P4E-1 The Dependence of Sonoporation on Cell Cycle Phase: Enhanced Effect During G2 and S-Phase

Microbubble-assisted sonoporation, the ability to change local tissue permeability with ultrasound, which can promote the delivery of drugs and genes into cells, has been investigated both in vitro and in vivo. Under the same experimental and acoustic conditions, cells of the same histological type may be permeabilized, killed or left unaffected. We hypothesize that this variability depends partly on the cell-cycle phase. Here we investigate the effect of the cell-cycle on cell membrane permeability and viability in an in vitro cell suspension model using fluorescent markers and flow cytometry. Murine fibrosarcoma KHT-C cells in suspension were exposed to varying ultrasound conditions in the presence of Definity microbubbles. Cells were exposed to low (Pneg=125kPa) and high (Pneg=570kPa) acoustic pressures at 500kHz frequency, 16 cycles and 3kHz pulse repetition frequency in the presence of 3.5% volume concentration of microbubbles. Cell permeability and viability were measured for different phases of the cell-cycle (Gl, S and G2) using flow cytometry. FITC- dextran was used to measure cell membrane permeability and Propidium Iodide was used to detect non-viable cells. Hoechst 33342 fluorescent marker was used to determine each cell´s cell- cycle phase. Data indicated that cells in different cell-cycle phases were permeabilized in different proportions. Cell permeability was 0.5%, 25% and 45%, and cell viability was 78.8%, 70.2% and 48.7% in untreated, 125 kPa-treated, and 570 kPa-treated samples, respectively. In the untreated sample, cell-cycle dependent viability after handling was 53.8% (Gl), 42.6% (S) and 3.6% (G2) (normalized with respect to a total viability of 78.8%). In 125kPa- treated samples, cell viability was 70.6% (Gl), 26.2% (S) and 1.7% (G2) (normalized to a total viability of 70.2%). Cell permeability was 20% (Gl), 15% (S) and 2% (G2). The number of permeabilized to viable cells, was 0.23, 0.4 and 0.55 in Gl, S and G2 phases, respectively (Pneg=125- kPa). Cells in S and G2 were more susceptible to ultrasound compared to cells in Gl phase. Cells treated at 570kPa demonstrated a similar distribution in cell viability (70.8% (Gl), 26.2% (S) and 2% (G2), normalized to total viability 48.7%), however, the distribution of cell permeability with respect to cell-cycle phase was different. In conclusion, cells in different cell-cycle phases demonstrated varied sensitivity to permeabilization when exposed to ultrasound and microbubbles. More cells were permeabilized and killed in S and G2 phases of their cell cycle. This may be related to differing cellular visco-elas- tic properties of the cell membrane that change during the cell cycle.