Article pubs.acs.org/IC
Red-Emitting Ruthenium(II) and Iridium(III) Complexes as Phosphorescent Probes for Methylglyoxal in Vitro and in Vivo Wenzhu Zhang,*,† Feiyue Zhang,† Yong-Lei Wang,‡ Bo Song,† Run Zhang,*,§ and Jingli Yuan† †
State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116024, P. R. China Applied Physical Chemistry, Department of Chemistry, KTH Royal Institute of Technology, SE-100 44 Stockholm, Sweden § Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, Queensland 4072, Australia ‡
S Supporting Information *
ABSTRACT: Transition-metal complexes, ruthenium(II) and iridium(III) complexes in particular, with fascinating triplet emissions are rapidly emerging as important phosphorescent dyes for application in the sensing and imaging of biological makers in live cells and organisms. In this contribution, two red-emitting transition-metal complexes, [Ru(bpy)2(DAphen)](PF6)2 and [Ir(ppy)2(DA-phen)](PF6) (bpy = 2,2′-bipyridine, DAphen = 4,5-diamino-1,10-phenanthroline, and ppy = 2-phenylpyridine), were designed and synthesized as phosphorescent probes for the highly sensitive and selective detection of methylglyoxal (MGO), an essential biomarker in the etiopathogenesis of several diseases. Both probes showed weak emissions in aqueous media because of the existence of an effective photoinduced-electron-transfer process, while their emissions could be remarkably enhanced upon the addition of MGO. The photophysical and electrochemical properties, as well as phosphorescent responses of the probes toward MGO, were examined. The ground- and excited-state properties of the probes and their reaction products with MGO, [Ru(bpy)2(MP-phen)](PF6)2 and [Ir(ppy)2(MP-phen)](PF6) (MP-phen = 2-methylpyrazino-1,10-phenanthroline), the sensing mechanism, and several important experimental facts were investigated and validated using density functional theory (DFT)/ time-dependent DFT computations. The results indicated that the phosphorescence switch-ON is due to the elimination of electron transfer and followed the reestablishment of emissive triplet excited states. To evaluate the feasibility of [Ru(bpy)2(DAphen)](PF6)2 and [Ir(ppy)2(DA-phen)](PF6) as bioprobes, their cytotoxicity was examined, and their applicability for visualizing intracellular and in vivo MGO was demonstrated.
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INTRODUCTION The fast development of biological research has catalyzed the design and synthesis of various biosensors, including molecular probes and nanosensors, for the quantitative determination of important biological markers in living cells and organisms.1−4 Because intracellular/in vivo bioimaging can be secured by using these “smart” biosensors as the probing agents, the dynamic interactions of biomarkers can be conveniently visualized under a microscope.5−11 As important luminophores, phosphorescent d6 transition-metal complexes, especially for ruthenium(II) and iridium(III) complexes, have recently received increasing attention in the field of biosensing and bioimaging because of their unique features in optical physics, chemistry, and electrochemistry, such as intense absorption and emission in the visible-light range, large Stokes shift, and high photostability and thermal and chemical stabilities.6,8,12−26 Our previous works have contributed to the development of ruthenium(II) and iridium(III) complex-based photoluminescence (PL) and electrochemiluminescence (ECL) probes for the detection of bioreactive molecules and ions27−29 and demonstrated their applications for the visualization of biospecies in living cells and in vivo.29−32 The mechanisms for tuning the photophysical, photochemical, and electrochemical properties of ruthenium(II) and iridium(III) com© XXXX American Chemical Society
plexes have also been extensively explored through modification of their coordination ligands.31−35 As part of our ongoing research in the design and synthesis of ruthenium(II) and iridium(III) complex-based molecular probes, we recently focused our interest toward the engineering of red-emitting phosphorescence probes for the determination of methylglyoxal (MGO) in a buffer, in live cells, and in vivo. MGO is an extremely reactive α-ketoaldehyde, which can be endogenously produced in all living cells by various metabolic pathways, such as dephosphorylation of glycolytic intermediates, metabolites of the polyol pathway, and aminoacetone metabolism.36−38 It has been reported that MGO is implicated in the etiopathogenesis of neurodegenerative diseases, like Alzheimer’s disease, in that MGO inhibits the growth of cells almost in all types of organisms.39,40 It is also well-known that other diseases, such as diabetes, kidney disease, oxidative stress, and uremia, are associated with the elevated levels of MGO. In tumor tissues, MGO is known as retine, serving as an important compound to inhibit the growth of cancer cells without poisoning the normal cells.41 Therefore, the sensitive and selective detection of MGO in situ in living samples is of great Received: October 9, 2016
