353. New Insights of Microbubbles-Cell Interactions During Sonoporation Process

Abstract

Upon ultrasound (US) exposure, gas microbubbles (MBs) can be expanded, moved and even destroyed. These properties offer the opportunity of site-specific local drug/gene delivery. Activation of MB under specific US beams induces a transient cell membrane permeabilization with a process known as sonoporation. Transient pores formed at the plasma membrane are supposed to be responsible for the intracellular delivery of molecules but also the outward transport of intracellular molecules. Endocytosis process has been shown to be involved during sonoporation. In the field of gene transfer, several studies including ours have reported an improvement of gene delivery by US assisted MB. A key to success of this technique lies in understanding mechanisms governing microbubble-cell interactions. Improving our knowledge will allow us to fully exploit this method for gene therapy purpose.Here, we investigate how MB and US behave towards cells under optimal conditions that allow an efficient gene transfer. Studies were performed on a specific set-up composed of sonoporation chamber mounted on a fluorescence confocal microscopy coupled to a high speed camera. Results obtained from our real time sonoporation indicate that, three events types could be recorded according to the acoustic parameters applied: 1) oscillating microbubbles that are stuck on the plasma membrane during the ultrasound stimulation corresponding to the cellular massage; 2) microbubbles entering into cells (translation); 3) microbubbles having a violent interaction with cells. All experiments were carried out at 1MHz of frequency. The first event occurs at 100 kPa a condition leading to low gene transfer. The second event is observed at 150 kPa after 600ms ultrasound stimulation, an optimal efficiency of gene transfer was obtained at this sound pressure range. The third event occurs for a sound pressure of 200kPa during which two phenomena were identified: either MBs were driven from the field of view due to excessive radiation force or a violent interaction of MB with the cell was observed. At 200kPa the gene transfer efficiency is ten times lower than at 150 kPa due likely to toxicity. It is tempting to correlate the entry of microbubbles phenomenon and efficiency of gene transfer meaning that the entry of the microbubble could promote the delivery of the plasmid into the cell. MBs were found to attach preferentially on lipid rafts of the plasma membrane. Most interestingly, sonoporation was able to modify chromatin compaction and a link between the plasma membrane and the effect on the nucleus has been investigated.