Transient permeabilization of cell membranes by ultrasound-exposed microbubbles is related to formation of hydrogen peroxide

Abstract

In the present study, we addressed the interactions among ultrasound, microbubbles, and living cells as well as consequent arising bioeffects. We specifically investigated whether hydrogen peroxide (H(2)O(2)) is involved in transient permeabilization of cell membranes in vitro after ultrasound exposure at low diagnostic power, in the presence of stable oscillating microbubbles, by measuring the generation of H(2)O(2) and Ca(2+) influx. Ultrasound, in the absence or presence of SonoVue microbubbles, was applied to H9c2 cells at 1.8 MHz with a mechanical index (MI) of 0.1 or 0.5 during 10 s. This was repeated every minute, for a total of five times. The production of H(2)O(2) was measured intracellularly with CM-H(2)DCFDA. Cell membrane permeability was assessed by measuring real-time changes in intracellular Ca(2+) concentration with fluo-4 using live-cell fluorescence microscopy. Ultrasound, in the presence of microbubbles, caused a significant increase in intracellular H(2)O(2) at MI 0.1 of 50% and MI 0.5 of 110% compared with control (P < 0.001). Furthermore, we found increases in intracellular Ca(2+) levels at both MI 0.1 and MI 0.5 in the presence of microbubbles, which was not detected in the absence of extracellular Ca(2+). In addition, in the presence of catalase, Ca(2+) influx immediately following ultrasound exposure was completely blocked at MI 0.1 (P < 0.01) and reduced by 50% at MI 0.5 (P < 0.001). Finally, cell viability was not significantly affected, not even 24 h later. These results implicate a role for H(2)O(2) in transient permeabilization of cell membranes induced by ultrasound-exposed microbubbles.

Effect of Focused Ultrasound Applied With an Ultrasound Contrast Agent on the Tight Junctional Integrity of the Brain Microvascular Endothelium

“…Recent high-speed imaging studies have shown that microbubbles oscillating in an ultrasound field can deform cell membranes and enhance permeability (van Wamel et al 2006). Others have associated this permeability with local hydrogen peroxide formation (Juffermans et al 2006). It is possible that the sonications in our experiments produced such membrane permeabilization at the luminal surface of selected ECs, leaving the neighboring cells intact.…”

Gene therapy progress and prospects: Ultrasound for gene transfer

“…40,44,45 More controversial is the potential role of extra and/or intracellular free radical production produced during transient cavitation. 25,46,47 We have reported that the presence of free radical scavengers in the transfection medium has no effect on transgene expression following UEGT, suggesting that free radicals are not required for sonoporation. 46 In contrast, Juffermans et al 25 found that scavenging hydrogen peroxide (H 2 O 2 ) with extracellular catalase largely prevented the increased calcium permeability induced by low MI (0.1-0.5) ultrasound (1-3.6 MHz) in H9c2 rat cardiomyoblast cells.…”

“…25,46,47 We have reported that the presence of free radical scavengers in the transfection medium has no effect on transgene expression following UEGT, suggesting that free radicals are not required for sonoporation. 46 In contrast, Juffermans et al 25 found that scavenging hydrogen peroxide (H 2 O 2 ) with extracellular catalase largely prevented the increased calcium permeability induced by low MI (0.1-0.5) ultrasound (1-3.6 MHz) in H9c2 rat cardiomyoblast cells. However, these authors felt that only stable cavitation was generated in their system, despite the detection of increased intracellular H 2 O 2 -somewhat surprisingly they did not report on extracellular levels.…”

“…38,39 Mechanistic studies implicate plasma membrane 'sonoporation' (reversible pore formation, up to 100 nm in effective diameter with a half-life of a few seconds) as the dominant mechanism underlying UEGT The prevailing dogma is that acoustic cavitation promotes UEGT principally through the generation of reversible pores in the plasma membrane ('sonoporation'), as evidenced by direct imaging and/or by analysis of increased plasma membrane permeability to marker compounds. 3,12,14,15,20,24,25,29,30,[40][41][42] Recent studies have focused upon the role of transient cavitation in sonoporation. Sundaram et al 12 examined the effect of lowfrequency (20-93 kHz) ultrasound on uptake of the fluorescent dye calcein (molecular weight 623 Da, radius 0.6 nm) into mouse 3T3 fibroblasts in the absence of echocontrast agents but at MI levels (mostly above 1) sufficient to induce cavitation in non-degassed culture medium.…”

Apoptosis induced by low-intensity ultrasound in vitro: Alteration of protein profile and potential molecular mechanism

“…The ultrasound irradiation can induce the apoptosis of tumor cells, including the gastric cancer cells, human leukemic cells HL-60, K562, Molt-4, U937 [3][4][5], and so on. The mechanism related to the apoptosis induced by ultrasound was suggested and three apoptotic signal processes were included.…”