A device for performing sonoporation on adherent cell cultures

Abstract

Sonoporation is a method for inducing a transient increase in the permeability of cell membranes to otherwise impermeable compounds using ultrasound. This technique has therapeutic potential as it allows for localized delivery of therapeutic agents in a noninvasive and non-cytotoxic manner. The discovery and testing of potential therapeutic agents that can be delivered using this technique requires performing studies on cell cultures in vitro. This thesis presents a prototype sonoporation device which aims to reduce the time and expertise required to perform sonoporation on adherent monolayer cell cultures. First, a prototype sonoporation device was designed and constructed. The device consisted of an array of six ultrasound transducers a xed below a cell culture stage. The six transducers were each constructed and electrically matched to 50 at an operating frequency of 1 MHz. The acoustic near- eld of each transducer was characterized using hydrophone scanning and the distance from the transducer at which the plane perpendicular to the beam path was most homogeneous was determined. The mean( s.d.) treatment distance was 15.9( 0.67) mm and the mean -3 dB width was 1.97( 0.22) mm. The electrical power required to produce 0.7 MPa on this plane was found for each transducer. The mean( s.d.) electrical power was 101( 12.2) W. Next, the prototype device was experimentally validated. Sonoporation was performed on cervical carcinoma-derived SiHa cells with 70-80% con uency at media temperatures of 37°C, 39.5°C, and 42°C. Pulsed ultrasound of 1 MHz, 4.8% duty cycle, 1.6 kHz pulse repetition frequency, and 0.7 MPa peak pressure was applied to induce sonoporation. Ultrasound contrast agent was added to the cell culture media (0.33% v/v) to provide cavitation nuclei during treatment. Plasmid DNA expressing green uorescent protein (GFP) was added to the cell culture (250 g/10 mL) to quantify successful permeabilization. While there were no signi cant e ects due to the temperature of the media, transfection was successfully performed using the prototype device given the positive expression of GFP in the cells 24 hours following treatment. The mean( s.d.) transfection e ciencies of the sonoporation treatment at 37°C, 39.5°C, and 42°C were 5.4( 0.92)%, 5.8( 1.3)%, and 5.3( 1.1)% respectively (n = 3 for each experimental group). Negative control treatments had transfection rates of < 1:5% on average and the detected levels of apoptosis among surviving cells was < 0:5% on average for all treatment groups. These results were in good agreement with those obtained using a di erent sonoporation experimental set-up on the same cell line with similar experimental parameters. Finally, the design of high-power ultrasound driving circuitry was explored in order to create an electrical device with the ability to provide independent, concurrent, and controlled excitation of the six transducers. A class DE half-bridge ampli er topology was chosen as the output power stage of this device. A design of a class DE ampli er was simulated using LTSpice with both a resistive 50 load and a Butterworth-Van Dyke equivalent circuit model of one of the six transducers, matched to 50 at 1 MHz. The ampli er was designed to deliver 150 W to a 50 resistive load at an output frequency of 1 MHz using a DC supply voltage of 96 V. The simulation of the ampli er using the transducer equivalent circuit yielded an output power of 134 W, a drain e ciency of 98.8%, a power-added e ciency of 89.0%, a gate power gain of 22.6 dB, and a total harmonic distortion at the output of 27.9%. The device presented here was shown to be e ective at performing sonoporation on adherent monolayer cell cultures and will reduce the time and expertise required to perform this technique in the future.