Development of a Novel Lysosome-Targeted Ruthenium (II) Complex

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Development of a Novel Lysosome-Targeted Ruthenium(II) Complex for Phosphorescence/Time-Gated Luminescence Assay of Biothiols Quankun Gao, Wenzhu Zhang, Bo Song, Run Zhang, Weihua Guo, and Jingli Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b04925 • Publication Date (Web): 21 Mar 2017 Downloaded from http://pubs.acs.org on March 24, 2017

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Analytical Chemistry

Development of a Novel Lysosome Lysosomeysosome-Targeted Ruthenium(II) Complex for Phosphorescence/T hosphorescence/Timeime-Gated Luminescence Assay of Biothiols Quankun Gao,† Wenzhu Zhang,† Bo Song,† Run Zhang,*,‡ Weihua Guo,† Jingli Yuan*,† † ‡

State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116024, China Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, QLD 4072, Australia

ABSTRACT: Considering the important roles of biothiols in lysosomes of live organisms, and unique photophysical/photochemical properties of ruthenium(II) complexes, a novel ruthenium(II) complex, Ru-2, has been developed as a molecular probe for phosphorescence and time-gated luminescence assay of biothiols in human sera, live cells and in vivo. Ru-2 is weakly luminescent due to the effective photo-induced electron transfer (PET) from Ru(II) luminophore to electron acceptor, 2,4dinitrobenzene-sulfonyl (DNBS). In the presence of biothiols, such as glutathione (GSH), cysteine (Cys) and homocysteine (Hcy), the emission of Ru-2 solution was switched ON, as the result of the cleavage of quencher to form the production, Ru-1. Ru-2 showed high selectivity and sensitivity for detection of biothiols under physiological conditions, with detection limits of 62 nM, 146 nM and 115 nM for GSH, Cys and Hcy, respectively. The emission lifetimes of Ru-1 and Ru-2 were measured to be 405 ns and 474 ns, respectively, which enabled them to be used for the background-free time-gated luminescence detection even in the presence of strongly fluorescent dye, rhodamine B. On the basis of this mode, the quantification of biothiols in human serum samples was achieved without interference of background autofluorescence. A morpholine moiety was introduced into the complex to ensure Ru-2 molecules to be driven into lysosomes of live cells. Ru-2 showed low cytotoxicity and excellent membrane permeability toward live cells. Using Ru-2 as an imaging agent, visualizations of biothiols in lysosomes of live cells and in Daphnia magna were successfully demonstrated. The results suggested the potential of Ru-2 for the biomedical diagnosis of biothiol-related human diseases.

Sensing and imaging of biothiols, including cysteine (Cys), glutathione (GSH) and homocysteine (Hcy), in biological systems have attracted considerable interests in recent years due to the essential roles of these biomolecules in maintaining intracellular redox status in living organisms.1,2 On the other hand, the imbalance of biothiols production is involved in various physiological and pathological processes.3 Specifically, the deficiency of Cys is associated with many diseases such as slow growth, hair depigmentation, edema, lethargy, liver damage, loss of muscle and fat, skin lesions, and weakness.4-7 The abnormal level of Hcy has also been identified to be able to cause or exacerbate a wide range of disorders, including cardiovascular and Alzheimer’s diseases.6,8,9 Among these biothiols, GSH is the most abundant intracellular non-protein thiol, serving as an important biomolecule in maintaining the intracellular redox activities, xenobiotic metabolism, intracellular signal transduction, and gene regulation.10 However, alterations in the level of intracellular GSH have been implicated in many diseases, such as liver damage, leucocyte loss, psoriasis, cancer, and HIV infection.11,12 Therefore, determination and monitoring of these biothiols at complicated biological conditions could be helpful for early diagnosis and prevention of the related diseases. Lysosome is well known as the main organelle for providing hydrolases for the degradation and recycling of macromol-

ecules.13 Biothiols, in lysosomes, are mainly formed through reduction of disulphide during proteolysis.14 It has been reported that GSH is involved in the stabilization of lysosome’s membranes, and Cys is an effective stimulator of albumin degradation in liver lysosomes.15 Therefore, optical molecular probes that can specifically respond to these biothiols within lysosomes of live cells are of particular importance for the better elucidation of biological functions of biothiols.16-18 To date, enormous efforts have been made to the development of effective methodologies for the detection of biothiols in vitro and in vivo, such as high performance liquid chromatography (HPLC),19 capillary zone electrophoresis (CZE),20 electrochemical,21 mass spectrometry,22 and fluorometric2,23 methods. Among them, fluorescence analysis method based on the use of responsive molecular probes has been focused currently due to its high sensitivity and selectivity, operational simplicity, and the capability in real-time monitoring of biomolecules in situ.9,24 On the basis of different reaction mechanisms, such as Michael addition reaction,25-29 cyclization with aldehydes,6,30,31 cleavage of sulfonamide and sulfonate esters,32-35 cleavage of selenium-nitrogen bonds,4 cleavage of disulfide bonds,36,37 and others,38-40 a variety of optical molecular probes for biothiols have been developed in recent years. Nevertheless, the probes that can be used both for the determination of biothiols at a complicated biological condition with

