From bubble dynamics to cellular responses: multi-scale imaging of acoustic droplet vaporization in three-dimensional fibrin hydrogels

Intracellular delivery of therapeutics is limited by the cell membrane. Acoustic droplet vaporization (ADV), the ultrasound-triggered conversion of perfluorocarbon droplets into bubbles, can permeabilize nearby cells, but the relationship between bubble mechanics and cellular responses in three-dimensional matrices is not well understood. Here, monodisperse perfluorohexane droplets (average radius: ≈ 6.35 µm) and normal human dermal fibroblasts were co-encapsulated in fibrin hydrogels, recapitulating a physiologically relevant tissue-like microenvironment, and exposed to a single ultrasound burst (2.5MHz, 6 cycles, 5 MPa peak negative pressure). Ultra-high-speed brightfield imaging at 10 million frames per second resolved bubble dynamics and cell-centroid displacement, and time-lapse confocal microscopy tracked intracellular calcium (Fluo-4) and propidium iodide (PI) uptake for 200 s. ADV bubbles reached a maximum expansion ratio of 4.7 ± 1.1 (maximum radius, R m a x ≈ 30 µm) and had near-circular projections at maximum expansion. Cell displacement and inferred matrix hoop strain decayed steeply with initial cell-to-droplet distance, d 0 . Comparison with incompressible spherical kinematics, evaluated per cell using its event’s maximum radius, gave root-mean-square errors of 4.4µm and 0.088, respectively. These estimates describe matrix deformation at cell locations rather than intracellular strain. Hoop strains exceeded 0.1 only within d 0 ≲ 60 µm ( ≈ 2 R m a x ) , and strain rates at the bubble wall reached ≈ 1 0 6 s −1 . PI uptake occurred predominantly within d 0 ≲ 35 µm ( ≈ R m a x ) , where inferred hoop strains were ≳ 0.2, whereas calcium responses extended to d 0 ≈ 110 µm ( ≈ 3.7 R m a x ) , well beyond the region of