Original ContributionPlasma Membrane Blebbing Dynamics Involved in the Reversibly Perforated Cell by Ultrasound-Driven Microbubbles
Introduction
The plasma membrane can be transiently perforated by microbubbles, which oscillate and collapse (i.e., sonoporation), providing a temporary window for transport of membrane-impermeant macromolecules into the cytoplasm (Ferrara et al. 2007; Yuan et al. 2015; Helfield et al. 2016). This is a promising strategy for drug delivery and gene therapy (Sboros 2008; Lentacker et al. 2014; Silvani et al. 2019). However, sonoporated cells can recover from membrane injury, by quickly resealing to prevent loss of the intracellular content (i.e., reversible sonoporation) on a time scale of seconds to minutes, and further restore their normal physiologic state by additional self-remodeling to regain functionality on a longer time scale of minutes to hours (Tzu-Yin et al. 2013; Qin et al. 2018). The capabilities of resealing and recovery dictate the short- and long-term fate of the sonoporated cells, thereby influencing delivery efficacy and biosafety. To promote the clinical application of this non-invasive and spatiotemporally controlled method, it is important to understand the biological mechanisms by which the cell reseals and recovers after sonoporation (Kooiman et al. 2014).
Early studies determined the resealing time to range from several to hundreds of seconds, depending on the degree of sonoporation, which is related to the acoustic cavitation dose (Mehier-Humbert et al. 2005, Qiu et al. 2010; Fan et al. 2012; Hu et al. 2013; Qin et al. 2016; van Rooij et al. 2016). According to a previous report, the degree of membrane injury may be dependent on resealing mechanism (McNeil and Steinhardt 2003). On this basis, small pores (tens of nanometers) might passively reseal owing to the inherent flowing qualities of lipid bilayers or endocytosis, whereas large pores (from hundreds of nanometers to micrometers) reseal by patch formation from exocytic compartments (McNeil and Steinhardt 2003; Andrews et al. 2015). Further, an extensively perforated membrane cannot be rapidly resealed, resulting in instant necrosis (i.e., irreversible sonoporation) (Hu et al. 2013). Importantly, chelation of extracellular calcium was found to terminate the resealing process, leading to cytosol loss and cell death, suggesting the essential role of calcium in the resealing of sonoporated cells (Deng et al. 2004; Fan et al. 2010; Hassan et al. 2010). By use of the immunostaining method, previous studies indirectly found that the luminal domain Lamp-1 of the lysosome inserted into the surface of the perforated membrane (Yang et al. 2008; De Cock et al. 2015). This suggests that lysosomes may be the main vesicles capable of rapid exocytosis and homotypic fusion to patch perforation in response to an elevated calcium level (Tam et al. 2010). However, although sonoporated cells retain viability in the short term after the completion of membrane resealing, concomitant bio-effects—such as plasma membrane potential depolarization (Qin et al. 2014), intracellular calcium waves and oscillations (Fan et al. 2010; Li et al. 2018) and change in intracellular reactive oxygen species homeostasis change (Jia et al. 2018)—are elicited as the initial signals triggering the related physiochemical pathways, resulting in secondary necrosis or apoptosis in the long term (Hassan et al. 2010). Some molecular pathways, such as the mitochondrial inherent pathway, have been identified to be involved in sonoporation-induced apoptosis (Danno et al. 2008; Sun et al. 2015). It is conceivable that by leveraging these biophysical mechanisms, we can promote resealing of the sonoporated membrane and regulate cell fate, thereby improving the efficiency of macromolecule delivery (Tzu-Yin et al. 2013; Kooiman et al. 2014).
In recent reports on cell recovery after sonoporation, membrane protrusions (blebs) and patches were observed at sites where the perforation had been resealed, with the bleb volume positively correlate with the diameter of the cavitating microbubbles (Leow et al. 2015). Moreover, blebs disappeared in dead cells when membrane resealing was inhibited via extracellular Ca2+ chelation, indicating that intracellular vesicle trafficking and blebs are related to the resealing and recovery of sonoporated cells (Leow et al. 2015; Jia et al. 2018a and 2018b). To our knowledge, blebs manifest mainly during physiologic cellular activities, such as cytokinesis, cell spreading and migration, or when cells are wounded by laser ablation (Kelly et al. 2009; Taneja and Burnette 2019), mechanical stimulation (e.g., micropipette aspiration) (Jimenez et al. 2014) or pore-forming toxins (PFTs) (Mesquita et al. 2017). However, very few studies have investigated the characteristics and functions of blebs in cells perforated by ultrasound-driven microbubbles.
