Phosphorescence Lifetime Imaging of Labile Zn2+ in Mitochondria via

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Phosphorescence Lifetime Imaging of Labile Zn2+ in Mitochondria via a Phosphorescent Iridium(III) Complex Changli Zhang,*,† Minsheng Liu,† Shaoxian Liu,† Hui Yang,† Qiang Zhao,‡ Zhipeng Liu,*,§ and Weijiang He*,∥ †

School of Environmental Science, Nanjing Xiaozhuang College, Nanjing 211171, PR China Key Laboratory for Organic Electronics and Information Displays and Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing University of Posts and Telecommunications (NUPT), Nanjing 210023, PR China § Institute of Advanced Materials (IAM), Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing Tech University (NanjingTech), Nanjing 211816, PR China ∥ State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, PR China

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ABSTRACT: Phosphorescence lifetime Zn2+ imaging possesses the advantage over normal fluorescence imaging in offering the more accurate temporal-spatial Zn2+ information. Herein, we report a new phosphorescent cyclometalated Ir(III) complex with a Zn2+-chelator bearing 1,10phenanthrolin acting as ancillary ligand, Zin-IrDPA, which displays the specific Zn2+-induced enhancement of phosphorescence and phosphorescence lifetime, and the mitochondria-targeting ability. Moreover, its Zn2+-induced phosphorescence lifetime enhancement factor is not affected by medium lipophilicity, viscosity, polarity, and especially ambient oxygen. The reversible tracking of introduced exogenous labile Zn2+ in MCF-7 and HeLa cells via phosphorescence imaging and phosphorescence lifetime imaging (PLIM) have been realized with Zin-IrDPA. Moreover, PLIM with Zin-IrDPA is able to track the SNOC-stimulated endogenous Zn2+ release in mitochondria.

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INTRODUCTION Similar to fluorescence lifetime imaging (FLIM), phosphorescence lifetime imaging (PLIM) is able to visualize the lifetime alteration of sensor luminescence in the presence of analyte, and the temporal−spatial information on analyte in living specimens can be determined avoiding the artifacts induced by light scattering and sensor concentration.1−3 This made PLIM attract great interest in recent years, with PLIM shows still the advantage of avoiding the interference from the short-lived intracellular autofluorescence via time-gated acquisition. Therefore, the accurate measurement for specific analyte is more feasible with PLIM. Various PLIM-based sensors have been developed to detect metal ions (e.g., Ca2+,4,5 Cu2+,6 and Hg2+),7 viscosity,8,9 oxygen,10 and pH11 in living samples. As one of the most important transition-metal ion in the human body, Zn2+ is closely involved in various biological processes including cellular metabolism, gene expression, apoptosis, and neurotransmission.12,13 Zn2+ disorder has also been associated with neurodegenerative diseases such as cerebral ischemia and Alzheimer’s disease.14 Fluorescent imaging of Zn2+ has demonstrated great success to clarify Zn2+ functions. A great number of fluorescent Zn2+ sensors © XXXX American Chemical Society

have been reported, and many have been successfully applied in tracking labile Zn2+ in intact zebrafish embryos,15,16 mapping zinc fluxes in the mammalian egg,17 monitoring labile Zn2+ release in rat hippocampus,18 sensing oxidative stress triggering Zn2+ release in mitochondria.19 However, even with the help of ratiometric and/or NIR imaging, the imaging interference induced by autofluorescence, photobleaching, light scattering, and fluctuation of local sensor concentration cannot be eliminated efficiently. PLIM of Zn2+ has been proved to be a potential way to overcome this obstacle. A few of PLIM Zn2+ sensors based on iridium and terbium complexes have been reported to show the promising Zn2+ imaging ability.20−23 This inspired us to explore PLIM Zn2+ sensor offering specific imaging ability such as the specific organelle targeting ability, since labile Zn2+ has been found to act as one of the essential signaling species to regulate the physiological processes of different organelles.24 Herein, we report a new phosphorescence sensor, ZinIrDPA (Figure 1), for phosphorescence lifetime Zn2+ imaging Received: May 9, 2018

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DOI: 10.1021/acs.inorgchem.8b01272 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

Figure 1. Synthesis of Zin-IrDPA and the proposed Zn2+ sensing mechanism.

