Iridium(III) Anthraquinone Complexes as TwoÐ² ... - Wiley Online Library

DOI: 10.1002/chem.201600310

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Iridium(III) Anthraquinone Complexes as Two-Photon Phosphorescence Probes for Mitochondria Imaging and Tracking under Hypoxia Lingli Sun, Yu Chen, Shi Kuang, Guanying Li, Ruilin Guan, Jiangping Liu, Liangnian Ji, and Hui Chao*[a] Abstract: In the present study, four mitochondria-specific and two-photon phosphorescence iridium(III) complexes, Ir1–Ir4, were developed for mitochondria imaging in hypoxic tumor cells. The iridium(III) complex has two anthraquinone groups that are hypoxia-sensitive moieties. The phosphorescence of the iridium(III) complex was quenched by the functions of the intramolecular quinone unit, and it was restored through two-electron bioreduction under hypoxia. When the probes were reduced by reductase to hydroquinone derivative products under hypoxia, a significant en-

Introduction Mitochondria, which are cellular dynamic organelles bound by two distinct membranes, are associated with various vital cellular physiological processes, including energy metabolism, cell signaling, apoptosis, and cellular homeostasis.[1] In solid tumor cells, these processes are interdependent and may occur under various stress conditions, such as diffusion-limited hypoxia, low levels of nutrients, and increased levels of waste products, among which low oxygen levels (hypoxia) are prominent.[2] In many solid tumors, for example, the median oxygen concentration has been reported to be approximately 4 %, and locally may even decrease to 0 %.[3, 4] Tumor cells respond to hypoxic conditions and adapt their mitochondrial metabolism to adjust their O2 demand to meet the limited supply.[5, 6] Perhaps the most important aspect of how cells respond to this unique microenvironment is the activity of the hypoxia-inducible factor 1a (HIF-1a), which is an important player in tumorigenesis and tumor progression.[7] Activation of this factor is strictly bound to mitochondrial functions, which, in turn, are related to oxygen levels. Therefore, in hypoxia, mitochondria act as oxygen sensors and convey signals to HIF-1a directly or indirectly.[2] Designing probes to image and track mitochondria [a] L. Sun, Y. Chen, S. Kuang, G. Li, R. Guan, J. Liu, Prof. L. Ji, Prof. Dr. H. Chao MOE Key Laboratory of Bioinorganic and Synthetic Chemistry School of Chemistry and Chemical Engineering Sun Yat-Sen University, Guangzhou 510275 (P.R. China) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201600310. Chem. Eur. J. 2016, 22, 8955 – 8965

hancement in phosphorescence intensity was observed under one- (l = 405 nm) and two-photon (l = 720 nm) excitation, with a two-photon absorption cross section of 76– 153 GM at l = 720 nm. More importantly, these probes possessed excellent specificity for mitochondria, which allowed imaging and tracking of the mitochondrial morphological changes in a hypoxic environment over a long period of time. Moreover, the probes can visualize hypoxic mitochondria in 3D multicellular spheroids and living zebrafish through two-photon phosphorescence imaging.

under hypoxic environments might help in the development of new therapeutic and diagnostic strategies for cancer. Several organic dyes, such as rhodamine 123, MitoTracker Green, and MitoTracker Red, are used as commercial mitochondria imaging agents. However, these commercial probes can barely meet the requirements for hypoxia detection. In recent decades, a variety of hypoxia-sensitive fluorescent molecular probes have been developed to detect hypoxia in living cells.[8–12] However, most reported hypoxia probes are normally aimed at the cellular level, not the organelle level, and have also not been applied to two-photon microscopy (TPM).[13] The TPM technique, which offers a number of advantages over one-photon microscopy (OPM), including noninvasive excitation, increased penetration depth, localized excitation, and prolonged observation time, is growing in popularity among biologists.[14] More recently, new two-photon probes for hypoxia detection[15–19] or mitochondria imaging[20–24] have been developed. To the best of our knowledge, there are two widely used mechanisms in hypoxia probe detection. One optical method was shown to directly detect oxygen in cells and tissues through oxygen-dependent quenching of phosphorescence and phosphorescence lifetime imaging microscopy, in particular.[17, 25–29] The other mechanism for the design of hypoxia-sensitive imaging probes was by exploiting the bioreductive microenvironment of hypoxia, resulting in excess expression of nitro reductase (NTR), azo reductase, quinone reductase, and so forth.[30] Several hypoxia-sensitive probes, the luminescence intensity of which changes depending on bioreduction under hypoxia, have been developed by employing nitro groups,[31–33]

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Full Paper azo groups,[8, 10, 34] quinone groups,[19, 35] or QSY-21[9] as the hypoxia-sensing moiety. We demonstrated previously that some iridium(III) complexes possessed high specificity for mitochondria and could be used for mitochondrial imaging and tracking without damaging mitochondrial membranes.[22–24, 36] More importantly, iridium(III) complexes have been developed as two-photon probes.[36–45] Herein, we designed and synthesized a series of two-photon phosphorescent iridium(III) complexes for mitochondria imaging and tracking under hypoxia with high sensitivity and specificity. We introduced two anthraquinone groups as ancillary ligands to the iridium(III) complexes (Figure 1). An-

Figure 1. Detection mechanism and chemical structures of the phosphorescent IrIII complexes Ir1–Ir4 synthesized and evaluated herein.

thraquinone groups are good electron acceptors and have a quenching function for fluorescence. Under hypoxic conditions, anthraquinone groups can convert into the hydroquinone form through two-electron reduction coupled with protonation,[46] whereupon the intense fluorescence emission is restored. The phosphorescence emission of the iridium(III) complex is quenched by anthraquinone groups, but turns on under high reductive stress in hypoxic tumor tissues, providing mitochondria specificity with minimal background interference. Hence, we investigated the capability of probes for mitochondrial imaging and tracking under hypoxia in vitro and in vivo by using adherent tumor cells, 3D multicellular spheroids (MCSs), and living zebrafish. Our results showed that these new probes could specifically illuminate mitochondria and monitor mitochondrial morphological changes under hypoxia in living cells. Furthermore, they can conveniently visualize inner hypoxic mitochondria in 3D MCSs and living zebrafish through two-photon phosphorescent imaging.

