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Simultaneously Inducing and Tracking Cancer Cell Metabolism Repression by Mitochondria-Immobilized Rhenium(I) Complex Jing Yang,†,§ Ji-Xian Zhao,†,§ Qian Cao,*,† Liang Hao,† Danxia Zhou,‡ Zhenji Gan,‡ Liang-Nian Ji,† and Zong-Wan Mao*,† †

MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-Sen University, Guangzhou 510275, China ‡ MOE Key Laboratory of Model Animals for Disease Study, Model Animal Research Center of Nanjing University, Nanjing 210061, China S Supporting Information *

ABSTRACT: Mitochondrial metabolism is essential for tumorigenesis, and the development of cancer is usually accompanied by alternations of mitochondrial function. Emerging studies have demonstrated that targeting mitochondria and mitochondrial metabolism is an effective strategy for cancer therapy. In this work, eight phosphorescent organometallic rhenium(I) complexes have been synthesized and explored as mitochondria-targeted theranostic agents, capable of inducing and tracking the therapeutic effect simultaneously. Complexes 1b− 4b can quickly and efficiently penetrate into A549 cells, specifically localizing within mitochondria, and their cytotoxicity is superior to cisplatin against the cancer cells screened. Notably, complex 3b [Re(CO)3(DIP) (py-3-CH2Cl)]+ containing thiol-reactive chloromethylpyridyl moiety for mitochondria immobilization shows higher cytotoxicity and selectivity against cancer cells than other Re(I) complexes without mitochondria-immobilization properties. Mechanistic studies show that complexes 1b−4b induce a cascade of mitochondria-dependent events including mitochondrial damage, mitochondrial respiration inhibition, cellular ATP depletion, reactive oxygen species (ROS) elevation, and caspase-dependent apoptosis. By comparison, mitochondria-immobilized 3b causes more effective repression of mitochondrial metabolism than mitochondrial-nonimmobilized complexes. The excellent phosphorescence and O2-sensitive lifetimes of mitochondriaimmobilized 3b can be utilized for real-time tracking of the morphological changes of mitochondria and mitochondrial respiration repression during therapy process, accordingly providing reliable information for understanding anticancer mechanisms. KEYWORDS: organometallic rhenium complex, mitochondria immobilization, metabolism repression, mitochondria dysfunction, PLIM, theranostic

1. INTRODUCTION

Emerging clinical evidence and laboratory-based experiments have demonstrated that targeting mitochondria and mitochondrial metabolism is an effective therapeutic strategy against cancer,7−9 and some targeted molecules have already attended clinical trials.10−16 These drugs exert their anticancer activities by disrupting the mitochondrial energy producing system, the biosynthesis function, and the redox homeostasis, ultimately leading to the activation of mitochondrial-dependent cell death signaling pathways, usually the apoptotic pathways because of the various apoptosis-promoting proteins existing in mitochondria.10−17 Thus, even when the endogenous apoptosis induction pathways were disrupted, mitochondria-targeted drugs can still trigger cellular apoptosis efficiently, which is more advanced than other conventional apoptosis-inducing

Mitochondria are well-known as the “cellular energy factory”, producing ATP and metabolites necessary for the cellular bioenergetic and biosynthetic demands.1 Recent studies provide genetic and pharmacologic evidence that mitochondrial metabolism is essential for oncogene revolution, tumorigenesis, and tumor growth.2,3 The occurrence and development of cancer are usually accompanied by alternations of mitochondrial function, e.g., increased oxidative stress and decreased mitochondrial membrane potential.4 Moreover, the mitochondrial metabolic pathways in cancer cells are different from those in normal cells. Cancer cells are primarily characterized by a metabolic phenotype of aerobic glycolysis for ATP generation while normal cells generally use oxidative phosphorylation as the major metabolic pathway.5,6 In this perspective, mitochondrial metabolism has emerged as a potentially fruitful arena for cancer therapy.1 © XXXX American Chemical Society

