A Mitochondria-Targetable Ratiometric Time-Gated Luminescence

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A Mitochondria-Targetable Ratiometric Time-Gated Luminescence Probe for Carbon Monoxide Based on Lanthanide Complexes Zhixin Tang, Bo Song, Hua Ma, Tianlie Luo, Lianying Guo, and Jingli Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05127 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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Analytical Chemistry

A Mitochondria-Targetable Ratiometric Time-Gated Luminescence Probe for Carbon Monoxide Based on Lanthanide Complexes

Zhixin Tanga, Bo Songa*, Hua Maa, Tianlie Luob, Lianying Guoc, Jingli Yuana

a

State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology,

Dalian 116024, China b

Key Laboratory of Industrial Ecology and Environmental Engineering (MOE), School of

Environmental Science and Technology, Dalian University of Technology, Linggong Road 2, Dalian 116024, China. c Department

of Pathophysiology, Dalian Medical University, Dalian 116044, P. R. China

*Corresponding authors. Tel: +86-411-84986041; E-mail: [email protected]

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ABSTRACT As a critical gasotransmitter, carbon monoxide (CO) has been demonstrated to be related with mitochondrial respiration, but the monitoring of CO in mitochondria remains a great challenge. In this work, a unique ratiometric time-gated luminescence (TGL) probe, Mito-NBTTA-Tb3+/Eu3+, that can specifically respond to mitochondrial CO has been developed. The probe was designed by incorporating a mitochondria-targeting moiety, triphenylphosphonium, into a CO-activatable terpyridine

polyacid

derivative,

4'-(4-nitrobenzyloxy-2,2':6',2''-terpyridine-6,6''-diyl)

bis(methylenenitrilo) tetrakis(acetic acid), for coordinating to Eu3+ and Tb3+ ions to construct lanthanide complex-based probe for ratiometric TGL detection of CO. Upon reaction with CO, accompanied by the conversion of nitro group to amino group, a 1,6-rearrangement-elimination reaction occurs, which leads to the cleavage of 4-nitrobenzyl group from Mito-NBTTA-Tb3+/Eu3+, resulting in the significant increase of Tb3+ emission at 540 nm and moderate decrease of Eu3+ emission at 610 nm. After the reaction, the I540/I610 ratio was found to be 48-fold enhanced. This feature allowed Mito-NBTTA-Tb3+/Eu3+ to be employed as a ratiometric TGL probe for CO detection with the I540/I610 ratio as a signal. In addition, the probe showed outstanding mitochondria-localization characteristic, which enabled the probe to be successfully applied to imaging CO within mitochondria of living cells under TGL and ratiometric modes. The application of Mito-NBTTA-Tb3+/Eu3+ was demonstrated by the visualization and quantitative detection of exogenous and endogenous CO in living cells and mouse liver tissue slices, as well as in living Daphnia magna and mice. All of the results suggested the potential of Mito-NBTTA-Tb3+/Eu3+ for the quantitative monitoring of CO in vitro and in vivo.


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INTRODUCTION Carbon monoxide (CO) has been increasingly approved as one of important gasotransmitter molecules in mammalian cells, although it was previously known as a pollutant or toxic molecule.1-3 In living bodies, CO plays a significant role in the regulation of vasodilation, neurotransmission, anti-apoptotic, anti-inflammatory and anti-proliferative activities.2,4 Emerging studies revealed that mitochondria could be a major organelle for the action of CO to regulate cellular respiration.5 Chance and co-workers demonstrated the inhibitory effect of CO on the cytochrome a3, an important link in the enzyme system of respiration.6 Furthermore, cellular respiration may also be impaired by inactivation of mitochondrial enzymes after CO exposure, and then normal mitochondrial function is disturbed.7,8 Therefore, the development of specific CO-responsive probes for directly tracking mitochondrial CO is of great significance. In the past decade, a number of fluorescent probes have been designed for the detection of CO in live cells, in which some transition metal species (mainly Pd2+ species)-mediated reactions were used for the recognition of CO.9-23 However, the detection using these probes requires the addition of cytotoxic heavy metal salts as carbonylation catalysts or Tsuji-Trost reaction mediators. Quite recently, Dhara group designed a lysosome-targetable fluorescent probe, LysoFP-NO2, for the imaging of CO in live cells through the transformation of the nitro group into an amino-functionalized system in the presence of CO.24 Unfortunately, all of above mentioned fluorescent probes were hardly suitable to be used for the in situ detection of CO in mitochondria of live cells. One of main challenges is the lack of mitochondria-specificity, which causes the signal disturbance from other organelles. Besides, neither of these intensity-based fluorescent probes could be used for the accurate quantification of intracellular CO, due to the effects of excitation fluctuations, sample environments and local probe concentrations on their emission intensities, as



