Communication pubs.acs.org/Organometallics
A Heterodinuclear Complex OsIr Exhibiting Near-Infrared Dual Luminescence Lights Up the Nucleoli of Living Cells Jitao Wang,† Shiguo Sun,*,† Daozhou Mu,† Jingyun Wang,‡ Wei Sun,† Xiaoqing Xiong,† Bo Qiao,† and Xiaojun Peng*,† †
State Key Laboratory of Fine Chemicals and ‡School of Life Science & Biotechnology, Dalian University of Technology, 2 Linggong Road, 116024 Dalian, People’s Republic of China S Supporting Information *
ABSTRACT: The heterodinuclear metal complex OsIr has been designed and synthesized. Upon irradiation with visible light, OsIr exhibits dual luminescence (534 and 721 nm) due to the coexistence of the iridium and osmium activating centers. The cellular uptake of OsIr examined by laser scanning confocal microscopy revealed an apparent nucleolar staining. The iridium moiety interacts with proteins and RNA to trigger a significant luminescence enhancement, whereas the osmium moiety displays a near-infrared luminescence; a ratiometric luminescence response between the two moieties was observed upon protein addition.
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of an iridium complex in the nuclei of living cells, which is associated with the interaction of the complex with proteins in the nucleus.12 All of these studies suggest that the iridium complex could be employed as an effective “intranuclear protein-localizing group”. On the other hand, osmium complexes have also received attention for their potential application in biological labeling and bioimaging.13 Murtaza et al. reported an osmium complex with a long emission lifetime that displays an emission maximum near 700 nm and can be used as a biophysical probe or in clinical applications,14 highlighting the potential of developing near-infrared nucleolus imaging agents. In our previous work, a flexible, saturated carbon chain was employed to covalently link the two ruthenium(II) moieties together, resulting in a synergistic increase in the properties of the two intramolecularly linked ruthenium activating centers.15 Continuing our research efforts in the same direction in the present study, the heterodinuclear metal complex [Os(bpy)3− (CH2)10−Ir(F2ppy)2]3+ (ppy = 1-phenylpyridine; bpy = 2,2′bipyridine), hereafter designated as OsIr (Figure 1), was
ince the discovery of the nucleolus over a century ago, its role in cellular cycle, aging, physiology, and homeostasis in eukaryotic cells has been widely accepted.1 The nucleolus plays a vital role in ribosome biogenesis and has been termed the “ribosome factory”.2 Despite this, its biological roles have not yet been fully elucidated; thus, studies aimed toward understanding nucleolar functions have assumed great importance. Since fluorescent technology offers tremendous advantages in terms of simplicity, sensitivity, and specificity,3 a great deal of attention has been paid to the development of fluorescent probes for understanding nucleolar structures and their diverse functions.4 Although a variety of organelle-specific dyes for staining live cells are widely available, there is currently only a single commercially available nucleolar imaging agent for live cells, “SYTO RNA-Select” (λexc 480 nm, λem 500 nm). Unfortunately, this organic dye emits at a short wavelength and suffers from the drawbacks of photobleaching and autofluorescence interference from biological samples; moreover, its structure has not yet been published.5 Of the various fluorophores, metal coordination complexes have attracted a great deal of attention for use as imaging agents because of their desirable properties, which include large Stokes shifts, pronounced photostability, color-tunable luminescence, and long emission lifetimes, presenting another approach for the development of nucleolar probes.6 Currently, luminescent ruthenium(II),7 europium,8 and iridium(III)9,11,12 complexes have been utilized for nuclear staining in mammalian cells. Various studies have shown that, among them, the iridium complexes are quickly taken up by living cells due to their affinity for intranuclear proteins.10−12 Lo and co-workers found that iridium dipyridoquinoxaline complexes could be taken up by MDCK cells and stain the nucleolus through interaction with the hydrophobic pockets of proteins.11 Li et al. reported the rapid (incubation time 50 nm), which is attributed to the overlap of ligand-centered π−π* transitions of both iridium(I) and osmium(II) moieties.11−14 Upon excitation at 480 nm at room temperature, OsIr exhibits dual-color emission (Figure 2, blue line), with peaks at 534 and 721 nm corresponding to the iridium and osmium activating centers, respectively. The control complex, Os, exhibits an emission peak at 721 nm (Figure S3 (Supporting Information)), whereas Ir shows strong emission at 534 nm upon excitation at 405 nm (Figure S4 (Supporting Information)). These observations demonstrate that both activating centers retain their individual absorption and emission characteristics, despite being covalently linked. A luminescence decay experiment has shown that OsIr (luminescence lifetime 37.8 ns) exhibited longer lifetime than RNA-Select (fluorescence lifetime 1.9 ns) (Figures S5 and S6 (Supporting Information)). Furthermore, OsIr displayed excellent photostability in DMSO after 25 min of irradiation under a 500 W iodine−tungsten lamp in comparison with SYTO RNA-Select (significant fading after 5 min exposure time, as shown in Figure S7 (Supporting Information)). MCF-7 cells were incubated with solutions of OsIr, Os, and Ir to examine the cellular uptake of these complexes. After incubation with OsIr (20 μM) for 1 or 2 h, MCF-7 cells were subjected to excitation wavelengths of 405 and 458 nm, and the