Results and Discussion Design and synthesis Due to outstanding mitochondrial specificity and photochemical properties,[47–49] including large Stokes shifts (hundreds of nm), long luminescence lifetimes (100 ns to ms), and lower photobleaching, phosphorescent iridium(III) complexes have Chem. Eur. J. 2016, 22, 8955 – 8965

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emerged as typical candidates for living-cell imaging.[50–53] Quinones are a class of fully conjugated cyclic dione compounds. In electron-transport chains, they can function as electron carriers and undergo two-electron reduction and protonation, leading to the corresponding hydroquinones.[54, 55] In biological systems, quinones can be reduced by coenzyme nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) in the presence of cellular reductases, such as NADH ubiquinone oxidoreductase and NADPH cytochrome P-450 reductase.[56, 57] Moreover, quinone groups are good electron acceptors and can be efficient quenchers of the singlet-state donor fluorescence of numerous fluorophores.[46] Based on this, we thought that conjugating quinone moieties directly onto iridium(III) complexes would be useful for the development of probes for hypoxic mitochondria imaging. The C^N ligand 2-(benzo[d]thiazol-2-yl)anthracene-9,10dione (AqSN) was synthesized in good yield by the reduction of 2-formyl-9,10-anthraquinone and 2-aminobenzenethiol with the catalyst p-toluenesulfonic acid (PTSA) in DMF. The IrIII complexes were prepared by coordinating bridge splitting reactions of the binuclear precursor [Ir(C^N)2Cl]2 with the N^N ligands (2,2’-bipyridine (bpy), 1,10-phenanthroline (phen), 1,10phenanthrolinethiazole (PhenS), 1,10-phenanthrolineselenazole (PhenSe)). The complexes were then purified by aluminum oxide chromatography by using CH3CN/CH3CH2OH as an eluent. All complexes were characterized by elemental analysis, ESI-MS, and 1H and 13C NMR spectroscopy (Figures S1–S9 in the Supporting Information). The photophysical properties of Ir1– Ir4 were investigated by means of spectroscopic techniques (Figure S10 in the Supporting Information and Table 1). The four complexes exhibited favorable stability when exposed to visible light at l = 405 nm (Figure S11 in the Supporting Information).

Table 1. Photophysical properties of Ir1–Ir4 before and after reduction with rat liver microsomes.[a]

Ir1 Ir2 Ir3 Ir4

labs [nm]

Before reduction lPL [nm] Fem

After reduction lPL [nm] Fem d[b] [GM]

360 362 361 360

623 620 619 613

592 594 582 577

0.009 0.006 0.002 0.002

0.110 0.088 0.056 0.052

86 76 96 153

[a] All data were obtained in methanol at 298 K. [b] Maximum TPA cross section at l = 720 nm.

Response toward hypoxia To examine the hypoxia sensitivity of probes in solution, we conducted an enzymatic assay with rat liver microsomes, which contained diverse reductases. Without NADPH, upon excitation at l = 405 nm, no phosphorescence signal could be observed under normoxic (20 % O2) or hypoxic (15, 10, 5, and 1 % O2) conditions (Figure S12 in the Supporting Information). The results suggest that the photoluminescence of iridium(III) complexes cannot be affected by oxygen, which rules out the

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Full Paper possibility of oxygen quenching. After adding NADPH (50 mm) as a cofactor for the reductases to solutions of IrIII complexes (5 mm) in the presence of rat liver microsomes, a drastic enhancement of phosphorescence intensity was observed only under hypoxia (11-fold for Ir1, 16-fold for Ir2, 19-fold for Ir3 and 18-fold for Ir4; Figure 2 a and Figure S13 in the Supporting Information). As shown in Table 1, the phosphorescence spectral peaks of complexes are apparently blueshifted by 30 nm (from l = 623 to 592 nm for Ir1). Next, we studied the reactivity of the probes toward reductases with rat liver microsomes. After enzymatic reactions under hypoxic conditions, a rapid increase in phosphorescence intensity of the probes was observed, and the phosphorescence intensity reached a plateau within 15 min (Figure 2 b and Figure S14 in the Supporting Information). On the other hand, under normoxic conditions, no

phosphorescence increase occurred. These results indicated that Ir1–Ir4 could detect hypoxia selectively and rapidly. In addition, when oxygen was bubbled into the hypoxic reaction solution, no change was observed for the phosphorescence intensity of Ir1–Ir4. This suggested that the reduction product was insensitive to oxygen and could not be oxidized by oxygen (Figure S15 in the Supporting Information). The turn-on response of probes for hypoxia was tested over a wide array of possible competitive species, including reducing reagents, such as hypochlorite (ClO¢), sulfite (SO32¢), bisulfite (HSO3¢), thiosulfate (S2O32¢), nitrite (NO2¢), ascorbic acid, cysteine (Cys), homocysteine (Hcy), dithiothreitol (DTT), glutathione (GSH), I¢ , Fe2 + , and Cu + . In the test system, 100 equivalents of analytes were added to the solution of complexes (5 mm) and the response was monitored under hypoxia for over 60 min. As shown in Figure 3, the emission intensity of Ir1

Figure 3. Phosphorescence intensity of Ir1 (5 mm) under hypoxia upon the addition of NADPH (50 mm, red bar) in the presence of different background reducing ions or agents (500 mm) in potassium phosphate buffer (pH 7.4). The buffer contained rat liver microsomes (0.25 mg mL¢1). 1) control, 2) DTT, 3) ascorbic acid, 4) Cys, 5) Hcy, 6) GSH, 7) S2O32¢, 8) NO2¢ , 9) ClO¢ , 10) SO32¢, 11) HSO3¢ , 12) I¢ , 13) Fe2 + , and 14) Cu + ; lex/em = 405/590 nm.

