title: Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells.
abstract: Cytosolic delivery of nanobodies for molecular target binding and fluorescent labeling in living cells.
  
  
  
  Fluorescently labeled nanobodies were formulated with sixteen different sequence-defined oligoaminoamides. The delivery of formulated anti-GFP nanobodies into different target protein-containing HeLa cell lines was investigated by flow cytometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy.
  
  
  
  The initial oligomer screen identified two cationizable four-arm structured oligomers (734, 735) which mediate intracellular nanobody delivery in a receptor-independent (734) or folate receptor facilitated (735) process. The presence of disulfide-forming cysteines in the oligomers was found critical for the formation of stable protein nanoparticles of around 20 nm diameter. Delivery of labeled GFP nanobodies or lamin nanobodies to their cellular targets was demonstrated by fluorescence microscopy including time lapse studies.
  
  
  
  Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as effective carriers for intracellular nanobody delivery, as exemplified by GFP or lamin binding in living cells. Due to the conserved nanobody core structure, the methods should be applicable for a broad range of nanobodies directed to different intracellular targets.
authors: Pharmaceutical Biotechnology, Center for System-Based Drug Research, and Center for Nanoscience (CeNS), Ludwig-Maximilians-Universität München, Butenandtstraße 5, 81377, Munich, Germany.
authors: Ruth
authors: R
authors: Röder
authors: Department of Biology II, Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Munich, Germany.
authors: Jonas
authors: J
authors: Helma
authors: Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-Universität München, Geschwister-Scholl-Platz 1, D-80539, Munich, Germany.
authors: Tobias
authors: T
authors: Preiß
authors: Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-Universität München, Geschwister-Scholl-Platz 1, D-80539, Munich, Germany.
authors: Joachim O
authors: JO
authors: Rädler
authors: Department of Biology II, Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Munich, Germany.
authors: Heinrich
authors: H
authors: Leonhardt
authors: Pharmaceutical Biotechnology, Center for System-Based Drug Research, and Center for Nanoscience (CeNS), Ludwig-Maximilians-Universität München, Butenandtstraße 5, 81377, Munich, Germany. ernst.wagner@cup.uni-muenchen.de.
authors: Ernst
authors: E
authors: Wagner
mesh_terms: D002460:Cell Line; D045744:Cell Line, Tumor; D002626:Chemistry, Pharmaceutical / Q000379:methods; D003593:Cytoplasm / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D005456:Fluorescent Dyes / Q000008:administration & dosage / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007624:KB Cells; D053758:Nanoparticles / Q000008:administration & dosage* / Q000378:metabolism; D021381:Protein Transport; D011506:Proteins / Q000008:administration & dosage* / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000008:administration & dosage* / Q000378:metabolism
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D061905:Single-Domain Antibodies
keywords: folate; nanobody; oligoaminoamides; protein delivery; receptor targeting