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well as the interference of background fluorescence.25,26 In view of above problems, the development of lanthanide complex-based ratiometric luminescence probes bearing a mitochondria-targeting moiety for detection of mitochondrial CO would be an ideal approach. This probe can be considered to have three favorable properties for detection of mitochondrial CO, i) the presence of mitochondria-targeting moiety allows the probe molecules to be drawn into the mitochondria after cellular uptake for responding to CO therein;27,28 ii) the long-live luminescence of the probe enables the detection to be performed under TGL mode to eliminate the interference of short-lived autofluorescence;29-31 iii) the impacts of excitation intensity, probe concentration and sample environment alterations on the detection can be eliminated by ratiometric mode through self-correction of two emissions at different wavelengths.28,32,33 Although this kind of probes can be anticipated to be a powerful tool for the accurate quantification of mitochondrial CO under ratiometric and TGL modes, to the best of our knowledge, the relative probe has not been explored until now. Herein we report the design and synthesis of lanthanide complex-based mitochondria-targetable ratiometric TGL probe, Mito-NBTTA-Tb3+/Eu3+, for detection of mitochondrial CO. Scheme 1 illustrate the molecular structure of Mito-NBTTA-Tb3+/Eu3+ and its TGL response mechanism towards CO. In the probe, the nitro group is a CO-reactive site,24 and positively charged triphenylphosphonium (TPP) acts as a mitochondria-anchoring motif to enable the probe molecules to be accumulated within mitochondria after cellular uptake.28 The probe composed of heterometallic Tb3+/Eu3+ complexes with the same antenna ligand allows the probe to be able to emit Tb3+ and Eu3+ emissions at different wavelengths under the same excitation wavelength.32,33 After being reacted with CO, the conversion of nitro group to amino group induces the cleavage of 4-nitrobenzyl moiety from the probe, affording the new complexes of Mito-HTTA-Tb3+/Eu3+. The


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above conversion simultaneously induce a remarkable enhancement of Tb3+ emission (main band at 540 nm) and a moderate weakening of Eu3+ emission (main band at 610 nm), to allow the CO detection to be performed using the TGL intensity ratio of Tb3+ emission to the Eu3+ emission, I540/I610, as the signal. In addition, Mito-NBTTA-Tb3+/Eu3+ showed excellent cell membrane permeability and mitochondria-targeting ability when it was used for loading live cells, which enabled the mitochondrial CO in living cells to be successfully imaged under TGL and ratiometric modes. The utility of the probe was comprehensively demonstrated via ratiometric and TGL detection and imaging of CO in vitro and in vivo. NO2

NH2

O

O-

O NH

N N N

Eu3+/Tb3+

N

CO2- -O2C O -O C 2

N

HN P(Ph)3 Mito-NBTTA-Tb3+/Eu3+ Eu : moderate luminescent Tb 3+: weakly luminescent 3+

N

N

CO

N N

Eu 3+/Tb3+

N

N

CO2- -O 2C O -O C 2

N

N

3+

Eu /Tb 3+

N

CO 2- -O 2C O -O C 2

N

HN

HN

P(Ph)3

P(Ph)3

Mito-HTTA-Tb3+/Eu3+

intermediate

3+

Eu : weakly luminescent Tb 3+: strongly luminescent

Scheme 1. Structure of the lanthanide complex-based probe, Mito-NBTTA-Tb3+/Eu3+ and its TGL response mechanism with CO.

EXPERIMENTAL SECTION Reagents. The starting material (compound 1, shown in Scheme S1) for the synthesis of the ligand, Mito-NBTTA was synthesized following the previous method.32 CORM-3, a commercially available

CO-releasing

molecule

with

a

chemical

name

of

tricarbonylchloro(glycinato)ruthenium(II), was purchased from Sigma-Aldrich. HeLa cells, RAW 264.7 cells and BALB/c nude mice were obtained from Dalian Medical University. Cultured Daphnia magna were obtained from Professor Jingwen Chen's group at the School of ACS Paragon Plus Environment