in mitochondria in living cells. In Zin-IrDPA, the phosphorescent cyclometalated iridium(III) complex was selected as chromophore because of its promising photophyscial properties including long lifetime (typically several microseconds), high phosphorescence quantum yield and the adjustable absorption wavelength.25 However, bis(pyridin-2-ylmethyl)amine (DPA) was integrated into its ancillary ligand due to its fine Zn2+ binding ability and reliability as Zn2+ chelator in many reported Zn2+ sensors.26 In this sensor, the photoinduced electron transfer (PeT) from DPA to the excited iridium species can be expected because of the higher oxidation potential of DPA,21 which would effectively decrease phosphorescence intensity and lifetime of the integrated cyclometalated iridium(III) complex. Therefore, Zn2+ binding to DPA would reduce the electron-donating ability of DPA and block the PeT process, and the Zn 2+ -triggered phosphorescence “turn-on” and lifetime increase can be expected. In fact, this new sensor shows not only Zn2+-specific enhancement of phosphorescence and phosphorescence lifetime but also the distinct mitochondria-targeting ability. The oxygen-independent Zn2+-induced lifetime enhancement factor provides this sensor the advantage over normal phosphorescent metal complex-based sensors. The oxidative stressinduced the endogenous mitochondrial Zn2+ releases has also been effectively visualized by PLIM with this new sensor.

Figure 2. (a) Emission spectra of Zin-IrDPA (10 μM) in PBS buffer (10 mM, pH 7.4, containing 1% C2H5OH) titrated by Zn(NO3)2 (1.25 mM) solution. Inset is the titration profile according to F/F0 at 580 nm. (b) Phosphorescence decay of Zin-IrDPA (10 μM) in same medium upon Zn(NO3)2 titration (1.25 mM) determined with nanosecond pulsed photoexcitation at 370 nm. Inset is photoluminescence lifetime of Zin-IrDPA calculated upon Zn(NO3)2 (1.25 mM) titration. (c and d) Time-resolved fluorescence spectra of ZinIrDPA (c) and Zin-IrDPA/Zn 2+ (1:1) (d). The time of phosphorescence reaching its maximum is set as 0 ns.

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RESULTS AND DISCUSSION Phosphorescent Behavior and Zn2+ Response of ZinIrDPA. Zin-IrDPA was prepared from intermediate Ir(III) complex 2, which was synthesized from the corresponding chloride-bridged dimer [(ppy)2Ir(μ-Cl)2(ppy)2] by reacting with DPA (Figure 1). This complex has been characterized with 1H and 13C NMR spectroscopies, as well as mass spectrometry. The phosphorescence spectrum of Zin-IrDPA in PBS (10 mM, pH 7.4, containing 1% C2H5OH) exhibits a phosphorescence band centered at 590 nm with an excitation maximum at 372 nm (Figure S1). Phosphorescence titration of ZinIrDPA in ambient condition by Zn2+ addition demonstrates a distinct emission enhancement with an enhancement factor of 1.8 at 580 nm, accompanied by an emission maximum blueshift from 590 to 580 nm (Figure 2a). In the meanwhile, the

quantum yield of Zn2+/Zin-IrDPA complex (Zn/S complex, S, Zin-IrDPA) was determined as 7.1%, which is 1.5-fold as that of Zin-IrDPA (4.7%). The blue-shifted emission and “turn-on” response is ascribed to the Zn2+ coordination-triggered amidoto-iminol tautomerization that inhibits PeT progress from DPA to Ir(III) center.27 The titration profile based on the emission intensity at 580 nm shows that the phosphorescence increases linearly with [Zn2+]total until the [Zn2+]total/[ZinIrDPA] ratio reaches 1:1. Even higher [Zn2+]total does not lead to any further evident change, suggesting a 1:1 stoichiometry B

DOI: 10.1021/acs.inorgchem.8b01272 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 3. Histogram of F/F0 (a) and τ/τ0 (b) of Zin-IrDPA (10 μM) according to the emission at 580 nm upon the addition of 1 equiv of transition metal cations, Ba2+ and Al3+, or 1000 equiv of Na+, K+, Ca2+, and Mg2+ in PBS buffer (10 mM, pH 7.4, containing 1% C2H5OH). Gray and cyan bars represent F/F0 or τ/τ0 values of free sensor and sensor in the presence of 1 equiv of Zn2+, respectively. Black bars represent the F/F0 or τ/τ0 values of sensor in the presence of 1 equiv of Cd2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Cu+, Ba2+, Al3+, Mn2+, or 1000 equiv of Na+, K+, Ca2+, and Mg2+. Red bars represent the F/F0 or τ/τ0 values of sensor determined after adding the indicated metal ions followed by addition of 1 equiv of Zn2+. λex = 370 nm. (c) Histogram of τZn/S/τZin‑IrDPA of Zin-IrDPA (S, 10 μM) in different aliphatic alcohol solvents: 1, methanol; 2, ethanol; 3, npropanol; 4, n-butanol; 5, n-hexanol; and 6, n-octanol. Zn/S complex, zinc complex of Zin-IrDPA. (d) Histogram of τZn/S/τZin‑IrDPA of Zin-IrDPA (S, 10 μM) in glycerol/water binary solvents of different glycerol content: 1, 20%; 2, 40%; 3, 50%; 4, 60%; 5, 80% (v/v). . (e and f) Histograms of τZn/S/τZin‑IrDPA (e) and FZn/S/FZin‑IrDPA (f) of Zin-IrDPA (S, 10 μM) in water solutions saturated with O2/Ar atmosphere of different oxygen content: 1, 0%; 2, 5%; 3, 10%;4, 15%; 5, 20% (v/v). All data were based on the emission at 580 nm upon excitation at 370 nm. Buffer, PBS buffer.

