Ultrasound-Induced Calcium Oscillations and Waves in Chinese Hamster Ovary Cells in the Presence of Microbubbles

Kumon, Ronald E.; Aehle, M.; Sabens, D.; Parikh, P.; Kourennyi, Dmitri E.; Deng, Cheri X.

doi:10.1529/biophysj.107.113365

Cited by 47 publications

(45 citation statements)

References 15 publications

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“…bubble excitation (36)(37)(38)(39), and in surrounding cells that were not sonoporated via calcium waves. The calcium waves were likely induced by factors released from the sonoporated cells (40,41).…”

Section: Resultsmentioning

confidence: 99%

Spatiotemporally controlled single cell sonoporation

Fan

Liu

Mayer

et al. 2012

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

216

215

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This paper presents unique approaches to enable control and quantification of ultrasound-mediated cell membrane disruption, or sonoporation, at the single-cell level. Ultrasound excitation of microbubbles that were targeted to the plasma membrane of HEK-293 cells generated spatially and temporally controlled membrane disruption with high repeatability. Using whole-cell patch clamp recording combined with fluorescence microscopy, we obtained time-resolved measurements of single-cell sonoporation and quantified the size and resealing rate of pores. We measured the intracellular diffusion coefficient of cytoplasmic RNA/DNA from sonoporation-induced transport of an intercalating fluorescent dye into and within single cells. We achieved spatiotemporally controlled delivery with subcellular precision and calcium signaling in targeted cells by selective excitation of microbubbles. Finally, we utilized sonoporation to deliver calcein, a membrane-impermeant substrate of multidrug resistance protein-1 (MRP1), into HEK-MRP1 cells, which overexpress MRP1, and monitored the calcein efflux by MRP1. This approach made it possible to measure the efflux rate in individual cells and to compare it directly to the efflux rate in parental control cells that do not express MRP1.D espite the development of various approaches for transporting membrane-impermeant compounds (such as fluorescent markers, DNA, RNA, siRNA, proteins, peptides, and amino acids) into living cells (1-3), efficient intracellular delivery of bioactive agents for biomedical applications with minimal adverse effects remains challenging. In addition, it is desirable yet difficult to achieve local perturbation of intracellular processes, which requires subcellular molecular localization inside the living cell (4, 5).Sonoporation uses ultrasound to induce transient disruption of cell membranes (6-8), thereby enabling transport of membraneimpermeant compounds into the cytoplasm of living cells (6,(9)(10)(11). Without the need to use viral vectors, sonoporation enables the delivery of a wide range of bioactive agents with minimal inflammatory and immunological responses for both in vitro studies and in vivo applications (12-16). In addition, ultrasound application can be targeted to a specific volume of tissue in vivo noninvasively. These unique characteristics make sonoporation a compelling and versatile technology for nonviral drug and gene delivery.Sonoporation is typically performed for bulk treatment of a tissue volume in vivo or a large number of cells in vitro, often facilitated by microbubbles that are either injected in the vasculature or mixed in solution with suspended or attached cells. Ultrasound application induces cavitation of the microbubbles (17), signified by rapid volume expansion/contraction and/or collapse (18). These effects generate localized fluid flow, shear stress, and other mechanical or physical impact capable of affecting cells and structures nearby (7,19,20).However, the detailed processes supporting sonoporationmediated transmembrane and ...

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Section: Resultsmentioning

confidence: 99%

Spatiotemporally controlled single cell sonoporation

Fan

Liu

Mayer

et al. 2012

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

216

215

View full text Add to dashboard Cite

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“…The influx of Ca 2+ might signal cell injury and trigger active mechanisms similar to those involved in cell repair of membrane injuries; such process rapidly seal membranes and ensure cell survival in sonoporation. The in-rush of Ca 2+ may also play role in signal transduction linked to downstream effects of sonoporation such as apoptosis [21] and calcium oscillation and calcium waves [22].…”

