Ultrasound-activated microbubbles as novel enhancers of radiotherapy in leukemia cells in vitro

The aim of this work was to develop a novel ultrasound-microbubble mediated vascular-disruption based method to enhance the effects of ionizing radiation on tumours. In this study, we hypothesized that ultrasound-activated microbubbles can be used as radioenhancers in an in vitro cell line. Acute myeloid leukemia (AML-5) cells in suspension were exposed to ultrasound pulses and ionizing radiation. Variations in insonating acoustic pressure (125-570 kPa), Definity microbubble concentration (0-3% v/v), ionizing radiation dose (0-8 Gy) and treatment order of ultrasound and radiation were investigated. The effect on the viability of cells was assessed using flow cytometry with propidium iodide immediately and 48 hours following treatment. Clonogenic viability of cells was assessed using a clonogenic assay and electron microscopy images were acquired of untreated, treated with ultrasound and microbubbles, treated with ionizing radiation and with the combined treatments. The results indicate that ultrasound-activated microbubbles can enhance the effect of ionizing radiation in AML-5 cells. Cell death increased by ~35% with the combined treatment of ultrasound and ionizing radiation (42%?6%) compared to ultrasound (77%?5%) and radiotherapy (71%?7%) 48 hours following the treatments. The ability of ultrasound and microbubbles to enhance therapeutic effect of radiotherapy depended on ultrasound pressure and microbubble concentration. Higher acoustic pressures (570 kPa) and microbubble concentrations (greater than 1.5% v/v) were more effective at increasing cell death in the combined treatment. Cell death increased with ionizing radiation dose in treatments with radiotherapy only and with combined treatments. The induced enhancement was not sensitive to the order of the treatments with one hour between treatments. Clonogenic survival assay showed a reduced viability in the combined treatment (~2%) compared to ultrasound and microbubble treatment (~30%) and ionizing radiation - - (~11%). Microscopy images of cells treated with both systems showed a more pronounced membrane deformations compared to images of cells with each treatment. In conclusion, ultrasound-activated microbubbles can enhance the therapeutic effects of ionizing radiation. In addition, preliminary data from animal experiments indicated that this effect could be reproduced to yield enhanced tumour cell kill in a xenograft model.