title: Viability of endothelial cells after ultrasound-mediated sonoporation: Influence of targeting, oscillation, and displacement of microbubbles.
abstract: Microbubbles (MBs) have been shown to create transient or lethal pores in cell membranes under the influence of ultrasound, known as ultrasound-mediated sonoporation. Several studies have reported enhanced drug delivery or local cell death induced by MBs that are either targeted to a specific biomarker (targeted microbubbles, tMBs) or that are not targeted (non-targeted microbubbles, ntMBs). However, both the exact mechanism and the optimal acoustic settings for sonoporation are still unknown. In this study we used real-time uptake patterns of propidium iodide, a fluorescent cell impermeable model drug, as a measure for sonoporation. Combined with high-speed optical recordings of MB displacement and ultra-high-speed recordings of MB oscillation, we aimed to identify differences in MB behavior responsible for either viable sonoporation or cell death. We compared ntMBs and tMBs with identical shell compositions exposed to long acoustic pulses (500-50,000cycles) at various pressures (150-500kPa). Propidium iodide uptake highly correlated with cell viability; when the fluorescence intensity still increased 120s after opening of the pore, this resulted in cell death. Higher acoustic pressures and longer cycles resulted in more displacing MBs and enhanced sonoporation. Non-displacing MBs were found to be the main contributor to cell death, while displacement of tMBs enhanced reversible sonoporation and preserved cell viability. Consequently, each therapeutic application requires different settings: non-displacing ntMBs or tMBs are advantageous for therapies requiring cell death, especially at 500kPa and 50,000cycles, whereas short acoustic pulses causing limited displacement should be used for drug delivery.
authors.affiliation: Department of Biomedical Engineering, Thorax Center, Erasmus MC, Room Ee2302, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands. Electronic address: tvrooij@gmail.com.
authors.forename: Tom
authors.initials: T
authors.lastname: van Rooij
authors.affiliation: Department of Biomedical Engineering, Thorax Center, Erasmus MC, Room Ee2302, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands. Electronic address: i.v.skachkov@gmail.com.
authors.forename: Ilya
authors.initials: I
authors.lastname: Skachkov
authors.affiliation: Department of Biomedical Engineering, Thorax Center, Erasmus MC, Room Ee2302, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands. Electronic address: d.beekers@erasmusmc.nl.
authors.forename: Inés
authors.initials: I
authors.lastname: Beekers
authors.affiliation: Department of Biomedical Engineering, Thorax Center, Erasmus MC, Room Ee2302, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands. Electronic address: k.lattwein@erasmusmc.nl.
authors.forename: Kirby R
authors.initials: KR
authors.lastname: Lattwein
authors.affiliation: Department of Biomedical Engineering, Thorax Center, Erasmus MC, Room Ee2302, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands. Electronic address: j.voorneveld@erasmusmc.nl.
authors.forename: Jason D
authors.initials: JD
authors.lastname: Voorneveld
authors.affiliation: Department of Biomedical Engineering, Thorax Center, Erasmus MC, Room Ee2302, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands. Electronic address: tjakokhuis@hotmail.com.
authors.forename: Tom J A
authors.initials: TJA
authors.lastname: Kokhuis
authors.affiliation: Department of Biomedical Engineering, Thorax Center, Erasmus MC, Room Ee2302, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands. Electronic address: d.bera@erasmusmc.nl.
authors.forename: Deep
authors.initials: D
authors.lastname: Bera
authors.affiliation: Department of Biomedical Engineering, Thorax Center, Erasmus MC, Room Ee2302, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands. Electronic address: ying.luan@kcl.ac.uk.
authors.forename: Ying
authors.initials: Y
authors.lastname: Luan
authors.affiliation: Department of Biomedical Engineering, Thorax Center, Erasmus MC, Room Ee2302, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands; Laboratory of Acoustical Wavefield Imaging, Faculty of Applied Sciences, Delft University of Technology, Building 22, Room B22-D218, Lorentzweg 1, 2628 CJ Delft, the Netherlands. Electronic address: a.vandersteen@erasmusmc.nl.
authors.forename: Antonius F W
authors.initials: AFW
authors.lastname: van der Steen
authors.affiliation: Department of Biomedical Engineering, Thorax Center, Erasmus MC, Room Ee2302, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands; Laboratory of Acoustical Wavefield Imaging, Faculty of Applied Sciences, Delft University of Technology, Building 22, Room B22-D218, Lorentzweg 1, 2628 CJ Delft, the Netherlands. Electronic address: n.dejong@erasmusmc.nl.
authors.forename: Nico
authors.initials: N
authors.lastname: de Jong
authors.affiliation: Department of Biomedical Engineering, Thorax Center, Erasmus MC, Room Ee2302, P.O. Box 2040, 3000 CA Rotterdam, the Netherlands. Electronic address: k.kooiman@erasmusmc.nl.
authors.forename: Klazina
authors.initials: K
authors.lastname: Kooiman
mesh_terms: D016923:Cell Death; D002470:Cell Survival; D003287:Contrast Media / Q000009:adverse effects; D016503:Drug Delivery Systems / Q000009:adverse effects / Q000379:methods*; D042783:Endothelial Cells / Q000166:cytology*; D005456:Fluorescent Dyes / Q000008:administration & dosage; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D045423:Microbubbles / Q000009:adverse effects; D011419:Propidium / Q000008:administration & dosage; D013010:Sonication / Q000009:adverse effects / Q000379:methods*
chemical_list: D003287:Contrast Media; D005456:Fluorescent Dyes; D011419:Propidium
keywords: Drug delivery; Endothelial cells; Microbubble behavior; Sonoporation; Therapy; Ultrasound contrast agents