Figure 2. a) Phosphorescence spectra of Ir1 (5 mm) treated under different conditions at 37 8C; incubation without NADPH for 30 min under normoxic (black line) or hypoxic (wine-colored line) conditions; incubation with NADPH (50 mm) for 30 min under normoxic conditions (blue line) or hypoxic conditions (red line); lex = 405 nm. Inset: photographs of a solution of Ir1 in the presence of rat liver microsomes and NADPH under normoxic (L) or hypoxic (R) conditions under excitation at l = 365 nm by a handheld UV lamp. b) Time-dependent changes in the phosphorescence intensity of Ir1 under hypoxia (red line) or normoxia (black line); NADPH (50 mm) was added as a cofactor for reductases at the point indicated by the arrow. Measurements were performed in potassium phosphate buffer (pH 7.4) containing rat liver microsomes (0.25 mg mL¢1). The hypoxic conditions were prepared by bubbling argon gas into the reaction solution for 30 min; lex/em = 405/590 nm. Chem. Eur. J. 2016, 22, 8955 – 8965

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did not increase significantly at high concentrations of these bioreducing agents. However, upon the addition of only 10.0 equivalents of NADPH to the mixture, a robust phosphorescence intensity enhancement was observed. Similar cases were observed for other probes (Figure S16 in the Supporting Information). These results suggested that the phosphorescence changes of the probes were only induced by reductase and NADPH under hypoxia, which could be used to detect hypoxia without interference from other biological reductants. Additionally, we studied the emission behavior of probes with or without NADPH under hypoxia in different pH environments. The pH titration curve (Figure S17 in the Supporting Information) also reveals that the phosphorescence “off–on” switch is operational within a pH range of 3–10, which demonstrates that the probes can work over a wide pH range without bias. It is reported that the anthraquinone group can be reduced to hydroanthraquinone by a two-electron reduction.[46] We fur-

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Figure 4. High-resolution mass spectra for Ir1 and its product after reduction.

ther confirmed the reduction product generated from Ir1–Ir4 under hypoxia by means of HRMS (Figure 4 and Figures S18– S20 in the Supporting Information). HRMS analysis was conducted by magnification of the iridium isotope signals. For example, the mass spectrum of the starting Ir1 complex, [C52H28N4O4S2Ir] + , showed a bivalence signal at m/z 1029.1, which corresponded to [Ir1¢Cl] + . After incubation with rat liver microsomes and NADPH under hypoxia, the reduction product was formed, as shown by the appearance of a signal at m/z 1033.2, which was calculated for [C52H32N4O4S2Ir] + . The HRMS results matched perfectly with the simulated spectra (Figure 4). These results confirmed our hypothesis of the anthraquinone reduction mechanism shown in Figure 1.

Two-photon absorption (TPA) cross sections The TPA properties of Ir1–Ir4 in the presence of rat liver microsomes and NADPH under hypoxic or normoxic conditions were investigated by using a two-photon-induced fluorescence method with rhodamine B as a reference (Figure 5). Under normoxia, almost no fluorescence signal was observed for twophoton excitation, whereas the emission spectra of the reduction product at l = 720 nm under hypoxia displayed similar emission spectra to those obtained under excitation at l = 360 nm for one-photon excitation (Figure S21 in the Supporting Information). The TPA cross sections of Ir1–Ir4 under hypoxic conditions were determined in the wavelength range from l = 700 to 870 nm. Over the measured range, the dmax value of Ir1–Ir4 under hypoxic conditions ranged from 76 to 153 GM (1 GM = 10¢50 cm4 photon¢1) at l = 720 nm (Table 1 and Figure 5 b). The output intensity of the two-photon-excited fluorescence was linearly dependent on the square of the input laser power (Figure S22 in the Supporting Information), thereby confirming nonlinear TPA. The TPA cross section of our probes is moderate compared with those of other iridium(III) Chem. Eur. J. 2016, 22, 8955 – 8965

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Figure 5. a) Emission spectra of Ir1–Ir4 under normoxic and hypoxic conditions (lex = 720). b) TPA cross sections of Ir1–Ir4 under hypoxia at different excitation wavelengths. The buffer contained rat liver microsomes (0.25 mg mL¢1) and 50 mm NADPH as a coenzyme.

complexes measured with a two-photon laser-induced phosphorescence method reported in the literature.[37–44]

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ing to relatively mild hypoxia, whereas that of Ir2–Ir4 increased at oxygen concentrations of 1 % or less.

To determine the potential of probes for imaging hypoxia in tumor cells, A549 cells were selected because they are known to express reductase.[19, 35] First, to evaluate the potential toxicity of the probes to the cells, a standard 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed under normoxia and hypoxia conditions. Considering that these complexes often produce singlet oxygen after photoirradiation due to triplet-state formation, the cell toxicity of the probes with photoirradiation under normoxia was also examined (Figure S23 in the Supporting Information). The results showed that cell viability was quite high (> 80 %), even after incubation with 10 mm probes at 37 8C for 12 h under normoxic (with or without photoirradiation) or hypoxic conditions; this indicated the low cytotoxicity and good biocompatibility of the probes under experiment conditions (5 mm for 30 min). In addition, photoirradiation did not have a clear effect on the cytotoxicity of the probes. We next applied these probes to living cells to monitor cellular hypoxia. In our experiments, cells were first grown at 37 8C under normoxic (20 % O2) and different hypoxic (15, 10, 5, and 1 % O2) conditions for 6 h and then incubated with probes (5 mm) for 30 min under the respective conditions. As shown in Figure 6, the one- and two-

Mitochondrial locations of Ir1–Ir4 We expect that the cationic charged iridium(III) complexes can accumulate in the mitochondria. Inductively coupled plasma (ICP) MS was carried out to verify our speculation. A549 cells were incubated with iridium(III) complexes (5 mm) under normoxic/hypoxic environments for 30 min, and the amount of iridium present in the mitochondria, nucleus, or cytoplasm (without mitochondria) was isolated and measured by ICP-MS. As shown in Figure 7, regardless of normoxia or hypoxia, the ma-

Figure 7. Subcellular distribution of iridium in A549 cell incubation with Ir1– Ir4 (5 mm) for 1 h under normoxic (left) and hypoxic (right) conditions. The amount of iridium in mitochondrial, cytoplasm, and nuclear fractions was measured by ICP-MS. Data were collected at least three times independently.

