Effects of extracellular calcium on cell membrane resealing in sonoporation

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 Ca²⁺ 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 [Ca²⁺] (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 Ca²⁺ 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 [Ca²⁺] above 0.54 mM. The cells resealed in 6-26 s at 1.8 mM Ca²⁺, but took longer at lower concentrations, up to 58-170 s at 0.54 mM Ca²⁺.