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Environmental Science and Technology, Dalian University of Technology. All other reagents were purchased from commercial sources and used directly. Synthesis of the ligand Mito-NBTTA and preparation of the stock solution of Mito-NBTTA-Tb3+/Eu3+. The synthesis procedure of the ligand, Mito-NBTTA is illustrated in Scheme S1 and the details of experiments are described in the Supplementary Information (SI). The experimental details for preparation of the stock solution of Mito-NBTTA-Tb3+/Eu3+ are also described in the SI. Transmission electronic microscopy study of the Mito-NBTTA-Tb3+ in the Mitochondria. Hela cells grown in cell culture flasks were loaded with 200 μM of Mito-NBTTA-Tb3+ for 2.0 h. After washing, the cells were fixed with 2.5% glutaraldehyde for 4 h at 4°C. The cells were then washed with 0.1 M PBS buffer and post-fixed with 1% OsO4 for 2 h at 4 °C. The above cell samples were dehydrated in a gradient ethanol series and infiltrated with Epon. Then they were embedded and cured in Araldite resin for 24 h at 37 °C, 24 h at 45 °C, and 24 h at 60 °C. Ultrathin sections (50~70 nm) were cut on an ultramicrotome and stained with uranyl acetate and lead citrate before observation under the JEM-2000EX transmission electron microscope (JEOL, Japan). Control samples were treated in the same way, except that they were not incubated with Mito-NBTTA-Tb3+. TGL imaging of CO in living mouse liver tissue slices. The mouse liver was excised from BALB/c mice (with a weight of ∼20 g) and stored at -20 0C for 24 h. The frozen liver tissue was cryosectioned via microtome at -20 0C into slices of 15 μm thicknesses. The slices in control group was

incubated

with

PBS

of

pH

7.4

containing

100

μM

Mito-NBTTA-Tb3+/Eu3+

(Mito-NBTTA/Tb3+/Eu3+ = 2/1/1) for 2 h at 37 0C; those in experimental group were further incubated with 300 μM of CORM-3 for 1.5 h at 37 0C. After washing with PBS buffer four times,


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the above slices were subjected to the TGL imaging measurements (exposure time, 4.2 s). Luminescence imaging of CO in BALB/c nude mice. Two BALB/c nude mice (~20 g bodyweight) was subcutaneously injected with 100 μL physiological saline solution containing Mito-NBTTA-Tb3+/Eu3+ (1.0 mM, Mito-NBTTA/Tb3+/Eu3+ = 2/1/1) into the left rear leg and the right rear leg (as control) of the mice. Then a physiological saline solution (100 μL) containing CORM-3 (5.0 mM) was sequentially injected into the left rear leg via subcutaneous injection. After 1.5 h, the mice were anesthetized with 3.6% chloral hydrate and placed into the imaging chamber for

luminescence

imaging.

Whole

body

luminescence

images

were

recorded

on

a

MesoQMR23-060H multi-functional in vivo imaging system (Molecular Devices, San Jose, CA. USA). For green luminescence signals: an excitation filter, 365 nm, and an emission filter, 525 ± 50 nm. For red luminescence signals: an excitation filter, 365 nm, and an emission filter, 630 ± 75 nm. The authors state that all animal studies were carried out at Specific Pathogen Free Animal Center at the Dalian Medical University according to the animal protocols (No. L2014014) approved by the Animal Ethics Committee (AEC).

RESULTS AND DISCUSSION Design and properties of the probe Over the last decade, although various fluorescent probes for CO have been developed, most of them are the intensity-based probes, and few of them can be specifically loaded into mitochondria of live cells for responding to CO therein.9-23 Recently, we demonstrated that the commonly used mitochondria-targeting moiety, triphenylphosphonium, could also be employed for the design of lanthanide complex-based mitochondria-targetable luminescent probes. By incorporating this moiety into the ligands of several lanthanide complexes, the mitochondria-targeting probes responsive to hypochlorous acid and biothiols were successfully developed for the TGL detection of ACS Paragon Plus Environment


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hypochlorous acid and biothiols in mitochondria of live cells.27,28 Inspired by preceding works, we developed a new lanthanide complex-based probe, Mito-NBTTA-Tb3+/Eu3+, for ratiometric TGL detection of CO in mitochondria of live cells. In this probe, owing to the existence of intramolecular photo-induced electron transfer (PET) process from the luminophor, terpyridine polyacid-Ln3+ to the 4-nitrobenzyl group,24 the complex Mito-NBTTA-Tb3+ is weakly luminescent, and Mito-NBTTA-Eu3+ shows moderate luminescence. Furthermore, the 4-nitrobenzyl group also serves as the CO recognition and reaction site. In the presence of CO and H2O, the nitro group can be easily and selectively reduced to amino group, followed by subsequent extrusion of carbon dioxide. 24,34 This conversion leads to the cleavage of 4-nitrobenzyl moiety from the probe through a 1,6-rearrangement-elimination reaction, affording the new complexes of Mito-HTTA-Tb3+/Eu3+ (Scheme 1). These provide opposite luminescence changes at 540 nm (increase, Tb3+ emission) and 610 nm (decrease, Eu3+ emission) for ratiometric TGL detection of CO. The proposed mechanistic steps of the conversion of nitro to amino compound triggered by CO were depicted in Scheme S2. Given the 4-nitrobenzyl group can also respond to nitroreductase, hypoxic

