Membrane Perforation and Recovery Dynamics in Microbubble-Mediated Sonoporation

Hu, Yaxin; Wan, Jennifer M. F.; Yu, Alfred C. H.

doi:10.1016/j.ultrasmedbio.2013.08.003

Cited by 165 publications

(181 citation statements)

References 36 publications

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“…While direct observations of sonoporation site closure have been reported [34], the mechanistic steps involved in the process are still unknown with the exception of a few hypotheses based on site patching and membrane self-reunion arguments [25]. In this work, we have gained insight into the recovery dynamics in sonoporation episodes that were generated by inertial cavitation of targeted microbubbles.…”

Section: Sonoporation-induced Membrane Blebbing: Summative Recapmentioning

confidence: 98%

“…As a lateral investigation, a subset of experimental trials was performed without the presence of extracellular Ca 2þ , which was previously observed to block the initiation of sonoporation site recovery [34]. This side study was intended to delineate whether the suppression of membrane resealing would affect bleb formation.…”

Section: Extracellular Calcium Ion Chelationmentioning

confidence: 99%

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Membrane blebbing as a recovery manoeuvre in site-specific sonoporation mediated by targeted microbubbles

Leow

Wan

2015

J. R. Soc. Interface.

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Site-specific perforation of the plasma membrane can be achieved through ultrasound-triggered cavitation of a single microbubble positioned adjacent to the cell. However, for this perforation approach (sonoporation), the recovery manoeuvres invoked by the cell are unknown. Here, we report new findings on how membrane blebbing can be a recovery manoeuvre that may take place in sonoporation episodes whose pores are of micrometres in diameter. Each sonoporation site was created using a protocol involving single-shot ultrasound exposure (frequency: 1 MHz; pulse length: 30 cycles; peak negative pressure: 0.45 MPa) which triggered inertial cavitation of a single targeted microbubble (diameter: 1-5 mm). Over this process, live confocal microscopy was conducted in situ to monitor membrane dynamics, model drug uptake kinetics and cytoplasmic calcium ion (Ca 2þ ) distribution. Results show that blebbing would occur at a recovering sonoporation site after its resealing, and it may emerge elsewhere along the membrane periphery. The bleb size was correlated with the pre-exposure microbubble diameter, and 99% of blebbing cases at sonoporation sites were inflicted by microbubbles larger than 1.5 mm diameter (analysed over 124 sonoporation episodes). Blebs were not observed at irreversible sonoporation sites or when sonoporation site repair was inhibited via extracellular Ca 2þ chelation. Functionally, the bleb volume was found to serve as a buffer compartment to accommodate the cytoplasmic Ca 2þ excess brought about by Ca 2þ influx during sonoporation. These findings suggest that membrane blebbing would help sonoporated cells restore homeostasis.

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Section: Sonoporation-induced Membrane Blebbing: Summative Recapmentioning

confidence: 98%

Section: Extracellular Calcium Ion Chelationmentioning

confidence: 99%

Membrane blebbing as a recovery manoeuvre in site-specific sonoporation mediated by targeted microbubbles

Leow

Wan

2015

J. R. Soc. Interface.

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“…This increased mean intensity might be also due to incomplete pore closure. Pores were reported to be resealed within seconds to minutes [24], while this recording stopped 25 sec after finishing ultrasound exposure. Upon collision with the cell membrane, a pore (dotted circle) is created.…”

Section: Real-time Confocal Microscopy During Ultrasound Radiationmentioning

confidence: 99%

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

Cock

Zagato

Braeckmans

et al. 2015

Journal of Controlled Release

210

247

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Although promising results are achieved in ultrasound mediated drug delivery, its underlying biophysical mechanisms remain to be elucidated. Pore formation as well as endocytosis has been reported during ultrasound application. Due to the plethora of ultrasound settings used in literature, it is extremely difficult to draw conclusions on which mechanism is actually involved. To our knowledge, we are the first to show that acoustic pressure influences which route of drug uptake is addressed, by inducing different microbubble-cell interactions. To investigate this, FITC-dextrans were used as model drugs and their uptake was analyzed by flow cytometry. In fluorescence intensity plots, two subpopulations arose in cells with FITCdextran uptake after ultrasound application, corresponding to cells having either low or high uptake. Following separation of the subpopulations by FACS sorting, confocal images indicated that the low uptake population showed endocytic uptake. The high uptake population represented uptake via pores. Moreover, the distribution of the subpopulations shifted to the high uptake population with increasing acoustic pressure. Real-time confocal recordings during ultrasound revealed that membrane deformation by microbubbles may be the trigger for endocytosis via mechanostimulation of the cytoskeleton. Pore formation was shown to be caused by microbubbles propelled towards the cell. These results provide a better insight in the role of acoustic pressure in microbubble-cell interactions and the possible consequences for drug uptake. In addition, it pinpoints the need of a more rational, microbubble behavior based choice of acoustic parameters in ultrasound mediated drug delivery experiments.

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“…Previously, others have investigated single bubble interaction with cell(s) for membrane poration using ultrasound contrast agents (7,15,18,40) or laser-generated single bubbles (14,41). However, cell membrane deformation was not characterized, and cell shape, orientation, and adhesion conditions were not carefully controlled, which could all substantially influence the cell response and bioeffects produced under mechanical stresses (29).…”

Section: Discussionmentioning

confidence: 99%

Cell membrane deformation and bioeffects produced by tandem bubble-induced jetting flow

Yuan

Yang

Zhong

2015

Proc. Natl. Acad. Sci. U.S.A.

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Cavitation with bubble-bubble interaction is a fundamental feature in therapeutic ultrasound. However, the causal relationships between bubble dynamics, associated flow motion, cell deformation, and resultant bioeffects are not well elucidated. Here, we report an experimental system for tandem bubble (TB; maximum diameter = 50 ± 2 μm) generation, jet formation, and subsequent interaction with single HeLa cells patterned on fibronectin-coated islands (32 × 32 μm) in a microfluidic chip. We have demonstrated that pinpoint membrane poration can be produced at the leading edge of the HeLa cell in standoff distance S d ≤ 30 μm, driven by the transient shear stress associated with TB-induced jetting flow. The cell membrane deformation associated with a maximum strain rate on the order of 10 4 s −1 was heterogeneous. The maximum area strain (e A,M ) decreased exponentially with S d (also influenced by adhesion pattern), a feature that allows us to create distinctly different treatment outcome (i.e., necrosis, repairable poration, or nonporation) in individual cells. More importantly, our results suggest that membrane poration and cell survival are better correlated with area strain integral ( R e 2 A dt) instead of e A,M , which is characteristic of the response of materials under high strain-rate loadings. For 50% cell survival the corresponding area strain integral was found to vary in the range of 56 ∼ 123 μs with e A,M in the range of 57 ∼ 87%. Finally, significant variations in individual cell's response were observed at the same S d , indicating the potential for using this method to probe mechanotransduction at the single cell level.microfluidics | cavitation bioeffects | single-cell analysis | high strain-rate | cell mechanics

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Membrane Perforation and Recovery Dynamics in Microbubble-Mediated Sonoporation

Cited by 165 publications

References 36 publications

Membrane blebbing as a recovery manoeuvre in site-specific sonoporation mediated by targeted microbubbles

Membrane blebbing as a recovery manoeuvre in site-specific sonoporation mediated by targeted microbubbles

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

Cell membrane deformation and bioeffects produced by tandem bubble-induced jetting flow

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