title: Intracellular O2 sensing probe based on cell-penetrating phosphorescent nanoparticles.
abstract: A new intracellular O(2) (icO(2)) sensing probe is presented, which comprises a nanoparticle (NP) formulation of a cationic polymer Eudragit RL-100 and a hydrophobic phosphorescent dye Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP). Using the time-resolved fluorescence (TR-F) plate reader set-up, cell loading was investigated in detail, particularly the effects of probe concentration, loading time, serum content in the medium, cell type, density, etc. The use of a fluorescent analogue of the probe in conjunction with confocal microscopy and flow cytometry analysis, revealed that cellular uptake of the NPs is driven by nonspecific energy-dependent endocytosis and that the probe localizes inside the cell close to the nucleus. Probe calibration in biological environment was performed, which allowed conversion of measured phosphorescence lifetime signals into icO(2) concentration (μM). Its analytical performance in icO(2) sensing experiments was demonstrated by monitoring metabolic responses of mouse embryonic fibroblast cells under ambient and hypoxic macroenvironment. The NP probe was seen to generate stable and reproducible signals in different types of mammalian cells and robust responses to their metabolic stimulation, thus allowing accurate quantitative analysis. High brightness and photostability allow its use in screening experiments with cell populations on a commercial TR-F reader, and for single cell analysis on a fluorescent microscope.
authors.affiliation: Biochemistry Department, University College Cork, Cavanagh Building, College Road, Cork, Ireland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Fercher
authors.forename: Sergey M
authors.initials: SM
authors.lastname: Borisov
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Zhdanov
authors.forename: Ingo
authors.initials: I
authors.lastname: Klimant
authors.forename: Dmitri B
authors.initials: DB
authors.lastname: Papkovsky
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry; D000818:Animals; D001692:Biological Transport; D002138:Calibration; D002460:Cell Line; D019069:Cell Respiration; D002470:Cell Survival; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism*; D051379:Mice; D024101:Mitochondrial Proteins / Q000378:metabolism; D057054:Molecular Imaging; D053758:Nanoparticles; D010100:Oxygen / Q000378:metabolism*; D011166:Porphyrins / Q000737:chemistry; D005464:Fluorobenzenes; D005466:Fluorocarbons
chemical_list: D000180:Acrylic Resins; D005456:Fluorescent Dyes; D024101:Mitochondrial Proteins; D010100:Oxygen; D011166:Porphyrins; C526738:meso-tetra(pentafluorophenyl)porphyrin; D005464:Fluorobenzenes; D005466:Fluorocarbons; C050528:Eudragit RS