status

of

cancer

cells,

we

investigated

the

35,36

a biomarker associated with

ratiometric

TGL

response

of

Mito-NBTTA-Tb3+/Eu3+ to nitroreductase. As shown in Figure S1, no significant changes on the ratiometric TGL signal of Mito-NBTTA-Tb3+/Eu3+ could be observed when the probe reacted with a considerable level of nitroreductase (< 0.2 μg mL-1). Due to the low content of nitroreductase in normoxic cells,

35,36

we infer that the CO detection using Mito-NBTTA-Tb3+/Eu3+ as a probe will

not be interfered by nitroreductase in normoxic environments. Furthermore, the covalently conjugated TTP group provides an overall positive charge for the probe molecule, resulting in excellent cell membrane permeability and selective accumulation in the mitochondria. The structure of Mito-NBTTA and its lanthanide (Eu3+ and Tb3+) complexes were well characterized by 1H NMR,


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13C

NMR and ESI-MS analyses (See Figure S7–S9 in the SI).

The luminescence properties of Mito-NBTTA-Eu3+ and Mito-NBTTA-Tb3+ and their reaction products with CO were investigated in 10 mM PBS buffer of pH 7.4. Before reaction with CO, Mito-NBTTA-Eu3+ showed moderate luminescence at 610 nm ( = 6.1%, τ = 1.31 ms), and Mito-NBTTA-Tb3+ exhibited very weak luminescence at 540 nm ( < 0.5%, τ = 0.96 ms). After reaction with CO (CORM-3 was used as a CO source) to afford Mito-HTTA-Eu3+ and Mito-HTTA-Tb3+ (the reaction products were verified by ESI-MS analysis. The MS peaks at m/z 975.2 and 983.3 proved the production of Mito-HTTA-Eu3+ and Mito-HTTA-Tb3+. See Figure S10 and S11 in the SI), the Tb3+ emission at 540 nm was significantly increased ( = 11.3%, τ = 1.72 ms), while the Eu3+ emission at 610 nm was ~2.8-fold weakened ( = 2.1%,τ = 1.21 ms) owing to the existence of an intramolecular charge transfer (ICT) in the deprotonated Mito-HTTA–Eu3+.37 These results reveal that Mito-NBTTA-Tb3+ and Mito-NBTTA-Eu3+ have different luminescence response behaviors to CO, and their emission intensity ratio, I540/I610, could be employed as a signal for the detection of CO. To confirm this, the mixture of Mito-NBTTA-Tb3+ and Mito-NBTTA-Eu3+, Mito-NBTTA-Tb3+/Eu3+ (Mito-NBTTA/Tb3+/Eu3+ = 2/1/1), was prepared, and its TGL emission spectra in the absence and presence of CO in PBS buffer (pH 7.4) were collected. It was found that Mito-NBTTA-Tb3+/Eu3+ displayed the same luminescence response behaviors at 540 nm and 610 nm as those of Mito-NBTTA-Tb3+ and Mito-NBTTA-Eu3+ in separate state (Figure S12 in the SI), which suggested the potential of Mito-NBTTA-Tb3+/Eu3+ as a ratiometric TGL probe for CO. To assess the capability of Mito-NBTTA-Tb3+/Eu3+ as a ratiometric TGL probe for the quantitative

detection

of

CO,

TGL

emission

spectra

of

Mito-NBTTA-Tb3+/Eu3+

(Mito-NBTTA/Tb3+/Eu3+ = 2/1/1, Ctotal = 5.0 M) upon reaction with various concentrations of CORM-3 (0.0, 3.0, 5.0, 10, 30, 50, 100, 150, 200, 250 and 300 M) in PBS buffer (pH 7.4) were



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collected and the results were shown in Figure 1. With increased concentration of CORM-3 from 0 to 300 M, the emission intensity of the probe solution at 540 nm was gradually increased with up to 17-fold enhancement, and that at 610 nm was gradually decreased with down to 2.8-fold weakness. Meanwhile, the I540/I610 ratio was ~48-fold increased from 0.13 to 6.37, showing a good linear correlation with the concentration of CORM-3 from 0.0 to 150 μM (Figure 1B). The detection limit (LOD), defined as the equation, LOD=3σ/k (σ is the standard deviation of the background signal and k is the slope of the calibration curve), was calculated to be 0.44 M, which indicates that Mito-NBTTA-Tb3+/Eu3+ can be employed as a ratiometric TGL probe for quantitative detection of CO under micromolar concentration level. In comparison with previously reported fluorescent probes for CO (Table S1), the response sensitivity of Mito-NBTTA-Tb3+/Eu3+ is comparable to that of others, but this probe enables the luminescence detection to be performed with ratiometric and TGL modes, to improve accuracy and precision of the measurement without interference of background autofluorescence especially in bioimaging.

