Evaluation of subharmonic emission from encapsulated microbubbles as an indicator for sonoporation of cell monolayers

Sonoporation is the ultrasound induced transient opening of cell membranes. Large scale oscillation of nearby microbubbles (MBs) is considered to be the primary effect in sonoporation whereas MB destruction correlates with lower sonoporation efficiency and increased cell damage. The scale of MB oscillation in a cloud can be determined by monitoring subharmonic emission. In this study, SW480 cells are grown in Opticell containers, which are filled with a growth medium containing SonoVue MBs and propidium iodide (PI). Each container is placed in water in the focus of a single element transducer emitting 10 cycles sine-bursts at 3.3 MHz. The peak negative pressure is varied from 75 to 750 kPa. Scattered signals are recorded by a broadband transducer to monitor subharmonic-to-fundamental ratio (SFR) during therapy. A fluorescence microscope is focused on the therapy region to monitor PI fluorescence during and after acoustic excitation. PI cannot overcome intact cell membranes and changes its fluorescence properties when bound to intracellular fluid. A cell is considered sonoporated, if it reveals a sharp rise of fluorescence intensity and a subsequent decrease. A cell is considered permanently stained, if fluorescence intensity is concentrated at the cell nucleus and rises to a plateau. For maximum peak negative excitation pressure, maximum sonoporation (4.9%) and maximum permanent poration (2.8%) is observed. Correcting the number of sonoporated cells by the number of permanently stained cells, best sonoporation efficiency is achieved for the amplitude of 520 kPa. The same holds for the SFR, which significantly rises to a maximum of -26,6 dB at 520 kPa, too. The relationship of sonoporation efficiency and SFR reveals that subharmonic emission from ultrasound contrast agent is an indicator for sonoporation efficiency of cell monolayers.