Ultrastructural modifications of cell membranes and organelles induced by sonoporation

Sonoporation increases transiently the native cell membrane permeability. However, the exact mechanism involved in the membrane permeabilization remains to be elucidated. While, no consensus is reached, the pore formation is usually hypothesized as having a central role in the membrane permeabilization induced by sonoporation. In this study, we investigate the sonoporation effect on the plasma membrane and organelles using electron microscopy. Adherent U-87 MG cells were insonated at 1 MHz, 1 W/cm² acoustic intensity, 20% duty cycle for 10 or 60 s. BR14^® microbubbles were added at a bubble/ cell ratio of 5. SYTOX^® Green was used as a permeabilization marker. With these US conditions in combination with BR14^®, flow cytometry results showed a permeabilization rate of 60%. The ultrastructural modifications of the cells were monitored by scanning and transmission electron microscopy (SEM/TEM) either immediately or 15 min post-sonoporation. Based on SEM images, the control cells (No US) showed a regular plasma membrane with microvilli, while the insonified cells (US+BR14^®) exhibited circular and dark spots on their surfaces, suggesting pore-like structures. The number of these structures increased in the presence of BR14^® and with the insonation time. The porelike structures size distribution is heterogeneous, and ranged from 10 nm to 160 nm. However, these structures might also correspond to caveolae or clathrin endocytic vesicles. To investigate the transient character of these structures, the cells were fixed 15 min after sonoporation. The results showed that the number of these pore-like structures decreased strongly indicating that the cells are still metabolically active. The maximal size of these pores is 100 nm and we suggest that small pores still require more time to reseal. Sonoporation effects on the organelles structure were investigated. Based on TEM images, insonified cells (US+BR14^®) presented a stimulation of endocytosis pathways compared to control cells. In addition, the sonoporated cells showed less dense cytoplasm, suggesting a decrease of their viscosity, which would facilitate the intracellular traffic of delivered molecules. In conclusion, these microscopic observations reveal that the sonoporation effects are not confined to the membrane only, but also occur at the intracellular level.