Sonoporation by Single-Shot Pulsed Ultrasound with Microbubbles Adjacent to Cells

Abstract

In this article, membrane perforation of endothelial cells with attached microbubbles caused by exposure to single-shot short pulsed ultrasound is described, and the mechanisms of membrane damage and repair are discussed. Real-time optical observations of cell-bubble interaction during sonoporation and successive scanning electron microscope observations of the membrane damage with knowledge of bubble locations revealed production of micron-sized membrane perforations at the bubble locations. High-speed observations of the microbubbles visualized production of liquid microjets during nonuniform contraction of bubbles, indicating that the jets are responsible for cell membrane damage. The resealing process of sonoporated cells visualized using fluorescence microscopy suggested that Ca2+-independent and Ca2+-triggered resealing mechanisms were involved in the rapid resealing process. In an experimental condition in which almost all cells have one adjacent bubble, 25.4% of the cells were damaged by exposure to single-shot pulsed ultrasound, and 15.9% (approximately 60% of the damaged cells) were resealed within 5 s. These results demonstrate that single-shot pulsed ultrasound is sufficient to achieve sonoporation when microbubbles are attached to cells.

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

“…In our set-up, ultrasound was applied based on a single-shot pulsing protocol similar to that adopted in other cellular-level studies on sonoporation [13]. The applied pulse was a 30-cycle tone burst with 1 MHz wave frequency (i.e.…”

“…In fostering localized induction of sonoporation, one technical advance that has played an important role is the advent of targeted microbubbles with binding affinity to antigen receptors on the cell type of interest [8,9], such as vascular endothelial growth factor (VEGF) receptors expressed on cancerous cells [10] or hypoxic endothelial cells [11]. These targeted microbubbles, often composed of a perfluorocarbon gas core encapsulated within an antibody-conjugated shell material [12], are in effect artificial gas bodies that would cavitate in response to ultrasound excitation, which could be as short as a single pulse firing [13]. As their binding is known to persist under shear-flow conditions [14,15], they can effectively reside adjacent to the plasma membrane to locally induce membrane disruption over their course of acoustic cavitation [16,17] and concurrently release drugs which may be preloaded onto the microbubble shell [18].…”

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

“…By voltage clamp techniques, an increase in transmembrane current simultaneously with ultrasound application was reported, indicating ion flux through pores [10]. Moreover, several papers demonstrated the uptake of cell-impermeable molecules when exposing cells to ultrasound [11][12][13]. However, more recently, Meijering et al [14] stated that besides pore formation, enhanced endocytosis also contributes to ultrasound mediated delivery.…”

A study on sonoporation of cells cultured on a soft collagen gel scaffold

“…We have shown that sonoporation induces a temporal increase in cell membrane permeability by exposure to single-shot short-pulsed ultrasound with a microbubble adjacent to a cell [1][2][3][4]. This is a promising technique for safe sonoporation in vivo because short-pulsed ultrasound, which is mainly used for diagnostic ultrasound, is safer than continuous-wave or long-burst ultrasound, which is mainly used for ultrasound therapies.…”