Frequency-Selective Microbubble Targeting <i>In Vitro</i>: A Step Toward Multicolor Ultrasound Molecular Imaging

Ultrasound molecular imaging (USMI) utilizing targeted microbubbles (tMBs) and primary acoustic radiation force (<i>F</i>_rad) 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 <i>F</i>_rad 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 (<i>f</i>₀) tuned by <i>F</i>_rad pulses. The goal of this study was to demonstrate frequency-selective tMB adhesion to receptor-bearing microvessel tubes <i>in vitro</i>. Size-isolated BLA tMBs of 1 and 5 μm diameter were synthesized with <i>f</i>₀ 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 α_vβ₃ integrin targeting. The MB gas volume fraction (φ_MB) 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 <i>F</i>_rad pulses at their respective <i>f</i>₀, both individually and in a cocktail. In the cocktail population, φ_MB of RGD-MB targeting increased 18-fold at 4 MHz compared to 7 MHz, while IELLQAR-MB targeting φ_MB 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 <i>in vivo</i> within a single scan.