Biomaterials 53 (2015) 522e531

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Ruthenium(II) anthraquinone complexes as two-photon luminescent probes for cycling hypoxia imaging in vivo Pingyu Zhang 1, Huaiyi Huang 1, Yu Chen, Jinquan Wang, Liangnian Ji, Hui Chao* MOE Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-Sen University, Guangzhou 510275, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 November 2014 Received in revised form 19 February 2015 Accepted 27 February 2015 Available online

Recently, cycling hypoxia, with cycles of hypoxia followed by reoxygenation, was found to induce the upregulation of HIF-1 activity in tumor cells and reduce the success rate of radiotherapy or chemotherapy. Thus, the ability to visualize cycling hypoxia in cells and in vivo is of great significance. To address this critical need, we have developed ruthenium(II) anthraquinone complexes as reversible two-photon luminescent probes; these probes are well suited for selectively and dynamically monitoring cycling hypoxia in live cells. Moreover, the probes can conveniently visualize inner hypoxia in 3D multicellular tumor spheroids and detect repeated cycles of hypoxia-reoxygenation in living zebrafish via two-photon luminescent imaging. The present study provides a powerful luminescent imaging tool for dynamically sensing of cycling hypoxia in vivo. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Hypoxia Cycling Two-photon Ru(II) complex Anthraquinone

1. Introduction Hypoxia, which is caused by an inadequate oxygen supply, is closely associated with various diseases, including cancer, cardiopathy, ischemia, and vascular diseases [1e3]. An immediate need exists for an approved diagnostic technology that can determine the hypoxia status of cancer lesions. There were two different kinds of mechanisms to detect hypoxia. One is that hypoxia induces a strong reducing environment, which accelerates a bioreductive reaction that does not proceed under normoxia [4]. This characteristic provides a convenient route for the design of hypoxiasensitive imaging probes. The other mechanism to detect O2 through quenching of the excited state of the given luminophore, reducing its emission intensity and lifetime. Some of recently works showed the progress of oxygen sensing and luminescence imaging in living cells [5e9]. In recent years, most hypoxia probes have required excitation by high-energy ultraviolet (UV) or visible light [9e14]. These probes cannot penetrate into hypoxic regions buried deeply in tumors and have even caused detrimental effects on healthy cells and organs.

* Corresponding author. Tel.: þ86 20 84110613; fax: þ86 20 84112245. E-mail address: [email protected] (H. Chao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2015.02.126 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

Moreover, the reported fluorescent organic probes are always subject to poor solubility and low photostability in bioenvironments [15]. One effective approach to overcoming these drawbacks and improving non-invasive in vivo fluorescent imaging is to utilize hypoxia-sensing systems that can be directly excited by a two-photon process. These hypoxia-sensing molecules exhibit near-infrared (NIR) or longer excitation wavelengths (two-photon), less phototoxicity, deeper penetration depths and less photobleaching [16,17]. As such, some laboratories have focused on the development of new two-photon imaging probes for hypoxia [18e21]. However, a strong need still exists for rationally designed molecules that provide efficient two-photon absorption (TPA), medium compatibility, photostability, membrane-crossing capabilities and low cytotoxicity. Tumors often have a poorly developed vasculature resulting in an inefficient delivery of oxygen. Chronic hypoxia, which exists in the regions of tumors beyond the diffusion distance of oxygen, is understood well. More recently, acute hypoxia or intermittent hypoxia, now known as cycling hypoxia, with cycles of hypoxia followed by reoxygenation, has received increased attention [22]. Cycling hypoxia has been reported to induce the up-regulation of HIF-1 transcription factor in tumor cells and to reduce the success rate of radiotherapy or chemotherapy [23]. However, most previously reported hypoxia-sensitive fluorescence probes are irreversible and cannot visualize the dynamic changes of cycling hypoxia in