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strong autofluorescence and visualization of biothiols in a specific organelle, such as lysosome, are scarce. Owing to its capability in effective discrimination of background noises from complicated biological samples and scattering lights from nearby optics, enormous efforts have been made for the development of time-gated luminescence bioassay technique.41-43 With the rapid development of this technique, a number of luminescent materials with long emission lifetimes have been reported as probes/chemosensors for timegated luminescence bioassays.44-46 Among these materials, lanthanide complexes or lanthanide-doped nanoparticles have particularly been investigated for the design of luminescence probes/chemosensors.47-49 Our group has also developed a series of functional Eu3+ and Tb3+ complexes for time-gated luminescence detections of various bioactive molecules.34,41,47,50 However, the optical excitation of these complexes at UV lights has become a major drawback for their applications in living systems. Taking advantage of long-lived phosphorescence lifetimes of ruthenium(II) complexes,43,51,52 we envision that the time-gated luminescence bioassay can also be achieved using these complexes as molecular probes under the excitation of visible lights. In this work, we developed a novel ruthenium(II)-bipyridine (bpy) complex derivative, Ru-2, as an optical molecular probe for phosphorescence/time-gated luminescence detection of biothiols in vitro, and for imaging of biothiols in lysosomes of live cells and in organisms. Ru-2 was designed by integrating phosphorescence quencher, biothiols-responsive linker, and lysosome-targeting moiety with a signaling unit, Ru(II)-bpy complex (Scheme 1). Phosphorescent Ru(II)-bpy complex was selected as the signaling moiety, owing to its desirable features including the intense visible absorption and emission, large Stokes shift, highphoto-, thermal and chemical stabilities, and very low cytotoxicity.53-60 In previous works, we have demonstrated that the MLCT emission properties of Ru(II) complexes could be modulated with photo-induced electron transfer (PET) mechanism by introducing strong electron acceptors or donors into the complexes.61,62 Thus, the designed complex, Ru-2, bearing strong electron acceptors, 2,4-dinitrobenzenesulfonyl (DNBS), was expected to be weakly phosphorescent. As the result of biothiols-induced cleavage of DNBS, the phosphorescence of the Ru(II) complex could be restored, thereby the phosphorescence analysis of biothiols could be performed. The emission lifetimes of the synthesized Ru(II) complexes, Ru-2 and Ru-1, were determined to be several hundred nanoseconds, which allowed them to be used for time-gated luminescence measurements without interferences of autofluorescence and scattering lights. Considering the effective lysosome-targetable feature of morpholine derivatives,57,58 a morpholine moiety was conjugated to one of bpy ligands in Ru-2, thereby the visualization of biothiols in lysosomes of live cells could be achieved. Using Ru-2 as a probe, phosphorescence/time-gated luminescence detection of biothiols in aqueous solutions and sera, and the imaging of biothiols in lysosomes of living cell samples and in Daphnia magna were successfully demonstrated.

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Scheme 1. Schematic representation of the design and response mechanism of Ru-2 toward biothiols.

EXPERIMENTAL SECTION Reagents and Instruments. 4-Bromomethtyl-4’-methyland bis(4-(4-methoxylphenyl)-2,2’2,2’-bipydine59 bipyridine)RuCl260 were synthesized by using the literature methods. 2-Morpholinoethylamine, 2,4-dinitrobenzenesulfonyl chloride, and BBr3 were purchased from Sigma Aldrich. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), Roswell Park Memorial Institute’s Medium (RPMI-1640), fetal bovine serum (FBS), L-glutamine, penicillin, streptomycin sulfate, and LysoTracker Green were purchased from Life Technologies. Human serum samples were collected from healthy people, and stored at -20 °C before use. Unless otherwise stated, all chemical materials were purchased from commercial sources and used without further purification. Deionized distilled water was used throughout. 1 H NMR and 13C NMR spectra were measured on a Bruker Avance spectrometer (400 MHz and 500 MHz for 1H NMR; 100 MHz for 13C NMR). Mass spectra were recorded on a HP1100 LC/MSD and LTQ Orbitrap XL mass spectrometer. Elemental analysis was carried out on a Vario-EL analyser. Absorption spectra were measured on a Perkin-Elmer Lambda 35 UV-vis spectrometer. Luminescence spectra were measured on a Perkin-Elmer LS 50B luminescence spectrometer with excitation and emission slits of 10 nm. Luminescence lifetimes and time-gated luminescence spectra were determined on an Edinburgh FS5 spectrofluorometer. The fluorescence cell imaging was performed with OLYMPUS FV-1000 inverted fluorescence microscope. Under the confocal fluorescence microscope, the Ru(II) complex was excited at 458 nm, and the emissions in 580-640 nm were collected. Bright field and phosphorescence images of Daphnia magna were recorded on a Nikon TE 2000-E fluorescence microscope. The microscope, equipped with a 100 W mercury lamp, Nikon B-2A filters and a color CCD camera system (RET-2000R-FCLR12-C, Qimaging Ltd), was used for phosphorescence imaging measurements with the exposure time of 160 ms for Daphnia magna and 2 s for HeLa, MCF-7 and HepG2 cells. Syntheses of Ru(II) Complexes. The synthesis procedure of Ru(II) complexes, 4, Ru-1 and Ru-2, were illustrated in Scheme 2. Ligand 3 was synthesized following the procedure shown in Scheme S1. The details of experiments were described as follows and in the Supporting Information.


