title: Sequence defined disulfide-linked shuttle for strongly enhanced intracellular protein delivery.
abstract: Intracellular protein transduction technology is opening the door for a promising alternative to gene therapy. Techniques have to address all critical steps, like efficient cell uptake, endolysosomal escape, low toxicity, while maintaining full functional activity of the delivered protein. Here, we present the use of a chemically precise, structure defined three-arm cationic oligomer carrier molecule for protein delivery. This carrier of exact and low molecular weight combines good cellular uptake with efficient endosomal escape and low toxicity. The protein cargo is covalently attached by a bioreversible disulfide linkage. Murine 3T3 fibroblasts could be transduced very efficiently with cargo nlsEGFP, which was tagged with a nuclear localization signal. We could show subcellular delivery of the nlsEGFP to the nucleus, confirming cytosolic delivery and expected subsequent subcellular trafficking. Transfection efficiency was concentration-dependent in a directly linear mode and 20-fold higher in comparison with HIV-TAT-nlsEGFP containing a functional TAT transduction domain. Furthermore, β-galactosidase as a model enzyme cargo, modified with the carrier oligomer, was transduced into neuroblastoma cells in enzymatically active form.
authors.affiliation: Pharmaceutical Biotechnology, Center for System-Based Drug Research, and Center for Nanoscience, Ludwig-Maximilians-University Munich , Butenandtstrasse 5-13, 81377 Munich, Germany.
authors.forename: Kevin
authors.initials: K
authors.lastname: Maier
authors.forename: Irene
authors.initials: I
authors.lastname: Martin
authors.forename: Ernst
authors.initials: E
authors.lastname: Wagner
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D003593:Cytoplasm / Q000378:metabolism*; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism*; D019913:Nuclear Localization Signals / Q000378:metabolism; D021381:Protein Transport; D001616:beta-Galactosidase / Q000378:metabolism*; D054322:tat Gene Products, Human Immunodeficiency Virus / Q000378:metabolism*
chemical_list: D004220:Disulfides; D019913:Nuclear Localization Signals; C485184:enhanced green fluorescent protein; D054322:tat Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins; D001616:beta-Galactosidase