Blebs of cells are driven by hydrostatic pressure generated by a contraction of the cortical actomyosin, which originates from the detachment of plasma membrane from the actin cortex. In general, two types of blebs (small dynamic and larger stable blebs) are found to be related to the fate of wounded cells (Charras 2008; Aoki et al. 2016). Dynamic blebbing, which comprises nucleation, growth, pausing and retraction phases, is closely linked to “healthy” cells and represents a cell's attempt to escape death (Charras and Paluch 2008; Idone et al. 2008; Jimenez et al. 2014). On the other hand, larger, stable blebs appear during cell necrosis (Barros et al. 2003). Mechanical-sensitive organelles were found to be involved in the blebbing behavior. For example, blebs originate from the delamination of the cell membrane from the cortex actin or fracture of the cortex actin, and then retract, driven by myosin II, after the actin cytoskeleton reassembled with the cell membrane (Charras et al. 2005, 2006, 2008; Paluch and Raz 2013). The cavitating microbubbles affect not only plasma membrane, but also submembrane structures. Our group also found that the actin cytoskeleton was disrupted on sonoporation by collapsing microbubbles (Jia 2018a, 2018b), further suggesting that the disrupted actin cytoskeleton at the perforation site may nucleate localized blebs. Moreover, a previous study found that dynamic blebs represent the mechanically wounded cells’ attempt to escape death (Babiychuk et al. 2011). However, the specific role of blebbing in the sonoporated cells still remains unclear. On the basis of these observations, we hypothesize that dynamic blebbing may be involved in the recovery of the sonoporated cells. To test this hypothesis, the aim of the present study was to determine the spatiotemporal dynamics of and mechanism underlying blebs in sonoporated cells and its relationship with reversible sonoporation by ultrasound-driven microbubbles.
We established an experimental platform for ultrasound exposure and real-time microscopic imaging at the single-cell level. Localized membrane perforation was induced by a single cavitating microbubble and different fluorescence probes were used to visualize membrane perforation, activities of the lipid components and spatiotemporal changes of lysosomes and the actin cytoskeleton. We then statistically determined the spatiotemporal relationship between blebs and the sonoporated cell and the underlying mechanism responsible for the spatiotemporal dynamics of bleb.
Section snippets
Cell culture and microbubble preparation
HeLa cervical cancer cells (CCL-2; ATCC, Manassas, VA, USA) were selected as a model to perform experiments. There have previously been used to investigate ultrasound-mediated bio-effects by our group and others (Fan et al. 2010; Qin et al. 2016; Jia et al. 2018). Dulbecco's Modified Eagle's Medium (Hyclone, Thermo Scientific Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco 10099; Invitrogen, Carlsbad, CA, USA) was used to culture HeLa cells at 37°C and 5% CO2. One day before
Spatiotemporal dynamics of localized blebs in reversibly sonoporated cells
Thirty-eight reversibly perforated cells induced by cavitating microbubbles were observed to determine the cellular responses. As a representative example, Figure 2a illustrates the spatiotemporal dynamics of blebbing in sonoporated cells. At 0 s (Fig. 2a), a single microbubble was adjacent to cell 1, and no microbubble was around cell 2. After the microbubble rapidly collapsed with the 13.33-μs ultrasonic pulse, notable red fluorescence was observed at the site of the microbubble (Fig. 2b; 6
Localized blebs specifically involved in the reversibly sonoporated cells
In the reversibly sonoporated cells, we found that quasi-hemispherical protrusions emerged at the sites of membrane perforation and then quickly expanded, forming localized blebs. After the blebs maintained their morphology for a relatively short time (tens of seconds to minutes), they slowly retracted and completely disappeared. Although the site of the blebs spatially coincided with those of the membrane perforation, the four successive dynamic phases (nucleation, expansion, pausing and
Conclusions
Dynamic blebs were found to be specifically associated with the reversibly sonoporated cells. These blebs occurred at the site of perforation, and their maximal diameter (about several microns) and lifetime (about tens of seconds to minutes) positively correlated with the degree of membrane perforation; these blebs persisted beyond the time required to reseal the membrane (about tens of seconds to minutes). By contrast, larger, stable blebs appeared in the irreversibly sonoporated cells. Our
Acknowledgments
This research is funded by the National Natural Science Foundation of China (Nos. 12074255 and 31630007), Program of Medicine and Engineering Cross Fund of Shanghai Jiao Tong University (Nos. YG2019ZDA27, ZH2018QNA21) and SMC Rising Star Fund of Shanghai Jiao Tong University (No. 16X100080028).
Conflict of interest disclosure
The authors have no conflicts of interest to declare.
References (55)
- et al.
High-resolution imaging of intracellular calcium fluctuations caused by oscillating microbubbles
Ultrasound Med Biol
(2020) - et al.
Life and times of a cellular bleb
Biophys J
(2008) - et al.
Effects of ultrasound on apoptosis induced by anti-CD20 antibody in CD20-positive B lymphoma cells
Ultrason Sonochem
(2008) - et al.
Ultrasound and microbubble mediated drug delivery: Acoustic pressure as determinant for uptake via membrane pores or endocytosis
J Control Release
(2015) - et al.
Ultrasound-induced cell membrane porosity
Ultrasound Med Biol
(2004) - et al.
Dealing with damage: Plasma membrane repair mechanisms
Biochimie
(2014) - et al.
Intracellular delivery and calcium transients generated in sonoporation facilitated by microbubbles
J Control Release
(2010) - et al.
Self-assembled liposome-loaded microbubbles: The missing link for safe and efficient ultrasound triggered drug-delivery
J Control Release
(2011) - et al.
