Lipophilic phosphorescent gold(I) clusters as selective probes for visualization of lipid droplets by two-photon microscopy

Abstract The development of organelle-specific phosphorescent multiphoton probes is a rapidly growing area in biomedical imaging. Phosphorescent properties allow spectral or time-resolved discrimination from autofluorescence, while the use of two-photon microscopy provides higher spatial resolution as well as deeper penetration into the sample compared to conventional microscopies. The main challenge in this area is the design of probes that combine a complex of desired properties. Here we present investigation of the phosphorescent homoleptic alkynyl gold(I) cluster, (AuC 2 R) 10 (R − 2,6-dimethyl-4-heptanol), which combines nonlinear optical properties with selectivity to lipid droplets of adipocytes and other cells. This label reveals high two-photon absorption and emission cross-sections in hexane, good solubility in triglycerides (vegetable oil) and microsecond lifetimes both in solution and in biological tissues. These features make it a promising probe for multiphoton bioimaging as demonstrated by successful visualization of various adipose tissues (subcutaneous fat, visceral adipose tissue, adipose tissue of mesentery) of different animals (pigeon, chicken, mouse) as well as lipid droplets in Hep G2 cells. The complex demonstrates high selectivity to lipid droplets in animal tissues and cells. The probe lifetimes in microsecond domain made possible its visualization using phosphorescent lifetime imaging (PLIM) techniques.

[1]  Deborah A. Brown,et al.  Fluorescent Detection of Lipid Droplets and Associated Proteins , 2007, Current protocols in cell biology.

[2]  W. Yang,et al.  Monodansylpentane as a Blue-Fluorescent Lipid-Droplet Marker for Multi-Color Live-Cell Imaging , 2012, PloS one.

[3]  S. Fowler,et al.  Nile red: a selective fluorescent stain for intracellular lipid droplets , 1985, The Journal of cell biology.

[4]  Y. Ouchi,et al.  Foam cell formation containing lipid droplets enriched with free cholesterol by hyperlipidemic serum. , 2001, Journal of lipid research.

[5]  J. Demas,et al.  Measurement of photoluminescence quantum yields. Review , 1971 .

[6]  Chunhui Huang,et al.  A nonemissive iridium(III) complex that specifically lights-up the nuclei of living cells. , 2011, Journal of the American Chemical Society.

[7]  N. Nukolova,et al.  HSA-based phosphorescent probe for two-photon in vitro visualization. , 2015, Journal of inorganic biochemistry.

[8]  J. Peychl,et al.  Live Cell Multicolor Imaging of Lipid Droplets with a New Dye, LD540 , 2009, Traffic.

[9]  Sanghee Lee,et al.  A Seoul-Fluor-based bioprobe for lipid droplets and its application in image-based high throughput screening. , 2012, Chemical communications.

[10]  Jae Hong Lee,et al.  Synthesis of a new fluorescent small molecule probe and its use for in vivo lipid imaging. , 2011, Chemical communications.

[11]  K. Y. Zhang,et al.  A phosphorescent rhenium(I) tricarbonyl polypyridine complex appended with a fructose pendant that exhibits photocytotoxicity and enhanced uptake by breast cancer cells , 2013 .

[12]  J. Zubieta,et al.  Targeting the Folate Receptor (FR): Imaging and Cytotoxicity of ReI Conjugates in FR‐Overexpressing Cancer Cells , 2008, ChemMedChem.

[13]  P. Chou,et al.  Intensely luminescent homoleptic alkynyl decanuclear gold(I) clusters and their cationic octanuclear phosphine derivatives. , 2012, Inorganic chemistry.

[14]  Hui Chao,et al.  Two-photon luminescent metal complexes for bioimaging and cancer phototherapy , 2016 .

[15]  A. Kimmel,et al.  On the Control of Lipolysis in Adipocytes , 1999, Annals of the New York Academy of Sciences.

[16]  Martin Oheim,et al.  Principles of two-photon excitation fluorescence microscopy and other nonlinear imaging approaches. , 2006, Advanced drug delivery reviews.

[17]  Catrin F. Williams,et al.  Bioconjugated Rhenium(I) Complexes with Amino Acid Derivatives: Synthesis, Photophysical Properties, and Cell Imaging Studies , 2012 .

[18]  Hazel A. Collins,et al.  Two-photon absorption and the design of two-photon dyes. , 2009, Angewandte Chemie.

[19]  H. Tam,et al.  A strong two-photon induced phosphorescent Golgi-specific in vitro marker based on a heteroleptic iridium complex. , 2012, Chemical communications.

[20]  T. Zimmermann.  Spectral imaging and linear unmixing in light microscopy. , 2005, Advances in biochemical engineering/biotechnology.

[21]  Flora L Thorp-Greenwood,et al.  Application of d6 transition metal complexes in fluorescence cell imaging. , 2010, Chemical communications.

[22]  T. Pakkanen,et al.  Halide-directed assembly of multicomponent systems: highly ordered Au(I)-Ag(I) molecular aggregates. , 2010, Angewandte Chemie.

[23]  L. Sironi,et al.  Luminescent Conjugates between Dinuclear Rhenium Complexes and Peptide Nucleic Acids (PNA): Synthesis, Photophysical Characterization, and Cell Uptake , 2012 .

[24]  Stanley W Botchway,et al.  Time-resolved and two-photon emission imaging microscopy of live cells with inert platinum complexes , 2008, Proceedings of the National Academy of Sciences.