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DOI: 10.1021/acs.inorgchem.6b02443 Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry
complexes on a Vario-EL analyzer. The measurements on the absorption features were performed using a PerkinElmer Lambda 35 UV−vis spectrometer. Luminescence spectra were recorded on a PerkinElmer LS 50B luminescence spectrometer. The excitation and emission slits were 10 nm for all luminescence measurements. The ECL properties were measured on a MPI-A ECL instrument system from Remex Electronics Instrument Ltd. Co., and all of the tests were conducted using a small quartz ECL cell at room temperature. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) were measured on a CHI 660D electrochemical analyzer (Shanghai Chen Hua Instrument Co., Ltd.). Visualization of MGO in live cells and D. magna was carried out on a Nikon TE 2000-E luminescence microscope. Red luminescence images (>520 nm) were obtained by excitation with a 100 W mercury lamp and Nikon excitation B-2A filters (450−490 nm, dichroic mirror, 505 nm). A color CCD camera system (RET-2000R-F-CLR-12-C, Qimaging Ltd.) was used for the bright-field and luminescence imaging measurements. The images were analyzed with ImageJ software, version 1.44p. Synthesis and Characterization of the Complexes. Synthesis of [Ir(ppy)2(DA-phen)](PF6). The synthesis pathway of [Ir(ppy)2(DAphen)](PF6) is shown in Scheme S1. Specifically, [Ir(ppy)2Cl]2 (0.25 g, 0.29 mmol) and DA-phen (99 mg, 0.47 mmol) were dissolved in 30 mL of CH3OH/CH2Cl2 (2:1, v/v). This mixture was then stirred at 60 °C for 4 h under an argon atmosphere. After evaporation to remove the solvents, the desired residue was purified by a silica column using CH2Cl2/methanol (30:1, v/v) as the eluent. The fractions containing the desired product were collected, and the solvent was removed under reduced pressure. Then, the solid was treated with a saturated solution of NH4PF6 in a small amount of CH3CN/H2O (1:1) to get [Ir(ppy)2(DA-phen)](PF6). Yield: 78%. 1H NMR (CD3CN): δ 4.75 (s, 4H), 6.38 (d, J = 8.0 Hz, 2H), 6.86 (t, J = 7.4 Hz, 2H), 6.95 (t, J = 7.4 Hz, 2H), 7.07 (t, J = 7.5 Hz, 2H), 7.41 (d, J = 5.2 Hz, 2H), 7.71− 7.85 (m, 6H), 8.05 (t, J = 6.6 Hz, 4H), 8.57 (d, J = 4.0 Hz, 2H). 13C NMR (CD3CN): δ 119.67, 122.43, 123.30, 124.79, 124.96, 125.35, 130.27, 130.94, 131,71, 138.29, 141.88, 144.28, 146.97, 149.28, 150.73, 167.52. Elem anal. Calcd for C34H26F6IrN6P: C, 47.72; H, 3.06; N, 9.82. Found: C, 47.78; H, 3.22; N, 9.34. ESI-HRMS ([M − PF6]+). Calcd: m/z 711.1848. Found: m/z 711.1832. ECL and Electrochemical Measurements. Three electrodes were used in ECL and electrochemical experiments, including a working electrode (3.0 mm glassy carbon electrode), an auxiliary electrode (0.3 mm platinum wire), and a reference electrode. For the measurements conducted in a 50 mM PBS solution, a KCl-saturated Ag/AgCl electrode was selected as the reference electrode, while in a CH3CN solution containing 0.1 M tetrabutylammonium hexafluorophosphate (TBAPF6), a Ag/AgNO3 (20 mM) electrode was used as the reference electrode. The working electrode was sonicated in 10% HNO3 for 1 min, followed by polishing with a Al2O3 slurry and washing with water for another 1 min before the experiments. The electrochemical experiments were performed in CH3CN (containing 0.1 M TBAPF6) or PBS (50 mM, pH 7.4). In ECL measurements, trin-propylamine (TPrA) was added to the PBS buffer or acetonitrile (CH3CN; containing 0.1 M TBAPF6) with a final concentration 10 mM. The voltage of the photomultiplier tube was set at 850 V. Theoretical Computation. The ground-state molecular structures of [Ir(ppy)2(DA-phen)]+, [Ir(ppy)2(MP-phen)]+, [Ru(bpy)2(DAphen)]2+, and [Ru(bpy)2(MP-phen)]2+ were initially optimized using DFT, and the excited-state-related computations were conducted by the TDDFT52 method using the optimized molecular structures in the ground state. Beck’s three-parameter hybrid functional with the Lee− Yang−Parr correlation functional (B3LYP)51 was used throughout. The LanL2DZ basis set53 was used for the Ir and Ru atoms in four complexes, whereas the 6-311*G(d) basis set54 was applied to the C, H, and N atoms. The polarized continuum model method55 was employed for the solvent effects (water) in optimization, absorbance, and emission calculations. All calculations in the present work were performed by the Gaussian 09 package of programs.56 Imaging of MGO in Living Cells. Stock solutions of [Ru(bpy)2(DA-phen)](PF6)2 (50 mM) and [Ir(ppy)2(DA-phen)](PF6) (50 mM) were prepared by dissolving certain amounts of ruthenium-
