title: Thiophene-based dyes for probing membranes.
abstract: We report the synthesis of four new cationic dipolar push–pull dyes, together with an evaluation of their photophysical and photobiological characteristics pertinent to imaging membranes by fluorescence and second harmonic generation (SHG). All four dyes consist of an N,N-diethylaniline electron-donor conjugated to a pyridinium electron-acceptor via a thiophene bridge, with either vinylene (–CH=CH–) or ethynylene (–C≡C–) linking groups, and with either singly-charged or doubly-charged pyridinium terminals. The absorption and fluorescence behavior of these dyes were compared to a commercially available fluorescent membrane stain, the styryl dye FM4-64. The hyperpolarizabilities of all dyes were compared using hyper-Rayleigh scattering at 800 nm. Cellular uptake, localization, toxicity and phototoxicity were evaluated using tissue cell cultures (HeLa, SK-OV-3 and MDA-231). Replacing the central alkene bridge of FM4-64 with a thiophene does not substantially change the absorption, fluorescence or hyperpolarizability, whereas changing the vinylene-links to ethynylenes shifts the absorption and fluorescence to shorter wavelengths, and reduces the hyperpolarizability by about a factor of two. SHG and fluorescence imaging experiments in live cells showed that the doubly-charged thiophene dyes localize in plasma membranes, and exhibit lower internalization rates compared to FM4-64, resulting in less signal from the cell cytosol. At a typical imaging concentration of 1 μM, the doubly-charged dyes showed no significant light or dark toxicity, whereas the singly-charged dyes are phototoxic even at 0.5 μM. The doubly-charged dyes showed phototoxicity at concentrations greater than 10 μM, although they do not generate singlet oxygen, indicating that the phototoxicity is type I rather than type II. The doubly-charged thiophene dyes are more effective than FM4-64 as SHG dyes for live cells.
authors.affiliation: Department of Chemistry, Oxford University, Chemistry Research Laboratory, Oxford, UK OX1 3TA. harry.anderson@chem.ox.ac.uk.
authors.forename: Ismael
authors.initials: I
authors.lastname: López-Duarte
authors.forename: Phoom
authors.initials: P
authors.lastname: Chairatana
authors.forename: Yilei
authors.initials: Y
authors.lastname: Wu
authors.forename: Javier
authors.initials: J
authors.lastname: Pérez-Moreno
authors.forename: Philip M
authors.initials: PM
authors.lastname: Bennett
authors.forename: James E
authors.initials: JE
authors.lastname: Reeve
authors.forename: Igor
authors.initials: I
authors.lastname: Boczarow
authors.forename: Wojciech
authors.initials: W
authors.lastname: Kaluza
authors.forename: Neveen A
authors.initials: NA
authors.lastname: Hosny
authors.forename: Samuel D
authors.initials: SD
authors.lastname: Stranks
authors.forename: Robin J
authors.initials: RJ
authors.lastname: Nicholas
authors.forename: Koen
authors.initials: K
authors.lastname: Clays
authors.forename: Marina K
authors.initials: MK
authors.lastname: Kuimova
authors.forename: Harry L
authors.initials: HL
authors.lastname: Anderson
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry*; D002470:Cell Survival / Q000187:drug effects; D004396:Coloring Agents / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D017711:Nonlinear Dynamics; D055620:Optical Phenomena; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013876:Thiophenes / Q000737:chemistry*; D053835:Unilamellar Liposomes / Q000737:chemistry
chemical_list: D004396:Coloring Agents; D013876:Thiophenes; D053835:Unilamellar Liposomes