Figure 6. Confocal images of adherent A549 cells treated with probes (5 mm) under normoxic (20 % O2) and different hypoxic (15, 10, 5, and 1 % O2) conditions. lex = 405 nm (OPM)/720 nm (TPM); for Ir1 and Ir2, lem = (590 20) nm; for Ir3 and Ir4, lem = (580 20) nm. Scale bars: 10 mm.

photon phosphorescence signal increases with a decrease in the O2 concentration from 20 to 1 %, which implies that Ir1–Ir4 can indicate the extent of relative hypoxia in the cells. Diphenyleneiodonium chloride (DPI), an inhibitor of electron transport in a wide range of flavoproteins, including NADPH,[58, 59] can suppress phosphorescence enhancement in cells under hypoxia (Figure S24 in the Supporting Information). This result indicates that these probes are reduced by flavoproteins, which are responsible for the metabolic activation of bioreductive compounds under hypoxia.[60] Moreover, fluorescence microscopy imaging showed that Ir1–Ir4 had different sensitivities to oxygen concentrations: the phosphorescence intensity of Ir1 increased at oxygen concentrations of 5 % or less, correspondChem. Eur. J. 2016, 22, 8955 – 8965

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jority of iridium was localized and accumulated in the mitochondrial fraction of the cells. In addition, colocalization experiments were performed by costaining A549 cells with a commercially available mitochondrial dye, Mito-tracker Green. A549 cells incubated with complexes (5 mm) under normoxia do not show phosphorescence signals. However, when the cells were incubated under hypoxia, a significant one- and two-photon phosphorescence signal was observed. The OPM/TPM image and the image of Mito-tracker Green merged perfectly with the high Pearson’s correlation coefficient (0.90 for Ir1, 0.88 for Ir2, 0.91 for Ir3, and 0.90 for Ir4; Figure 8). The ICP-MS and colocalization results consistently showed that Ir1–Ir4 could specifically localize in the mitochondria of living cells. Tracking mitochondrial morphological changes under hypoxia Mitochondria form a highly dynamic tubular network, the morphology of which is regulated by frequent fission and fusion events. Mitochondrial morphology was thought to be associated with metabolic transition, apoptosis, mitophagy, and macroautophagy,[61] which are crucially involved in various pathologies, including cancer. Consequently, mitochondrion-tracking reagents and techniques are significant in biomedical research and diagnostic applications. Several organic molecular[62–64] and

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Figure 8. TPM images of A549 cells incubated with Ir1–Ir4 (5 mm) under hypoxic (1 % O2) conditions for 30 min at 37 8C, followed by 50 nm of Mitotracker Green. Column a: bright-field images; column b: images of cells costained with Mito-tracker Green; column c: OPM confocal phosphorescence images of Ir1–Ir4; column d: TPM confocal phosphorescence images of Ir1– Ir4; column e: overlay images of columns a–d; and column f: the overlap coefficient of columns b and d and Pearson’s colocalization coefficients. Ir1– Ir4: lex = 405 nm (OPM)/720 nm (TPM); Ir, Ir2: lem = (590 20) nm; Ir, Ir2: lem = (580 20) nm; Mito-tracker Green: lex/lem = (488 10)/(520 10) nm; scale bars: 10 mm.

iridium(III) complexes[22–24] have been developed in our laboratory to track mitochondrial morphological changes in living cells. Nevertheless, none of these reported probes can image the dynamic changes of mitochondria and detect hypoxia. In view of their high mitochondrial selectivity, herein, we employed Ir1–Ir4 to monitor the mitochondrial morphological change processes under hypoxia in A549 cells. In normoxic environments, the mitochondria in cells appear as network-like, linear, and typical tubular structures (Figure S25a and b in the Supporting Information). After incubation under hypoxia for 6 h, the mitochondria become clearly round (Figure S25c and d in the Supporting Information), which is consistent with previous results of mitochondrial hypoxic injury treated by a spe-

cial stimulation of CoCl2.[62] To further evaluate whether Ir1–Ir4 could be utilized to track mitochondrial morphological changes under hypoxia in detail, a series of videos were applied to record these processes. As shown in Figure 9 (Video S1 in the Supporting Information), the change in mitochondrial morphology induced by a membrane potential uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), can be observed in hypoxic A549 cells (1 % O2) treated with Ir1. The mitochondria are gradually transformed into small, dispersed fragments or even hollow spheres after treatment by CCCP, which is in agreement with morphology alternation of mitochondria during cell apoptosis. Similar phenomena were observed for Ir2–Ir4 (Figures S26–S28 and Videos S2–S4 in the Supporting Information). These results demonstrate that Ir1– Ir4 have excellent specificity to mitochondria and sensitivity to hypoxic microenvironments in living cells. Complexes Ir1–Ir4 represent potential candidates for mitochondria tracking agents under hypoxia. One- and two-photon imaging of hypoxic mitochondria in 3D MCSs Three-dimensional cell cultures, which include organotypic explants, 3D scaffolds, and MCSs, are thought to offer an improvement over monolayer culture because they preserve cell– cell and cell–extracellular matrix (ECM) interactions that affect cellular phenotype, gene expression, and a range of cellular functions.[65] The 3D MCSs, which are microscale, spherical cell clusters formed by self-assembly, are one of the most common and versatile methods of culturing cells in 3D.[66] One valuable characteristic of 3D MCSs is their diffusion limit of approximately 150–200 mm to many molecules, particularly oxygen.[66, 67] As a result of transport limitations, spheroids generally display gradients of oxygen at diameters between 300 and 500 mm with central hypoxia. Therefore, 3D MCSs are excellent models for hypoxia detection by generating a hypoxic environment without specialized hypoxic chambers. More recently, hypoxia detection probes were introduced for in vitro

