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.
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Fluorescence correlation spectroscopy
Heavy-chain only camelid antibody
Succinoyl tetraethylene pentamine
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ACKNOWLEDGMENTS AND DISCLOSURES
We thank Dr. Dongsheng He, Dr. Edith Salcher, Dr. Claudia Scholz and Philipp Klein for the synthesis of the different oligomers. We also thank Dr. Katharina von Gersdorff for the generation of the HeLa_Actin-GFP and HeLa_Tubulin-GFP cell lines. The German Research Foundation is gratefully acknowledged for financial support of related research by the authors within the Cluster of Excellence Nanosystems Initiative Munich (NIM). Prof. Dr. Heinrich Leonhardt also acknowledges the support by additional grants from the German Research Foundation (SPP1623; LE 721/13-1).
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Röder, R., Helma, J., Preiß, T. et al. Intracellular Delivery of Nanobodies for Imaging of Target Proteins in Live Cells. Pharm Res 34, 161–174 (2017). https://doi.org/10.1007/s11095-016-2052-8
- protein delivery
- receptor targeting