Microbubbles interact with ultrasound to induce transient microscopic pores in the cellular plasma membrane in a highly localized thermo-mechanical process called sonoporation. Theranostic applications of in vitro sonoporation include molecular delivery (e.g., transfection, drug loading and cell labeling), as well as molecular extraction for measuring intracellular biomarkers, such as proteins and mRNA. Prior research focusing mainly on the effects of acoustic forcing with polydisperse microbubbles has identified a “soft limit” of sonoporation efficiency at 50% when including dead and lysed cells. We show here that this limit can be exceeded with the judicious use of monodisperse microbubbles driven by a physiotherapy device (1.0 MHz, 2.0 W/cm2, 10% duty cycle). We first examined the effects of microbubble size and found that small-diameter microbubbles (2 µm) deliver more instantaneous power than larger microbubbles (4 & 6 µm). However, owing to rapid fragmentation and a short half-life (0.7 s for 2 µm; 13.3 s for 6 µm), they also deliver less energy over the sonoporation time. This translates to a higher ratio of FITC-dextran (70 kDa) uptake to cell death/lysis (4:1 for 2 µm; 1:2 for 6 µm) in suspended HeLa cells after a single sonoporation. Sequential sonoporations (up to four) were consequently employed to increase molecular delivery. Peak uptake was found to be 66.1 ± 1.2% (n=3) after two sonoporations when properly accounting for cell lysis (7.0 ± 5.6%) and death (17.9 ± 2.0%), thus overcoming the previously reported soft limit. Substitution of TRITC-dextran (70 kDa) on the second sonoporation confirmed the effects were multiplicative. Overall, this study demonstrates the possibility of utilizing monodisperse small-diameter microbubbles as a means to achieve multiple low-energy sonoporation bursts for efficient in vitro cellular uptake and sequential molecular delivery.

Ultrasound contrast agents (UCAs) are shell encapsulated microbubbles developed originally for ultrasound imaging enhancement. More recently, UCAs are being exploited for therapeutic applications such as drug and gene delivery. Ultrasound transducer pulses can induce spherical (radial) UCA oscillations, translation, and nonspherical shape oscillations, the latter of which can lead to breakup. Breakup can facilitate drug or gene delivery, but should be minimized for imaging purposes to increase residence time and maximize diagnostic effect. Therefore, an understanding of the interplay between the acoustic driving and shape mode stability of UCAs is important for both diagnostic and therapeutic applications. The present work couples a radial model of a lipid-coated microbubble with a model for bubble translation and nonspherical shape oscillation to predict shape mode stability for ultrasound driving frequencies and pressure amplitudes of clinical interest. In addition, calculations of the stability of individual shape modes, residence time, maximum radius, and translation are provided with respect to acoustic driving parameters and compared to an unshelled bubble. The effects of shell elasticity, shell viscosity, and initial radius on stability are investigated. The results show greater stability at higher values of shell elasticity and viscosity and at smaller radius, and provide guidance for optimizing shell design and ultrasound driving parameters with respect to shape stability.

Ultrasound contrast agents (UCAs) are shell encapsulated, gas-filled microbubbles developed originally for ultrasound imaging enhancement. UCAs are approximately 1–10 micrometers in diameter with a shell typically comprised of lipid, protein, or polymer. When injected into the bloodstream, the high compressibility of these microbubbles, relative to the surrounding blood and tissue, and their highly nonlinear response to ultrasound, leads to strong enhancement of the blood-tissue contrast in the resulting ultrasound image. While UCAs have been commercially available since the early 1990’s [1] for ultrasound imaging, they are more recently being exploited for therapeutic applications, for example, as vehicles for drug delivery and gene therapy, and thermal and mechanical tissue ablation. The effectiveness of UCAs in therapeutic applications depends strongly on the nonspherical character of the bubble oscillation, which can effect the breakup and release of therapeutic agents from the UCA, as well as the formation of high-speed jets near the tissue interface. In this work, two different models for nonspherical oscillation of UCAs are presented: one for small shape oscillations of a lipid-coated bubble, and one for large nonspherical oscillations of a polymer-coated bubble. Nonspherical shape mode stability and dynamics are investigated with each model for ranges of ultrasonic frequency and amplitude relevant to medical applications.