RESEARCH PAPER Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells Ruth Röder 1 & Jonas Helma 2 & Tobias Preiß 3 & Joachim O. Rädler 3 & Heinrich Leonhardt 2 & Ernst Wagner 1 Received: 1 August 2016 /Accepted: 10 October 2016 # Springer Science+Business Media New York 2016 ABSTRACT Purpose Cytosolic delivery of nanobodies for molecular tar- get binding and fluorescent labeling in living cells. Methods 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 cy- tometry and fluorescence microscopy. Nanoparticle formation was analyzed by fluorescence correlation spectroscopy. Results 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 nanopar- ticles 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. Conclusion Two sequence-defined oligoaminoamides with or without folate for receptor targeting were identified as ef- fective 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. KEY WORDS folate . nanobody . oligoaminoamides . protein delivery . receptor targeting ABBREVIATIONS FCS Fluorescence correlation spectroscopy FolA Folic acid HcAb Heavy-chain only camelid antibody Nb Nanobody PEG Polyethyleneglycol Stp Succinoyl tetraethylene pentamine INTRODUCTION Nanobodies are single domain- antibody fragments (VHH) derived from heavy-chain only camelid antibodies (HcAb) ( 1 ). Especially with regard to their possible use as biopharmaceuticals (2,3) or imaging tools (4–6), nanobodies have significant advantages compared to standard antibodies. They can be easily screened for affinity and specificity; due to their compact, single domain structure, they stay chemically active in the reducing environment of the cell; they can be produced in prokaryotic systems in high yield and be easily chemically and genetically modified (7–9). Additionally, due to their small size (15 kDa), nanobodies can better diffuse through tissues or also intracellularly across nuclear pores, and also bind and inhibit targets such as enzymes which are addressed by standard antibodies to a far lesser extend (10,11). Potential intracellular molecular targets can be accessed in fixed and permeabilized cells only. Intracellular protein delivery, also called Bprotein transduction^ (12) may expand possible Electronic supplementary material The online version of this article (doi:10.1007/s11095-016-2052-8) contains supplementary material, which is available to authorized users. * Ernst Wagner ernst.wagner@cup.uni-muenchen.de 1 Pharmaceutical Biotechnology, Center for System-Based Drug Research, and Center for Nanoscience (CeNS), Ludwig-Maximilians-Universität München, Butenandtstraße 5, 81377 Munich, Germany 2 Department of Biology II, Center for Integrated Protein Science Munich, Ludwig-Maximilians-Universität München, Munich, Germany 3 Faculty of Physics and Center for NanoScience, Ludwig-Maximilians-Universität München, Geschwister-Scholl-Platz 1, D-80539 Munich, Germany Pharm Res DOI 10.1007/s11095-016-2052-8