Abstract
Sonoporation via microbubble-mediated ultrasound exposure has shown potential in drug and gene delivery. However, there is a general lack of mechanistic knowledge on sonoporation-induced cellular impact after membrane resealing, and this issue has made it challenging to apply sonoporation efficiently in practice. Here, we present new evidence on how sonoporation, without endangering immediate cell viability, may disrupt downstream cellular hemostasis in ways that are distinguished from the bioeffects observed in other sonicated and unsonoporated cells. Sonoporation was realized on HL-60 leukemia cells by delivering pulsed ultrasound (1 MHz frequency, 0.50 MPa peak negative pressure; 10% duty cycle; 30 s exposure period; 29.1 J/cm2 acoustic energy density) in the presence of lipid-shelled microbubbles (1:1 cell-to-bubble ratio). Results showed that 54.6% of sonoporated cells, despite remaining initially viable, underwent apoptosis or necrosis at 24 h after sonoporation. Anti-proliferation behavior was also observed in sonoporated cells as their subpopulation size was reduced by 43.8% over 24 h. Preceding these cytotoxic events, the percentages of sonoporated cells in different cell cycle phases were found to be altered by 12 h after exposure. As well, for sonoporated cells, their expressions of cytoprotective genes in the heat shock protein-70 (HSP-70) family were upregulated by at least 4.1 fold at 3 h after exposure. Taken altogether, these findings indicate that sonoporated cells attempted to restore homeostasis after membrane resealing, but many of them ultimately failed to recover. Such mechanistic knowledge should be taken into account to devise more efficient sonoporation-mediated therapeutic protocols.
Highlights
arrest[23], and morphological changes[24, 25]
We have sought to test the overall hypothesis that various downstream bioeffects of sonoporated cells, such as proliferation behavior and heat shock protein-70 (HSP-70) expression levels, are different from those for cells that merely received ultrasound exposure
To investigate the potential downstream molecular bioeffects of ultrasound exposure in the presence of microbubbles, we evaluated the post-exposure transcription levels of three HSP-70 genes (HSPA1A, HSPA1B and HSPA6) by quantitative polymerase chain reaction analysis
Summary
arrest[23], and morphological changes[24, 25]. Yet, it remains unclear as to whether these biological effects are specific to sonoporation or whether they are merely attributed to ultrasound exposure. The focus of our investigation is on analyzing sonoporated cells’ post-resealing viability, proliferation trend, cell-cycle distribution, and heat shock protein-70 (HSP-70) expression level, the last of which is known to be upregulated when cells are stressed[26]. Such an analysis allows us to more precisely identify whether, after membrane resealing has taken place, sonoporation may induce cellular impact that could be cytoprotective or cytotoxic in nature. We have sought to test the overall hypothesis that various downstream bioeffects of sonoporated cells, such as proliferation behavior and HSP-70 expression levels, are different from those for cells that merely received ultrasound exposure. In sonoporated cells, alterations in cell proliferation kinetics and HSP-70 expressions may emerge hours after ultrasound exposure when membrane integrity has long been restored
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