title: Enhancing endosomal escape of transduced proteins by photochemical internalisation.
abstract: Induced internalisation of functional proteins into cultured cells has become an important aspect in a rising number of in vitro and in vivo assays. The endo-lysosomal entrapment of the transduced proteins remains the major problem in all transduction protocols. In this study we compared the efficiency, cytotoxicity and protein targeting of different commercially available transduction reagents by transducing a well-studied fluorescently labelled protein (Atto488-bovine serum albumin) into cultured human sarcoma cells. The amount of internalised protein and toxicity differed between the different reagents, but the percentage of transduced cells was consistently high. Furthermore, in all protocols the signals of the transduced Atto488-BSA were predominantly punctual consistent with an endosomal localisation. To overcome the endosomal entrapment, the transduction protocols were combined with a photochemical internalisation (PCI) treatment. Using this combination revealed that an endosomal disruption is highly effective in cell penetrating peptide (CPP) mediated transduction, whereas lipid-mediated transductions lead to a lower signal spreading throughout the cytosol. No change in the signal distribution could be achieved in treatments using non-lipid polymers as a transduction reagent. Therefore, the combination of protein transduction protocols based on CPPs with the endosomolytic treatment PCI can facilitate protein transduction experiments in vitro.
authors.affiliation: Institute of Human Genetics, University of Ulm, Ulm, Germany. kevin.mellert@uni-ulm.de
authors.forename: Kevin
authors.initials: K
authors.lastname: Mellert
authors.forename: Markus
authors.initials: M
authors.lastname: Lamla
authors.forename: Klaus
authors.initials: K
authors.lastname: Scheffzek
authors.forename: Rainer
authors.initials: R
authors.lastname: Wittig
authors.forename: Dieter
authors.initials: D
authors.lastname: Kaufmann
mesh_terms: D000818:Animals; D002417:Cattle; D016923:Cell Death; D045744:Cell Line, Tumor; D002675:Child, Preschool; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000378:metabolism; D055668:Photochemical Processes; D012710:Serum Albumin, Bovine / Q000378:metabolism*; D012815:Signal Processing, Computer-Assisted; D014161:Transduction, Genetic; D055815:Young Adult
chemical_list: D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D012710:Serum Albumin, Bovine