Figure 4. Cellular uptake of Os, Ir, and Os + Ir. (a) Luminescence microscopy images of MCF-7 breast cancer cells incubated with Os (20 μM, 2 h). From left to right: optical, luminescence (excited at 458 nm and collected at 655−755 nm), and overlay images. (b) Incubation with Ir (20 μM, 2 h). From left to right: optical, luminescence (excited at 405 nm and collected at 510−560 nm), and overlay images. (c) MCF-7 breast cancer cells incubated for 3 h with 20 μM each of Os and Ir. From left to right: Ir and Os luminescence and overlay images. Scale bar: 20 μm. 2682
dx.doi.org/10.1021/om500357x | Organometallics 2014, 33, 2681−2684
Organometallics
Communication
costaining with Os and Ir (20 μM, 3 h) did not result in any luminescence in the nucleolus, although the cytoplasmic luminescence was very bright (Figure 4c). Taken together, these results indicate that the localization of the complex OsIr in the nucleolus can be ascribed to the covalent interactions between osmium(II) and iridium(I) activating centers. The localization of the complex OsIr at the nucleolus and cytoplasm was further confirmed by colocalization experiments. Upon treatment of fixed and permeabilized MCF-7 cells with OsIr and the commercially available nucleolar stain SYTO RNA-Select, bright yellow spots could be observed in the nucleolus in the overlay image (Figure S8a (Supporting Information)), denoting colocalization. The results of the experiments examining the colocalization of OsIr with the DNA stain Hoechst 33258 on fixed cells are shown in Figure S8b (Supporting Information); the red luminescence observed from the nucleolus and cytoplasm provides further evidence that OsIr is a nucleolar stain. For further confirmation that the complex OsIr stains nucleoli, MCF-7 cells were fixed in methanol (Figure 5a) and
accumulates in the cytoplasm and nucleolus, possibly by binding intranucelar proteins and interacting with RNA. We therefore utilized luminescence-based techniques to investigate the interactions of OsIr with certain biomolecules, including bovine serum albumin (BSA),11,12,16 ct-DNA, and RNA. As shown in Figure S9A (Supporting Information), a significant enhancement of emission at 534 nm was observed upon BSA addition, whereas the emission at 721 nm was quenched. Importantly, the two luminescent moieties in OsIr were found to exhibit a ratiometric response upon the addition of BSA (Figure S10 (Supporting Information)). The complex Os, on the other hand, displayed no change in emission intensity, and the luminescence of the complex Ir was quenched upon BSA addition (Figure S9B,C (Supporting Information)). These observations suggest that the change in emission intensities of OsIr, Os, and Ir complexes is caused by the difference in hydrophobicity of the local environment after binding with BSA.11,17 The interactions of these complexes with RNA and ct-DNA have also been investigated by emission titrations. As shown in Figure S11A (Supporting Information), the addition of RNA to OsIr induced enhancement of luminescence intensity at both 534 and 721 nm. In contrast, the complexes Os and Ir did not show any luminescence enhancement (Figure S11B,C (Supporting Information)). Spectra were also obtained in the presence or absence of a 6-fold excess of RNA and ct-DNA (Figure S12 (Supporting Information)), which revealed that OsIr showed a much higher affinity for RNA than for ct-DNA. Furthermore, upon pretreatment of OsIr with excess BSA, a more significant increase in luminescence was observed in the presence of 6-fold excess RNA but not ct-DNA (Figure S13 (Supporting Information)). Taken together, these results suggest that the nucleolar luminescence enhancement was due to the interactions of OsIr with both proteins and RNA in the nucleolus. This is also in agreement with the results obtained upon RNase treatment, where the fixed MCF-7 cells exhibited relatively lower nucleolar staining after treatment with RNase. To determine the influence of hydrophobic environment on the emission intensities of OsIr, Os, and Ir complexes, studies were carried out in [H2O]:[1,4-dioxane] mixtures ranging from 5:5 to 0:10. As shown in Figure S14 (Supporting Information), the emission intensities of Ir and Os gradually decreased with increasing solvent polarity; this observation suggests that the more hydrophobic the surroundings, the relatively weaker the emission intensities for Ir and Os. In contrast, the emission intensity of OsIr showed an obvious increase at 534 nm, whereas the emission intensity at 721 nm decreased significantly. From these results, it can be further inferred that hydrophobic interactions mediate the association of OsIr with proteins. In conclusion, the complex OsIr with dual-emissive properties (green and near-infrared) has been designed for use as a nucleolar stain. Cell imaging studies showed that OsIr permeates the cell membrane and accumulates in the nucleolus and cytoplasm, as opposed to the case for Os and Ir complexes, suggesting that OsIr can be employed as a dual-luminescent probe for the nucleolus. Emission titrations revealed that OsIr binds to the intranucelar proteins and interacts with RNA but not DNA. In summary, the complex OsIr has the potential to be employed in studies involving living cells.
