and in a cocktail. In conclusion, this study presents the first demonstration of frequency-selective targeting of two different receptors by two different tMB reporters, representing a significant step towards multicolor USMI and the potential for simultaneous imaging of multiple biomarkers in vivo within a single scan.