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Scheme 2. Synthesis procedure of Ru-1 and Ru-2. Synthesis of the Ru(II) complex 4. A mixture of ligand 3 (62 mg, 0.2 mmol) and bis(4-(4-methoxylphenyl)-2,2’bipyridine)RuCl2·2H2O (146 mg, 0.2 mmol) in 30 mL EtOH was refluxed overnight with stirring. After the solvent was evaporated, the residue was purified by silica gel column chromatography using CH3CN-H2O-KNO3(sat.) (100:6:1, v/v/v) as eluent. The fractions containing the target product were collected, and the solvent was evaporated. The resulting solid was dissolved in a small amount of EtOH-H2O (1:1), and a saturated aqueous solution of NH4PF6 was then added to give a red precipitate. The product was filtered and washed with small amount of water. The complex 4 was obtained (99 mg, 40% yield). 1H NMR (400 MHz, CD3CN): δ = 8.67-8.70 (m, 4H), 8.49 (s, 1H), 8.43 (s, 1H), 8.08 (t, J = 8.0 Hz, 1H), 7.86 (d, J = 8.0 Hz, 4H), 7.61-7.78 (m, 8H), 7.38-7.44 (m, 3H), 7.27 (d, J = 4.0 Hz, 1H), 7.12 (d, J = 8.0 Hz, 4H), 4.03 (s, 2H), 3.87 (s, 6H), 3.82 (t, J = 4.0 Hz, 4H), 3.07 (d, J = 4.0 Hz, 6H), 2.99 (t, J = 6.0 Hz, 2H), 2.58 (s, 3H). ESI-MS (m/z): 1083.3 ([M-PF6]+), 542.2 ([M-PF6+H]2+), 469.3 ([M-2PF6]2+). Synthesis of Ru-1. Under argon atmosphere, BBr3 (200 mg, 0.8 mmol) in 10 mL of dry CH3CN was added dropwise to a solution of 4 (123 mg, 0.1 mmol) in 25 mL of dry CH3CN at 0 °C. After stirring for 6 h at room temperature, 30 mL of water was added, and the mixture was stirred for another 30 min. The residue was dried and then purified by silica gel column chromatography using CH3CN-H2O-KNO3(sat.) (100:10:1, v/v/v) as eluent. The fractions containing the target product were collected, and the solvent was evaporated. The resulting solid was dissolved in a small amount of CH3CN-H2O (1:1), and then a saturated aqueous solution of NH4PF6 was added to give the complex Ru-1 as a red precipitate. Complex Ru-1 was filtered, washed with small amount of water, and dried (72.4 mg, 76% yield). 1H NMR (400 MHz, CD3CN): δ = 8.658.67 (m, 4H), 8.55-8.56 (m, 1H), 8.47-8.49 (m, 1H), 8.04-8.09 (m, 2H), 7.54-7.80 (m, 12H), 7.37-7.41 (m, 3H), 7.26-7.28 (m, 1H), 6.99-7.02 (m, 4H), 4.06-4.07 (m, 2H), 3.83-3.87 (m, 4H), 3.05-3.13 (m, 8H), 2.56 (s, 3H). 13C NMR (100 MHz, CD3CN) ： 159.2, 157.1, 157.0, 156.8, 156.2, 151.3, 151.1, 150.9, 150.5, 150.2, 148.9, 137.3, 128.7, 128.2, 127.2, 126.7, 126.4, 124.8, 124.0, 123.7, 123.0, 120.6, 116.0, 64.4, 56.0, 52.3, 50.7, 42.9, 20.0. Elemental analysis calcd. (%) for C50H48F12N8O3P2Ru·0.4NH4PF6·5H2O: C 44.31, H 4.43, N 8.68; found (%): C 44.34, H 4.06, N 8.31. ESI-MS (m/z): 1055.4 ([M-PF6]+), 455.4 ([M-2PF6]2+). Synthesis of Ru-2. To a solution of Ru-1 (180 mg, 0.15 mmol) in dry CH2Cl2 (30 mL), Et3N (30 mg, 0.3 mmol) was added. The resulting solution was stirred in an ice-water bath for 15 min. Then, 2,4-dinitrobenzenesulfonyl chloride (85 mg, 0.32 mmol) was added, and the mixture was stirred for 1 h at room temperature. The solvent was evaporated, and the crude product was purified by silica gel column chromatography using CH3CN-H2O-KNO3(sat.) (100:8:0.5, v/v/v) as eluent. The purified product was dissolved in 10 mL anhydrous