Figure 9. Phosphorescent images of CCCP-treated (10 mm) living A549 cells under hypoxic (1 % O2) conditions stained with Ir1 (5 mm) with increasing scan time (the scan is time shown in the upper panel); lex = 405; lem = (590 20) nm. Chem. Eur. J. 2016, 22, 8955 – 8965

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Full Paper applications to analyze localized oxygen gradients in 3D tissue models.[19, 29, 34, 68] Another aim of the project was to visualize hypoxic mitochondria in 3D MCSs. A549 MCSs were incubated with Ir1 (5 mm) for 6 h, and then imaged under one- and two-photon confocal laser scanning microscopy. As shown in Figure 10 a,

(BDM), which could completely abolish cardiac contractility and resulted in cerebral anoxia.[69] It was reported that the reductases were expressed in zebrafish and tissue-specific distribution of reductases was observed with the high level present in the brains of zebrafish.[70] No phosphorescence signal could be observed in preincubated five-day-old zebrafish with 5 mm IrIII complexes for 1 h. After the introduction of BDM (15 mm), the phosphorescence intensity in the brain rapidly increased due to cerebral anoxia, and after another 6 min, the phosphorescence intensity was saturated (Figure 11 and Figure S33 in

Figure 11. Two-photon confocal images of the head of a living five-day-old zebrafish after incubation of Ir1 followed by adding BDM, as described in the text. The phosphorescence images were recorded under excitation of l = 720 nm with a 10 Õ objective lens.

Figure 10. a) OPM (left) and TPM (right) images of 3D MCSs incubated with Ir1 (5 mm) for 6 h; scale bars: 100 mm. b) Z-stack images of large 3D spheroids with intervals of 30 mm; lex = 405 nm (OPM)/720 nm (TPM), lem = (590 20) nm. The images were recorded under a 10 Õ objective lens.

the one- and two-photon phosphorescence signals were routinely detected throughout the central region of the spheroid and up to 100–150 mm from the outer edge of the spheroid, which was consistent with the limitation of oxygen diffusion in 3D MCSs.[67] The z-axis phosphorescent images can describe the phosphorescence intensity spatial distribution in 3D MCSs more clearly. These images of the spheroids showed much stronger phosphorescence in deep-layer cells by using twophoton excitation rather than one-photon excitation, which indicated the deeper penetration of two-photon excitation light (Figure 10 b). Similar results were observed with Ir2–Ir4 (Figures S29–S31 in the Supporting Information). In addition, complexes Ir1–Ir4 did not exhibit clear inhibition to the growth of the spheroids (Figure S32 in the Supporting Information). These results suggest that the probes are sensitive enough to distinguish the hypoxic core and illuminate the mitochondria of the hypoxia cells in the 3D MCSs. Two-photon imaging of hypoxic mitochondria in zebrafish Encouraged by the satisfactory response characteristics of the probes in vitro, we attempted to visualize the hypoxic mitochondria in the intact animal in vivo by using a cerebral anoxia model of the zebrafish. The zebrafish model of cerebral anoxia was prepared by adding 15 mm 2,3-butanedione monoxime Chem. Eur. J. 2016, 22, 8955 – 8965

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the Supporting Information). Moreover, the phosphorescence enhancement was only detected by two-photon excitation (Figure 12 and Figures S34–S36 in the Supporting Information), and we considered this observation to be due to the advantage of two-photon excitation in penetration depth. These results indicate that the developed Ir1–Ir4 complexes can be useful tools to image hypoxic mitochondria through twophoton excitation imaging not only in living cells, but also in living animals.

Figure 12. One- and two-photon confocal images of the head of a five-dayold zebrafish. The living zebrafish was incubated with Ir1 for 1 h, followed by adding BDM (15 mm). The excitation wavelengths for one and two photons were l = 405 and 720 nm, respectively. The images were recorded under a 10 Õ objective lens. BF = bright field.

Conclusion We developed four iridium(III) complex based two-photon phosphorescent mitochondrial-specific hypoxia probes. Owing

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Full Paper to quenching by anthraquinone groups, complexes Ir1–Ir4 showed almost no phosphorescence. Incubated with NADPH under hypoxia, the reductases activated the probes Ir1–Ir4 to release the phosphorescent hydroquinone derivatives, and the intensity of the emissions increased dramatically. More importantly, the reduction product showed a significant TPA cross section. These probes could distinguish hypoxic mitochondria in adherent cells, 3D MCSs, and living zebrafish. Most attractively, complexes Ir1–Ir4 have potential applications for realtime tracking of mitochondrial changes in morphology under hypoxic environments; this offers a new method to recognize mitochondria-related physiological and pathological changes, and possibly offers novel medical strategies for diagnosis. We anticipate that Ir1–Ir4 could be used as valuable tools to explore the response of organisms to hypoxia and mitochondria regulation effects under hypoxia in solid tumors, which is essential for the development of novel imaging and treatment modalities, with the promise of improving targeted cancer therapy and overcoming therapeutic resistance induced by hypoxia.

2-(Benzo[d]thiazol-2-yl)anthracene-9,10-dione (AqSN) PTSA (10 % mmol) was added to a mixture of 2-formyl-9,10-anthraquinone (0.236 g, 1.0 mmol) and 2-aminobenzenethiol (0.15 g, 1.2 mmol) in DMF (5 mL), and the reaction mixture was stirred at 100 8C in darkness overnight. After cooling to room temperature, a yellow–green precipitate was collected by vacuum filtration and washed with water and ethanol (0.21 g, 61.6 %). 1H NMR (400 MHz, CDCl3): d = 8.96 (s, 1 H), 8.63 (d, J = 8.0 Hz, 1 H), 8.47 (d, J = 8.0 Hz, 1 H), 8.42–8.35 (m, 2 H), 8.19 (d, J = 8.0 Hz, 1 H), 8.00 (d, J = 8.0 Hz, 1 H), 7.87 (dd, J = 5.2, 3.6 Hz, 2 H), 7.58 (t, J = 7.6 Hz, 1 H), 7.49 ppm (t, J = 7.6 Hz, 1 H); ESI-MS (CH3OH): m/z: 342.1 [M + H] + ; elemental analysis calcd (%) for C21H11NO2S: C 73.88, H 3.25, N 4.10; found: C 73.85, H 3.21, N 4.11.