P. Zhang et al. / Biomaterials 53 (2015) 522e531

real time. Until now, only two examples of one-photon fluorescent sensors have been successfully applied to the reversible detection of hypoxia levels [12,24,25]. Therefore, the development of a twophoton fluorescent sensor that can probe cycling hypoxia in both cells and deep living organisms with high sensitivity and selectivity is still challenging. In order to imaging cycling hypoxia in vivo, a better alternative strategy is design the fluorescent probes which not only can be reversibly bioreduced under cycling hypoxia but also possess large two-photon absorption cross sections for deeper penetration in vivo. Herein, we report reversible two-photon luminescent probes for imaging cycling hypoxia in live cells and in vivo. It is, to the best of our knowledge, the first example of two-photon fluorescent probes for cycling-hypoxia imaging in vivo. 2. Materials and methods 2.1. Materials and general instruments The complex cis-[Ru(bpy)2Cl2]$2H2O [26] was synthesized according to literature methods. Ruthenium chloride hydrate (Alfa Aesar, USA), bpy (2,20 -bipyridine, Sigma Aldrich, USA), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-um bromide, Sigma Aldrich, USA), DMSO (dimethyl sulfoxide, Sigma Aldrich, USA), Rhodamine B (SigmaeAldrich, USA) were used as received. All the compounds tested were dissolved in DMSO just before the experiments, and the final concentration of DMSO was kept at 0.1% (v/v). Microanalysis (C, H, and N) was carried out with a Vario EL cube elemental analyzer. Infrared spectra were obtained with a Nicolet 170SX-FTIR spectrophotometer and KBr discs. 1H NMR spectra were recorded on a Brückner AVANCE III 400 MHz NMR spectrometer at room temperature. All chemical shifts are given relative to tetramethylsilane (TMS). High resolution electrospray ionization mass spectra (HRMS) were recorded by ESI-Q-TOF maxis 4G (Bruker Daltonics). UVeVis spectra were recorded on a PerkineElmer Lambda 850 spectrophotometer. Emission spectra were recorded on a PerkineElmer LS 55 spectrofluorophotometer at room temperature. 2.2. Synthesis 2.2.1. Synthesis of ligands L1e3 2-(anthracene-9,10-dione-2-yl)-1-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline (L1). A mixture of 9,10-phenanthrenequinone (0.212 g, 1.0 mmol), 2-formyl-9,10anthraquinone (0.236 g, 1.0 mmol), aniline (0.13 g, 1.2 mmol) and ammonium acetate (0.949 g, 12.23 mmol) was refluxing in glacial acetic acid (10 mL) for 24 h under an argon atmosphere. After cooling to room temperature, a pale yellow mixture was obtained and poured into a methanol solution under stirring. The separated solid was filtered off, washed with methanol, and dried to give a pale yellow solid. The solid was purified by column chromatography (petroleum ether: CH2Cl2, 1:1) on silica gel. A white powder was finally obtained after it was stirred in refluxing ethanol, subsequently filtered, and dried in vacuum. Yield: 0.35 g, 70.1%. Anal. Calcd for C33H18N4O2：C, 78.87; H, 3.61; N, 11.15; Found: C, 78.98; H, 3.94; N, 11.28; 1H NMR (400 MHz, CD3Cl-d6): d 9.60 (dd, J ¼ 8.0 Hz, 2H), 9.32 (d, J ¼ 8.1 Hz, 1H), 8.46 (d, J ¼ 7.7 Hz, 1H), 8.28 (m, 2H), 8.16 (dd, J ¼ 7.9 Hz, 2H), 7.81 (dd, J ¼ 8.1 Hz, 3H), 7.61 (m, 3H), 7.30 (m, 4H). 13C NMR (101 MHz, CD3Cl-d6): d 182.25, 182.12, 154.48, 154.43, 150.55, 150.46, 149.94, 149.85, 149.64, 149.57, 138.25, 136.22, 133.6, 133.56, 133.24, 133.12, 132.92, 132.78, 130.56, 130.46, 128.67, 128.59, 126.78, 126.70, 126.61, 125.82, 125.55, 124.84, 124.61, 124.55, 124.47, 122.62, 122.54 ppm. ESI-MS (CH3OH) m/z: 503 [MþH]þ. 2-(anthracene-9,10-dione-2-yl)-(1-p-tolyl)-1H-imidazo[4,5-f][1,10]phenanthroline (L2). This ligand was synthesized in a manner identical to that described for ligand L1, with p-toluidine (0.128 g, 1.2 mmol) in place of aniline. Yield: 0.40 g, 77.5%. Anal. Calcd for C34H20N4O2：C, 79.06; H, 3.90; N, 10.85; Found: C, 79.15; H, 4.17; N, 10.92; 1 H NMR (400 MHz, CD3Cl-d6): d 9.29 (dd, J ¼ 8.1 Hz, 2H), 9.14 (d, J ¼ 8.0 Hz, 1H), 8.53 (d, J ¼ 8.0 Hz, 1H), 8.28 (dd, J ¼ 7.9 Hz, 3H), 8.08 (dd, J ¼ 8.0 Hz, 1H), 7.88 (dd, J ¼ 7.8 Hz, 1H), 7.80 (m, 2H), 7.48 (m, 6H), 2.62 (s, 3H). 13C NMR (101 MHz, CD3Cl-d6): d 182.30, 182.24, 154.55, 154.47, 151.45, 151.36, 149.82, 149.76, 149.66, 149.52, 139.75, 138.70, 134.26, 134.13, 133.64, 133.52, 131.85, 131.76, 130.45, 130.37, 129.27, 129.19, 128.18, 127.32, 125.32, 125.12, 125.05, 124.84, 124.61, 124.65, 124.57, 123.72, 123.64, 21.67 ppm. ESI-MS (CH3OH) m/z: 517 [MþH]þ. 2-(anthracene-9,10-dione-2-yl)-(4-tert-butylphenyl)-1H-imidazo[4,5-f][1,10]phenanthroline (L3). This ligand was synthesized in a manner identical to that described for ligand L1, with 4-tert-butylaniline (0.18 g, 1.2 mmol) in place of aniline. Yield: 0.42 g, 76.4%. Anal. Calcd for C37H26N4O2: C, 79.55; H, 4.69; N, 10.03; Found: C, 79.68; H, 4.81; N, 10.15; 1H NMR (400 MHz, CD3Cl-d6): d 9.27 (dd, J ¼ 8.3 Hz, 2H), 9.10 (dd, J ¼ 8.0 Hz, 1H), 8.36 (d, J ¼ 7.9 Hz, 1H), 8.19 (m, 3H), 8.10 (dd, J ¼ 7.8 Hz, 1H), 7.82 (dd, J ¼ 7.9 Hz, 1H), 7.74 (m, 4H), 7.56 (m, 3H), 7.36 (dd, J ¼ 8.0 Hz, 1H), 1.51 (s, 9H). 13C NMR (101 MHz, CD3Cl-d6): d 182.24, 182.15, 154.33, 154.27, 151.39, 151.32, 149.77, 149.68, 149.54, 149.46, 138.66, 138.58, 134.65, 134.43, 133.74, 133.62, 131.54, 131.43,