CH3CN to remove the excess KNO3 by filtration. After evaporation, the product was dissolved in a small amount of CH3CN-H2O (1:1), and then a saturated aqueous solution of NH4PF6 was added to give a red precipitate. The product was filtered, and washed with small amount of water. Complex Ru-2 was obtained (154 mg, 62% yield). 1H NMR (400 MHz, CD3CN): δ = 8.77 (d, J = 2.0 Hz, 2H), 8.67 (t, J = 9.4 Hz, 4H), 8.51-8.54 (m, 2H), 8.45 (d, J = 0.8 Hz, 1H), 8.39 (s, 1H), 8.228.25 (m, 2H), 8.09 (t, J = 8.0 Hz, 2H), 7.89-7.92 (m, 4H), 7.71-7.79 (m, 5H), 7.59-7.62 (m, 3H),7.40-7.45 (m, 6H), 7.267.28 (m, 2H), 4.03 (s, 2H), 3.84 (t, J = 4.8 Hz, 4H), 2.973.11(m, 8H), 2.56 (s, 3H). 13C NMR (100 MHz, CD3CN)： 157.4, 156.8, 156.7, 156.0, 151.7, 151.6, 151.5, 151.2, 152.0, 150.5, 150.0, 148.4, 147.5, 137.5, 135.6, 133.6, 131.8, 129.3, 128.2, 127.5, 126.9, 126.4, 124.8, 124.7, 124.3, 123.1, 122.9, 121.8, 120.6, 63.7, 55.5, 51.9, 50.4, 42.0, 20.0. Elemental analysis calcd. (%) for C62H52F12N12O15P2RuS2·6.5H2O: C 41.90, H 3.69, N 9.46; found (%): C 41.70, H 3.29, N 9.13. ESI-MS (m/z): 1515.1 ([M-PF6]+), 685.3 ([M-2PF6]2+). Time-Gated Luminescence Detection. Time-gated emission spectra were determined with a time-correlated single photon counting (TCSPC) technique on the Edinburgh FS5 spectrofluorometer. Under excitation of 450 nm, the emission signals from 525 nm to 800 nm were recorded with intervals of 5 nm. Rhodamine B, with a short emission lifetime, was used as a fluorescent interference. After it (5.0 µM) was added into the solution of Ru-2 (10 µM) in 50 mM HEPES-DMSO buffer (1:1, pH 7.0), the time-gated emission spectra of the mixture were acquired with a delay time of 100 ns, in the absence and presence of biothiols. Phosphorescence Imaging of Biothiols in HeLa Cells. HeLa cells were typically seeded at a density of 5 × 104 cells/mL in a 20 mm cover glass bottom culture dishes for the confocal microscopy imaging. After incubation for 24 h at 37 °C in a 5% CO2/95% air incubator, the cells were washed with PBS buffer, and then incubated with 2.0 mL PBS solution of Ru-2 (100 µM, containing 0.5% DMSO and 1.0 mg/mL cremophor C040) for 2 h. The cells were washed with PBS for three times, and then subjected to the phosphorescence imaging measurements on the confocal microscope. For the lysosome colocalization imaging of HeLa cells, the Ru-2-loaded cells were further stained with LysoTracker Green according to the protocol from Life Technologies. After washing with PBS for three times, the cells were subjected to the imaging experiments on the confocal microscope. Phosphorescence Imaging of Biothiols in Daphnia magna. Cultured Daphnia magna were obtained from the School of Environmental Science and Technology at Dalian University of Technology. After newborn Daphnia magna (age < 48 h) were incubated with Ru-2 (50 µM) in nonchlorinated tap water at 25 °C for 1 h, the Daphnia magna were washed four times with nonchlorinated tap water, and then subjected to the phosphorescence imaging measurements.

RESULTS AND DISCUSSION DISCUSSION Design and Characterization of the Ru(II) Complexes. Considering of the important roles of biothiols in lysosomes of live cells and the desirable photophysical and photochemical properties of Ru(II) complexes, in this work, a novel Ru(II) complex, Ru-2, was designed as a molecular probe for phos-



Phosphorescence Response of Ru-2 towards Biothiols. The temporal dynamics of the phosphorescence responses of Ru-2 towards GSH, Cys and Hcy were investigated in 50 mM HEPES-DMSO (1:1, pH 7.0) by monitoring the changes of phosphorescence intensity at 620 nm. Upon the addition of Cys, the phosphorescence intensity of Ru-2 was gradually increased, and reached a plateau after 30 min. For the reactions of Ru-2 with GSH and Hcy, the changes of phosphorescence intensity reached to the maximum value after 60 min and 90 min, respectively (Figure S13). Therefore, further spectrometric analyses were conducted after incubation of Ru2 with amino acids for 90 min. The phosphorescence titrations of Ru-2 with different biothiols, including GSH, Cys and Hcy, were conducted, and the changes of excitation and emission spectra were recorded (Figure 1, S14, and S15). As shown in Figure 1, Ru-2 exhibited extremely weak emission in the absence of GSH. Upon the addition of increasing concentration of GSH, the phosphorescence intensity of the solution was gradually increased. A good linear correlation was obtained between the phosphorescence intensity and the concentration of GSH ranging from 4.0 to 25 µM. Following the reported method defined by IUPAC, the detection limit for GSH was calculated to be 62 nM based on the concentration corresponding to three standard deviations of the background signal. Under the same test condition, the phosphorescence responses of Ru-2 towards Cys and Hcy were then examined, and the results were shown in Figure S14 and S15. The detection limits for Cys and Hcy were determined to be 146 nM and 115 nM, respectively. The above results reveal that Ru-2 could serve as a highly sensitive probe for the phosphorescence detection of biothiols. 300