for Zn2+ binding of Zin-IrDPA. The result obtained from phosphorescence Job’s plot confirms also the 1:1 Zn2+ binding stoichiometry (Figure S2). The dissociation constant of Zn/S complex was calculated to be 1.9 × 10−6 M according to the phosphorescence titration profile at 580 nm (Figure S3). Phosphorescence decay of Zin-IrDPA at 580 nm with nanosecond pulsed excitation at 370 nm was studied in PBS solution (Figure 2b). The average phosphorescence lifetime of free Zin-IrDPA was determined to be 66.4 ns, which is contributed by two lifetimes of 59 and 101 ns. Zn2+ titration (0−1 equiv) discloses the distinct linear increment of lifetime from 66.4 to 123.5 ns. The subsequent Zn2+ addition (from 1.0 to 1.6 equiv) resulted in the stable lifetime of Zn/S complex, which is consistent with the titration profile based on the phosphorescence intensity at 580 nm. Moreover, the phosphorescence “turn-on” response and the lifetime enhancement of Zin-IrDPA observed upon Zn2+ titration was also studied via time-resolved emission spectra (TRES) (Figure 2c,d). For free Zin-IrDPA, its phosphorescence emission decays subsequently after reaching to its emission intensity maximum, and the intensity remained only 10% after 150 ns in

comparison with its emission maximum (Figure 2c). Upon Zn2+ binding, Zin-IrDPA shows an enhanced emission maximum when compared with that of free sensor, and an enhancement factor of 1.6-fold was observed. After 150 ns, the subsequent decay makes the remained intensity be only 25% of its maximum, showing the phosphorescence decay upon Zn2+ binding is distinctly slower than that of free sensor (Figure 2d). On the basis of the above results, the Zn2+-triggered distinct increment of phosphorescence lifetime and intensity make Zin-IrDPA have the great potential for Zn2+ imaging via the time-resolved photoluminescence technique (TRPT).28 The Zn2+ sensing details of Zin-IrDPA were further investigated via UV−vis and ESI mass spectra. The UV−vis spectrum of Zin-IrDPA demonstrates several intense absorption bands at 220−290 nm (ε = 3.6 × 104 to 5.4 × 104 M−1 cm−1) and the minor absorption bands at 370−450 nm (ε < 0.7 × 104 M−1 cm−1) (Figure S4). The high energy bands could be assigned as the spin-allowed interligand LC (π → π*) transitions, while the minor bands are due to the mixed singlet and triplet metal-to-ligand charge-transfer (1MLCT and 3 MLCT).29 When Zn2+ was added, the clear isosbestic points C

DOI: 10.1021/acs.inorgchem.8b01272 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 4. (a−d) Confocal phosphorescence images of MCF-7 cells stained by 50 nM MitoTracker Deep Red 633 and 10 μM Zin-IrDPA: (a) phosphorescence image of the cells obtained from Zin-IrDPA channel (λex, 405 nm, λobs, 500−650 nm); (b) fluorescence image of cells in (a) obtained from MitoTracker channel (λex, 633 nm, λobs, 650−720 nm); (c) overlay image of (a) and (b); (d) bright-field image of cells in (a−c). (e−h) Phosphorescence lifetime images of MCF-7 cells: (e) image of MCF-7 cells stained via 30 min of Zin-IrDPA (10 μM) incubation at room temperature; (f) image of the stained MCF-7 cells in (e) underwent subsequent incubation with 10 μM Zn(NO3)2/pyrithione (1:1) solution followed by rinse with 10 μM Zin-IrDPA solution; (g) image of the Zn2+-incubated cells in (f) treated further with 25 μM TPEN. (h) Histogram of the average phosphorescent lifetime corresponding to images shown in (e−g): S, average lifetime of cells in (e); Zn2+, average lifetime of cells in (f); TPEN, average lifetime of cells in (g).

