title: Dynamic Fluorescence Microscopy of Cellular Uptake of Intercalating Model Drugs by Ultrasound-Activated Microbubbles.
abstract: The combination of ultrasound and microbubbles can facilitate cellular uptake of (model) drugs via transient permeabilization of the cell membrane. By using fluorescent molecules, this process can be studied conveniently with confocal fluorescence microscopy. This study aimed to investigate the relation between cellular uptake and fluorescence intensity increase of intercalating model drugs.
  
  
  
  SYTOX Green, an intercalating fluorescent dye that displays >500-fold fluorescence enhancement upon binding to nucleic acids, was used as a model drug for ultrasound-induced cellular uptake. SYTOX Green uptake was monitored in high spatiotemporal resolution to qualitatively assess the relation between uptake and fluorescence intensity in individual cells. In addition, the kinetics of fluorescence enhancement were studied as a function of experimental parameters, in particular, laser duty cycle (DC), SYTOX Green concentration and cell line.
  
  
  
  Ultrasound-induced intracellular SYTOX Green uptake resulted in local fluorescence enhancement, spreading throughout the cell and ultimately accumulating in the nucleus during the 9-min acquisition. The temporal evolution of SYTOX Green fluorescence was substantially influenced by laser duty cycle: continuous laser (100 % DC) induced a 6.4-fold higher photobleaching compared to pulsed laser (3.3 % DC), thus overestimating the fluorescence kinetics. A positive correlation of fluorescence kinetics and SYTOX Green concentration was found, increasing from 0.6 × 10-3to 2.2 × 10-3s-1for 1 and 20 μM, respectively. Finally, C6 cells displayed a 2.4-fold higher fluorescence rate constant than FaDu cells.
  
  
  
  These data show that the temporal behavior of intracellular SYTOX Green fluorescence enhancement depends substantially on nuclear accumulation and not just on cellular uptake. In addition, it is strongly influenced by the experimental conditions, such as the laser duty cycle, SYTOX Green concentration, and cell line.
authors.affiliation: Imaging Division, UMC Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
authors.forename: B H A
authors.initials: BHA
authors.lastname: Lammertink
authors.affiliation: Imaging Division, UMC Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
authors.forename: R
authors.initials: R
authors.lastname: Deckers
authors.affiliation: Imaging Division, UMC Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Derieppe
authors.affiliation: Department of Pharmaceutics, Ghent University, Ghent, Belgium.
authors.forename: I
authors.initials: I
authors.lastname: De Cock
authors.affiliation: Department of Pharmaceutics, Ghent University, Ghent, Belgium.
authors.forename: I
authors.initials: I
authors.lastname: Lentacker
authors.affiliation: Pharmaceutics Department, Utrecht University, Utrecht, The Netherlands.|Targeted Therapeutics, MIRA Institute, University of Twente, Enschede, The Netherlands.
authors.forename: G
authors.initials: G
authors.lastname: Storm
authors.affiliation: Imaging Division, UMC Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
authors.forename: C T W
authors.initials: CTW
authors.lastname: Moonen
authors.affiliation: Imaging Division, UMC Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands. c.bos@umcutrecht.nl.
authors.forename: C
authors.initials: C
authors.lastname: Bos
mesh_terms: D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D002470:Cell Survival; D006801:Humans; D007364:Intercalating Agents / Q000378:metabolism*; D007700:Kinetics; D045423:Microbubbles; D008856:Microscopy, Fluorescence / Q000379:methods*; D009930:Organic Chemicals / Q000378:metabolism; D038761:Photobleaching; D012815:Signal Processing, Computer-Assisted; D014465:Ultrasonics
chemical_list: D007364:Intercalating Agents; D009930:Organic Chemicals; C402795:SYTOX Green
keywords: Confocal microscopy; Drug delivery; Fluorescence; Microbubbles; Model drug; Ultrasound