Synthesis of the cyclometalated IrIII chlorobridged dimer The cyclometalated IrIII chlorobridged dimer [{Ir(AqSN)2Cl2}2] was synthesized in a similar manner to previously reported methods.[73, 74] A mixture of 2-ethoxyethanol and water (3:1, v/v) was added to a flask containing IrCl3 (0.5 mmol) and the C^N ligand (AqSN; 1.2 mmol). The mixture was heated at reflux for 24 h. After cooling, the dark-red precipitate was filtered to give the crude cyclometalated IrIII chlorobridged dimer.

Experimental Section General

Synthesis of [Ir(AqSN)2(bpy)]Cl (Ir1)

All reagents were purchased from commercial sources and used without further purification, unless otherwise specified. Milli-Q water was used throughout all experiments. IrCl3 (Alfa Aesar, USA), bpy (Sigma Aldrich, USA), phen (Sigma Aldrich, USA), MTT (Sigma Aldrich, USA), DPI (Sigma Aldrich, USA), and DMSO (Sigma Aldrich, USA) were used as received. The commercially available mitochondrial imaging agent MitoTrackerÒ Green FM was purchased from Invitrogen. The ligands PhenSe[71] and PhenS[72] were prepared according to previously reported methods. All IrIII complexes were dissolved in DMSO preceding the experiments; the calculated quantities of the IrIII complex solutions were then added to the appropriate medium to yield a final DMSO concentration of less than 1 % (v/v). A549 cells were purchased from the Committee on Type Culture Collection of Chinese Academy of Sciences.

A mixture of [{Ir(AqSN)2Cl2}2] (91 mg, 0.05 mmol) and bpy (23 mg, 0.15 mmol) in MeOH/CHCl3 (20 mL; 1:1, v/v) was heated at 68 8C under argon for 6 h. The reaction mixture was then cooled to room temperature and evaporated under reduced pressure. The crude product was purified by column chromatography on alumina by using CH3CN/CH3CH2OH (10:1, v/v) as the eluent (81 mg, 78.72 %). 1H NMR (400 MHz, [D6]DMSO): d = 8.94 (d, J = 8.4 Hz, 2 H), 8.68 (s, 2 H), 8.49 (d, J = 8.0 Hz, 2 H), 8.38 (t, J = 8.4 Hz, 2 H), 8.18 (d, J = 6.4 Hz, 4 H), 8.00–7.95 (m, 2 H), 7.87 (p, J = 7.2 Hz, 4 H), 7.79–7.73 (m, 2 H), 7.61 (t, J = 7.6 Hz, 2 H), 7.31 (t, J = 8.0 Hz, 2 H), 7.13 (s, 2 H), 6.20 ppm (d, J = 8.4 Hz, 2 H); 13C NMR (126 MHz, [D6]DMSO): d = 182.7, 182.5, 180.1, 159.2, 156.2, 151.6, 148.8, 146.3, 141.6, 135.1, 135.0, 133.6, 133.5, 133.3, 132.8, 130.4, 130.1, 130.0, 129.4, 128.7, 127.7, 127.1, 127.0, 125.8, 125.6, 124.7, 117.8, 55.2 ppm; ESI-MS (CH3OH): m/z: 1029.2 [M¢Cl] + ; elemental analysis calcd (%) for C52H28N4O4S2Ir: C 60.69, H 2.74, N 5.44; found: C 60.59, H 2.71, N 5.38.

The animal use protocol titled “Iridium(III) Antraquinone Complexes as Two-Photon Phosphorescence Probes for Mitochondria Imaging and Tracking under Hypoxia” has been reviewed and approved by the Animal Ethical and Welfare Committee (AEWC) of the SYSU.

Synthesis of [Ir(AqSN)2(phen)]Cl (Ir2) Physical measurements Microanalysis (C, H, and N) was performed by using a Perkin–Elmer 240Q elemental analyzer. 1H NMR spectra were recorded on a Bruker AVANCE III400 MHz NMR spectrometer and 13C NMR spectra were recorded on a 500 MHz superconducting Fourier transform NMR spectrometer (Varian, INOVA500NB) at room temperature. All chemical shifts are given relative to tetramethylsilane (TMS). ESI mass spectra were recorded on an LCQ system (FinniganMAT, USA). HRMS (ESI) results were recorded by using an ESI-QTOF maxis 4G (Bruker Daltonics) spectrometer. UV/Vis spectra were recorded on a Perkin–Elmer Lambda 850 spectrophotometer. Emission spectra were recorded on a Perkin–Elmer LS 55 spectrofluorophotometer at room temperature. Chem. Eur. J. 2016, 22, 8955 – 8965

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This complex was synthesized in a manner identical to that described for the preparation of Ir1, except that phen (27 mg, 0.15 mmol) was used instead of bpy. Yield: 88 mg, 83.57 %; 1H NMR (400 MHz, [D6]DMSO): d = 9.02 (d, J = 8.0 Hz, 1 H), 8.72 (s, 1 H), 8.57 (d, J = 4.4 Hz, 1 H), 8.40 (d, J = 8.0 Hz, 2 H), 8.20 (d, J = 7.2 Hz, 1 H), 8.10 (dd, J = 8.0, 5.2 Hz, 1 H), 8.02–7.95 (m, 1 H), 7.88 (p, J = 7.2 Hz, 2 H), 7.48 (t, J = 7.6 Hz, 1 H), 7.24 (s, 1 H), 7.06 (t, J = 7.6 Hz, 1 H), 5.84 ppm (d, J = 8.4 Hz, 1 H); 13C NMR (126 MHz, [D6]DMSO): d = 182.8, 182.6, 180.3, 158.6, 152.7, 148.8, 146.9, 146.6, 140.6, 135.1, 135.0, 133.6, 133.5, 133.3, 132.7, 131.4, 130.8, 130.2, 129.4, 129.1, 129.0, 128.7, 128.2, 127.5, 127.1, 127.0, 125.8, 125.7, 124.7, 117.4, 55.4 ppm; ESI-MS (CH3OH): m/z: 1053.9 [M¢Cl] + ; elemental analysis calcd (%) for C54H28N4O4S2Ir: C 61.58, H 2.68, N 5.32; found: C 61.51, H 2.65, N 5.38.

