High Efficiency Molecular Delivery with Sequential Low-Energy Sonoporation Bursts

Abstract

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.

An evaluation of the sonoporation potential of low-boiling point phase-change ultrasound contrast agents in vitro

“…The peak sonoporation efficiency we achieved (36%) is similar to what has previously been reported for microbubble sonoporation (28–39% efficiency) [6, 30, 31], albeit with lower cell viability (70% viability for PCCA sonoporation vs. 90–96% viability for MB sonoporation [6, 30, 31]). However, these microbubble sonoporation studies employ unique strategies to increase sonoporation efficiency and minimize cell death, making it difficult to make direct comparisons.…”

“…For example, McLaughlan et al achieved their highest viable sonoporation using a combination of (1) targeted microbubbles that increase cell-microbubble interactions and (2) chirp frequency excitation to maximize the response of their polydisperse microbubbles [6]. Song et al found that using monodisperse 2.0-μm microbubbles resulted in the highest sonoporation and viability after a single ultrasound treatment [31]. We believe that with further optimization of our PCCA-mediated sonoporation methods, we will be able to match the sonoporation efficiencies and viabilities achieved with microbubbles.…”

The Role of Ultrasound-Driven Microbubble Dynamics in Drug Delivery: From Microbubble Fundamentals to Clinical Translation

“…Larger shear stress have been shown to correlate with higher propidium iodide uptake, which shows that the pores either stay open longer or are larger. 27 Likewise, larger holes 129 , higher sonoporation efficiency 40 and spatiotemporal control over pore formation 106 can be achieved by using two successive sonoporation pulses with a different pressure.…”

Synergistic Antifungal Effect of Amphotericin B-Loaded Poly(Lactic-Co-Glycolic Acid) Nanoparticles and Ultrasound against Candida albicans Biofilms

“…During biofilm formation, surface-adherent extracellular matrices of the biofilm act as a barrier to antibiotic diffusion, which limits the exposure of intracellular bacteria in the biofilms to superficial antibiotics. Ultrasound (US) has been widely acknowledged as a promising approach to overcome these obstacles by enabling increased permeability for gene/drug delivery (13,14). Ultrasound exposure induces sonoporation, which may transiently disrupt the cell membrane and increase the permeability of the membrane to promote transportation of membrane-impermeable agents, such as nanoparticles or antibodies, into cells or tissues in preclinical studies.…”