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130.24, 130.17, 128.85, 128.29, 128.09, 127.96, 126.64, 126.52, 126.35, 124.67, 124.56, 124.43, 124.36, 123.56, 123.45, 34.42, 31.32 ppm. ESI-MS (CH3OH) m/z: 559 [MþH]þ. 2.2.2. Synthesis of ruthenium(II) complexes RuL1e3 [Ru(bpy)2(L1)](ClO4)2 (RuL1). A mixture of cis-[Ru(bpy)2Cl2]$2H2O (0.096 g, 0.2 mmol) and L1 (0.101 g, 0.2 mmol) in ethylene glycol (10 mL) were heated to 120  C for 8 h under nitrogen. The cooled reaction mixture was diluted with water (50 ml). Saturated aqueous sodium perchlorate solution was added under vigorous stirring and filtered. The dark red solid was collected and washed with small amounts of water, and diethyl ether, then dried under a vacuum and purified by column chromatography on alumina with acetonitrile-toluene (5:1 v/v) as eluant. The solvent was removed under reduced pressure, and red microcrystals were obtained. Yield: 0.178 g, 82%. Anal. Calcd for C53H34N8Cl2O10Ru (%): C, 57.10; H, 3.07; N, 10.05; Found (%): C, 57.21; H, 3.20; N, 10.09. 1H NMR (400 MHz, DMSO-d6): d 9.30 (d, J ¼ 8.3 Hz, 1H), 8.87 (t, J ¼ 8.1 Hz, 4H), 8.42 (d, J ¼ 8.0 Hz, 1H), 8.20 (m, 9H), 8.04 (m, 2H), 7.96 (t, 2H), 7.91 (d, 2H), 7.84 (m, 5H), 7.67 (dd, J ¼ 7.8 Hz, 1H), 7.60 (m, 4H), 7.49 (d, J ¼ 8.0 Hz, 1H), 7.39 (t, J ¼ 8.2 Hz, 2H). 13C NMR (101 MHz, DMSO-d6) d 182.35, 182.31, 157.20, 157.12, 157.04, 151.95, 151.92, 151.87, 151.81, 151.41, 150.81, 146.30, 146.00, 138.53, 138.47, 138.40, 136.81, 136.71, 135.19, 135.11, 134.77, 134.63, 133.76, 133.55, 133.40, 132.02, 131.59, 131.14, 129.12, 128.90, 128.60, 128.40, 128.28, 128.20, 127.64, 127.41, 127.29, 127.27, 126.41, 125.94, 124.98, 124.88, 121.94 ppm. ESI-MS (CH3OH) m/z: 458 [Me2ClO4]2þ, 915.9 [Me2ClO4 þH]þ. [Ru(bpy)2(L2)](ClO4)2 (RuL2). This complex was synthesized in a manner identical to that described for complex RuL1, with L2 (0.103 g, 0.2 mmol) in place of L1. Yield: 0.167 g, 74%. Anal. Calcd for C54H36N8Cl2O10Ru (%): C, 57.45; H, 3.21; N, 9.93; Found (%): C, 57.62; H, 3.48; N, 10.06. 1H NMR (400 MHz, DMSO-d6): d 9.29 (d, J ¼ 8.0 Hz, 1H), 8.87 (t, J ¼ 8.3 Hz, 4H), 8.47 (d, J ¼ 9.1 Hz, 1H), 8.19 (m, 9H), 8.05 (t, 2H), 7.96 (m, 2H), 7.84 (dd, J ¼ 7.8 Hz, 2H), 7.77 (m, 2H), 7.69 (dd, J ¼ 7.8 Hz, 1H), 7.59 (m, 7H), 7.39 (m, 2H), 2.50 (s, 3H). 13C NMR (101 MHz, DMSO-d6) d 182.37, 157.20, 157.12, 157.04, 152.15, 151.91, 151.86, 151.82, 151.36, 150.76, 146.28, 146.25, 145.98, 141.73, 138.53, 138.47, 138.40, 136.78, 135.20, 135.12, 134.74, 134.67, 134.07, 133.75, 133.58, 133.44, 131.96, 131.15, 128.91, 128.80, 128.64, 128.40, 128.29, 128.21, 127.65, 127.60, 127.32, 127.28, 126.42, 125.96, 124.98, 124.88, 122.00, 21.57 ppm. ESI-MS (CH3OH) m/z: 465[Me2ClO4]2þ, 930.0 [Me2ClO4þH]þ. [Ru(bpy)2(L3)](ClO4)2 (RuL3). This complex was synthesized in a manner identical to that described for complex RuL1, with L3 (0.112 g, 0.2 mmol) in place of L1. Yield: 0.187 g, 80%. Anal. Calcd for C57H42N8Cl2O10Ru (%): C, 58.47; H, 3.62; N, 9.57; Found (%): C, 58.58; H, 3.81; N, 9.62. 1H NMR (400 MHz, DMSO-d6): d 9.29 (d, J ¼ 8.0 Hz, 1H), 8.87 (t, J ¼ 8.3 Hz, 4H), 8.30 (d, J ¼ 9.1 Hz, 1H), 8.19 (m, 9H), 8.04 (m, 2H), 7.95 (m, 2H), 7.82 (m, 6H), 7.68 (dd, J ¼ 7.8 Hz, 1H), 7.59 (m, 5H), 7.39 (t, J ¼ 7.8 Hz, 2H), 1.44 (s, 9H). 13C NMR (101 MHz, DMSO-d6) d 182.37, 182.21, 171.87, 157.20, 157.12, 157.04, 157.02, 154.88, 152.04, 151.93, 151.84, 151.39, 150.75, 146.34, 146.01, 138.54, 138.49, 138.40, 136.90, 135.14, 134.71, 134.62, 134.07, 133.73, 133.55, 133.42, 131.14, 128.87, 128.59, 128.54, 128.41, 128.35, 128.28, 128.21, 127.60, 127.27, 127.23, 126.36, 125.96, 125.67, 124.98, 124.89, 122.03, 35.43, 31.51 ppm. ESI-MS (CH3OH) m/z: 486 [M-2ClO4]2þ, 972.0 [Me2ClO4þH]þ. 2.3. Determination of two-photon absorption cross sections The two-photon absorption spectra of probes were determined over a broad spectral region by the typical two-photon induced luminescence (TPL) method relative to Rhodamine B in methanol as the standard. The two-photon luminescence data were acquired using an Opolette™ 355II (pulse width 100 fs, 80 MHz repetition rate, tuning range 750e1050 nm, Spectra Physics Inc., USA). Two-photon luminescence measurements were performed in fluorometric quartz cuvettes. The experimental luminescence excitation and detection conditions were conducted with negligible reabsorption processes, which can affect TPA measurements. The quadratic dependence of two-photon induced luminescence intensity on the excitation power was verified at an excitation wavelength of 800 nm. The two-photon absorption cross section of the probes was calculated at each wavelength according to equation (1) [27]. d2 ¼ d1