Section: Discussionmentioning

confidence: 99%

“…The US generated pores on the plasma membrane must reseal to prevent the loss of intracellular contents to ensure cell survival, thereby limiting efficient inward transmembrane passage of desired extracellular agents within a time window before the completion of pore resealing. Furthermore, repair of the membrane disruption is necessary to avoid intracellular overload of ions that might be toxic to the cell or serve as the triggering sources for other irreversible and reversible cellular processes such as apoptosis [21] and calcium oscillation [22], making the rate of resealing one of the key factors determining the uptake efficiency and post-ultrasound cell fate.…”

Section: Introductionmentioning

confidence: 99%

Effects of extracellular calcium on cell membrane resealing in sonoporation

Zhou

Shi

Cui

et al. 2008

Journal of Controlled Release

109

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Sonoporation has been exploited as a promising strategy for intracellular drug and gene delivery. The technique uses ultrasound to generate pores on the cell membrane to allow entry of extracellular agents into the cell. Resealing of these non-specific pores is a key factor determining both the uptake and post-ultrasound cell survival. This study examined the effects of extracellular Ca 2+ on membrane resealing in sonoporation, using Xenopus oocytes as a model system. The cells were exposed to tone burst ultrasound (1.06 MHz, duration 0.2 s, acoustic pressure 0.3 MPa) in the presence of 0.1% Definity ® at various extracellular [Ca 2+ ] (0-3 mM). Sonoporation inception and resealing in a single cell were monitored in real time via the transmembrane current of the cell under voltage clamp. The time-resolved measurements of transmembrane current revealed the involvement of two or more Ca 2+ related processes with different rate constants and characteristics. Rapid resealing occurred immediately after ultrasound application followed by a much slower resealing process. Complete resealing required [Ca 2+ ] above 0.54 mM. The cells resealed in 6-26 s at 1.8 mM Ca 2+ , but took longer at lower concentrations, up to 58 ~170 s at 0.54 mM Ca 2+ .

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“…Pore formation was also investigated in Xenopus oocytes by measuring cell membrane potential changes using a voltage clamp technique [33]. The changes in cell transmembrane current can be attributed to Ca 2+ influx during pore formation [34][35][36][37], and are a clear indication of cell membrane disruption.…”

Section: Biophysical Aspects Of Stable Cavitationmentioning

confidence: 99%

Understanding ultrasound induced sonoporation: Definitions and underlying mechanisms

Lentacker

Cock

Deckers

et al. 2014

Advanced Drug Delivery Reviews

672

592

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In the past two decades, research has underlined the potential of ultrasound and microbubbles to enhance drug delivery. However, there is less consensus on the biophysical and biological mechanisms leading to this enhanced delivery. Sonoporation, i.e. the formation of temporary pores in the cell membrane, as well as enhanced endocytosis is reported. Because of the variety of ultrasound settings used -and corresponding microbubble behavior, a clear overview is missing. Therefore, in this review, the mechanisms contributing to sonoporation are categorized according to three ultrasound settings: i) low intensity ultrasound leading to stable cavitation of microbubbles, ii) high intensity ultrasound leading to inertial cavitation with microbubble collapse, and iii) ultrasound application in the absence of microbubbles. Using low intensity ultrasound, the endocytotic uptake of several drugs could be stimulated, while short but intense ultrasound pulses can be applied to induce pore formation and the direct cytoplasmic uptake of drugs. Ultrasound intensities may be adapted to create pore sizes correlating with drug size. Small molecules are able to diffuse passively through small pores created by low intensity ultrasound treatment. However, delivery of larger drugs such as nanoparticles and gene complexes, will require higher ultrasound intensities in order to allow direct cytoplasmic entry.2

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Ultrasound-Induced Calcium Oscillations and Waves in Chinese Hamster Ovary Cells in the Presence of Microbubbles

Cited by 47 publications

References 15 publications

Spatiotemporally controlled single cell sonoporation

Spatiotemporally controlled single cell sonoporation

Effects of extracellular calcium on cell membrane resealing in sonoporation

Understanding ultrasound induced sonoporation: Definitions and underlying mechanisms

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