Monitoring and Modeling of Microbubble Behavior during Ultrasound Mediated Transfection of Cell Monolayers

摘要

The large scale oscillation of insonified microbubbles (MBs) is considered to be the primary effect for sonoporation and thus enhances cell transfection in gene therapy. MB destruction on the other hand is suspected to lead to lower transfection rates. For future in vivo therapy, online acoustic monitoring could be used to identify these effects and to determine optimal pulse sequence parameters adaptively. As a first step, we monitor MB cloud behavior optically and acoustically during ultrasound mediated transfection of cell monolayers. Opticell~R containers are used to grow monolayers of 293T cells. The containers are filled with a medium containing GFP expressing plasmid DNA and SonoVue~R MBs. Each container is placed in water in the focus of a single element transducer emitting 5 cycles sine-bursts at 1.1 MHz repeated 150 times at 3 Hz. The peak negative pressure varies from 0.29 to 1.53 MPa. A second transducer (1 MHz center frequency) detects transmitted signals on the opposite side. The transducers horizontally scan the entire cell monolayer in a rectangular grid with a spacing of 6 mm. Transmitted and backscattered therapy signals are recorded. For optical MB monitoring, a microscope coupled to a high speed camera is used. The transfection rate is determined by flow cytometry after incubating the cells for 48 hours. The acoustical transmission results reveal MB destruction, which is confirmed by optical MB monitoring. Furthermore, an exponential model of MB destruction in suspensions can be fitted to the monolayer situation. A correlation of the point in time of the maximum of the backscattered signal with the point in time of maximum bubble expansion can be identified. Transfection efficiency, bubble extension and the maximum of the backscattered signals at MB resonance frequency rise with increasing peak negative pressure. In this study, the correlation of sonoporation efficiency and MB extension at cell layers is demonstrated by online monitoring. MB cloud dynamics are acoustically monitored and identified during sonoporation therapy for different excitation peak negative pressures. This is a first step towards adaptively optimizing transfection efficiency in sonoporation therapy by online acoustic monitoring.