2.4. Ultrasound Responsiveness of Regenerated Nanobubbles

To confirm that the nanoparticles present after lyophilization and dissolution were indeed US-responsive bubbles, their size distribution was measured before and after sonication using a method based on a previous report [36]. Briefly, the NB suspension (100 μL) was placed within an acoustically transparent film based 96 multi-well cell culture polystyrene plate (lumox multiwell; Sarstedt, Nümbrecht, NRW, DE). The culture plate was fixed above the surface of the US transducer using an acoustic transmission gel (Aquasonic 100 gel; Parker Lab, Fairfield, NJ, USA). US was applied using a sonoporator (SP100; Sonidel Limited, Dublin, Ireland) with a transducer (diameter 1.6 cm), driving frequency of 1 MHz, burst rate of 100 Hz, and duty ratio of 50% and intensity of 1.0, 2.0 or 5.0 W/cm² for 30 s (Figure 1A). The US irradiation method was identical to a previously described micro-scale in vitro sonoporation system using a 96 multi-well plate that included cultured cells. The diameters of the NBs were measured after sonication at various intensities (0, 1, 2, and 5 W/cm²) for 30 s. Changes in the number and distribution of NB particles before and after US irradiation were measured using NTA, flow cytometry (FCM), and RMM, as described above.

Schematic representation of sonication treatment of nanobubbles (NBs) or sonoporation in 96-well plates. (A) Sonication treatment of NBs by ultrasonic irradiation to medium. (B–F) Method of sonoporation. (B) The incubation medium is removed from the well (white arrow) of a 96 multi-well plate seeded with human squamous carcinoma (HSC-2) cells. (C) Each well is filled with medium containing NBs (white arrow). (D) Transfection by ultrasonic irradiation. mRNA is transferred into the cytoplasm (white arrow). (E-1) Aspiration of sonicated medium and (E-2) addition of new incubation medium. (F) After a 24 h incubation period, the supernatant was collected for a reporter assay. (G) Arrangement of wells of seeded cells (indicated with color) and ultrasonic irradiation area (inside of dashed circle) on a 96 multi-well plate.