Ultrastructural sonoporation bio-effects: Comparative study on two human cancer cell lines

Sonoporation increases transiently the cell membrane permeability, enabling the therapeutic compounds internalization into the cells. Several investigations reported heterogeneities in the permeabilization and transfection efficacy depending on the ultrasound (US) settings and cell type. Here, we compare the sonoporation effects on two human cell lines, glioblastoma and breast cancer using scanning electron microscopy (SEM). Adherent U-87 MG and MDA-MB-231 cells were insonated at 1 W/cm², during 60 s at 10% or 20% duty cycle, in the presence of BR14^® microbubbles, added at a microbubble-cell ratio of 5. SYTOX^® Green, a non-permeant fluorescent dye was used at 1μM, to quantify the membrane permeabilization using flow cytometry. The ultrastructural changes of the cell membrane morphology were monitored by SEM. Flow cytometry results show that the percentage of permeabilized U-87 MG cells reaches 60%, while this value doesn´t exceed 40% for MDA-MB-231 cells. These results indicate that the percentage of permeabilized cells depends on the cell type. SEM observations were carried out to elucidate the differences in permeabilization rate between the two cell lines. The SEM analysis reveals that control cells show regular plasma membrane morphology. Their insonation in the presence of BR14^® induce the formation of dark holes on their membrane surfaces (named here pore-like structures). However, the quantitative analysis of the SEM micrographs highlights noticeable differences in morphological changes post-sonoporation between the two cell lines. Thus, the mean number of pore-like structures is more abundant on U-87 MG cell membrane than on MDA-MB-231 cell membrane (645 vs. 290). In addition, the mean size of pore-like structures depends on the cell line. Indeed, the mean size on MDA-MB-231 cells was 40 ± 1.2 nm (30-60 nm) while this value reached 80 ± 0.9 nm (10 to 160 nm) for U-8- MG cells. In conclusion, the study confirms that the pore-like structures observed post sonoporation are directly associated to the cell permeabilization rate. Moreover, the observed differences in the permeabilization levels between both cell lines could be attributed to the differences in the number and size of pore-like structures that were seen on the cell membrane. This difference may be due to the fibroblastic nature of the U-87 MG cells in comparison to MDA-MB-231 cells.