Figure 1. (A) TGL emission spectra (λex = 330 nm) of Mito-NBTTA-Tb3+/Eu3+ reacted with various concentrations of CORM-3 in 10 mM PBS buffer at pH 7.4 (the inset displays the luminescence colors of the solutions under a 365 nm UV lamp). (B) Calibration curve for ratiometric TGL detection of CORM-3 using Mito-NBTTA-Tb3+/Eu3+ as a probe.


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The kinetic curves of ratiometric TGL responses of Mito-NBTTA-Tb3+/Eu3+ to the additions of various concentrations of CORM-3 were investigated in PBS buffer at pH 7.4. As shown in Figure 2A, the I540/I610 ratios reach maximum values within 60 min in all cases, which is similar to the reported result of fluorescence response rate of LysoFP-NO2 to CORM-3,24 indicating that the reaction between Mito-NBTTA-Tb3+/Eu3+ and CORM-3-released CO can be completed in 60 min. Figure 2B shows the effects of pH on the reaction of the probe with CO. In the presence of CORM-3, the I540/I610 ratio of the probe increases gradually with the increase of pH from 4.0 to 8.0, and then keeps stable relatively. This result reveals that a weakly basic medium is beneficial to the CO-induced cleavage of 4-nitrobenzyl moiety from the probe. Considering the use of the probe for cell imaging, a PBS buffer (pH 7.4) was used for the characterizations of the probe and its reaction products with CO. The response specificity of the probe to CO was examined by adding various potential interfering species (500 M) into the solution of Mito-NBTTA-Tb3+/Eu3+, respectively. After incubating for 60 min, the TGL emission spectra of the solutions were recorded, and then their I540/I610 ratios were calculated. As shown in Figure 2C, excepted for CORM-3, all of species including HClO, H2O2, H2S, gluthathione, cysteine, homocysteine, t-BuOOH, •OH, ascorbic acid, O2-•, histidine, glutamic acid, arginine, SO32-, NO, ONOO-, and singlet oxygen could not induce the increase of the I540/I610 ratio of the probe, which demonstrates that the ratiometric TGL response of the probe to CO is highly specific without disturbances of the tested interferents at a high concentration level.



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Figure 2. (A) Time course of ratiometric TGL responses of Mito-NBTTA-Tb3+/Eu3+ (Mito-NBTTA/Tb3+/Eu3+ = 2/1/1, Ctotal = 5.0 M) to the additions of different concentrations of CORM-3 (0, 10, 30, 100 and 200 M). (B) Effects of pH on the I540/I610 ratio of Mito-NBTTA-Tb3+/Eu3+ (Mito-NBTTA/Tb3+/Eu3+ = 2/1/1, Ctotal = 5.0 M) in the presence (red line) and absence (black line) of CORM-3. (C) The I540/I610 ratios of Mito-NBTTA-Tb3+/Eu3+ (Mito-NBTTA/Tb3+/Eu3+ = 2/1/1, Ctotal = 5.0 M) upon reactions with various reactive species (500 μM) in 10 mM PBS buffer of pH 7.4 (1: blank; 2: HClO; 3: H2O2; 4: H2S; 5: gluthathione; 6: cysteine; 7: homocysteine; 8: t-BuOOH; 9: •OH; 10: ascorbic acid; 11: O2-•; 12: histidine; 13: glutamic acid; 14: arginine; 15: SO32-; 16: NO; 17: ONOO-; 18: singlet oxygen; 19: CORM-3).