The role of Ca2+ in ultrasound-elicited bioeffects: Progress, perspectives and prospects
Drug Discov Today
(2010) - et al.
Membrane perforation and recovery dynamics in microbubble-mediated sonoporation
Ultrasound Med Biol
(2013)
New insights in the actin cytoskeleton dynamics of the sonoporated human umbilical vein endothelia
Generation of reactive oxygen species in heterogeneously sonoporated cells by microbubbles with single-pulse ultrasound
Ultrasound Med Biol
Acoustic behavior of microbubbles and implications for drug delivery
Adv Drug Deliv Rev
Understanding ultrasound induced sonoporation: Definitions and underlying mechanisms
Adv Drug Deliv Rev
Effect of acoustic parameters on the cavitation behavior of SonoVue microbubbles induced by pulsed ultrasound
Ultrason Sonochem
Plasma membrane poration induced by ultrasound exposure: Implication for drug delivery
J Control Release
Cell blebbing and membrane area homeostasis in spreading and retracting cells
Biophys J
The role and regulation of blebs in cell migration
Curr Opin Cell Biol
Sonoporation-induced depolarization of plasma membrane potential: Analysis of heterogeneous impact
Ultrasound Med Biol
Effect of non-acoustic parameters on heterogeneous sonoporation mediated by single-pulse ultrasound and microbubbles
Ultrason Sonochem
Mechanistic understanding the bioeffects of ultrasound-driven microbubbles to enhance macromolecule delivery
J Control Release
The correlation between acoustic cavitation and sonoporation involved in ultrasound-mediated DNA transfection with polyethylenimine (PEI) in vitro
J Control Release
Plasma membrane repair is mediated by Ca2+-regulated exocytosis of lysosomes
Cell
Response of contrast agents to ultrasound
Adv Drug Deliver Rev
Real-time detection of intracellular reactive oxygen species and mitochondrial membrane potential in THP-1 macrophages during ultrasonic irradiation for optimal sonodynamic therapy
Ultrasonics Sonochem
Viability of endothelial cells after ultrasound-mediated sonoporation: Influence of targeting, oscillation, and displacement of microbubbles
J Control Release
Experimental study on cell self-sealing during sonoporation
J Control Release
Cited by (12)
Ultrasound-responsive microbubbles and nanodroplets: A pathway to targeted drug delivery
2024, Advanced Drug Delivery ReviewsBarrier-breaking effects of ultrasonic cavitation for drug delivery and biomarker release
2023, Ultrasonics SonochemistryFaster calcium recovery and membrane resealing in repeated sonoporation for delivery improvement
2022, Journal of Controlled ReleaseCitation Excerpt :The three-dimensional positioner was adjusted to achieve an 8-mm distance (in the far acoustic field of the transducer) along the acoustic axis between the surface of the transducer and the exposed region of interest. Further details are described in our previous reports [15,23]. A 1.5-MHz and 13.33-μs single-pulse sinusoidal signal with a fixed voltage amplitude was output by an arbitrary waveform generator (33120A, Agilent, Palo Alto, CA, USA) and then amplified by a radio-frequency amplifier (2100 L, Electronics & Innovation, Rochester, NY, USA) to drive the transducer.
Sonoporation of Immune Cells: Heterogeneous Impact on Lymphocytes, Monocytes and Granulocytes
2022, Ultrasound in Medicine and BiologyCitation Excerpt :MB-US exposure may also induce localized hemorrhage (Dalecki et al. 1999; Feril and Kondo 2004), microvascular leakage (Izadifar et al. 2017) and other potential adverse bio-effects (Shankar et al. 2011). With respect to the cell morphology, significant size reduction (Chen et al. 2013) and membrane blebbing (Leow et al. 2015; Jia et al. 2021) have been observed in vitro after MB-US exposure. These multifaceted effects should be taken into account when devising sonoporation-based immunomodulation treatments.
Spatiotemporal Dynamics and Mechanisms of Actin Cytoskeletal Re-modeling in Cells Perforated by Ultrasound-Driven Microbubbles
2022, Ultrasound in Medicine and BiologyCitation Excerpt :Because of the lipophilicity of the lipid-shelled microbubbles, they could passively contact the cells through buoyancy. More details for microbubble-cell contact were reported in our previous articles (Jia et al. 2018, 2021). To minimize the influence of multiple acoustic and non-acoustic parameters on the degree of sonoporation, a single cell with fewer than three microbubbles was exposed to single-pulse ultrasound (Qin et al. 2016).
Improving temporal stability of stable cavitation activity of circulating microbubbles using a closed-loop controller based on pulse-length regulation
2022, Ultrasonics SonochemistryCitation Excerpt :Furthermore, to achieve controllable bioeffects and minimize any undesirable results, some specific bioeffects induced by SC, after being quantitated using advanced imaging technologies, can act as state observers and be combined with the proposed controller to regulate the PL of the incident acoustic waves. For example, in sonoporation-based therapy, certain biomarkers (e.g., calcein) could be used as sonoporation tracers [39–41]. The intracellular calcein determined via real-time imaging could act as a state observer to regulate the emitted PL, thus producing the desired bioeffects.