Vibrational Responses of Bound and Nonbound Targeted Lipid-Coated Single Microbubbles

One of the main challenges for ultrasound molecular imaging is acoustically distinguishing nonbound microbubbles from those bound to their molecular target. In this in vitro study, we compared two types of in-house produced targeted lipid-coated microbubbles, either consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, C16:0 (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine, C18:0 (DSPC) as the main lipid, using the Brandaris 128 ultrahigh-speed camera to determine vibrational response differences between bound and nonbound biotinylated microbubbles. In contrast to previous studies that studied vibrational differences upon binding, we used a covalently bound model biomarker (i.e., streptavidin) rather than physisorption, to ensure binding of the biomarker to the membrane. The microbubbles were insonified at frequencies between 1 and 4 MHz at pressures of 50 and 150 kPa. This paper shows lower acoustic stability of bound microbubbles, of which DPPC-based microbubbles deflated most. For DPPC microbubbles with diameters between 2 and 4 μm driven at 50 kPa, resonance frequencies of bound microbubbles were all higher than 1.8 MHz, whereas those of nonbound microbubbles were significantly lower. In addition, the relative radial excursions at resonance were also higher for bound DPPC microbubbles. These differences did not persist when the pressure was increased to 150 kPa, except for the acoustic stability which further decreased. No differences in resonance frequencies were observed between bound and nonbound DSPC microbubbles. Nonlinear responses in terms of emissions at the subharmonic and second harmonic frequencies were similar for bound and nonbound microbubbles at both pressures. In conclusion, we identified differences in vibrational responses of bound DPPC microbubbles with diameters between 2 and 4 μm that distinguish them from nonbound ones.