title: A bioanalytical assay to distinguish cellular uptake routes for liposomes.
abstract: Lipid-based nanoparticles are frequently used for drug or DNA delivery into mammalian cells. However it is difficult to determine whether such particles are taken up via endocytosis or fusion to the plasma membrane. Here, we propose a simple and reliable analytical method to do so based on the unique spectral properties of the fluorescent tracer BODIPY FL. At high local concentrations, this dye displays an additional red-shifted emission peak that is absent at low concentrations. In dye-loaded liposomes taken up by endocytosis, the local dye concentration did not significantly change upon internalization. Accordingly, unchanged fluorescence spectra were detected. When cells were incubated with liposomes able to fuse with the plasma membrane of mammalian cells, a reduction of local dye concentration and much weaker emission in the red-shifted peak were observed. The ratio of intensities in both fluorescence channels was shown to be a reliable indicator of the cellular uptake mechanism.
authors.affiliation: ICS-7: Biomechanics, Forschungszentrum Jülich GmbH, Institute of Complex Systems, Jülich, 52425, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Braun
authors.affiliation: ICS-7: Biomechanics, Forschungszentrum Jülich GmbH, Institute of Complex Systems, Jülich, 52425, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Kleusch
authors.affiliation: ICS-7: Biomechanics, Forschungszentrum Jülich GmbH, Institute of Complex Systems, Jülich, 52425, Germany.
authors.forename: Elena
authors.initials: E
authors.lastname: Naumovska
authors.affiliation: ICS-7: Biomechanics, Forschungszentrum Jülich GmbH, Institute of Complex Systems, Jülich, 52425, Germany.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Merkel
authors.affiliation: ICS-7: Biomechanics, Forschungszentrum Jülich GmbH, Institute of Complex Systems, Jülich, 52425, Germany.
authors.forename: Agnes
authors.initials: A
authors.lastname: Csiszár
mesh_terms: D000818:Animals; D001681:Biological Assay; D016466:CHO Cells; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism*; D003412:Cricetulus; D004705:Endocytosis; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D008561:Membrane Fusion; D051379:Mice; D018613:Microscopy, Confocal; D011162:Porphobilinogen / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D061251:Primary Cell Culture
chemical_list: C538930:3-anilino difluoroboron dipyrromethene; D005456:Fluorescent Dyes; D008081:Liposomes; D011162:Porphobilinogen
keywords: BODIPY FL fluorescence; confocal microscopy; endocytosis; flow cytometry; fusogenic liposomes; membrane fusion