[25]  M. Drobizhev,et al.  Two-photon absorption standards in the 550-1600 nm excitation wavelength range. , 2008, Optics express.

[26]  R. Forster,et al.  Multimodal cell imaging by ruthenium polypyridyl labelled cell penetrating peptides. , 2010, Chemical communications.

[27]  C. Thiele,et al.  Cell biology of lipid droplets. , 2008, Current opinion in cell biology.

[28]  D. Brooks,et al.  Modulation of the organelle specificity in Re(I) tetrazolato complexes leads to labeling of lipid droplets , 2014 .

[29]  Wolfgang Becker,et al.  Combined fluorescence and phosphorescence lifetime imaging , 2016 .

[30]  K. K. Lo,et al.  Applications of luminescent inorganic and organometallic transition metal complexes as biomolecular and cellular probes. , 2012, Dalton transactions.

[31]  Yumin Zhang,et al.  Visible light excitable Zn2+ fluorescent sensor derived from an intramolecular charge transfer fluorophore and its in vitro and in vivo application. , 2009, Journal of the American Chemical Society.

[32]  T. Pakkanen,et al.  Self-assembly of supramolecular luminescent Au(I)-Cu(I) complexes: "wrapping" an Au6Cu6 cluster in a [Au3(diphosphine)3]3+ "belt". , 2008, Angewandte Chemie.

[33]  Qiang Zhao,et al.  Phosphorescent heavy-metal complexes for bioimaging. , 2011, Chemical Society reviews.

[34]  U. Eriksson,et al.  Imaging of neutral lipids by oil red O for analyzing the metabolic status in health and disease , 2013, Nature Protocols.

[35]  B. Davidson,et al.  Human liver cancer cells and endothelial cells incorporate iodised oil. , 1996, British Journal of Cancer.

[36]  R. Pepperkok,et al.  Spectral imaging and its applications in live cell microscopy , 2003, FEBS letters.

[37]  W. Webb,et al.  Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm , 1996 .

[38]  J. Barton,et al.  Fluorescein redirects a ruthenium-octaarginine conjugate to the nucleus. , 2009, Journal of the American Chemical Society.

[39]  P. Chou,et al.  Rational reductive fusion of two heterometallic clusters: formation of a highly stable, intensely phosphorescent Au-Ag aggregate and application in two-photon imaging in human mesenchymal stem cells. , 2010, Chemical communications.

[40]  Y. Lam,et al.  A bioaccumulative cyclometalated platinum(II) complex with two-photon-induced emission for live cell imaging. , 2009, Inorganic chemistry.

[41]  P. Chou,et al.  Water-soluble noncovalent adducts of the heterometallic copper subgroup complexes and human serum albumin with remarkable luminescent properties. , 2014, Chemical communications.

[42]  W. Denk,et al.  Two-photon laser scanning fluorescence microscopy. , 1990, Science.

[43]  J. L. Ramírez-Zacarías,et al.  Quantitation of adipose conversion and triglycerides by staining intracytoplasmic lipids with oil red O , 1992, Histochemistry.

[44]  S. Soper,et al.  Synthesis and properties of cell-targeted Zn(II)-phthalocyanine-peptide conjugates. , 2007, Bioconjugate chemistry.

[45]  Joseph W. Perry,et al.  Two-photon absorption: an overview of measurements and principles , 2010 .

[46]  R. Coleman,et al.  Neutral lipid storage disease: a genetic disorder with abnormalities in the regulation of phospholipid metabolism. , 1998, Journal of lipid research.

[47]  C. Bagowski,et al.  Naphthalimide gold(I) phosphine complexes as anticancer metallodrugs. , 2009, Dalton transactions.

[48]  David Ogden,et al.  From one-photon to two-photon probes: "caged" compounds, actuators, and photoswitches. , 2013, Angewandte Chemie.

[49]  Guanying Li,et al.  Mitochondria-specific phosphorescent imaging and tracking in living cells with an AIPE-active iridium(III) complex. , 2013, Chemical communications.

[50]  A. Bhattacharjee,et al.  New fluoranthene FLUN-550 as a fluorescent probe for selective staining and quantification of intracellular lipid droplets. , 2014, Organic letters.

[51]  R. J. Arthur,et al.  3-Chloromethylpyridyl bipyridine fac-tricarbonyl rhenium : a thiol-reactive luminophore for fluorescence microscopy accumulates in mitochondria , 2008 .

[52]  Kenneth Kam-Wing Lo,et al.  Luminescent rhenium(I) polypyridine complexes appended with an α-D-glucose moiety as novel biomolecular and cellular probes. , 2011, Chemistry.

[53]  D. Murphy.  The biogenesis and functions of lipid bodies in animals, plants and microorganisms. , 2001, Progress in lipid research.

[54]  G. Prencipe,et al.  Luminescent conjugates between dinuclear rhenium(I) complexes and peptide nucleic acids (PNA) for cell imaging and DNA targeting. , 2010, Chemical communications.

[55]  P. Chou,et al.  An intensely and oxygen independent phosphorescent gold(I)-silver(I) complex: "trapping" an Au8Ag10 oligomer by two gold-alkynyl-diphosphine molecules. , 2009, Chemical communications.

[56]  A. Warth,et al.  Lipid droplet-associated PAT-proteins show frequent and differential expression in neoplastic steatogenesis , 2010, Modern Pathology.

[57]  W. Webb,et al.  Design of organic molecules with large two-photon absorption cross sections. , 1998, Science.