Co2+, Ni2+, and Cu2+ (1 equiv) only leads to minor decrement of the Zn2+-induced lifetime enhancement factor. This suggests that lifetime sensing for Zn2+ might be a more reliable sensing mode. Zin-IrDPA displays the stable emission from pH 4.0 to 10.0, although the minor decrease can be observed at 4 < pH > 10.27 Moreover, its Zn2+ complex at 580 nm displays also the stable emission at pH 7.0−10.0, whereas the medium pH lower than 7.0 induces the distinct emission decrease due to the Zn2+ dissociation from DPA ligand (Figure S6).30 All these results indicate that Zin-IrDPA is able to sense Zn2+ specifically in physiological conditions. Medium-Independence of Lifetime Zn2+ Sensing Ability of Zin-IrDPA. Since phosphorescence lifetime is normally sensitive to microenvironment factors such as medium polarity, lipophilicity, viscosity (rigidity), and molecular oxygen, the influence of these factors on the Zn2+-induced lifetime enhancement factor of Zin-IrDPA was investigated. The lipophilicity and polarity influence was determined in aliphatic alcohols with different n-alkyl chains (C1−C4, C6, and C8 chains). Although phosphorescence lifetimes of Zin-IrDPA and its zinc complex (formed in situ by mixing Zin-IrDPA and Zn2+ in a ratio of 1:1) increase with the alkyl chain length of solvents (Figure S7), the Zn2+-induced lifetime enhancement factors, τZn/S/τZin‑IrDPA, are almost identical in these alcohols (Figure 3c). This result indicates that the medium lipophilicity and polarity shows no distinct influence on the Zn2+-induced lifetime enhancement factor. The medium rigidity influence was determined in glycerol/water binary solvents with different glycerol content (20−80%) to regulate medium viscosity. The result in these solvents of different viscosity discloses only negligible difference in the related Zn2+-induced lifetime enhancement factors (Figures 3d and S8), implying the lifetime Zn2+ sensing ability of Zin-IrDPA show no depend-

at 245 and 231 nm imply the conversion of free Zin-IrDPA to a single Zn2+-complex. The linear decrease of the absorption at 261 nm was observed until the [Zn2+]total/[Zin-IrDPA] ratio attains to 1:1. This 1:1 Zn2+ binding mode of the Zn/S complex was confirmed by the mass spectrum of Zn/S complex in methanol. The main signal at m/z 1033.58 was assigned as [Zn2+/Zin-IrDPA-H + H2O + OH]+ (Figure S5).26 Phosphorescent Sensing Selectivity of Zin-IrDPA. The sensing selectivity of Zin-IrDPA toward Zn2+ was evaluated by adding 1 equiv of various metal ions including Cd2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Cu+, Ba2+, Al3+, and Mn2+, respectively. As shown in Figure 3a, Cd2+ also leads to an emission enhancement with a factor of 1.3-fold besides Zn2+ triggering the emission enhancement. The addition of other metal cations does not increase the emission intensity of Zin-IrDPA, and the distinct emission decrease were found upon the addition of Fe2+, Cu2+, and Co2+. Moreover, the Zn2+-induced emission enhancement is almost stable even in the presence of 1 equiv of Cd2+, Fe3+, Cu+, Ba2+, Al3+, and Mn2+. In contrast, K+, Na+, Ca2+, and Mg2+, which are abundant in cells, do not affect the sensor’s Zn2+ response even when the [Mn+]/[Zn2+] ratio attains 1000:1. It was observed that 1 equiv of Fe2+, Co2+, Ni2+, or Cu2+ decreases the Zn2+-induced emission enhancement distinctly especially in the case of Co2+, Ni2+, and Cu2+, yet the scarcity of intracellular Co2+ and the intracellular level of labile Fe2+, Cu2+, and Ni2+ being distinctly lower than labile Zn2+ makes them not interfere with the sensor’s Zn2+ sensing ability. Investigation on the Zn2+-induced lifetime response (Figure 3b) discloses that the Zn2+-induced lifetime enhancement factor is almost stable in the presence of other metal cations (1000 equiv of Na+, K+, Ca2+, and Mg2+, and 1 equiv of Cd2+, Fe2+, Fe3+, Cu+, Ba2+, and Al3+). Even the presence of D

DOI: 10.1021/acs.inorgchem.8b01272 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 5. Phosphorescence lifetime imaging for labile Zn2+ in MCF-7 cells stimulated by 10 mM SNOC. (a) Image of MCF-7 cells after incubation with 10 μM Zin-IrDPA at room temperature for 30 min; (b) image for the stained MCF-7 cells exposed to 10 mM SNOC solution for 20 min; (c) image for the SNOC-stimulated MCF-7 cells treated further with 50 μM TPEN solution. (d) Histogram of average phosphorescence lifetime in the cells shown in (a−c): S, average lifetime of cells in (a); SNOC, average lifetime of cells in (b); TPEN, average lifetime of cells in (c).

