The effects of power on–off durations of pulsed ultrasound on the destruction of cancer cells

Figures & data

Figure 1. Schematic of the experimental setup. HeLa cells were seeded at a concentration of 3.5 × 10⁴ cells/well in the six wells of a 24-well plate shown in gray.

Table I.  Parameters for experiments involving a fixed duty cycle of 50% with variable on- and off-times.

	PRF (Hz)	Total ultrasound time (s)	Total ultrasound on-time (s)	On-time	Off-time	On-time : off-time
1–1	2	20	10	250 ms	250 ms	1
1–2	20	20	10	25 ms	25 ms	1
1–3	100	20	10	5 ms	5 ms	1
1–4	200	20	10	2.5 ms	2.5 ms	1
1–5	1000	20	10	0.5 ms	0.5 ms	1
1–6	2000	20	10	250 µs	250 µs	1
1–7	4000	20	10	125 µs	125 µs	1

Table II.  Parameters for experiments involving a fixed off-time (25 ms) with variable on-times.

	PRF (Hz)	Total ultrasound time (s)	Total ultrasound on-time (s)	On-time (ms)	Off-time (ms)	On-time: off-time
2–1	39.21	510	10	0.5	25	0.02
2–2	36.36	110	10	25	25	0.10
2–3	33.33	60	10	5	25	1.00
2–4	2000	20	10	25	25	1.00
2–5	3.64	11	10	250	25	10.00

Table III.  Parameters for experiments involving a fixed on-time with variable off-times.

	PRF (Hz)	Total ultrasound time (s)	Total ultrasound on-time (s)	On-time (ms)	Off-time (ms)	On-time: off-time
3–1	2000.00	20	10	0.25	0.25	1.0
3–2	363.64	110	10	0.25	2.5	0.1
3–3	200.00	20	10	2.5	2.5	1.0
3–4	36.36	110	10	2.5	25	0.1
3–5	100.00	20	10	5.00	5.00	1.0
3–6	18.18	110	10	5.00	50.00	0.1
3–7	20.00	20	10	25.00	25.00	1.0
3–8	3.64	110	10	25.00	250.00	0.1
3–9	2.00	20	10	250.00	250.00	1.0
3–10	0.36	110	10	250.00	2500.00	0.1

Figure 2. Effect of the PRF (fixed duty cycle with variable on-/off-times) on the percentage of viable cultured HeLa cells. Significant cell destruction compared with controls (p < 0.05, two-tailed t-test) is evident for PRFs between 2 and 2000 Hz. The temperature did not differ significantly among the groups for PRF equal to 2000 and 4000 Hz (#, p > 0.05, two-tailed t-test), but significantly difference in cell survival was found between them (*, p < 0.05, two-tailed t-test).

Figure 3. Effect of the on-time (and variable off-times) on the percentage of viable HeLa cells. Significant cell destruction compared with controls (p < 0.05, two-tailed t-test) is evident for on-times of 5, 25 and 250 ms. No significant cell destruction is evident for on-time of 0.5 and 2.5 ms. However, the temperature decreased with the on-time. Significant difference was found in cell destruction (*) between on-times of 5 and 2.5, but not in temperature elevation (#).

Figure 4. Effect of the off-times (pairs of a fixed on-time with an off-time that was either the same or 10 times longer) on the percentage of viable HeLa cells at 28 h after sonication. Significant cell destruction but with a temperature increase of less than 1.5°C compared with controls (p < 0.05, two-tailed t-test) is evident for on/-off-times of 25/250 and 250/2500 ms/ms were found.

Figure 5. Effect of the fixed on-times but varied off-times on the survival ratio of different types of cultured cells at 4 h after sonication. * The survival ratio is significantly different from controls (p < 0.05, two-tailed t-test). Control: cells without ultrasound exposure. Each column represents the average of three trials of the combined result from nine samples per trial.

Figure 6. Ultrasound induces apoptotic cell death as a function of time, determined by flow cytometer. Hela cell line was sonicated using five paired parameters (on-off ratios of 1 and 0.1, with different on- and off-times). Four (a) and 28 (b) hours after the ultrasound exposure, the relative percentage of cell apoptosis, including both early apoptosis and secondary necrotic cells, were determined using the FITC-labeled annexin V kit. * : p < 0.05 (n = 3).

Figure 7. The correlation between the maximal temperature elevation and the percentage of surviving cells relative to controls.