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phorescence/time-gated luminescence detection of biothiols in aqueous solutions and sera, and for imaging of biothiols in lysosomes of live cells and in vivo. The probe Ru-2 was designed with a “luminophore-responsive linker-quencher” approach (Scheme 1). In addition, a morpholine moiety was conjugated to this molecular probe through a C-N bond,63,64 which enables the localization of Ru-2 in lysosomes of live cells for visualization of biothiols therein. Following our previous success in the design of optimized Ru(II) complex molecular probes, i.e., OFF-to-ON contrast ratio is depended on the symmetry of the complexes,65 Ru-2 was designed to bear two dinitrobenzenesulfonyl (DNBS) quenchers. This strategy allows the OFF-to-ON ratio of luminescence enhancement is at a maximum level. In the presence of biothiols, the cleavage of 2,4-dinitrophenyl of Ru-2 occurs through a thiol-induced SNAr substitution reaction, which allows the complex, Ru-1, to be generated, accompanied by the remarkable enhancement of the MLCT-based phosphorescence as the result of the corruption of PET process. As shown in Scheme 2 and Scheme S1, Ru-2 was synthesized by four step reactions. Briefly, complex 4 was synthesized by the complexation of ligand 3 with bis(4-(4methoxylphenyl)-2,2’-bipyridine)RuCl2. After treating with BBr3 in anhydrous CH3CN, the methyl ether bonds in complex 4 were cleaved to form Ru-1. Then, Ru-2 was obtained by reacting Ru-1 with 2,4-dinitrobenzenesulfonyl chloride in CH2Cl2 in the presence of Et3N. The chemical structures of the synthesized ligand and Ru(II) complexes were well confirmed by NMR, ESI-MS and elemental analyses (Figure S1-S10 in the Supporting Information). The proposed sensing mechanism, the cleavage of DNBS with the production of Ru-1, was then confirmed by ESI-MS analysis of the solution of Ru-2 in the presence of GSH. As shown in Figure S11, the MS peak at m/z 455.0 was attributed to the peak of [(Ru-1)-2PF6]2+, and that at m/z 1055.2 could be assigned to the peak of [(Ru-1)PF6]+. The UV-vis absorption spectra of Ru-2 and its reaction product with biothiols, Ru-1, were initially measured. As shown in Figure S12, both Ru-2 and Ru-1 displayed identical visible absorption maximum at 459 nm, which can be assigned to the absorption of metal-to-ligand charge transfer (MLCT) transition of the Ru(II) complexes.53 No obvious differences in absorption spectra in the range of 400-600 nm were observed for Ru-2 and Ru-1, corroborating that the phosphorescence OFF-ON is modulated by the PET mechanism.62,65 The phosphorescence emission properties of Ru-1 and Ru-2 were then measured, and the results were listed in Table S1. After excitation at 459 nm, both Ru-1 and Ru-2 exhibited MLCT-based emissions with the same maximum emission wavelength at 620 nm. As expected, Ru-2 showed very weak phosphorescence emission due to the presence of efficient intramolecular PET process in the complex. The phosphorescence quantum yield of Ru-2 was determined to be 0.12%. In contrast, Ru-1 exhibited intense phosphorescence emission with a quantum yield of 1.94%. These results suggest that the phosphorescence of Ru-2 could be switched ON in the presence of biothiols owing to the formation of Ru-1. The phosphorescence emission lifetimes of Ru-1 and Ru-2 were determined to be 405 ns and 474 ns in 50 mM HEPES-DMSO buffer (1:1, v/v, pH 7.0), respectively, which could be attributed to the typical long-lived MLCT-based phosphorescence of the Ru(II)-bipyridine complexes.

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Figure 1. (A) Excitation and emission spectra of Ru-2 (10 µM) in the presence of different concentrations of GSH (0.0, 1.0, 2.0, 4.0, 6.0, 8.0, 10, 15, 20, 25, 30, 35, 40, 45, 50 and 55 µM) in 50 mM HEPES-DMSO buffer (1:1, pH 7.0). (B) Linear correlation of emission intensity against GSH concentration.