Figure 5. Treatment of (a) fixed MCF-7 cells with (b) Dnase, (c) RNase, and (d) both DNase and RNase. OsIr was used at a concentration of 20 μM. From left to right: green channel (510−560 nm), near-infrared channel (685−755 nm), and overlay images. Scale bar: 20 μm.
treated with deoxyribonuclease (DNase). It was observed that nucleolar staining by the complex OsIr remained almost unaltered, whereas the intensity in the nucleoplasm was dramatically diminished, due to the selective hydrolysis of DNA in the cell by DNase (Figure 5b). On the other hand, upon treatment with ribonuclease A (RNase A), the fixed MCF-7 cells showed weaker nucleolar staining with OsIr (Figure 5c). MCF-7 cells were treated further with both DNase and Rnase. As shown in Figure 5d, the intensity in the nucleoplasm was dramatically diminished, while the nucleolus showed weaker luminescence staining with OsIr, suggesting that the emissions observed upon microscopic examination did not entirely originate from interactions of the complex with RNA. Taken together, these results show that the heterodinuclear metal complex OsIr is effectively taken up by MCF-7 cells and 2683
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(9) Ma, D. L.; Wong, W. L.; Chung, W. H.; Chan, F. Y.; So, P. K.; Lai, T. S.; Zhou, Z. Y.; Leung, Y. C.; Wong, K. Y. Angew. Chem. 2008, 47, 3735. (10) Ma, D.; Wong, W.; Chung, W.; Chan, F.; So, P.; Lai, T.; Zhou, Z.; Leung, Y.; Wong, K. Angew. Chem., Int. Ed. 2008, 47, 3735. (11) Zhang, K. Y.; Li, S. P.; Zhu, N.; Or, I. W.; Cheung, M. S.; Lam, Y. W.; Lo, K. K. Inorg. Chem. 2010, 49, 2530. (12) Li, C.; Yu, M.; Sun, Y.; Wu, Y.; Huang, C.; Li, F. J. Am. Chem. Soc. 2011, 133, 11231. (13) (a) Rijt, S. H.; Kostrhunova, H.; Brabec, V.; Sadler, P. J. Bioconjugate Chem. 2011, 22, 218. (b) Yang, T.; Xia, A.; Liu, Q.; Shi, M.; Wu, H.; Xiong, L.; Huang, C.; Li, F. J. Mater. Chem. 2011, 21, 5360. (14) Murtaza, Z.; Herman, P.; Lakowicz, J. R. Biophys. Chem. 1999, 80, 143. (15) (a) Sun, S.; Yang, Y.; Liu, F.; Pang, Y.; Fan, J.; Sun, L.; Peng, X. Anal. Chem. 2009, 81, 10227. (b) Sun, S.; Li, F.; Liu, F.; Yang, X.; Fan, J.; Song, F.; Sun, L.; Peng, X. Dalton Trans. 2012, 41, 12434. (16) (a) Saha, S.; Majumdar, R.; Roy, M.; Dighe, R. R.; Chakravarty, A. R. Inorg. Chem. 2009, 48, 2652. (b) Maity, B.; Roy, M.; Saha, S.; Chakravarty, A. R. Organometallics 2009, 28, 1495. (17) (a) He, X. M.; Carter, C. D. Nature 1992, 358, 209. (b) Lo, K.; Leung, A. Sci. China: Chem. 2010, 53, 2091.
ASSOCIATED CONTENT
S Supporting Information *
Text and figures giving detailed synthetic and experimental procedures, NMR and HRMS characterization of probes, and absorption, emission, and luminescence spectra. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Authors
*E-mail for S.S.:
[email protected]. *E-mail for X.P.:
[email protected]; Tel/Fax: +86-41184986306. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was financially supported by the NSF of China (Grant Nos. 21072024, 21272030, 21136002), the National Key Technology R&D Program (Grant No. 2011BAE07B06), and the National Basic Research Program of China (Grant Nos. 2009CB724700 and 2013CB733702).