f1 C1 I2 n2 f2 C2 I1 n1

(1)

where I is the integrated luminescence intensity, C is the concentration, n is the refractive index, and F is the quantum yield. Subscript ‘1’ stands for reference samples, and ‘2’ stands for samples. 2.4. 2D cancer cell culture A549 cancer cell lines were obtained from the Cell Bank (Cell Institute, Sinica Academica Shanghai, Shanghai, China). Cells were routinely maintained in DMEM or RPMI 1640 (Roswell Park Memorial Institute medium, Gibco) supplemented with 10% FBS (fetal bovine serum, Gibco), 50 U/mL streptomycin and 50 ng/mL penicillin. The cancer cells were cultured at 37  C in 20% O2, 10%, 1%, 0.1% O2 incubator. 2.5. Generation of 3D multicellular tumor spheroids A549 cellular spheroids (MCTSs) were generated using the liquid overlay method [28]. Briefly, the cells were incubated at 37  C in a 5% CO2 humidified

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atmosphere. When the cells reached approximately 80% confluence, they were harvested by trypsinization and resuspended in DMEM supplemented with 10% (v/ v) FBS. Single cell suspension was seeded to flat-bottom standard 96-well plates. The flat-bottom standard 96-well plates were previously coated with 50 mL of a sterile 1.0% (wt/vol) solution of agarose in DMEM to generate a non-adherent surface. To generate multicellular spheroids, 6000 cells suspended in 150 mL of culture media were added into each agarose-coated well, and the plates were left for 72 h in a 37  C humidified incubator with 5% CO2 until spheroids formed. For subsequent drug treatments and imaging procedures, spheroids were carefully transferred from 96well plates to fluorodish cell culture dishes. Spheroids with an average diameter of ~500e600 mm after 3 days growth were used for the experiments. 2.6. One- and two-photon luminescent imaging on 2D cancer cells and MCTSs The 2D A549 cells were seeded at a density of 2  106 cells/mL in culture media. After 24 h, the A549 cells were treated with Ru complex (10 mM) and incubated in a humidified mini-incubator (Zeiss, Incubator PM S1), which provided an atmosphere of 5% CO2 (CO2 gas cylinder) and different O2 concentration (N2 gas cylinder) at a constant temperature of 37  C and connected with the confocal microscope. After 1 h, the cells were imaged on a Zeiss LSM 710 NLO confocal microscope (63 oil immersion objective). MCTSs of 500e600 mm diameter were treated with Ru(II) complexes (10 mM) for 6 h and the DMSO volume was less than 0.1% (v/v). The images of spheroids were collected using a on a Zeiss LSM 710 NLO confocal microscope (10 objective). For one-photon images, the excitation wavelength of the laser was 458 nm. For twophoton images, the excitation wavelength of the laser was 800 nm. The emission spectra were integrated over the range of 560e670 nm. 2.7. Cellular uptake analysis Exponentially grown A549 cells were plated at a density of 1  106 cells per mL in a volume of 10 mL DMEM medium, Ru (10 mM) was added to the culture medium and incubated for varying amounts of time. After digestion by trypsinEDTA solution, A549 cells were counted and divided into two parts. One part: the nuclei were extracted using a nucleus extraction kit; the second part: the cytoplasms and mitochondria were extracted using a mitochondria extraction kit (Shanghai Sangon Biological Engineering Technology & Services Co. Ltd.). The samples were digested by 60% HNO3 at RT for one day. Each sample diluted with Milli Q water to obtain 3% HNO3 sample solutions. The standards for calibration were freshly prepared by diluting this stock solution with 3% HNO3 in Milli Q water. The ruthenium concentration in the two parts was determined by ICP-MS (Thermo Elemental Co., Ltd.). 2.8. Cell viability assay on 2D cancer cells and MCTSs The cytotoxicity of ruthenium complexes toward 2D A549 cancer cells was studied by MTT assay. 1  104 cells/well were seeded in 96-well flat bottomed multi-well plate with supplemented culture medium (180 mL/well) followed by incubation with 5% CO2/95% air at 37  C for 24 h. Test complexes were then added to the wells to achieve final concentrations ranging from 106 to 104 M. Control cell wells were prepared without the complexes. Wells containing culture medium without cells were used as blanks. Upon completion of the incubation for 24 h in a hypoxic (1% O2) incubator, stock MTT dye solution (15 mL, 5 mg/mL) was added to each well. After 4 h incubation, move out the culture medium then DMSO (150 mL) was added to solubilize the MTT formazan. The optical density of each well was then measured on a microplate spectrophotometer at a wavelength of 595 nm. The IC50 value was determined from plots of 50% viability against dose of compound added. Data were presented as averages of three independent experiments standard deviations. The cytotoxicity of ruthenium complexes towards MCTSs was measured by ATP concentration with CellTiterGlo kit (Promega) [29]. MCTSs of 500e600 mm diameter were treated by carefully replacing 50% of the medium with drug-supplemented standard medium using a 8-channel pipettor. In parallel, for the untreated reference MCTSs, we replaced 50% of medium of the solvent-containing or solvent-free medium. MCTSs were treated with drug concentration and the DMSO volume was less than 0.1% (v/v). The MCTSs were then allowed to incubate for another 12 h, 24 h or 48 h. The cytotoxicity of ruthenium complexes towards MCTSs were measured by ATP concentration with CellTiterGlo kit (Promega). 2.9. Imaging of zebrafish Zebrafish were kept at 28  C. For mating, male and female zebrafish were maintained in one tank on a 12 h light/12 h dark cycle, and the spawning of eggs was then triggered by light stimulation in the morning. Almost all of the eggs were fertilized immediately. Five-day-old zebrafish were maintained in E3 embryo media (15  103 M NaCl, 0.5  103 M KCl, 1  103 M MgSO4, 1  103 M CaCl2, 0.15  103 M KH2PO4, 0.05  103 M Na2HPO4, 0.7  103 M NaHCO3, 105% methylene blue; pH 7.5). The 5-day-old zebrafish were incubated with Ru(II) complexes (10 mM) in E3 media for 1 h at 28  C. After washing with PBS to remove the residual complexes, the zebrafish were further incubated with DBM. Such process