monitor the mitochondrial [Zn2+] fluctuation induced by exogenous Zn2+ incubation. Since the mitochondria are oxygen-rich organelles, the oxygen-independent Zn2+-induced lifetime enhancement factor of Zin-IrDPA is especially important for mitochondrial Zn2+ imaging. In fact, Zin-IrDPA shows the excellent lifetime imaging ability for mitochondrial labile Zn2+ (Figures 4e−h and S10h−k). As shown in Figure 4e, the cells stained by ZinIrDPA display the faint green color in mitochondria in the lifetime image, and the average phosphorescence lifetime (τp) is about 628 ± 6 ns, which is distinctly longer than that in aqueous solution.32 After exogenous Zn2+ introduction, the cells appear as mainly yellow in mitochondria, and τp is estimated as ∼ 722 ± 3.9 ns, indicating the enhanced labile Zn2+ level (Figure 4f). The following incubation with TPEN recovers the green color in lifetime image with an average τp of 561 ± 4.5 ns. The distinct decrease of τp in Figure 4g confirms that the phosphorescence lifetime enhancement in cells upon Zn2+ incubation is resulted from Zn2+ binding of Zin-IrDPA. The lifetime enhancement factor (setting lifetime after TPEN treatment as the original lifetime of this sensor in mitochondria) is ∼1.29 upon Zn2+ incubation, while that for MCF-7 cells at rest is 1.12, showing that PLIM with ZinIrDPA is able to visualize the introduced exogenous Zn2+ in MCF-7 cells. Although the phosphorescence imaging displays the similar response in the procedure (Figure S10a−d), the lifetime Zn2+ imaging is more reliable, since lifetime signal shows no dependence on local sensor concentration, and the Zn2+-induced lifetime enhancement factor of Zin-IrDPA shows no dependence on medium lipophilicity, polarity, viscosity and oxygen content. The endogenous labile Zn2+ lifetime imaging in mitochondria with Zin-IrDPA was further investigated (Figures 5, S12, and S13). S-Nitrosocysteine (SNOC) is able to stimulate intracellular Zn2+ release in mitochondria via redox stress,19,33 and our previous study has realized the ratiometric monitoring of the stimulated release.19 To explore the endogenous labile Zn2+ imaging ability of PLIM with Zin-IrDPA, the Zin-IrDPAstained MCF-7 cells were incubated with SNOC (10 mM) to trigger the mitochondrial labile Zn2+ fluctuation. After 20 min of incubation, the average phosphorescence lifetime in cells was enhanced from 626 ± 2.5 to 698 ± 3.8 ns. Moreover, this enhancement can be reversed and eliminated efficiently by subsequent TPEN treatment, and the average lifetime is decrease to 540 ± 2.9 ns, which is even lower than that observed in the original cells. All the results imply that PLIM

ence on medium viscosity. The influence of oxygen was investigated in aqueous solutions saturated with O2/Ar atmospheres of different O2 content. The lifetimes of both Zin-IrDPA and its complex decrease slightly with the increased oxygen concentration (0−20% oxygen, v/v, Figure S9a), yet it is Zn2+-induce lifetime enhancement factor is stable even when O2 content increases from 0 to 20% (Figure 3e). This provides this new phosphorescent sensor the additional advantage over other phosphorescent metal complexes in lifetime sensing for Zn2+. As shown in Figure 3f, the Zn2+induced emission intensity enhancement factor in aqueous solutions increases obviously with the oxygen content increasing from 0 to 20%, when compared with the stable Zn2+-induced lifetime enhancement factor. This indicates lifetime Zn2+ sensing ability of Zin-IrPA is more reliable than its intensity-based sensing ability for Zn2+. It is noted that the Zn2+-induced lifetime enhancement factors τZn/S/τZin‑IrDPA determined in these different media systems are slightly different from each other and that in PBS, despite the stable Zn2+-induced lifetime enhancement factors in each medium system. Phosphorescent Zn2+ Imaging with Zin-IrDPA in Living Cells. The phosphorescence Zn2+ imaging in living MCF-7 cells (human breast adenocarcinoma cell line) was investigated (Figure S10). After incubating with Zin-IrDPA, weak phosphorescence was observed within cells indicating the fine cell membrane permeability of this sensor (Figure S10a). The distribution of Zin-IrDPA in MCF-7 cells was further studied by colocalization with mitochondrial marker Mito 633 (Figure 4a−d). Overlay of the green image of Zin-IrDPA channel and the red image of Mito 633 channel displays the uniformly distributed yellow punctuated pattern inside the cells. The Pearson’s coefficient is 0.75, implying the mitochondria-targeting ability of this sensor. The mitochondrial location of Zin-IrDPA may be due to the positive charge and the suitable lipophilicity/hydrophilicity balance of this complex.31 When exogenous Zn2+ was introduced via incubation the cells with Zn(NO3)2-pyrithione solution, distinct emission enhancement was observed (Figure S10b). Moreover, the enhanced phosphorescence can be depressed effectively after treating the cells with cell membrane permeable Zn2+ chelator, N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) (Figure S10c). The same results were obtained on HeLa cells (human cervical cancer cell line) (Figure S11). All the data indicate Zin-IrDPA is able to E