Ratiometric TGL imaging of CO in live cells Before using for cell imaging, the cytotoxicity of Mito-NBTTA-Tb3+ to HeLa cells was evaluated by using the MTT assay method.38 As shown in Figure S13, over 90% HeLa cells remained viable after incubation with up to 300 M of Mito-NBTTA-Tb3+ for 24 h. This result reveals that Mito-NBTTA-Ln3+ is biocompatible with low cytotoxicity when it is used for loading live cells. To confirm the mitochondria-localization characteristic of the probe, HeLa cells were stained with 100 M of Mito-NBTTA-Tb3+ for 2 h at 37 °C, and then treated with 300 M of CORM-3 for 1.5 h, followed by a 30 min incubation with 100 nM of Mito-Tracker Deep Red (MTDR). After rinsing with PBS four times, the cells were imaged on a confocal laser scanning microscope ACS Paragon Plus Environment

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(CLSM). As shown in Figure 3, the cells showed green and red emission signals in channel 1 and channel 2 attributed to the emissions of the Tb3+ complex and MTDR, respectively (Figure 3B and 3C). The merged image of the red and green emission signals showed significant overlaps in the cells (Figure 3D). The intensity profiles of the linear region of interest across cells varied in very close synchrony (Figure 3E), and intensity correlation plots of red and green emission signals in cells (Figure 3F) had the overlap coefficient (0.95) and Pearson's correlation coefficient (P, 0.95) near to 1.0. Furthermore, co-localization experiments of Mito-NBTTA-Tb3+ with other fluorescent organelle indictors such as Golgi-Track Red, ER-Track Red and Lyso-Track Red were performed in HeLa cells for further verification of mitochondria-targetable feature of the probe. As shown in Figure S14, the red and green emission signals did not show remarkable overlaps in co-stained cells. Upon analysis of intensity correlation plots of red and green emission signals in co-stained cells, the P values of Mito-NBTTA-Tb3+ for Golgi, ER and lysosomes turn out to be 0.50, 0.61 and 0.74, respectively. All of these results revealed the majority of Mito-NBTTA-Tb3+ localized in mitochondria of the cells, suggesting the feasibility of the probe for imaging mitochondrial CO in live cells. Such a conclusion was further proved by transmission electron microscopy (TEM) of Mito-NBTTA-Tb3+-loaded cells owing to the high electron density of the complex (it results in darkening contrast in TEM image) accumulated in mitochondria. Compared to the control cells (Figure 3G and 3H), the Mito-NBTTA-Tb3+-loaded cells exhibited obvious darkening contrast in the mitochondria of as-prepared cells due to the mitochondrial accumulation of the complex (Figure 3I and 3J). These findings are in agreement with the above co-localization CLSM imaging, and strongly suggest that Mito-NBTTA-Tb3+ is located within mitochondria after cellular uptake.



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Figure 3. Intracellular localization analysis of Mito-NBTTA-Tb3+ in HeLa cells. Bright-field (A) and fluorescence (B and C) images of HeLa cells loaded with Mito-NBTTA-Tb3+, treated with CORM-3 and further incubated with MTDR (B: channel 1, λex = 405 nm, λem = 520-560 nm; C: channel 2, λex = 635 nm, λem = 650-680 nm); (D) overlay of B and C; (E) fluorescence intensity profiles of MTDR (red line) and the Tb3+ complex (green line) in the interest linear region (line 1) across HeLa cells in D; (F) fluorescence intensity correlation plots of HeLa cells co-loaded with the Tb3+ complex and MTDR; (G) TEM image of HeLa cells stained solely with osmium tetroxide; (H) magnified image of red box in image G, where the red ellipses indicate mitochondria of the cell; (I) TEM image of HeLa cells incubated with osmium tetroxide and Mito-NBTTA-Tb3+; (J) magnified image of red box in image I, where the red ellipses indicate mitochondria of the cell.

To estimate the capability of Mito-NBTTA-Tb3+/Eu3+ as a ratiometric TGL probe for imaging mitochondrial CO in live cells, HeLa cells were stained with Mito-NBTTA-Tb3+/Eu3+ for 2 h at 37 °C, and then incubated with different concentrations of CORM-3 (100, 200 and 300 M) for another

1.5

h

before

use

for

TGL

imaging.

As

shown

in

Figure

4,

the

Mito-NBTTA-Tb3+/Eu3+-loaded HeLa cells exhibited no green TGL signals, and only red TGL signals were observed in the cells. However, when the cells were further incubated with increased ACS Paragon Plus Environment

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concentration of CORM-3, green and red TGL signals from cells were gradually increased and decreased, respectively. Using ImageJ software, the ratiometric (Igreen/Ired) images of the as-prepared cells were obtained. It is clearly observed that the Igreen/Ired value in mitochondria of cells gradually enhanced from 0.12 to 5.05 with the increase of CORM-3 concentration (Figure S15), which indicates that Mito-NBTTA-Tb3+/Eu3+ could visualize mitochondrial CO and quantitatively detect it with ratiometric TGL imaging technique.