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Full Paper Synthesis of [Ir(AqSN)2(PhenS)]Cl (Ir3) This complex was synthesized in a manner identical to that described for the preparation of Ir1, except that PhenS (36 mg, 0.15 mmol) was used instead of bpy. Yield: 93 mg, 83.71 %; 1H NMR (400 MHz, [D6]DMSO): d = 9.40 (d, J = 6.8 Hz, 2 H), 8.72 (s, 2 H), 8.62 (d, J = 4.0 Hz, 2 H), 8.42 (d, J = 8.0 Hz, 2 H), 8.23–8.16 (m, 4 H), 7.99 (d, J = 9.2 Hz, 2 H), 7.93–7.84 (m, 4 H), 7.51 (t, J = 7.6 Hz, 2 H), 7.18 (s, 2 H), 7.14 (t, J = 7.6 Hz, 2 H), 6.05 ppm (d, J = 8.4 Hz, 2 H); 13C NMR (126 MHz, [D6]DMSO): d = 182.7, 182.5, 180.3, 158.1, 153.5, 151.1, 149.5, 148.9, 146.5, 137.0, 135.1, 135.0, 133.7, 133.5, 133.3, 132.7, 130.6, 130.3, 129.9, 129.5, 129.4, 128.7, 127.5, 127.1, 127.0, 126.1, 125.6, 124.7, 118.1, 55.2 ppm; ESI-MS (CH3OH): m/z: 1110.9 [M¢Cl] + ; elemental analysis calcd (%) for C54H26N6O4S3Ir: C 58.37, H 2.36, N 7.56; found: C 58.30, H 2.41, N 7.58.

Synthesis of [Ir(AqSN)2(PhenSe)]Cl (Ir4) This complex was synthesized in a manner identical to that described for the preparation of Ir1, except that PhenSe (43 mg, 0.15 mmol) was used instead of bpy. Yield: 90 mg, 77.72 %; 1H NMR (400 MHz, [D6]DMSO): d = 9.40 (d, J = 6.8 Hz, 2 H), 8.72 (s, 2 H), 8.62 (d, J = 4.0 Hz, 2 H), 8.42 (d, J = 8.0 Hz, 2 H), 8.23–8.16 (m, 4 H), 7.99 (d, J = 9.2 Hz, 2 H), 7.93–7.84 (m, 4 H), 7.51 (t, J = 7.6 Hz, 2 H), 7.18 (s, 2 H), 7.14 (t, J = 7.6 Hz, 2 H), 6.05 ppm (d, J = 8.4 Hz, 2 H); 13C NMR (126 MHz, [D6]DMSO): d = 182.7, 182.5, 180.1, 158.3, 154.5, 152.9, 149.8, 148.9, 146.5, 137.4, 135.1, 135.0, 133.7, 133.5, 133.3, 132.7, 130.6, 130.3, 129.8, 129.5, 129.4, 128.7, 128.3, 127.5, 127.1, 127.0, 125.8, 125.5, 124.7, 118.1, 55.2 ppm;. ESI-MS (CH3OH): m/z: 1158.9 [M¢Cl] + ; elemental analysis calcd (%) for C54H26N6O4S2SeIr: C 56.00, H 2.26, N 7.26; found: C 56.09, H 2.21, N 7.30.

Determination of TPA cross sections The TPA spectra of probes were determined over a broad spectral region by the typical two-photon laser-induced fluorescence method.[75] Two-photon fluorescence measurements were performed in fluorometric quartz cuvettes at a concentration of 20 mm in CH3OH at 298 K. The experimental fluorescence excitation and detection conditions were conducted with negligible reabsorption processes. The TPA cross section of the probes was calculated at each wavelength according to Equation (1)

C I n

ð1Þ

d2 ¼ d1 12 C12 I21 n21

in which d is the TPA cross section, F is the quantum yield, c is the concentration, I is the integrated fluorescence intensity, and n is the refractive index. Subscript 1 stands for reference and 2 stands for sample. In our experiments, we ensured that the excitation flux and excitation wavelengths were the same for both the sample and reference. Rhodamine B was utilized as the reference to measure the TPA cross sections, d, of iridium complexes.[76]

In vitro enzyme assay Microsomes were prepared according to the method reported by Omura and Sato.[77] Rats (Wistar, male, 6–7 weeks) fasted overnight and were sacrificed by an intraperitoneal injection of 80 mg kg¢1 pentobarbital sodium. The liver was diluted with 0.1 m potassium phosphate buffer at pH 7.4 for an assay and the final concentration was 0.25 mg mL¢1. Argon gas bubbled into the solution of probes (5 mm) for 30 min to create the hypoxic environment. Rat liver microsomes and 50 mm NADPH were added and incubated at 37 8C Chem. Eur. J. 2016, 22, 8955 – 8965

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for 30 min. The emission intensity was collected from l = 450 to 800 nm with excitation at l = 405 nm. The time-dependent phosphorescence intensity change for Ir1, shown in Figure 2, under normoxia and hypoxia was collected at l = 590 nm (l = 590 nm for Ir2, l = 580 nm for Ir3 and Ir4). The reaction mixture after the in vitro enzyme assay was extracted by chloroform and analyzed by HRMS.

