Controlled permeation of cell membrane by single bubble acoustic cavitation

Abstract

Sonoporation is the membrane disruption generated by ultrasound and has been exploited as a non-viral strategy for drug and gene delivery. Acoustic cavitation of microbubbles has been recognized to play an important role in sonoporation. However, due to the lack of adequate techniques for precise control of cavitation activities and real-time assessment of the resulting sub-micron process of sonoporation, limited knowledge has been available regarding the detail processes and correlation of cavitation with membrane disruption at the single cell level. In the current study, we developed a combined approach including optical, acoustic, and electrophysiological techniques to enable synchronized manipulation, imaging, and measurement of cavitation of single bubbles and the resulting cell membrane disruption in real-time. Using a self-focused femtosecond laser and high frequency (7.44 MHz) pulses, a single microbubble was generated and positioned at a desired distance from the membrane of a Xenopus oocyte. Cavitation of the bubble was achieved by applying a low frequency (1.5 MHz) ultrasound pulse (duration 13.3 or 40 µs) to induce bubble collapse. Disruption of the cell membrane was assessed by the increase in the transmembrane current (TMC) of the cell under voltage clamp. Simultaneous high-speed bright field imaging of cavitation and measurements of the TMC were obtained to correlate the ultrasound-generated bubble activities with the cell membrane poration. The change in membrane permeability was directly associated with the formation of a sub-micrometer pore from a local membrane rupture generated by bubble collapse or bubble compression depending on ultrasound amplitude and duration. The impact of the bubble collapse on membrane permeation decreased rapidly with increasing distance (D) between the bubble (diameter d) and the cell membrane. The effective range of cavitation impact on membrane poration was determined to be D/d = 0.75. The maximum mean radius of the pores was estimated from the measured TMC to be 0.106 ± 0.032 µm (n = 70) for acoustic pressure of 1.5 MPa (duration 13.3 µs), and increased to 0.171 ± 0.030 µm (n = 125) for acoustic pressure of 1.7 MPa and to 0.182 ± 0.052 µm (n=112) for a pulse duration of 40 µs (1.5 MPa). These results from controlled cell membrane permeation by cavitation of single bubbles revealed insights and key factors affecting sonoporation at the single cell level.

Ultrasound and microbubble mediated drug delivery: Acoustic pressure as determinant for uptake via membrane pores or endocytosis

“…When encountering a cell, this radiation force compresses the microbubble onto the cell membrane, resulting in membrane indentation without disruption. Fan et al [41] and Zhou et al [42] reported similar observations. The secondary radiation force causes microbubble clustering [43], as illustrated in Figure 5.…”

Spatiotemporally controlled single cell sonoporation

“…Recently, several studies (7,21,22) addressed this challenge using various innovative approaches for generating and trapping a single microbubble to induce sonoporation of a single cell, but required complex laser systems for controlling bubbles and only worked with one cell at a time. No quantification of membrane poration and delivery was performed in these studies.…”

Ultrasound-mediated cavitation does not decrease the activity of small molecule, antibody or viral-based medicines

“…26 Cells have the capacity to repair and reseal these holes, meaning the process is not necessarily cytotoxic. In contrast, lipid-/protein-coated viruses do not have this repair capacity, and so it is important to assess the impact of cavitation upon therapeutically useful enveloped viruses such as VV.…”