The role of inertial cavitation of ultrasound constrast agents in producing sonoporation

The objective of this project is to elucidate the relationship between ultrasound contrast agents (UCA) and sonoporation by varying the peak rarefactional pressure (P_r) in a threshold type study. The results of sonoporation in the presence of Optison™ or Definity® are directly compared to the collapse thresholds of the respective contrast agent to uncover the role inertial cavitation plays in sonoporation. Chinese Hamster Ovarian (CHO) cells were grown as a monolayer in a 96-microwell plate. Each well was filled with an exposure medium consisting of 0.05 mL Fluorescein isothiocyanate-dextran (FITC-dextran), 8.8 μL Optison™ or 0.57 μL Definity®, and Phosphate Buffered Saline. CHO cells were exposed for 30 s to pulsed ultrasound at 3 MHz center frequency, 5-cycle pulse duration, and 10-Hz pulse repetition frequency. P_r was varied over a range from 10 kPa to 3.5 MPa. Flow cytometery was used to determine the percentage of positively labeled cells. Over the P_r range applied, the sonoporated cells in the presence of Optison™ increased from 0.63% to 10.21%, with a maximum occurring at 2.4 MPa. Above 2.4 MPa, a significant drop in sonoporation activity was observed. Sonoporation in the presence of Definity® presented the same trend, with sonoporated cells increasing from 5.26% to 26.39%, with a maximum occurring 172 kPa. Above 172 kPa, a drop in sonoporation activity was observed. These results illustrate that sonoporation is not due inertial cavitation of the UCA. Instead the evidence directly suggests that the sonoporation effect was caused by linear and/or nonlinear oscillation of the UCA as these responses occur at lower P_r where sonoporation activity was present. Moreover, at higher pressures, the UCAs are rapidly collapsing and as such, are likely not present for enough time to significantly oscillate, thus minimal sonoporation activity was observed.