Cellular uptake and distribution (ICP-MS) To quantify the levels of iridium in different subcellular compartments, ICP-MS was employed, as described in our previous work.[78] Exponentially growing A549 cells were cultured in 25 cm2 culture plates (Corning) under normoxic (20 % O2) or hypoxic (1 % O2) environments and treated with the iridium(III) complexes at a concentration of 5 mm for 1 h. After digestion, A549 cells were counted and divided into two equal parts for the extraction of the nuclei and cytoplasm by using a nucleus extraction kit and a mitochondria extraction kit (Pierce, Thermo), respectively. All samples were digested by 60 % HNO3 at room temperature for 24 h. Each sample was diluted with Milli-Q water to achieve a final volume of 10 mL containing 3 % HNO3. The concentration of iridium in the three domains was determined by using an inductively coupled plasma mass spectrometer (Thermo Elemental Co., Ltd.). Data were reported as the means standard deviation (n = 3).

Generation of 3D multicellular tumor spheroids The 3D MCSs were formed in agarose-coated 96-well plates, as previously described.[79] Briefly, A549 cells in the exponential growth phase were dissociated by a solution of trypsin/ethylenediaminetetraacetic acid (EDTA) to gain single-cell suspensions. A 2.5 Õ 104 cells mL¢1 A549 single cell suspension (150 mL) was seeded onto agarose-coated (sterile, 0.75 % (w/v) in PBS) 96-well imaging plates. The cells were incubated in a 37 8C humidified incubator for 96 h without motion, resulting in the formation of single spheroids with diameters of 400–500 mm per well.

One- and two-photon cellular imaging The cells were seeded at a density of 2 Õ 106 cells mL¢1 in Dulbecco’s modified Eagle medium (DMEM) and cultured in an incubator (37 8C, 5 % CO2 and 20 % O2). The A549 cells were seeded on 35 mm glass-bottomed dishes and cultured overnight before the assay. For phosphorescence microscopy, the cells were incubated under normoxic (20 % O2) and hypoxic (15, 10, 5, and 1 % O2) conditions for 6 h at 37 8C. The cells were further incubated with 5 mm probes in DMEM for 30 min under the same conditions as those used for preincubation and incubated with Mito-tracker Green for another 30 min. When necessary, the hypoxic cells were incubated with 10 mm CCCP for 30 min, and then treated with 5 mm of complexes for another 30 min. Cell images were captured on a Zeiss LSM 710 NLO confocal microscope (63 Õ oil immersion objective) and analyzed by using Axio Vision 4.2 software (Carl Zeiss). MCSs (400–500 mm in diameter) were treated with 5 mm probes for 6 h to make sure the complexes penetrated throughout the spheroids. The cell spheroids were gently washed twice with phosphate buffer and imaged with confocal laser scanning microscopy (10 Õ /NA 0.40 air objective lens). For one-photon images, the excitation wavelength of the laser was l = 405 nm. For two-photon images, the excitation wavelength of the laser was l = 720 nm.

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Full Paper Reductase inhibition assay with DPI DPI was dissolved in Milli-Q water to make a 20 mm stock solution. A549 cells in the presence or absence of DPI (100 mm) were incubated under hypoxia (1 % O2 concentration) for 6 h. Next, the cells were washed with phosphate-buffered saline (PBS; pH 7.4) and treated with 5 mm complexes for 30 min. After careful washing three times with PBS, the cells were subjected to phosphorescence imaging measurements.

Cell viability assay The measurement of cell viability toward adherent cells was studied by MTT assay. Briefly, A549 cells were seeded in 96-well microplates (Corning) at a density of 1 Õ 105 cells mL¢1 in medium (100 mL) containing 10 % fetal bovine serum (FBS). After 24 h of cell attachment, cells were cultured in the medium with indicated concentrations of Ir1–Ir4 and incubated for 12 h in normoxia and hypoxia (1 % O2) incubators. For cell toxicity measurements of the probes with photoirradiation under normoxia, the cells were irradiated by a light-emitting diode (LED) area light source (l = 405 nm, 20 mW cm¢2) for 10 min. Cells in the culture medium without complexes were used as controls. Four replicate wells were used for each control and test concentration. Then, the medium was replaced with fresh DMEM, and stock MTT dye solution (20 mL, 5 mg mL¢1) was added to each well and incubated for another 4 h. The medium containing MTT was carefully removed and DMSO (200 mL) was added to dissolve the formazan crystals generated in the living cells. The optical density of each well was then measured on a microplate spectrophotometer at l = 595 nm. The cytotoxicity of the probes towards the MCSs was evaluated by using a cell spheroid growth inhibition assay.[80] The MCSs with diameters of 400–500 mm were incubated with 5 mm IrIII complexes for 24 h. As an indication of proliferation, the diameters of the MCSs were measured with an inverted fluorescence microscope (Zeiss) after 6, 12, and 24 h of incubation.

Hypoxic mitochondria imaging of zebrafish Zebrafish were raised and kept under standard laboratory conditions at 28.5 8C. Zebrafish embryos were obtained from spontaneous spawning and maintained in E3 embryo media. In phosphorescence imaging experiments, zebrafish grown for 5 days were incubated with 5 mm of Ir1–Ir4 in E3 embryo media for 1 h at 28 8C, and then washed with PBS (pH 7.4) to remove the remaining complexes. The zebrafish were further incubated with 15 mm BDM, which resulted in cerebral anoxia. Phosphorescence imaging experiments were performed on a Zeiss LSM 710 NLO confocal microscope (10 Õ objective) for excitation at l = 405 (one-photon images) and 720 nm (two-photon images). E3 medium: 15 mm NaCl, 0.5 mm KCl, 1 mm MgSO4, 1 mm CaCl2, 0.15 mm KH2PO4, 0.05 mm NaH2PO4, 0.7 mm NaHCO3, 10¢5 % methylene blue; pH 7.5.

Acknowledgements This work was supported by the 973 Program (no. 2015CB856301) and the National Science Foundation of China (nos. 21171177, 21471164, and 21525105). Keywords: biological activity · hypoxia · imaging agents · iridium · photochemistry Chem. Eur. J. 2016, 22, 8955 – 8965

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