The objective of this project is to elucidate the relationship between ultrasound contrast agents (UCAs) and sonoporation. Sonoporation is an ultrasound-induced, transient cell membrane permeability change, which allows for the uptake of normally impermeable macromolecules. Specifically, this study will determine the role that inertial cavitation plays in eliciting sonoporation. The inertial cavitation thresholds of the UCA, Optison ™ , are directly compared to the results of sonoporation in order to determine the involvement of inertial cavitation in sonoporation. Chinese Hamster Ovary (CHO) cells were exposed as a monolayer, in a solution of Optison ™ , 500,000 Da Fluorescein isothiocyanate-dextran (FITC-dextran), and Phosphate Buffered Saline (PBS) to 30 seconds of pulsed ultrasound (US) at 3.15-MHz center frequency, 5-cycle pulse duration, and 10-Hz pulse repetition frequency. The peak rarefactional pressure (P r ) was varied over a range from 120 kPa to 3.5 MPa, and five independent replicates were performed at each pressure. As the P r was increased, from 120 kPa to 3.5 MPa, the fraction of sonoporated cells among the total viable population increased from 0.63% to 10.21%, with the maximum occurring at 2.4 MPa. The inertial cavitation threshold for Optison ™ at these exposure conditions has previously been shown to be in the range 0.77-0.83 MPa, at which sonoporation activity was found to be 50% of its maximum level. Furthermore, significant sonoporation activity was observed at pressure levels below the threshold for inertial cavitation of Optison ™ . Above 2.4 MPa, a significant drop in sonoporation activity occurred, corresponding to pressures where >95% of the Optison ™ was collapsing. These results demonstrate that sonoporation is not directly due to inertial cavitation of the UCA, rather that the effect was related to linear and/or nonlinear oscillation of the UCA occurring at pressure levels below the inertial cavitation threshold.

Objectives Sonoporation uses ultrasound (US) and ultrasound contrast agents (UCAs) to enhance cell permeabilization, thereby allowing delivery of therapeutic compounds non-invasively into specific target cells. The objective of this study was to elucidate the biophysical mechanism of sonoporation, specifically the role of UCAs as well as exposure frequency. The inertial cavitation (IC) thresholds of the lipid-shelled octafluoropropane UCA were directly compared to the levels of generated sonoporation to determine the involvement of UCAs in producing sonoporation. Methods Chinese hamster ovary cells were exposed as a monolayer in a solution of the UCA, 500,000-Da fluorescein isothiocyanate-dextran, and phosphate-buffered saline to 30 seconds of pulsed US (pulse duration, 5 cycles; pulse repetition frequency, 10 Hz) at 3 frequencies (0.92, 3.2, and 5.6 MHz). The peak rarefactional pressure (Pr) was varied over a range from 4 kPa to 4.1 MPa, and 5 to 7 independent replicates were performed at each pressure. Results The experimental observations demonstrated that IC was likely not the physical mechanism for sonoporation. Sonoporation activity was observed at pressure levels below the threshold for IC of the UCA (1.27 ± 0.32 MPa at 0.92 MHz, 0.84 ± 0.19 MPa at 3.2 MHz, and 2.57 ± 0.26 MPa at 5.6 MHz) for all 3 frequencies examined. The Pr values at which the peak sonoporation activity occurred were 1.4 MPa at 0.92 MHz, 0.25 MPa at 3.2 MHz, and 2.3 MPa at 5.6 MHz. The UCA collapse thresholds followed a similar trend. A 1-way analysis of variance test confirmed that sonoporation activity differed among the 3 frequencies examined (P = 10−8). Conclusions These results thus suggest that sonoporation is related to linear and/or nonlinear oscillation of the UCA occurring at pressure levels below the IC threshold.

Sonoporation uses ultrasound, with the aid of ultrasound contrast agents (UCAs), to enhance cell permeabilization, thereby allowing delivery of therapeutic compounds noninvasively into specific target cells. The objective of this study was to determine if a computational model describing shear stress on a cell membrane due to microstreaming would successfully reflect sonoporation activity with respect to the peak rarefactional pressure. The theoretical models were compared to the sonoporation results from Chinese hamster ovary cells using Definity(®) at 0.9, 3.15, and 5.6 MHz and were found to accurately describe the maximum sonoporation activity, the pressure where a decrease in sonoporation activity occurs, and relative differences between maximum activity and the activity after that decrease. Therefore, the model supports the experimental findings that shear stress on cell membranes secondary to oscillating UCAs results in 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 TM 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 (FITCdextran), 8.8 µL Optison TM 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 TM 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.

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