importance for investigating its function in live cells. The methods developed could be of further assistance to the disease diagnosis and evaluation of the treatment. Although several traditional instrument-based analysis methods, such as high-performance liquid chromatography, gas chromatography−mass spectrometry, and liquid chromatography−mass spectrometry, have been developed for MGO detection in bulk solutions, the in situ detection of MGO in vivo remains a challenge because of the lack of methods for onsite MGO analysis.36,42−44 To address this challenge, Wang et al. recently developed a methyldiaminobenzene/boron dipyrromethene dye as the fluorescence probe for visualizing MGO in HeLa cells.45 Nevertheless, the probe, especially the one with excellent optical and electrochemical properties, for the detection of MGO in vivo is under development. In this work, two red-emitting phosphorescence ruthenium(II) and iridium(III) complexes, [Ru(bpy)2(DA-phen)](PF6)2 and [Ir(ppy)2(DA-phen)](PF6) (bpy = bipyridine, DA-phen = 4,5-diamino-1,10-phenanthroline, and ppy = 2-phenylpyridine), were reported for the detection of MGO in live cells and organisms. The complexes were first synthesized, and their photophysical and electrochemcial properties were extensively investigated in subsequent studies. As expected, both complexes showed weak emission in a phosphate-buffered saline (PBS) buffer because of the existence of an intramolecular photoinduced-electron-transfer (PET) process. As a result of the specific reaction with MGO, their phosphorescence emissions were remarkably enhanced, which allows the concentration of MGO to be determined. Quantum-chemical computations were then performed to assign the electronic transitions in [Ru(bpy)2(DA-phen)](PF6)2 and [Ir(ppy)2(DA-phen)](PF6) and their reaction products with MGO, [Ru(bpy)2(MPphen)](PF6)2, and [Ir(ppy)2(MP-phen)](PF6). The PETbased mechanism of phosphorescence turn-ON was analyzed by density functional theory (DFT)/time-dependent DFT (TDDFT) computations. Both [Ru(bpy)2(DA-phen)](PF6)2 and [Ir(ppy)2(DA-phen)](PF6) showed low cytotoxicity, which allows them to be adopted for visualization of MGO in live cells and bodies.
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EXPERIMENTAL SECTION
Materials and Physical Measurements. Iridium(III) chloride hydrate (IrCl3·3H2O), ruthenium(III) chloride (RuCl3·3H2O), sodium hypochlorite (NaOCl), hydrogen peroxide (H2O2), 1phenyl-1,2-propanedione (PPD), glyoxlic acid (GOA), o-phthaladehyde (OPA), ethyl pyruvate, methylglyoxal (MGO), 2,3-butadione, 1,10-phenanthroline, and 3-morpholinosydnonimine (a ONOO− donor), calf-thymus DNA, and various metal ions were obtained from Sigma-Aldrich and used without further purification. The concentration of MGO was confirmed by a Friedemann titration method.46 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), L-glutamine, penicillin, and streptomycin sulfate were obtained from Life Technologies. cis-Ru(bpy)2Cl2·2H2O,47 [Ir(ppy)2Cl]2,48,49 4,5-diamino-1,10-phenanthroline (DA-phen),50 and [Ru(bpy)2(DA-phen)](PF6)250 were synthesized according to previous literature reports. Cultured Daphnia magna was received from the School of Environmental Science and Technology, Dalian University of Technology, Dalian, P. R. China. Water was purified by deionization and used throughout all in vitro and in vivo studies. 1 H and 13C NMR spectra were obtained using a Bruker Avance NMR spectrometer (400 MHz for 1H and 100 MHz for 13C). The molecular masses of all ligands and complexes were acquired on a HP1100 LC/MSD mass spectrometer. Elemental analysis was performed to examine the composition and purity of the synthesized B
DOI: 10.1021/acs.inorgchem.6b02443 Inorg. Chem. XXXX, XXX, XXX−XXX
Article
Inorganic Chemistry (II) and iridium(III) complexes in dimethyl sulfoxide (DMSO) for bioimaging. RAW 264.7 macrophage cells (1 × 105 cells/mL) were grown in a DMEM culture medium (containing 10% FBS, 1% penicillin, and 1% streptomycin) in a 25 cm glass culture bottle. The bottle was placed in in a 5% CO2/95% air incubator for 12 h at 37 °C. Then, the cells were incubated with a fresh medium containing [Ru(bpy)2(DA-phen)]2+ (final concentration, 50 μM) or [Ir(ppy)2(DA-phen)]+ (final concentration, 50 μM) for another 3 h. The probe-stained cells were washed with PBS three times, followed by treatment with 50 μM MGO in PBS for 3 h. Luminescence imaging was then carried out after the cells were washed with PBS three times. Imaging of MGO in D. magna. D. magna was cultured according to our previously reported method.29 The newborn D. magna (age