was repeated for three times. The zebrafish were imaged by Zeiss LSM 710 NLO confocal microscope (10 objective).

3. Results and discussion 3.1. Design and synthesis In the present study, we focus on ruthenium(II) polypyridyl complexes because of their outstanding photochemical properties, such as their exceptional nonlinear optical properties, high luminescence, large Stokes shifts, high photostability and relatively long lifetimes, as well as their small molecular weight, which allows them to easily penetrate cell membranes for live cell staining processes [30e34]. The quinone group is selected as the hypoxiasensing moiety; this group can convert into its hydroquinone form in protic media by exchanging two protons and two electrons. Quinones are good electron acceptors and efficient quenchers of single-state donor fluorescence of numerous fluorophores. In contrast, hydroquinones are good donors and do not quench nearby fluorophores [35]. Our design strategy is to combine a luminescent ruthenium(II) complex (sensitizer) with a redox-active anthraquinone moiety (quencher) and thereby develop a reversible twophoton luminescent probe to study cycling hypoxia in vivo using high-resolution spatial imaging (Scheme 1). With this in mind, we synthesised three ruthenium(II) anthraquinone complexes [Ru(bpy)2(L1)]2þ (RuL1), [Ru(bpy)2(L2)]2þ (RuL2), and [Ru(bpy)2(L3)]2þ (RuL3) (bpy ¼ 2,20 -bipyridine, L1 ¼ 2(anthracene-9,10-dione-2-yl)-1-phenyl-1H-imidazo[4,5-f][1,10]ph enanthroline, L2 ¼ 2-(anthracene-9,10-dione-2-yl)-(1-p-tolyl)-1Himidazo[4,5-f][1,10]phenanthroline, L3 ¼ 2-(anthracene-9,10dione-2-yl)-(4-tert-butylphenyl)-1H-imidazo[4,5-f][1,10]phenanth roline). The Ru(II) complexes were obtained in yields ranging from 74% to 82% by the direct reaction of L1e3 with appropriate molar ratios of cis-Ru(bpy)2Cl2 in ethylene glycol. The crude products were purified by aluminum oxide chromatography with CH3CN and toluene as the eluents (Scheme 1). All complexes were characterized by elemental analysis, ESI-MS, 1H NMR and 13C NMR in the Supporting Information (Fig. S1eS9). 3.2. Response toward hypoxia To examine whether these ruthenium(II) complexes can detect hypoxia in the solution, we first conducted an in vitro assay using rat liver microsomes (0.25 mg/mL), which contain various reductases. Without NADPH, upon excitation at 458 nm, no luminescence of the Ru(II) complexes could be observed in air-saturated (normoxia, 20% pO2) or Ar atmosphere (hypoxia, 10%, 5%, 1%, 0.1% pO2) condition (Fig. 1 and Fig. S10, S11). The results clearly ruled out the possibility of oxygen-quenched photoluminescence of ruthenium(II) complexes. After 50 mM NADPH was added as a cofactor, a similar weak luminescence was observed with NADPH incubation under normoxia. In contrast, under hypoxia, a remarkable enhancement (more than 80-fold) in luminescence intensity of RuL3 was observed (Fig. 1). It was reported that NADPH catalyzes the reduction of quinone derivatives under hypoxic condition but not under normoxic condition [36,37]. Then the luminescence intensity rapidly decreased to the initial level after exposure to air (Fig. 2). This “off-on” luminescent switching process was completely reversible and repeatable. The absorption spectra of the ruthenium(II) complexes exhibited a hypochromic effect for hypoxia (Fig. S12). In addition, the remarkable difference in the luminescence lifetimes (t) and quantum yields (F) of ruthenium(II) complexes also confirmed such a luminescence switch. The luminescence lifetimes (t) and quantum yield (F) cannot be detected under normoxia. After incubated with NADPH under hypoxia, the

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Scheme 1. Schematic illustration of the ruthenium(II) anthraquinone complexes as two-photon luminescent probes for cycling hypoxia imaging.