The phosphorescence response specificity of Ru-2 towards biothiols over other amino acids was then investigated. After the solution of Ru-2 was treated with different amino acids, the phosphorescence intensities at 620 nm were measured. As shown in Figure 2, significant increases in the phosphorescence intensity were observed after Ru-2 was reacted with three kinds of biothiols, whereas no obvious changes in phosphorescence emission were detected after Ru-2 was reacted with other amino acids. Competition experiments were then conducted, in which GSH was added into the solution containing Ru-2 and other amino acids. In this case, the phosphorescence intensity was increased to the comparable level of Ru-2 solution reacted with GSH only. Specific phosphorescence response of Ru-2 towards biothiols was further evaluated in a mixture containing Arg, Gly, His, Tyr, Asp, Pro, Trp, Glu, Ser, Leu, Thr, Ala, Val at the concentrations of 50, 250, 500, 1000, and 2500 µM, respectively. As shown in Figure S16, no


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significant changes in phosphorescence spectra were observed upon the addition of various non-thiol amino acids. However, remarkable enhancement in phosphorescence intensity was noticed upon the addition of GSH (50 µM), suggesting the high specificity of the phosphorescence response of Ru-2 towards biothiols. These results clearly indicate that Ru-2 can be used as a probe to discriminate biothiols from other amino acids, and the specific phosphorescence response of Ru-2 towards biothiols is not disturbed by the competitive amino acids. The effects of pH on the phosphorescence intensities of Ru2 and Ru-1 were examined. As shown in Figure S17, the phosphorescence intensity of Ru-2 showed a pH-independent behavior in the range of pH 3-12, while that of Ru-1 was relatively stable only in the range of pH 3-7. The phosphorescence intensity of Ru-1 was gradually decreased upon the further increase of pH value, which could be attributed to the deprotonation of the hydroxyl group in Ru-1. Nevertheless, the results suggest that Ru-2could be a useful probe for detection of biothiols under the physiological pH condition. 300 250 200 150 100 50 0 bla nk Ar g Gl y His Ty r As p Pr o Trp Gl u Se r Le u Th r Ala Va l Hc y Cy s GS H

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Figure 2. Phosphorescence intensities of Ru-2 (10 µM) at 620 nm in the presence of different amino acids (50 µM, black bars). The grey bars show the phosphorescence intensities of Ru-2 (10 µM) at 620 nm in the co-presence of GSH (50 µM) and other amino acids (50 µM). Time-Gated Luminescence Assay of Biothiols Using Ru2 as a Probe. Considering the long phosphorescence lifetimes of Ru(II) complexes (Table S1, Figure S18), time-gated luminescence assay of biothiols using Ru-2 as a probe was demonstrated by the detection of GSH in buffer solution containing rhodamine B as a strong background interference. Figure S19 illustrated the time-gated phosphorescence emission spectra of the solutions containing Ru-2 and rhodamine B in the absence and presence of GSH. Relatively quick phosphorescence lifetime decay process of the solution containing Ru-2 and rhodamine B was noticed, while the lifetime decay process was slowdown in the presence of GSH. The slow decay process over the range of emission wavelength could be attributed to the production of Ru-1 after the reaction of Ru-2 with GSH. Under excitation at 450 nm, strong fluorescence emission of rhodamine B was clearly observed from the solution of Ru-2 and rhodamine B. Upon the addition of GSH, no obvious changes in emission spectra were observed, and the phosphorescence emission of Ru-2 could not be distinguished and identified due to the effect of strong emission of rhodamine B (Figure S19b). However, when the emission spectra were recorded with time-gated mode (100 ns delay), no obvious emission band of rhodamine B was observed from the solution

of Ru-2 and rhodamine B. After reaction with GSH, significant enhancement in time-gated luminescence intensity was observed, which indicates that the fluorescence emission of rhodamine B has been effectively removed (Figure S19c). These results highlighted the advantage of Ru-2 in the application of background-free luminescence detection of biothiols. Upon the addition of GSH, the enhancements of phosphorescence and time-gated luminescence of Ru-2 were then investigated. As shown in Figure S20, the emission of Ru-2 was very weak both under phosphorescence and time-gated luminescence modes. In the presence of 10 µM, 30 µM and 50 µM of GSH, the emission intensity of the solution at 620 nm was 8.6-fold, 26.8-fold and 28.8-fold enhanced, respectively. Under the same condition, time-gated luminescence intensity of the solution after 100 ns delay was elevated to be 12.1-fold, 40.6-fold and 44.3-fold enhanced, respectively. The corresponding signal-to-noise ratios were 53.8%, 51.8% and 40.7% increased, suggesting the superiority of Ru-2 as a time-gated luminescence probe for the detection of biothiols. Time-gated luminescence titration of Ru-2 with GSH was then conducted, and the changes of emission spectra of Ru-2 upon reaction with different concentrations of GSH were recorded. Similar to the results of Figure 1, upon the addition of increasing concentration of GSH, the time-gated emission intensity of the solution was gradually increased (Figure S21). Correspondingly, the luminescence enhancement of the solution showed a good linear correlation with the GSH concentration in the range of 4.0 µM to 15 µM. These results reveal that Ru-2 is suitable to be used as a probe for the sensitive timegated luminescence detection of biothiols in complicated biological samples. Quantitative time-gated luminescence assay of biothiols in human serum samples was conducted to further evaluate the potential application of Ru-2 for clinical analysis. Human sera were 40-fold diluted with the buffer, and then reacted with Ru-2 for the measurements. As shown in Figure 3A, the serum sample itself exhibited clear fluorescence emission at 620 nm under excitation at 450 nm. Upon reaction with Ru-2, no significant changes in emission at 620 nm were observed. Furthermore, in the presence of GSH, although the enhancement in phosphorescence emission of Ru-2 could be observed, quantitative analysis of biothiols is impossible due to the effect of strong background fluorescence from human sera. These results indicate that Ru-2 is difficult to be used for the quantitative detection of biothiols in human sera with normal phosphorescence mode. However, when time-gated luminescence mode (100 ns delay) was used for the detection, no emissions could be observed from serum samples, while significant enhancement in the luminescence intensity was observed in the presence of Ru-2 (Figure 3B), which could be attributed to the reaction of Ru-2 with biothiols in the serum. Furthermore, the time-gated luminescence intensity was further increased upon the addition of another 10 µM GSH (Figure 3B), which demonstrated the practical applicability of Ru2 for the quantitative detection of biothiols in complicated serum samples under time-gated luminescence mode. Using this method, the concentration level of total biothiols (40-fold diluted sera were used for the detection, and the calibration curve of GSH shown in Figure S21B was used for the concentration calculation) in human sera was evaluated to be 244.5 ± 8.9 µM, which is well in agreement with the results reported in the literature.66-68 In addition, as the results shown in Table S2,


