title: Ultrasound and microbubble-targeted delivery of macromolecules is regulated by induction of endocytosis and pore formation.
abstract: Contrast microbubbles in combination with ultrasound (US) are promising vehicles for local drug and gene delivery. However, the exact mechanisms behind intracellular delivery of therapeutic compounds remain to be resolved. We hypothesized that endocytosis and pore formation are involved during US and microbubble targeted delivery (UMTD) of therapeutic compounds. Therefore, primary endothelial cells were subjected to UMTD of fluorescent dextrans (4.4 to 500 kDa) using 1 MHz pulsed US with 0.22-MPa peak-negative pressure, during 30 seconds. Fluorescence microscopy showed homogeneous distribution of 4.4- and 70-kDa dextrans through the cytosol, and localization of 155- and 500-kDa dextrans in distinct vesicles after UMTD. After ATP depletion, reduced uptake of 4.4-kDa dextran and no uptake of 500-kDa dextran was observed after UMTD. Independently inhibiting clathrin- and caveolae-mediated endocytosis, as well as macropinocytosis significantly decreased intracellular delivery of 4.4- to 500-kDa dextrans. Furthermore, 3D fluorescence microscopy demonstrated dextran vesicles (500 kDa) to colocalize with caveolin-1 and especially clathrin. Finally, after UMTD of dextran (500 kDa) into rat femoral artery endothelium in vivo, dextran molecules were again localized in vesicles that partially colocalized with caveolin-1 and clathrin. Together, these data indicated uptake of molecules via endocytosis after UMTD. In addition to triggering endocytosis, UMTD also evoked transient pore formation, as demonstrated by the influx of calcium ions and cellular release of preloaded dextrans after US and microbubble exposure. In conclusion, these data demonstrate that endocytosis is a key mechanism in UMTD besides transient pore formation, with the contribution of endocytosis being dependent on molecular size.
authors.affiliation: Department of Clinical Pharmacology, University Medical Center Groningen, University of Groningen, The Netherlands.
authors.forename: Bernadet D M
authors.initials: BD
authors.lastname: Meijering
authors.forename: Lynda J M
authors.initials: LJ
authors.lastname: Juffermans
authors.forename: Annemieke
authors.initials: A
authors.lastname: van Wamel
authors.forename: Rob H
authors.initials: RH
authors.lastname: Henning
authors.forename: Inge S
authors.initials: IS
authors.lastname: Zuhorn
authors.forename: Marcia
authors.initials: M
authors.lastname: Emmer
authors.forename: Amanda M G
authors.initials: AM
authors.lastname: Versteilen
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Paulus
authors.forename: Wiek H
authors.initials: WH
authors.lastname: van Gilst
authors.forename: Klazina
authors.initials: K
authors.lastname: Kooiman
authors.forename: Nico
authors.initials: N
authors.lastname: de Jong
authors.forename: René J P
authors.initials: RJ
authors.lastname: Musters
authors.forename: Leo E
authors.initials: LE
authors.lastname: Deelman
authors.forename: Otto
authors.initials: O
authors.lastname: Kamp
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D001692:Biological Transport; D002417:Cattle; D021941:Caveolae / Q000378:metabolism*; D051242:Caveolin 1 / Q000378:metabolism; D002478:Cells, Cultured; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000378:metabolism; D003287:Contrast Media / Q000008:administration & dosage; D003600:Cytosol / Q000378:metabolism; D003911:Dextrans / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism*; D016503:Drug Delivery Systems; D004705:Endocytosis / Q000187:drug effects; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism*; D005263:Femoral Artery / Q000378:metabolism; D005372:Filipin / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000737:chemistry / Q000378:metabolism*; D021621:Imaging, Three-Dimensional; D007262:Infusions, Intravenous; D045423:Microbubbles; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D010743:Phospholipids / Q000008:administration & dosage; D010873:Pinocytosis; D011312:Pressure; D051381:Rats; D017208:Rats, Wistar; D013459:Sulfur Hexafluoride / Q000008:administration & dosage; D013997:Time Factors; D022161:Transport Vesicles / Q000378:metabolism; D014465:Ultrasonics; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D051242:Caveolin 1; D002966:Clathrin; D003287:Contrast Media; D003911:Dextrans; D005456:Fluorescent Dyes; D010743:Phospholipids; C420843:contrast agent BR1; D005372:Filipin; D000255:Adenosine Triphosphate; D002746:Chlorpromazine; D013459:Sulfur Hexafluoride; D000077191:Wortmannin