Intracellular delivery and calcium transients generated in sonoporation facilitated by microbubbles

Fan, Zhenzhen; Kumon, Ronald E.; Park, J.; Deng, Cheri X.

doi:10.1016/j.jconrel.2009.09.031

Cited by 148 publications

(143 citation statements)

References 67 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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“…Thus, the shear stress induced by sonoporation could contribute to the observed phenotypical changes. In addition, sonoporation was reported to influence Ca 2+ signaling within cells [28,30]. Importantly, increased intracellular Ca 2+ concentrations were also reported as one of the early events after LPS stimulation of mouse bone marrow-derived DCs [31].…”

Section: Discussionmentioning

confidence: 99%

The potential of antigen and TriMix sonoporation using mRNA-loaded microbubbles for ultrasound-triggered cancer immunotherapy

Dewitte

Lint

Heirman

et al. 2014

Journal of Controlled Release

103

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Dendritic cell (DC)-based cancer vaccines, where the patient's own immune system is harnessed to target and destroy tumor tissue, has emerged as a potent therapeutic strategy. In the development of such DC vaccines, it is crucial to load the DCs with tumor antigens, and to simultaneously activate them to become more potent antigen-presenting cells. For this, we report on microbubbles, loaded with both antigen mRNA as well as immunomodulating TriMix mRNA, which can be used for the ultrasound-triggered transfection of DCs. In vivo experiments with in vitro sonoporated DCs, show the effective induction of antigen-specific T cells, resulting in specific lysis of antigen-expressing cells. Especially in a therapeutic setting, sonoporation with TriMix has a significant added value, resulting in a significant reduction of tumor outgrowth and a marked increase in overall survival. What is more, complete tumor regression was observed in 30% of the antigen+TriMix DC vaccinated animals, which also displayed long-term antigen-specific immunological memory. As a result, DC sonoporation using microbubbles loaded with a combination of antigen and TriMix mRNA can elicit powerful immune responses in vivo, and might serve as a potential tool for further in vivo DC vaccination applications.

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“…Upon the application of single-shot ultrasound pulsing, membrane disruption was expectedly observed near the pre-exposure microbubble position as labelled in figure 3b. However, the sonoporation site (with 3.9 mm gap size) had remained unsealed over time because of the absence of Ca 2þ influx, which is a known condition that would trigger emergency repair of the severed membrane [28,29]. Throughout this period, the membrane periphery did not exhibit bleb-like protrusive morphology (figure 3c-h).…”

Section: No Blebbing At the Sonoporation Site If Extracellular Ca 2þ mentioning

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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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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Intracellular delivery and calcium transients generated in sonoporation facilitated by microbubbles

Cited by 148 publications

References 67 publications

Spatiotemporally controlled single cell sonoporation

Spatiotemporally controlled single cell sonoporation

The potential of antigen and TriMix sonoporation using mRNA-loaded microbubbles for ultrasound-triggered cancer immunotherapy

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

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