Figure 4. Ratiometric TGL images of HeLa cells loaded with different concentrations of CORM-3 using Mito-NBTTA-Tb3+/Eu3+ (Mito-NBTTA/Tb3+/Eu3+ = 2/1/1, Ctotal = 100 M) as a probe. Green filter image, 540 ± 25 nm; red filter image, >590 nm; ratiometric image, Igreen/Ired.

It has been reported that heme or lipopolysaccharide (LPS) can induce the expression of heme oxygenase-1 to facilitate the production of CO in live cells.20,39 Thus we attempted to use the as-prepared probe for visualization the generation of endogenous CO in mitochondria of live cells.



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After HeLa cells were pre-treated with heme (300 μM) for different times (1, 5 and 10 h), the stimulated cells were further incubated with Mito-NBTTA-Tb3+/Eu3+ for 1.5 h, and then their TGL images were collected. As shown in Figure S16, accompanied by the time increase of HeLa cells incubated with heme, the intracellular CO level was gradually increased, which induced the increase and decrease of green and red TGL signals in as-prepared cells, respectively. Correspondingly, the ratiometric images clearly showed the increase of the Igreen/Ired value from 0.33 to 3.77 with the increase of heme-stimulation time (Figure S17). Then the TGL images of RAW 264.7 cells pre-treated with different concentrations of LPS (100 and 200 ng/mL) for 18 h and further incubated with Mito-NBTTA-Tb3+/Eu3+ for 1.5 h were recorded. As shown in Figure S18, the cells without pre-treatment of LPS showed only red luminescence signals. However, for the cells pre-treated with LPS, the green and red TGL signals were obviously increased and decreased, respectively. And correspondingly, the Igreen/Ired value was enhanced from 0.11 to 3.75 (Figure S19). Furthermore, both changes of TGL signals and Igreen/Ired values exhibited dose-dependent relationships with the amount of LPS. All of above results proved the practicability of Mito-NBTTA-Tb3+/Eu3+ for the ratiometric TGL imaging and quantification of endogenous CO produced in mitochondria of live cells after heme- and LPS-stimulations. The photostability of Mito-NBTTA-Tb3+/Eu3+ was investigated in the probe-loaded cells. The Mito-NBTTA-Tb3+/Eu3+ stained HeLa cells were continuously irradiated for 30 min using 60-W Xenon lamp (exciting light source of the microscope) as an excitation source. The luminescence images were recorded at every 5-min interval for a period of 30 min. As shown in Figure S20 and S21, the emission intensities of Tb3+ and Eu3+ in HeLa cells did not display observable changes during the irradiation, suggesting that no photobleaching of the probe occurred in the cell imaging experiment.


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Ratiometric TGL imaging of CO in mouse liver tissues To further show the advantage of the probe for ratiometric TGL imaging of CO in complicated biological samples, the liver tissue slices of a mouse were prepared by cryosectioning the frozen liver into slices of 15 μm thicknesses. After the slices were loaded with 100 μM Mito-NBTTA-Tb3+/Eu3+ for 2 h, they were further treated with 300 μM CORM-3 for 1.5 h and their luminescence images were collected. As shown in Figure 5, for the slice incubated with Mito-NBTTA-Tb3+/Eu3+ alone, only red TGL signals were exhibited, and green TGL signals could hardly be found from the tissue (Figure 5A). In contrast, the slice incubated with Mito-NBTTA-Tb3+/Eu3+ and CORM-3 displayed remarkable green TGL signals and invisible red TGL signals (Figure 5B). The quantitative analysis of ratiometric images (last column in Figure 5) indicated

that

the

Igreen/Ired

value

was

increased

around

20.5-fold

after

the

Mito-NBTTA-Tb3+/Eu3+-stained slice was incubated with CORM-3, which demonstrated the feasibility of the probe for the quantitative TGL imaging of CO in complicated biosamples.

Figure 5. Ratiometric TGL images of cryosectioned mouse liver tissue slices loaded with Mito-NBTTA-Tb3+/Eu3+ (Mito-NBTTA/Tb3+/Eu3+ = 2/1/1, Ctotal = 100 M) for 2 h (A), and further ACS Paragon Plus Environment


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incubated with CORM-3 (300 M) for 1.5 h (B). Green filter image, 540 ± 25 nm; red filter image, >590 nm; ratiometric image, Igreen/Ired.