luminescence lifetimes (t) of ruthenium(II) complexes increased to 138e160 ns and the luminescence quantum yield (F) was 0.40e0.92% (Table 1). According to previous reports, anthraquinone can be selectively reduced to hydro-anthraquinone by a two-electron reduction [38]. The reduced product of RuL3 was isolated in a vacuum-glove-box and characterized using high-resolution mass spectrum (HRMS), 1 H NMR, 13C NMR and IR spectroscopy (Fig. 3 and Fig. S13eS16). The HRMS was conducted by zoom-scanning the ruthenium isotope peaks. The mass spectrum of the starting RuL3 ([C57H42N8O2Ru]2þ) showed a bivalence peak at 486.1274, corresponding to [M2ClO4]2þ (Fig. 3). After reaction with NADPH under hypoxia, the reduced product was found, as shown by the appearance of a peak at 488.1470 for [C57H46N8O2Ru]2þ (Fig. 3). The HRMS results matched perfectly with the simulation. Similar cases were observed in RuL1 and RuL2 (Fig. S13). The eOH proton signal was observed at d 5.2 in the 1H NMR spectrum of the reduced product (Fig. S14). In the 13C NMR spectrum of the reduced product of RuL3, peaks at 79.70 and 79.71 ppm were observed and they were related to the

Fig. 2. Reversible luminescence changes of 10 mM RuL1e3 under normoxia and hypoxia in PBS buffer. The buffer contained rat liver microsomes (0.25 mg/mL), and 50 mM NADPH was added as a coenzyme. The transition of hypoxia to normoxia was achieved by exposure to air. The transition of normoxia to hypoxia was achieved by exposure to argon. lex ¼ 458 nm; lem ¼ 615 nm.

two CeOH in the reduced complex (Fig. S15). The IR spectrum of the reduced product exhibited obvious absorption peaks between 3400 and 3500 cm1 (suggesting the presence of an eOH group) along with the disappearance of absorption peak at 1680 cm1 (suggesting the elimination of a carbonyl group, Fig. S16). These results suggested that the reduced reaction mechanism was achieved by the redox control of the quinone/hydroquinone pair (Scheme 1).

Table 1 Photoluminescence Data for RuL1e3 (10 mM) in normoxia and hypoxia condition in the presence or absence of rat liver microsomes (0.25 mg/mL) and 50 mM NADPH.a Complexes

Fig. 1. Luminescence spectra of RuL3 (10 mM) after treatment with different conditions in at 37  C in phosphate buffer (pH 7.4, contained rat liver microsomes (0.25 mg/mL); incubated without NADPH for 30 min under normoxia (black curve) or hypoxic conditions (blue curve); incubated with NADPH (50 mM) for 30 min under normoxia conditions (pink curve) or hypoxic conditions (red curve). The Luminescence spectra were measured with excitation at 458 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

RuL1 RuL2 RuL3

Without NADPH

With NADPH

Normoxia

Hypoxia

t (ns)

F (%)

t (ns)

F (%)

Normoxia

t (ns)

F (%)

Hypoxia

t (ns)

F (%)

n.d. n.d. n.d.

n.d. n.d. n.d.

n.d. n.d. n.d.

n.d. n.d. n.d.

n.d. n.d. n.d.

n.d. n.d. n.d.

138 145 160

0.40 0.54 0.92

a Room temperature; lex ¼ 458 nm; t refer to the luminescence lifetime; F refer the luminescence quantum yield.

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Fig. 3. HRMS of RuL3 and its product after reduction.

For biological applications, a chemosensor should operate in a wide range of pH conditions and in the presence of biologically relevant analytes. The emission spectra of the RuL1e3 complexes measured under different pH conditions ranging from 4 to 10 with or without NADPH (50 mM) under hypoxia (Fig. S17) indicated that

the luminescence “offeon” switch was operational within a pH range of 4e10. We further examined the specificity of RuL3 for hypoxia by measuring its luminescence intensity after the addition of various anions and bio-relative species at high concentrations (500 mM) (Fig. 4). Among the 10 additional analytes tested (Cl, I, 2 2    2 NO 2 , CN , SCN , S2O3 , SO3 , S , HSO3 and H2O2), very limited responses were observed. Considering that the anthraquinone group in RuL3 might be reduced inside cells, some possible biological reducing agents, such as ascorbic acid, cysteine (Cys), homocysteine (Hcy), reduced glutathione (GSH), dithiothreitol (DTT), and 2-mercaptoethanol (BME), were also tested. The emission intensity of RuL3 did not increase significantly at high concentrations (500 mM, the concentration of GSH was 10 mM) of these bioreducing agents. However, upon the addition of only 5.0 equivalents of NADPH to the mixture, the luminescence increased significantly. These results suggest that the ruthenium(II) anthraquinone complex is a sensitive and selective probe for hypoxia detection. 3.3. Two -photon luminescence response toward hypoxia

Fig. 4. Relative emission intensity of RuL3 (10 mM) under hypoxia upon the addition of NADPH (50 mM, gray bar) in the presence of different background reducing ions or agents (500 mM, The concentration of GSH was 10 mM, red bar) in potassium phosphate buffer (pH ¼ 7.3). The buffer contained rat liver microsomes (0.25 mg/mL). Bar   2 2 2 (1) control, (2) Cl, (3) I, (4) NO 2 , (5) CN , (6) SCN , (7) S2O3 , (8) SO3 , (9) S , (10) HSO 3 , (11) H2O2, (12) ascorbic acid, (13) Cys, (14) Hcy, (15) GSH, (16) DTT, (17) BME. (lex ¼ 458 nm and lem ¼ 615 nm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