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REFERENCES
(1) (a) Boisvert, F. M.; van Koningsbruggen, S.; Navascues, J.; Lamond, A. I. Nat. Rev. Mol. Cell Biol. 2007, 8, 574. (b) Wang, L.; Ni, S.; Zheng, C. J. Med. Microbiol. 2012, 61, 1637. (c) Parlato, R.; Kreiner, G. J. Mol. Med. 2013, 91, 541. (2) (a) Lam, Y. W.; Trinkle-Mulcahy, L. A.; Lamond, I. J. Cell Sci. 2005, 118, 1335. (b) Németh, A.; Längst, G. Trends Genet. 2011, 27, 149. (c) Leung, A. K. L.; Andersen, J. S.; Mann, M.; Lamond, A. I. Biochem. J. 2003, 376, 553. (3) Gonçalves, M. S. T. Chem. Rev. 2009, 109, 190. (4) (a) Kim, Q.; Li, Y.; Namm, J.; Kulkarni, A.; Rosania, G. R.; Ahn, Y. H.; Chang, Y. T. Chem. Biol. 2006, 13, 615. (b) Liu, X.; Sun, Y.; Zhang, Y.; Miao, F.; Wang, G.; Zhao, H.; Yu, X.; Liu, H.; Wong, W. Y. Org. Biomol. Chem. 2011, 9, 3615. (c) Stevens, N.; O’Connor, N.; Vishwasrao, H.; Samaroo, D.; K, E. R.; Akins, D. L.; Drain, C. M.; Turro, N. J. J. Am. Chem. Soc. 2008, 130, 7182. (d) Volmer, R.; MazelSanchez, B.; Volmer, C.; Soubies, S. M.; Guerin, J. L. Virol. J. 2010, 7, 63. (5) Haugland, R. P.; Spence, M. T. Z.; Johnson, I. D. The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies; Life Technologies: Grand Island, NY, 2005. (6) (a) Gill, M. R.; Garcia-Lara, J.; Foster, S. J.; Smythe, C.; Battaglia, G.; Thomas, J. A. Nat. Chem. 2009, 1, 662. (b) Lo, K. K.; Lee, P. K.; Lau, J. S. Organometallics 2008, 27, 2998. (c) Ma, D. L.; He, H. Z.; Leung, K. H.; Chan, D. S.; Leung, C. H. Angew. Chem. 2013, 52, 7666. (d) Pierroz, V.; Joshi, T.; Leonidova, A.; Mari, C.; Schur, J.; Ott, I.; Spiccia, L.; Ferrari, S.; Gasser, G. J. Am. Chem. Soc. 2012, 134, 20376. (e) Steunenberg, P.; Ruggi, A.; van den Berg, N. S.; Buckle, T.; Kuil, J.; van Leeuwen, F. W.; Velders, A. H. Inorg. chem. 2012, 51, 2105. (f) Thorp-Greenwood, F. L. Organometallics 2012, 31, 5686. (g) He, H.; Leung, K.; Wang, W.; Chan, D.; Leung, C.; Ma, D. Chem. commun. 2014, 50, 5313. (h) Leung, K.; He, H.; Ma, V.; Zhong, H.; Chan, D. S.; Zhou, J.; Mergny, J.; Leung, C.; Ma, D. Chem. Commun. 2013, 49, 5630. (i) He, H.; Chan, D.; Leung, C.; Ma, D. Chem. Commun. 2012, 48, 9462. (j) Leung, K.; He, H.; Ma, V.; Chan, D.; Leung, C.; Ma, D. Chem. Commun. 2013, 49, 771. (7) (a) O’Connor, N. A.; Stevens, N.; Samaroo, D.; Solomon, M. R.; Marti, A. A.; Dyer, J.; Vishwasrao, H.; Akins, D. L.; Kandel, E. R.; Turro, N. J. Chem. Commun. 2009, 2640. (b) Puckett, C. A.; Barton, J. K. J. Am. Chem. Soc. 2009, 131, 8738. (c) Zhang, R.; Ye, Z.; Yin, Y.; Wang, G.; Jin, D.; Yuan, J.; Piper, J. A. Bioconjugate Chem. 2012, 23, 725. (8) Yu, J.; Parker, D.; Pal, R.; Poole, R.; Cann, M. J. Am. Chem. Soc. 2006, 128, 2294. 2684
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