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

Abstract

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.

Acoustic Characteristics of Microbubbles: Effect of Acoustic Pressure and Pulse Duration

“…It is believed when a bubble is driven at this resonant frequency, it reaches the highest oscillation amplitude and may provide more effective therapeutic re- (44). The subharmonic emissions for Sonovue MB was studied at 3 and 30 cycle pulse duration and have shown that the emissions were higher at longer pulse duration of 30 cycles (45). Beside these the time dynamics of MBs at these varying acoustic conditions hasn't been investigated which is the focus of this thesis.…”

“…These unique markers of the MB characteristics are associated with bubble dynamics. Investigations on sub-harmonics imaging (54; 44) and in therapy (45) have shown that subharmonic emission can be exploited as indicator of sonoporation. Although the mechanism behind subharmonic-ultraharmonic emissions are not well understood, four possi-ble explanation have been suggested for free bubbles: (i) transient cavitation, (ii) subharmonic oscillation of bubbles excited at resonance, (iii) resonant oscillations of bubbles excited at their second harmonic and (iv) non-radial modes of oscillation.…”

Acoustic Characteristics of Microbubbles: Effect of Acoustic Pressure and Pulse Duration

“…The subharmonic emission of 5 different agents at high frequency (25 MHz) have been investigated and have shown that with increase of pulse duration the subharmonic emissions are enhanced for all agents (44). The subharmonic emissions for Sonovue MB was studied at 3 and 30 cycle pulse duration and have shown that the emissions were higher at longer pulse duration of 30 cycles (45). Beside these the time dynamics of MBs at these varying acoustic conditions hasn't been investigated which is the focus of this thesis.…”

Performance evaluation of an automatic controlled sonoporation therapy device

“…This is increased by the presence of microbubbles (MBs). Effective temporal membrane permeability implicates large scale MB oscillation with subsequent dissolution [1], [2]. High excitation pressure amplitudes cause violent MB destruction which often results in high numbers of dead cells, whereas low pressure amplitudes cause low sonoporation rates [3].…”