DOI: 10.1021/acs.inorgchem.8b01272 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry with Zin-IrDPA is able to track endogenous labile Zn2+ in mitochondria in a reversible manner. If the sensor lifetime after TPEN treatment is set as the original lifetime, then the Zn2+induced lifetime enhancement factor after SNOC stimulation is ∼1.29, yet that for cells at rest is ∼1.16. The increase of average Zn2+-induced lifetime enhancement factor implies distinctly the SNOC stimulated labile Zn2+ release in mitochondria.

(d, 1H, J = 4.25 Hz), 7.91−7.87 (m, 3H), 7.71−7.66 (m, 7H), 7.52 (d, 1H, J = 5.6 Hz), 7.41 (d, 2H, J = 7.75 Hz), 7.30 (d, 1H, J = 5.7 Hz), 7.21−7.19 (m, 2H), 7.09−6.93 (m, 5H), 6.86 (t, 1H, J = 6.3 Hz), 6.44−6.37 (q, 2H, J = 7.55 Hz), 4.06 (q, 4H, J = 15.4 Hz), 3.66− 3.75 (q, 2H, J = 17.55 Hz). 13C NMR (75 MHz, CDCl3) δ (ppm): 156.8, 150.6, 149.3, 149.0, 148.8, 148.0, 137.5, 137.4, 136.9, 133.9, 131.5, 131.3, 130.5, 130.1, 126.0, 125.7, 124.2, 124.1, 123.3, 123.2, 122.6, 122.5, 122.3, 122.1, 119.0, 118.8, 118.3, 59.98, 58.34. ESI mass (positive mode, m/z): Calcd 935.28. Found 935.50 for [M]+. Absorption and Phosphorescence Spectroscopic Determination of Zin-IrDPA. The stock solution of Zin-IrDPA (1 × 10−3 mol L−1) was prepared by directly dissolving the sensor in C2H5OH. For spectroscopic determination, the stock solution was diluted with PBS to the desired concentration (1 × 10−5 mol L−1). For Zn2+ titration, 2.5 μL aliquots of Zn2+ aqueous solution (Zn (NO3)2, 1.25 × 10−3 mol L−1) were added into 3 mL of diluted Zin-IrDPA solution. The measurements were carried out in 2 min after Zn2+ addition. All spectra were determined at 298 K. Air-saturated solutions (1 × 10−5 mol L−1) were utilized to determine the phosphorescence lifetimes. Phosphorescence decay traces were acquired based on TCSPC techniques using a Fluorolog TCSCP instrument (Horiba, USA). A 370 nm NanoLED (Horiba, USA) was used as the excitation source. The phosphorescence signals were obtained using an automatically motorized monochromator. Time-resolved emission spectra were acquired through timecorrelated single photon counting methods by using the same instrument and excitation source. The phosphorescence signal from 400 to 800 nm was obtained with the automatic monochromator with a step of 4 nm. Phosphorescence decay profiles were analyzed (Horiba Jobin Yvon IBH software) using two exponential decay model. The transient photoluminescence experiment was performed in duplicate with fresh samples. The Zn2+-specific sensing selectivity of this sensor was investigated by determining both phosphorescent intensity and lifetime of this sensor in the presence of different metal cations. Therefore, the phosphorescence spectra and lifetime of Zin-IrDPA (10 μM) in PBS buffer (10 mM, pH 7.4, containing 1% C2H5OH) were determined in the presence of 1 equiv of Zn2+, Cd2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Cu+, Ba2+, Al3+, of Mn2+ or 1000 equiv of Na+, K+, Ca2+, and Mg2+. In addition, the Zn2+ sensing behavior was also determined by adding respectively the above-mentioned metal cations followed by adding 1 equiv of Zn2+. Quantum yield of Zin-IrDPA and its Zn2+ complex were determined in aqueous solutions (10 mM PBS, pH 7.4) by using Ru(bpy)32+ salts (Φf = 0.04, λex = 406 nm, H2O) as reference.36 The phosphorescent pH-dependence of Zin-IrDPA and Zn/S complex was determined in aqueous solutions (1% C2H5OH, 100 mM KNO3), and the phosphorescence spectra were determined immediately after the solution pH values were adjusted to the desired value by NaOH and HNO3 solutions. The excitation wavelength was 372 nm. The experiments were carried out at 298 K. Determination of Phosphorescence Lifetime of Zin-IrDPA and Its Zinc Complex in Different Solvent Systems. To determine the influence of oxygen, medium lipophilicity, viscosity, and polarity on the sensor’s lifetime sensing ability for Zn2+, the phosphorescence lifetime determination of Zin-IrDPA and its zinc complex was carried out in different solvent systems based on the emission at 580 nm upon excitation at 370 nm. To determine the influence of medium lipophilicity and polarity, the aliphatic alcohols of different alkyl chains (n-C1−C4, C6, and C8 chains) was utilized as the solvents, zinc complex of Zin-IrDPA was formed in situ by adding 1 equiv of Zn2+ into Zin-IrDPA solution. Similarly, the glycerol− water binary solvents with different glycerol content (20−80%, v/v) were adopted to determine the solvent viscosity effect on the lifetime Zn2+ sensing behaviors. As to the oxygen effect on the lifetime Zn2+ sensing ability of this sensor, phosphorescence lifetime of Zin-IrDPA (10 μM) and its zinc complex (10 μM) were determined respectively in aqueous solutions saturated with O2/Ar atmospheres of different oxygen content (0−20%, v/v). Cell Imaging. HeLa and MCF-7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum,

