Plasma Membrane Blebbing Dynamics Involved in the Reversibly Perforated Cell by Ultrasound-Driven Microbubbles

Abstract

The perforation of plasma membrane by ultrasound-driven microbubbles (i.e., sonoporation) provides a temporary window for transporting macromolecules into the cytoplasm that is promising with respect to drug delivery and gene therapy. To improve the efficacy of delivery while ensuring biosafety, membrane resealing and cell recovery are required to help sonoporated cells defy membrane injury and regain their normal function. Blebs are found to accompany the recovery of sonoporated cells. However, the spatiotemporal characteristics of blebs and the underlying mechanisms remain unclear. With a customized platform for ultrasound exposure and 2-D/3-D live single-cell imaging, localized membrane perforation was induced with ultrasound-driven microbubbles, and the cellular responses were monitored using multiple fluorescent probes. The results indicated that localized blebs undergoing four phases (nucleation, expansion, pausing and retraction) on a time scale of tens of seconds to minutes were specifically involved in the reversibly sonoporated cells. The blebs spatially correlated with the membrane perforation site and temporally lagged (about tens of seconds to minutes) the resealing of perforated membrane. Their diameter (about several microns) and lifetime (about tens of seconds to minutes) positively correlated with the degree of sonoporation. Further studies revealed that intracellular calcium transients might be an upstream signal for triggering blebbing nucleation; exocytotic lysosomes not only contributed to resealing of the perforated membrane, but also to the increasing bleb volume during expansion; and actin components accumulation facilitated bleb retraction. These results provide new insight into the short-term strategies that the sonoporated cell employs to recover on membrane perforation and to remodel membrane structure and a biophysical foundation for sonoporation-based therapy.

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.