Ratiometric Luminescence imaging of CO in vivo The practical usage of Mito-NBTTA-Tb3+/Eu3+ for ratiometric TGL imaging of CO in vivo was further assessed. At first, D. magna, a commonly used laboratory animal in ecotoxicology,40 were employed as a model animal for imaging the CO generation upon stimulation with CORM-3. As shown in Figure 6, after incubation with Mito-NBTTA-Tb3+/Eu3+ for 30 min, the gut of D. magna showed only red TGL signals, and no green TGL signals could be observed. However, when the D. magna were sequentially treated with 300 M CORM-3 for 1.5 h, green TGL signals were clearly observed from the gut of D. magna, while red TGL signals almost disappeared. Thus the ratiometric (Igreen/Ired) images of the D. magna were obtained, and the Igreen/Ired value of the probe located in the gut was increased from 0.31 to 8.97. These results implied that Mito-NBTTA-Tb3+/Eu3+ and CORM-3 were mainly distributed in the gut of D. magna, and herein the probe was reacted with CORM-3-released CO to present a ratiometric TGL response. Furthermore, in contrast to the steady-state image (last column in Figure 6), the images recorded under TGL mode showed high contrast without any interferences of autofluorescence, which highlighted the merit of TGL imaging for autofluorescence-rich samples.


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Figure 6. Ratiometric TGL and steady-state luminescence images of D. magna incubated with Mito-NBTTA-Tb3+/Eu3+ (Mito-NBTTA/Tb3+/Eu3+ = 2/1/1, Ctotal = 100 M) for 30 min (A), and followed by an incubation with 300 μM CORM-3 for 1.5 h (B). Green filter image, 540 ± 25 nm; red filter image, >590 nm; ratiometric image, Igreen/Ired.

The ratiometric TGL imaging of CO in a live mouse was further performed using Mito-NBTTA-Tb3+/Eu3+ as a probe. After administration of anesthesia, a physiological saline solution (100 L) containing Mito-NBTTA-Tb3+/Eu3+ (1.0 mM) was injected subcutaneously into the left rear leg of a BALB/c nude mouse, and then the mouse was sequentially injected with CORM-3 (100 L, 5.0 mM in physiological saline solution) into the same area via subcutaneous injection. As the control, the same Mito-NBTTA-Tb3+/Eu3+ solution was injected subcutaneously into the right rear leg of the mouse. After 1.5 h, the luminescence images of the mouse were recorded on an in vivo imaging system. As shown in Figure 7, the right rear leg loaded with the probe displayed remarkable red luminescence signals and weak green luminescence signals, whereas the left rear leg loaded with the probe and CORM-3 exhibited weak red luminescence signals and strong green luminescence signals. The ratiometric (Igreen/Ired) image of the mouse



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showed that the Igreen/Ired value in left rear leg was 5.8-fold higher than that in right rear leg, which proved the applicability of Mito-NBTTA-Tb3+/Eu3+ for the quantitative sensing and imaging of CO in living animals.

Figure 7. Ratiometric luminescence images of CO in a BALB/c nude mouse. The right rear leg was injected with 100 μL of 1.0 mM Mito-NBTTA-Tb3+/Eu3+, and the left rear leg was injected with 100 μL of 1.0 mM Mito-NBTTA-Tb3+/Eu3+ and 100 L of 5.0 mM CORM-3. The images were recorded after the injection for 1.5 h. Green filter image, 525 ± 50 nm; red filter image, 630 ± 75 nm; ratiometric image, Igreen/Ired.

Conclusions In summary, a mitochondria-targetable ratiometric TGL probe, Mito-NBTTA-Tb3+/Eu3+, has been engineered for the quantitative monitoring and visualization of CO in living samples. This probe exhibited good biocompatibility and highly specific localization performance in mitochondria of live cells with sensitive, selective and ratiometric TGL response towards CO, which enabled the probe to be successfully employed to the ratiometric TGL imaging of mitochondrial CO generation in live cells after heme- and LPS-stimulations. Besides, the quantitative imaging of CO in mouse liver tissue slices as well as in live D. magna and mice verified the potential of Mito-NBTTA-Tb3+/Eu3+ for the monitoring of CO in live bodies. We envision that the proposed ACS Paragon Plus Environment

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probe could be a powerful tool for the quantification of CO in vitro and in vivo to reveal the roles of CO in physiology and relative diseases.

Supporting information The Supporting Information is available free of charge on the ACS Publications website at DOI: More experimental materials and instruments, synthesis and characterization of the ligand, experimental procedure for preparation of the stock solution of Mito-NBTTA-Tb3+/Eu3+, TGL detection of CO and selectivity experiment, details for MTT assay, co-localization experiments, imaging of CO in living cells and Daphnia magna, supplementary figures and table.

Acknowledgments We gratefully acknowledge the financial supports from the National Natural Science Foundation of China (Grant nos. 21475016 and 21775015).

Notes The authors declare no competing financial interest.

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A Mitochondria-Targetable Ratiometric Time-Gated Luminescence

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