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CONCLUSION In conclusion, a phosphorescent sensor, Zin-IrDPA, for phosphorescence lifetime Zn2+ imaging was developed from cyclometalated Ir(III) complex. The sensor exhibits the Zn2+specific enhancement of phosphorescence intensity and phosphorescence lifetime in medium of near neutral pH. Moreover, its Zn2+-induced phosphorescence lifetime enhancement factor is not affected by medium lipophilicity, viscosity, polarity, and especially oxygen concentration. With the confirmed 1:1 Zn2+ binding stoichiometry, Zin-IrDPA shows still the mitochondria targeting ability. The sensor’s intracellular Zn2+ imaging ability was confirmed via both phosphorescence and phosphorescence lifetime imaging in MCF-7 cells. Considering the oxygen-independent Zn2+induced lifetime enhancement factor, this sensor shows its specific advantage in mitochondrial Zn2+ tracking, since mitochondria are oxygen-rich organelle. Moreover, the endogenous labile Zn2+ fluctuation in mitochondria triggered by SNOC stimulation has been visualized by Zin-IrDPA via PLIM. All the data imply the promising potential of this new PLIM agent to explore Zn2+ homeostasis in mitochondria of living cells, favoring the clarification of Zn2+ roles in mitochondria physiological processes. As there is still no reliable method to control and determine the labile Zn2+ concentration in living cells, it is still difficult to determine the sensor’s calibration curve of intracellular Zn2+ concentration vs the determined phosphorescence lifetime. Therefore, the exact Zn2+ quantification is still challenging and requires reliable method to acquire such as a calibration curve.

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EXPERIMENTAL SECTION

Materials and General Methods. All the solvents were of analytic grade. The stock solutions of metal ions for fluorescence discrimination were prepared from MnCl2, CoCl2·6H2O, Zn(NO3)2· 7H2O, CaCl2, NaCl, CuSO4, [(CH3CN)4Cu]PF6, NiCl2·6H2O, KCl, CdCl2·2.5H2O, AlCl3·6H2O, MgCl2·6H2O using doubly distilled water. The 1H NMR and 13C NMR spectra were recorded on a Bruker DRX-500 spectrometer with tetramethylsilane, Si(CH3)4 as internal standard in CDCl3. Mass spectral data were determined with an LCQ (ESI Mass, Thermo Finnigan) mass spectrometer. Synthesis of Zin-IrDPA. The synthesis of Zin-IrDPA was realized according to the scheme shown in Figure 1. Compounds 1 and 2 were synthesized according to the reported procedure.34,35 Therefore, compound 2 (183 mg; 0.2 mmol), triethylamine (40 mg, 0.5 mmol), and bis(pyridin-2-ylmethyl)amine (48 mg, 0.24 mmol) were mixed in 5 mL of THF in a round-bottomed flask with stirring. The mixture was heated at 60 °C for 10 h. After being cooled to room temperature, the mixture was evaporated to remove solvent by rotary evaporator in vacuo. Then, the resulted residue was dissolved in 10 mL of CH2Cl2. After being rinsed with brine (3 × 10 mL), the organic phase was then dried over MgSO4. The solvent was then removed again and the residue was purified by chromatography on silica gel using CH2Cl2/ CH3OH (20:1 v/v) as eluent. The product was obtained as yellow solid in the yield of 82%. 1H NMR (500 MHz, CDCl3) δ (ppm): 11.62 (s, 1H), 9.23 (d, 1H, J = 7.25 Hz), 8.65 (s, 1H), 8.54 (d, 2H, J = 4.35 Hz), 8.44 (d, 1H, J = 7.85 Hz), 8.34 (d, 1H, J = 4.5 Hz), 8.16 F

