Frequency-Dependent Evaluation of the Role of Definity in Producing Sonoporation of Chinese Hamster Ovary Cells

Objectives-Sonoporation uses ultrasound (US) and 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 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 (P-r) 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.