The response of RuL1e3 to hypoxia was detected using a twophoton excitation window. Fig. 5a displays the TPA cross-section of RuL1e3þNADPH under hypoxic conditions from 750 to 1050 nm. With reference to Rhodamine B, the greatest TPA d of RuL1e3 under hypoxic conditions occurred at 800 nm and was €pperteMayer approximately 150e200 Go (GM) units (1 GM ¼ 1  1050 cm4,s1,photon1, 147 GM for RuL1þNADPH, 168 GM for RuL2þNADPH, 196 GM for RuL3þNADPH), which is larger than those of some recently reported two-photon bioavailable molecular probes [39,40]. The emission spectra of RuL1e3 under hypoxia conditions indicate strong two-photon

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under hypoxia conditions (Fig. S19). In contrast, the A549 cells treated with the probes under normoxic conditions (20% O2) exhibited negligible intracellular luminescence. The reversibility of the probe was then assessed. No luminescence was observed under normoxia conditions (Fig. 7a). After incubation under hypoxia conditions for another 0.5 h, the red emission intensity in the cells dramatically increased (Fig. 7b). The same changes in the luminescent intensity of the cells were observed when the normoxiaehypoxia cycle was repeated three times (Fig. 7cef). Taken together, the data indicate that the probes can report multiple hypoxiaenormoxia cycles via a reversible two-photon optical response. ICP-MS data indicated that ruthenium mainly accumulated in the mitochondria (Fig. S20). Then, we studied co-localization of the RuL3 (red emission) with MitoTracker Green or Lyso Tracker Green (green emission) (Fig. S21). The overlap coefficient with MitoTracker was found to be 70%, but the overlap coefficient with Lyso Tracker was only 20% (analyzed by the implemented ZEN software). These values suggested that most of ruthenium(II) complexes were accumulated in mitochondria and only a few in lysosome or cytoplasmic matrix. In addition, RuL1e3 did not exhibit obvious cytotoxicity towards A549 cells via an MTT assay after 24 h of treatment (Fig. S22). 3.5. Two-photon luminescent imaging for hypoxia in 3D spheroid tumour models

Fig. 5. (a) Two-photon absorption cross-section of RuL1e3 with NADPH under hypoxia was obtained by using excitation wavelengths of 750e1050 nm. The emission wavelength was 615 nm. (b) The two-photon luminescence spectra of RuL1e3 (10 mM) with excitation by 800 nm fs laser pulses in PBS buffer under normoxia and hypoxia conditions. The buffer contained rat liver microsomes (0.25 mg/mL) and 50 mM NADPH as a coenzyme.

luminescence; however, no luminescence was observed under normoxia when the two-photon excitation window at 800 nm was used (Fig. 5b). The two-photon process was further confirmed by a power-dependence experiment. A logelog linear relationship was observed between the emission intensity and the incident power, with a slope of ~2.0 for the RuL1e3 complexes' response toward hypoxia (Fig. S18). 3.4. Intracellular imaging for hypoxia With the probes that satisfied the need for hypoxia detection in live cells, we then monitored intracellular hypoxia using a twophoton fluorescence confocal microscope. The probes (10 mM) were loaded into A549 cells and incubated in a humidified miniincubator, which provided an atmosphere of 5% CO2 (CO2 gas cylinder) and different O2 concentration (N2 gas cylinder, 20%, 5%, 1%, 0.1% O2) at a constant temperature of 37  C and connected with the confocal microscope. After 1 h, cell imaging was then carried out by the confocal microscopy. Strong two-photon luminescent intensity was detected from the cells incubated with RuL1e3 at low oxygen concentrations (1 and 0.1% O2) (Fig. 6). One-photon and twophoton luminescence were both strong in the A549 cancer cells

Two-dimensional (2D) cultures of adherent cells are routinely used in numerous areas of the biomedical and life sciences. However, this model presents significant limitations in reproducing the complexity and pathophysiology of in vivo solid tumors [41]. Multicellular tumor spheroids (MCTSs) are heterogeneous cellular aggregates and have been gradually accepted as a valid 3D cancer model that lies between a cell monolayer and a solid tumor [42e46]. Thus, to probe the hypoxia under more challenging and biologically relevant conditions, the inner hypoxia in a 3D tumor model was visualized with RuL1e3 by one- and two-photon fluorescence microscopy by generating A549 MCTSs (Fig. 8). The A549 multicellular spheroids were incubated with 10 mM RuL3 for 6 h and were imaged under a confocal laser scanning microscope. A significant enhancement in luminescence intensity was observed on the surface of spheroids to a depth of ~70 mm for one-photon excitation and to ~130 mm for two photon excitation (Fig. 8c), as expected on the basis of the known limitation of oxygen diffusion [47]. The luminescent images were captured every 3.4 mm along the Z-axis (Fig. 8b). The spheroids exhibited a much stronger luminescence in the deeper layer cells when two-photon excitation was used compared to when one-photon excitation was used, indicating deeper penetration of the two-photon excitation light. Similar results were observed with RuL1 and RuL2 (Fig. S23). Moreover, RuL1e3 did inhibit the spheroids' growth or exhibit obvious cytotoxicity toward the MCTSs (Fig. S24). We conclude that the twophoton probe is an excellent technique for hypoxia detection in tumor or living organs in vivo because of its ability to deeply penetrate tissue. However, a one-photon probe cannot penetrate into inner hypoxic regions, which hinders its application in vivo. 3.6. Two-photon luminescence imaging of cycling hypoxia in vivo To validate the application of the probe in intra-vital imaging, it was used to detect hypoxia deep inside live zebrafish. The experiments were carried out using a cerebral anoxia model of zebrafish. The incubation of zebrafish with 15 mM 2,3-butanedione monoxime (BDM) is known to completely abolish cardiac contractility, which results in cerebral anoxia [48]. In addition, the effects of BDM

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Fig. 6. Two-photon luminescence confocal microscopy images of A549 cancer cells incubated with RuL1e3 (aeRuL1; beRuL2; ceRuL3) at various oxygen concentrations for 1 h. The excitation and emission wavelengths were 800 nm and 560e680 nm, respectively. Scale bar: 30 mm.

