Frequency-Selective Microbubble Targeting : A Step Toward Multicolor Ultrasound Molecular Imaging.

Ultrasound molecular imaging (USMI) utilizing targeted microbubbles (tMBs) and primary acoustic radiation force () pulses has demonstrated enhanced sensitivity in recent studies. However, current USMI techniques are limited to a single ligand-receptor pair per imaging scan. With the advent of the buried-ligand architecture (BLA), "cloaked" ligand-receptor binding and tMB adhesion can be activated by pulses, enabling multicolor USMI. This approach permits the selective activation of two or more tMB species, each binding to its cognate receptors based on distinct resonance frequencies () tuned by pulses. The goal of this study was to demonstrate frequency-selective tMB adhesion to receptor-bearing microvessel tubes . Size-isolated BLA tMBs of 1 and 5 μm diameter were synthesized with equal to 7 and 4 MHz, respectively (within the frequency limits of our ultrasound probe). The 1 μm tMBs were conjugated with IELLQAR peptide for P-selectin targeting, while the 5 μm tMBs were conjugated with cyclo-RGD peptide for αβ integrin targeting. The MB gas volume fraction (φ) was used to unify size and concentration into a single parameter. Frequency-selective tMB binding was quantified using fluorescence 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 tMB species to their cognate receptors when activated by pulses at their respective , both individually and in a cocktail. In the cocktail population, φ of RGD-MB targeting increased 18-fold at 4 MHz compared to 7 MHz, while IELLQAR-MB targeting φ increased 5-fold at 7 MHz compared to 4 MHz. In conclusion, this study presents the first demonstration of frequency-selective targeting of two different receptor species by two different tMB species, representing a significant step toward multicolor USMI and the potential for simultaneous imaging of multiple biomarkers within a single scan.