Analytical Chemistry the recoveries of the added GSH in serum samples were in the range of 89.4% to 95.5%, which indicates that the time-gated luminescence assay method with Ru-2 as a probe is highly accurate. A

Serum Ru-2+Serum Ru-2 Ru-2+Serum+GSH (10 µM)

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Serum Ru-2+Serum Ru-2+Serum+GSH (10 µM)

Ru-2

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feasibility of Ru-2 for visualization of biothiols was then evaluated in breast cancer cells, MCF-7, and human liver cancer cells, HepG2. As shown in Figure S24, clear red intracellular phosphorescence was observed after the cells were stained with Ru-2. These results corroborate that Ru-2 is a cell membrane permeable probe, which enables it to be favourably useful for the imaging of biothiols in various live cells.

50

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Figure 3. Phosphorescence (A, 0 ns delay) and time-gated luminescence (B, 100 ns delay) spectra of human serum samples (40-fold diluted) before and after reacting with Ru-2 (10 µM) or Ru-2 (10 µM) and GSH (10 µM). λex = 450 nm. Imaging of Biothiols in Biological Samples. On the basis of excellent sensing performance of Ru-2 to biothiols, the feasibility of Ru-2 for the imaging of intracellular biothiols was evaluated. Prior to the cell imaging, the cytotoxicity of Ru-2 was investigated by using the MTT assay method. As shown in Figure S22, no significant decrease in the cell proliferation was found after the cells were treated with different concentrations of Ru-2 (50, 100, 150 and 200 µM) for 24 h. Even a high concentration of Ru-2 (200 µM) was used to stain the cells, the cell viability was still greater than 85%, which revealed the good biocompatibility of Ru-2 for the cell imaging application. The phototoxicity of Ru-2 was then investigated in live HeLa cells. The cell death tests were conducted by microscopy imaging of the cells under irradiation with equipped 100 W mercury lamp. Specifically, the cells were loaded with Ru-2 and then irradiated under the mercury lamp for 0, 2, 4, 6, and 10 min, then the nuclei of dead cells were stained with propidium iodide (PI). As shown in Figure S23, no red emission from nuclei could be observed after the irradiation for 6 min, indicating that the cells were alive after these experiments. The PI-stained nuclei were observed upon increasing the irradiation time to 10 min, indicating that the significant phototoxicity occurred after the 6 min irradiation. It should be noted that the imaging measurements of biothiols presented in this work were done all within 1 min light irradiation (exposure time of 160 ms for Daphnia magna and 2 s for HeLa, MCF-7 and HepG2 cells). Therefore, the phototoxicity of Ru(II) complexes can be ignored. After HeLa cells were incubated with Ru-2 (100 µM) for 2 h and washed with PBS buffer, their phosphorescence images were recorded on the microscope. As shown in Figure 4A, HeLa cells showed intense red phosphorescence signals after incubation with Ru-2, which demonstrated that Ru-2 could be loaded into live HeLa cells for reacting with intracellular biothiols to afford the specific red phosphorescence signals. In a control experiment, HeLa cells were pretreated with 1.0 mM of N-methylmaleimide (NEM) for 30 min to eliminate biothiols,45 and then incubated with Ru-2 (100 µM) for 2 h before the imaging measurement. As shown in Figure 4B, no red phosphorescence signals could be observed from the NEMpretreated HeLa cells, indicating that the red phosphorescence signals observed in Figure 4A were indeed originated from the reaction of Ru-2 with endogenous biothiols in HeLa cells. The

