Role of Cell Culture Scaffold Stiffness on Sonoporation Efficiency

Objective Sonoporation employs ultrasound-driven microbubble oscillations to permeabilize cell membranes, offering the potential for intracellular drug delivery. However, its clinical adaption remains limited, primarily due to an incomplete understanding of the mechanisms through which oscillating bubbles disrupt cell membranes. Most mechanistic sonoporation studies have focused on cell monolayers cultured on rigid plastic scaffolds. Methods Here we investigated the influence of scaffold stiffness on sonoporation outcome using simultaneous ultra-high-speed imaging (10 million frames/s) to capture bubble dynamics and high-resolution confocal microscopy to assess cell membrane response and model drug uptake. Monodisperse 2.3 μm radius microbubbles were used to sonoporate single human umbilical vein endothelial cells cultured on either a soft hydrogel scaffold or a rigid polymer membrane. Ultrasound driving frequency and acoustic pressure amplitude were varied, while the pulse length was fixed at 15 cycles. Results Our results show that the slope of sonoporation efficiency versus microbubble radial excursion curve decreased by a factor of 30 when using the soft scaffold versus the rigid one, despite no apparent differences in microbubble dynamics. Furthermore, the nearly identical sonoporation efficiency versus radial excursion curves across the employed ultrasound frequencies of 0.5, 1.0 and 2.0 MHz suggest that acoustic radiation forces and normal or shear stresses are unlikely to be the primary mechanisms driving the observed differences. Additionally, membrane pore size increased with frequency for the rigid scaffold but decreased for the soft scaffold. Conclusion Our findings highlight the critical role of mechanical scaffold properties in determining sonoporation outcomes, where softer scaffolds result in reduced membrane disruption and altered pore formation dynamics despite unchanged bubble oscillation behavior.