title: 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/cm(2), 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.
authors.affiliation: 1. Department of Mechanical Engineering, University of Colorado, Boulder, CO 80309.
authors.forename: Kang-Ho
authors.initials: KH
authors.lastname: Song
authors.affiliation: 1. Department of Mechanical Engineering, University of Colorado, Boulder, CO 80309.
authors.forename: Alexander C
authors.initials: AC
authors.lastname: Fan
authors.affiliation: 2. Department of Mechanical and Aerospace Engineering, University of Colorado, Colorado Springs, CO 80918.
authors.forename: John T
authors.initials: JT
authors.lastname: Brlansky
authors.affiliation: 3. Department of Medicine, University of Colorado Anschutz Medical Center, Aurora, CO 80045.
authors.forename: Tammy
authors.initials: T
authors.lastname: Trudeau
authors.affiliation: 3. Department of Medicine, University of Colorado Anschutz Medical Center, Aurora, CO 80045.
authors.forename: Arthur
authors.initials: A
authors.lastname: Gutierrez-Hartmann
authors.affiliation: 2. Department of Mechanical and Aerospace Engineering, University of Colorado, Colorado Springs, CO 80918.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Calvisi
authors.affiliation: 1. Department of Mechanical Engineering, University of Colorado, Boulder, CO 80309.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Borden
mesh_terms: D003911:Dextrans / Q000032:analysis / Q000493:pharmacokinetics; D016503:Drug Delivery Systems / Q000379:methods*; D004847:Epithelial Cells / Q000187:drug effects* / Q000528:radiation effects*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000032:analysis / Q000493:pharmacokinetics; D006367:HeLa Cells; D006801:Humans; D045423:Microbubbles; D012235:Rhodamines / Q000032:analysis / Q000493:pharmacokinetics; D014463:Ultrasonography / Q000379:methods*
chemical_list: D003911:Dextrans; D012235:Rhodamines; C015219:fluorescein isothiocyanate dextran; C009434:tetramethylrhodamine isothiocyanate; D016650:Fluorescein-5-isothiocyanate
keywords: cell uptake; cell viability; drug delivery; drug release; microbubbles; ultrasound contrast agents