DOI: 10.1021/acs.inorgchem.8b01272 Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry

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penicillin (100 units/ml), streptomycin (100 mg/mL), and 5% CO2 at 37 °C. Phosphorescent imaging for intracellular Zn2+ via ZinIrDPA staining (10 μM, 30 min, 25 °C) was carried out for both cell lines. After removing the incubation media and rinsing twice with 1× PBS, the cells were stained with Zin-IrDPA solution (10 μM, in 1× PBS containing 1% C2H5OH) for 30 min at 25 °C. Then, the cells were washed two times with PBS and imaged with Zeiss LSM 710 microscope equipped with a 63× oil-immersion objective. The imaging was carried out upon excitation at 405 nm, and images were collected with a band path from 500 to 650 nm. For the imaging of cells with the introduced exogenous Zn2+, exogenous Zn2+ was introduced by incubating the cells with 10 μM Zn(NO3)2/2mercaptopyridine-N-oxide (1:1) solution. Then the cells were dyed with Zin-IrDPA solution in a procedure similar to that described above and imaged. After the imaging, the cells of exogenous Zn2+ were treated with 25 μM TPEN solution (prepared by diluting the TPEN stock solution with 1× PBS) for 10 min to scavenge Zn2+. Then, the cells were rinsed with 1× PBS followed by imaging. Co-localization experiments were carried out by co-staining the cells with MitoTracker Deep Red 633 (Invitrogen) and Zin-IrDPA. The cells were incubated in 50 nM MitoTracker Deep Red 633 solution at 25 °C for 15 min, then the cells washed twice with PBS. After being rinsed, the cells were incubated with Zin-IrDPA solution (10 μM) for 30 min at 25 °C. Then, the cells were washed twice with PBS before imaging. The MitoTracker Deep Red 633 marked images were obtained upon irradiation at 633 nm with a band path from 650 to 720 nm, while the Zin-IrDPA stained images were obtained using the same imaging parameters shown above. The monitoring of mitochondrial Zn2+ release upon SNOC stimulation was carried out in MCF-7 cells. Therefore, MCF-7 cells were first dyed with Zin-IrDPA with the same procedure described above. Then, the cells were incubated with SNOC (10 mM, 20 min) at 25 °C, and the Zn2+ release was tracked by imaging with confocal microscope Zeiss LSM 710. After that, the cells were treated with 50 μM TPEN solution to scavenge the intracellular Zn2+, and then the cells were imaged with the same imaging parameters. The PLIM image setup is integrated with an Olympus IX81 laser scanning confocal microscope. The luminescence signal was detected by the system of the confocal microscope, and correlative calculation of the data was performed by professional software that was provided by PicoQuant Company. The light from the pulse diode laser head (PicoQuant, PDL 800-D) with excitation wavelength of 405 nm and frequency of 0.5 MHz was focused onto the sample with a 40×/NA 0.95 objective lens for single-photon excitation.

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ACKNOWLEDGMENTS We thank National Basic Research Program of China (No. 2015CB856300), National Natural Science Foundation of China (Nos. 21401106, 21501085, and 21571099), Key University Science Research Project of Jiangsu Province (17KJA150004), and the Natural Science Foundation of Jiangsu (No. BK20150054) for financial support.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.8b01272. NMR and ESI mass spectra of Zin-IrDPA, Photo-

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Article

physical data, Additional cell imaging data (PDF)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (C.Z.). *E-mail: [email protected] (Z.L.). *E-mail: [email protected] (W.H.). ORCID

Qiang Zhao: 0000-0002-3788-4757 Weijiang He: 0000-0002-3157-5769 Notes

The authors declare no competing financial interest. G

DOI: 10.1021/acs.inorgchem.8b01272 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

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DOI: 10.1021/acs.inorgchem.8b01272 Inorg. Chem. XXXX, XXX, XXX−XXX