Fig. 7. Two-photon luminescence images of A549 cells loaded with RuL3 and exposed to cycles of normoxia (20% O2) and hypoxia (1% O2). (a) Cells were incubated under normoxia conditions for 1 h in DMEM containing 10 mM RuL3. (b) The cells were incubated under hypoxia conditions for an additional 0.5 h after (a). (cef) Subsequently, the cells were incubated under normoxia (c, e) and hypoxia (d, f) conditions. The process was repeated up to three times. lex ¼ 800 nm; lem ¼ 560e680 nm. Scale bar: 30 mm.

on contractility can be reversed after BDM washing. When 10 mM ruthenium(II) complexes were pre-incubated in 5-day-old zebrafish for 1 h, no luminescence was observed. After the BDM was introduced, the two-photon luminescent intensity in the brain obviously increased because of cerebral anoxia, and after another 5 min, the luminescent intensity saturated (Fig. 9a). In contrast, when BDM was washed out with water, the luminescent intensity

decreased because the oxygen concentration was recovered (Fig. 9b). A second and third injection of BDM resulted in another decrease of O2 and an increase in luminescence (Fig. 9cee and Fig. S25). This luminescent quenching and recovery under normoxic and hypoxic conditions was reversible in living zebrafish. However, such changes could not be imaged by one-photon fluorescence confocal microscopy (Fig. 10). Therefore, these two-

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Fig. 8. (a) One- and two-photon luminescence images of the 3D tumor spheroids after incubation with the RuL3 (10 mM) complex for 6 h. (b) The one- and two-photon Z-stack images were taken of every 3.4 mm section from the top to bottom. (c) The one- and two-photon 3D Z-stack images of an intact spheroid. The images were taken under a 10 objective. lex ¼ 460 nm (one-photon) or 800 nm (two-photon); lem ¼ 560e680 nm.

Fig. 9. Two-photon confocal luminescence images of the head of 5-day-old zebrafish. 1-Phenyl-2-thiourea (PTU) was added to the incubation medium to suppress the development of the pigment. (a) TPM of living zebrafish after incubation of RuL3 (10 mM) for 1 h followed by the addition of BDM (15 mM). (b) After fresh water was added, red emission in the brain was quenched. (cee) The process was repeated three times. The images were taken using a 10 objective. lex ¼ 800 nm; lem ¼ 560e680 nm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

photon probes are valuable tools for hypoxia imaging in the biological systems. 4. Conclusion In summary, we have developed three reversible two-photon luminescent probes, RuL1e3, which take advantage of the redox

switch of the quinone/hydroquinone pair to detect hypoxia selectively and dynamically. Because of the quenching by anthraquinone after photoexcitation, RuL1e3 exhibited no luminescence. Reduction of RuL1e3 by reductases under hypoxia conditions generated the two-photon luminescent ruthenium(II) hydro-anthraquinone complexes, and thus produced a large enhancement of the luminescence intensity. These probes can be further utilized for

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Fig. 10. One- and two-photon confocal luminescence images of the head of 5-day-old zebrafish. The living zebrafish incubated with RuL3 for 1 h, followed by adding BDM (15 mM). The wavelengths for one- and two-photon excitation were 458 and 800 nm, respectively. The images were taken under 10 objective.

visualizing inner hypoxia in 3D tumor spheroids and for detecting repeated cycles of hypoxia-reoxygenation in living zebrafish because of the high resolution and deep tissue penetration of twophoton imaging. The outstanding features of these probes make them valuable tools for imaging cycling hypoxia in live cells and in vivo. Acknowledgments This work was supported by the 973 Program (Nos. 2014CB845604 and 2015CB856301), the National Science Foundation of China (Nos. 21172273, 21171177, and 21471164), Program for Changjiang Scholars and Innovative Research Team in University of China (No. IRT1298). Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2015.02.126. References [1] Kizaka-Kondoh S, Inoue M, Harada H, Hiraoka M. Tumor hypoxia: a target for selective cancer therapy. Cancer Sci 2003;94:1021e8. [2] Thambi T, Deepagan VG, Yoon HY, et al. Hypoxia-responsive polymeric nanoparticles for tumor-targeted drug delivery. Biomaterials 2014;35: 1735e43. [3] Anada T, Fukuda J, Sai Y, et al. An oxygen-permeable spheroid culture system for the prevention of central hypoxia and necrosis of spheroids. Biomaterials 2012;33:8430e41. [4] Brown JM, Wilson WR. Exploiting tumour hypoxia in cancer treatment. Nat Rev Cancer 2004;4:437e47. [5] Son A, Kawasaki A, Hara D, Ito T, Tanabe K. Phosphorescent ruthenium complexes with a nitroimidazole unit that image oxygen fluctuation in tumor tissue. Chem Eur J 2015;21:2527e36. [6] Martin A, Byrne A, Burke CS, Forster RJ, Keyes TE. Peptide-bridged dinuclear Ru(II) complex for mitochondrial targeted monitoring of dynamic changes to oxygen concentration and ROS generation in live mammalian cells. J Am Chem Soc 2014;136:15300e9. [7] Varchola J, Huntosova V, Jancura D, et al. Temperature and oxygenconcentration dependence of singlet oxygen production by RuPhen as induced by quasi-continuous excitation. Photochem Photobiol Sci 2014;13: 1781e7.

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