Induction of apoptosis in sonoporation and ultrasonic gene transfer

The role of apoptosis in sonoporation and ultrasound-enhanced gene transfection of cell suspensions was examined in vitro. Suspensions of HL-60 and of CHO-K1 cells were exposed to 2.25-MHz continuous ultrasound for 1 min in a 60-rpm rotating-tube exposure system, with ultrasound contrast media added to ensure nucleation of cavitation. Cell necrosis was measured by trypan blue dye exclusion (using a hemacytometer) and by propidium iodide nuclear staining (using flow cytometry). Apoptosis was detected by the annexin V method with Alexa Fluor 350 as the fluorescent label, and confirmed by Hoechst 33342 nuclear staining. Sonoporation cell loading was assessed by uptake of large fluorescent-dextran molecules from the medium. Transfection was demonstrated by expression of green fluorescent protein (GFP) from plasmids transferred into the cells by the treatment. Cell scoring was performed by flow cytometry, with necrotic cell events excluded. For HL-60 cells at 0.4 MPa, cell loading and transfection was significantly increased relative to shams at 2, 6 and 24 h post exposure, peaking at 19.0 +/- 5.5% and 9.6 +/- 4.2% of non-necrotic cells, respectively, at 6 h. However, about one third of the treatment-positive cells were identified as apoptotic. The cell loading and gene transfer effects increased for increasing peak rarefactional pressure amplitude, reaching 24.4 +/- 7.7% and 12.7 +/- 5.1% of non-necrotic cells, respectively, for 0.6-MPa exposure. However, the lethal cellular injury caused by cavitation in the rotating tube system reduced the overall apparent efficacy of cell loading and gene transfer to 5.1 +/- 2.1% and 2.1 +/- 0.9%, respectively, after accounting for necrosis and apoptosis. Similar tests with CHO cells showed increased sonoporation but mostly cell death by necrosis, rather than apoptosis. The induction of apoptosis by cavitation treatments should be considered as a possible confounding factor, in addition to necrosis, in sonoporation and ultrasonic gene transfer research.