Figure 4. Bright-field, phosphorescence and merged images (excitation at 458 nm, emission channel, 580-640 nm) of HeLa cells incubated with Ru-2 (100 µM) for 2 h (A), and those of HeLa cells pretreated with NEM (1.0 mM) for 30 min and incubated with Ru-2 (100 µM) for 2 h (B). Scale bars: 20 µm. It was noticed that the red phosphorescence signals observed in Figure 4A mainly localized in an isolated juxtanuclear area in the cytoplasm of the cells, which might be attributed to the lysosome-targetable feature of Ru-2. Thus, to clarify the nature of intracellular localization of Ru-2, a colocalization experiment of Ru-2 and a commercially available lysosome-speciﬁc fluorescent indicator, LysoTracker Green, was carried out in HeLa cells. After Ru-2-loaded HeLa cells were further incubated with LysoTracker Green, the cells were imaged on a confocal laser scanning microscope. As shown in Figure 5, good overlapping of red emission of the Ru(II) complex and green emission of LysoTracker Green was observed (overlapping, >85%). The intensity profiles of two kinds of emissions in the interested linear region across HeLa cells also varied in close synchrony. These results confirmed the majority of Ru-2 molecules within lysosomes of the cells, and suggested the potential of Ru-2 as a probe for the in situ detection of lysosomal biothiols in live cells.

Figure 5. Confocal luminescence images of the Ru-2-loaded HeLa cells incubated with LysoTracker Green. (A) Red emis-


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sion channel, (B) green emission channel, (C) merged image of (A) and (B), (D) merged image of bright-field image and (C), (E) intensity profiles of two kinds of emissions in the interested linear region across HeLa cells in (C). Excitation at 458 nm was used for the red channel (580-640nm), and excitation at 488 nm, for the green channel (500-540 nm). Scale bars: 10 µm. The feasibility of Ru-2 as a probe for the in vivo imaging was then evaluated by the visualization of biothiols in live Daphnia magna. As shown in Figure 6A, after Daphnia magna were incubated with Ru-2 (50 µM) for 1 h, intense red phosphorescence signals were observed mainly from the regions of esophagus and gut of Daphnia magna. For a control group, Daphnia magna were pretreated with 100 µM of NEM for 40 min, and then incubated with Ru-2 (50 µM) for 1 h. In this case, the images of Daphnia magna showed almost unobservable phosphorescence signals (Figure 6B), demonstrating that the red phosphorescence signals in Figure 6A were indeed originated from the reaction of Ru-2 with endogenous biothiols in Daphnia magna. Under the microscope equipped with a camcorder, the heart rate of Daphnia magna was recorded after the experiments. Then, the heart rate of each individual Daphnia magna was calculated to be around 200 beats per minute, which is in consistence with that of the reported live Daphnia magna,69 demonstrating that the growth of Daphnia magna was not significantly affected by the experiments of Ru-2-staining and the followed imaging detection of biothiols. All of these results suggested the potential of Ru-2 for the in vivo applications.

towards biothiols in aqueous media, such as high sensitivity and selectivity, enabled Ru-2 to be used for the detection of biothiols in live samples. Low cytotoxicity and cell membrane permeability were initially studied, followed by the successful visualization of biothiols in lysosomes of live cells and in Daphnia magna. The results of this effort suggest that Ru-2 could serve as an effective molecular probe for the investigation of biothiols’ levels in human sera and in live organisms, which could be anticipated to be a useful tool for the diagnosis and monitoring of diseases associated with the imbalance of intracellular redox status.

ASSOCIATED CONTENT Supporting Information General information, synthesis and characterization of ligand 3, the complex 4, Ru-1 and Ru-2, absorption, phosphorescence and time-gated luminescence spectra of Ru-2 and Ru-1, cytotoxicity assay results of Ru-2, and imaging of biothiols in live MCF-7 and HepG2 cells. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author * [email protected] (R. Zhang), Tel: +61 7 3346 3806, Fax: +61 7 3346 3978 *[email protected] (J. Yuan), Tel (Fax): +86 411 8498 6041

Author Contributions The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Figure 6. Bright-field, luminescence and merged images of biothiols in Daphnia magna using Ru-2 as a probe. (A) Daphnia magna were incubated with Ru-2 (50 µM) for 1 h at 25 °C, (B) Daphnia magna were pretreated with NEM (100 µM) for 40 min and then incubated with Ru-2 (50 µM) for 1 h at 25 °C. Scale bar: 200 µm.

CONCLUSIONS In the present contribution, a novel Ru(II) complex, Ru-2, has been successfully designed and synthesized for the phosphorescence and time-gated luminescence detection of biothiols in aqueous solutions and human sera, and for the imaging of biothiols in lysosomes of live cells and in live Daphnia magna. Ru-2 showed almost non-phosphorescence due to the presence of effective PET process in the complex. Triggered by biothiols, the formation of Ru-1 led to the significant enhancement in phosphorescence intensity, and thus biothiols were determined. The long-lived phosphorescence emissions of Ru-1 and Ru-2 allowed time-gated luminescence assay of biothiols to be performed without interferences from background noises. The excellent sensing performance of Ru-2

We gratefully acknowledge the financial supports from the National Natural Science Foundation of China (Grant Nos. 21475015, 21477011), and the open project fund of State Key Laboratory of Fine Chemicals, Dalian University of Technology (KF1209).

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Development of a Novel Lysosome-Targeted Ruthenium (II) Complex

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