|University of Illinois at Urbana-Champaign | Department of Electrical and Computer Engineering | Department of Bioengineering
Department of Statistics | Coordinated Science Laboratory | Beckman Institute | Food Science and Human Nutrition | Division of Nutritional Sciences | College of Engineering
|Thursday, October 19th, 2017|
SonoporationBy Monica M. Forbes, M.D./Ph.D. Student
Sonoporation utilizes the interaction of ultrasound (US) with UCAs to temporarily permeabilize the cell membrane allowing for the uptake of DNA, drugs, and other therapeutic compounds from the extracellular environment. This membrane alteration is transient, leaving the compound trapped inside the cell after US exposure. Sonoporation, unlike other methods of transfection or chemotherapy, combines the capability of enhancing gene and drug transfer with the possibility of restricting this effect to the desired area and the desired time. Thus, sonoporation is a promising drug delivery and gene therapy technique, limited only by lack of understanding regarding the biophysical mechanism that results in the cell membrane permeability change; which is what this project seeks to elucidate.
The objective of this project is to elucidate the relationship between ultrasound contrast agents (UCA) and sonoporation. Inertial cavitation (IC) of UCAs is the assumed mechanism; however, most of the data provided in the literature are circumstantial evidence and do not definitively indicate that IC is the cause of the sonoporation. UCAs can also produce microstreaming, shear stresses, and liquid jets as a result of linear and nonlinear oscillations at pressure levels well below the threshold for IC. Because these physical phenomena could also cause biological effects, a rigorous study to determine the biophysical mechanism of sonoporation must be conducted. Therefore, the long-term objective of this project is to elucidate the relationship between the UCA and sonoporation.
To date, the inertial cavitation thresholds of the UCA, OptisonTM, have been directly compared to the results of sonoporation to determine the involvement of inertial cavitation in sonoporation. Chinese Hamster Ovarian (CHO) Cells were grown in a monolayer configuration in a 96-microwell plate. Each well was filled with an exposure medium consisting of 3.7 µL OptisonTM, 0.05 mL Fluorescein isothiocyanate-dextran (FITC-dextran), with an average molecular weight of 500,000, and 0.32 mL Phosphate Buffered Saline (PBS). FITC-dextran is normally unable to cross the cell membrane and was used as the marker for cell membrane permeability change.
CHO cells were exposed for 30 seconds to pulsed ultrasound (US) at 3 MHz center frequency, 5 cycle pulse duration, and 10 Hz pulse-repetition frequency. The peak rarefactional pressure was varied over a range of 120 kPa to 3.5 MPa, and five samples were performed at each pressure. Flow cytometry was used to determine the percentage of positively labeled cells. To assess cell viability, propidium iodide (PI) was added to the suspension. FITC-dextran positive cells were considered to have undergone sonoporation.
Over the pressure range 120 kPa to 3.5 MPa, the percentage of sonoporated cells varied from 0.63% to 10.21%, with the sonoporation activity increasing as the pressure increased up to 2.7 MPa. The minimum inertial cavitation threshold for OptisonTM at these exposure conditions has been shown to be 0.83 MPa. At this pressure, sonoporation had already reached over 50% of the way to the maximum activity. Additionally, at pressure levels where inertial cavitation of OptisonTM was not occurring, significant sonoporation activity was observed. At pressures above 2.7 MPa a significant drop in sonoporation activity occurred. This drop corresponds to the pressure where 100% of the OptisonTM was collapsing.
We have shown that sonoporation is not due to inertial cavitation of the UCA. It is hypothesized that the sonoporation effect was caused instead by linear or nonlinear oscillation of the UCA, as these responses occur at lower pressure levels. Additionally, at higher pressures where the bubbles are collapsing, no oscillation occurs, and thus no sonoporation activity is observed.
|Bioacoustics Research Lab.|