Nonlinear Response of Phospholipid-Shelled Ultrasound Contrast Agents: Ambient Conditions Affect Temporal Evolution

Non-linear cavitation response from ultrasound contrast agents (UCAs) is sensitive to ambient conditions. Recent studies have shown that the cavitation response can exhibit a delayed onset of non-linear spectral peaks and can evolve over time, on the order of several tens of minutes. However, the effect of ambient conditions on the temporal evolution of UCAs has not been explored previously. Elucidating this behavior is essential for standardizing acoustic measurement protocols. This study characterized the impact of temperature, hydrostatic overpressure and the presence of dissolved protein in the solution on the temporal evolution of the non-linear acoustic response of two clinically approved UCAs, Definity and SonoVue, and a modified agent, octafluoropropane-substituted SonoVue (OFP-SonoVue). These ambient conditions are relevant for understanding how the UCA response may be affected in conditions relevant to laboratory studies as well as in vivo.

A custom 2 MHz single-element transducer (focal number of 1.26, fractional bandwidth 4.26%) was used to excite the diluted bulk UCA suspension at a peak rarefactional pressure of 470 kPa. A 50-cycle sine burst excitation was employed at a pulse repetition frequency of 1 kHz. A confocally aligned broadband transducer with a center frequency of 20 MHz, 6 mm aperture and a fractional bandwidth of 92.69% was used to receive the scattered echoes. The cavitation response from the UCAs was first evaluated at 25°C and 37°C in de-ionized water at atmospheric pressure. Next, the effect of static overpressure (40 mm Hg) was determined at 25°C. Finally, the effect of dissolved protein content was assessed by performing measurements in a 0.5% w/v bovine serum albumin solution at room temperature and atmospheric pressure.

The agents exhibited starkly different non-linear responses over time under the tested ambient conditions. Definity demonstrated sub-harmonic onset at 16, 6, 0 and 0 min after the start of ultrasound exposure at 25°C, 37°C, with overpressure and with added protein, respectively. In comparison, SonoVue exhibited an instantaneous or near-instantaneous sub-harmonic onset following ultrasound exposure (at 0, 0, 1 and 0 min). For the modified OFP-SonoVue agent, the corresponding sub-harmonic onset times were 0, 0, 5 and 0 min. The time to attain peak responses for various non-linear modes (sub-harmonic, ultra-harmonic and second harmonic) and their respective amplitudes also varied considerably across ambient conditions for the three agents.

Definity demonstrated a strong sensitivity to ambient conditions, whereas SonoVue displayed greater robustness across environments. Results from OFP-SonoVue suggest that the temporal response at physiological temperature and in protein-rich media is primarily determined by the lipid shell, whereas with overpressure, it is predominantly filling gas dependent. These results highlight fundamental differences in the response of these UCAs and help understand sources of variability in UCA response for specific applications in imaging, sensing and therapy.