Received: February 6, 2017 Accepted: April 3, 2017 Published: April 3, 2017 A

DOI: 10.1021/acsami.7b01764 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces drugs and may overcome cancer resistance.17,18 On the other hand, a series of fluorescent mitochondria-targeted molecules have been synthesized for probing specific chemicals in mitochondria and tracking the dynamic changes of mitochondria.19,20 Inspired by these investigations, rational design of emissive mitochondria-targeted multifunctional theranostic agents for cancer treatment are highly sought, which can monitor the changes in mitochondria during therapeutic process, giving insights into their anticancer mechanisms.21−23 With the success of platinum-based anticancer drugs in the past few decades has emerged the development of other nonplatinum metallodrugs. Recently, organometallic rhenium compounds have become a kind of new promising anticancer drug candidate, showing comparable or superior anticancer activity compared with cisplatin.24−28 Most reported cytotoxic and luminescent Re organometallic complexes are based on the Re(CO)3 core, which makes the synthesis procedure and the introduction of functional ligands easily accessible and chemically robust. These Re complexes can quickly and efficiently penetrate into cells, and their cytotoxicity has been found highly proportional to their lipophilicity.24−26 Very recently several rhenium complexes have also been reported possessing photodynamic and photoactive therapeutic potentials.29−32 On the other hand, phosphorescent Re complexes have been widely explored as bioimaging and biosensing agents exhibiting good photostability, long-lived emission states, high quantum yields, and large Stokes shifts, and the emission can be tuned by varying the ligands.33−37 Therefore, phosphorescent Re(I) complexes possess intrinsic advantages for the construction of novel multifunctional theranostic platforms. By integrating the anticancer activities and the bioimaging capabilities, Re complex can simultaneously induce and monitor the therapeutic effects. In this regard, eight organometallic Re(I) compounds [Re(CO)3(N−N)L]PF6 (N−N = 1,10-phenanthroline (phen) or 4,7-diphenyl-1,10-phenanthroline (DIP), Scheme 1) were

designed and synthesized. The bidentate N−N ligand tunes the lipophilicity of the compounds, and the monodentate ligand L is expected to contribute to the mitochondria affinity. It is worth mentioning that the reactive chloromethyl moiety present in compounds 3a and 3b has the reactivity with biologically relevant thiols, accordingly covalently binding with thiol-containing proteins, such as bovine serum albumin (BSA, contains one free cysteine residue) and glutathione (GSH).22,38 Therefore, these two rhenium compounds are expected to be not only accumulating but also immobilized within mitochondria by covalent interactions with thiol-containing proteins. If so, their metabolism repression efficacy and mitochondrial tracking capability will be enhanced because of the prolonged retention time within mitochondria even when the membrane potential is already lost. Mitochondria-immobilized cyclometalated iridium(III) complexes have been recently reported.22,39 In this work, the anticancer properties of both mitochondria-immobilized and -nonimmobilized Re(I) complexes were compared, including the in vitro cytotoxicity, cancer cell selectivity, metabolism repression, mitochondria damage, cellular ATP depletion, reactive oxygen species elevation, and induction of apoptosis. Their capability for mitochondrial immobilization, in situ tracking of mitochondrial morphology, and cancer cell metabolism repression was also explored depending on their excellent phosphorescence and O2-sensitive lifetimes by using confocal and PLIM microscopy.

2. RESULTS AND DISCUSSION 2.1. Synthesis and Photophysical Properties. In the present work, we synthesized eight phosphorescent rhenium(I) complexes, [Re(CO)3(N−N)L](PF6) (Scheme 1), in which the bidentate N−N ligand was 1,10-phenanthroline (phen, 1a− 4a) and 4,7-diphenyl-1,10-phenanthroline (DIP, 1b−4b), respectively, and the monodentate ligand L was various pyridine analogues. These complexes were obtained by reacting 10 equiv of excess L with the dechlorination product of Re(CO)3(N−N)Cl precursors in THF followed by anion exchange with NH4PF6. Among them the synthesis route of complexes 3a and 3b was an exception, through direct reaction of 2a and 2b, respectively, with SO2Cl2 in a minimum amount of CH2Cl2. All of the complexes were purified by recrystallization and characterized by ESI-MS (Figures S1−S8, Supporting Information (SI)), 1H NMR spectroscopy (Figures S9−S16, SI), and elemental analysis. Complexes 2a and 3a were characterized by X-ray crystallography, the crystallographic parameters and selected bond angles/length of which were described in Tables S1−S4 (SI), respectively. The perspective view (Scheme 1) showed that 2a and 3a adopted octahedral geometries, in which the N−N ligand and two carbonyl groups were coplanar, and the L and another carbonyl group were distributed at the axial position. These findings are consistent with previously reported organometallic rhenium complexes.24−26 All of these Re(I) compounds were stable for at least 48 h in PBS (1% DMSO) at room temperature as monitored by UV−visible spectroscopy. 1H NMR spectra of compound 3b in DMSO-d6 and D2O (v/v, 7/3) did not show any changes after 48 h incubation at room temperature, further demonstrating that the reactive chloromethyl group did not undergo hydrolysis at least in 48 h (Figure S17, SI). The electronic absorption spectra of Re(I) complexes in CH2Cl2, PBS, and CH3CN at 298 K exhibited intense absorption bands at ca. 250−320 nm and relatively weak absorptions at ca. 320−450 nm, which were mainly attributed

Scheme 1. (A) Chemical Structures of Re(I) Complexes in This Work (Counter Ion, PF6); (B) X-ray Crystal Structures of 2a and 3a (H Atoms and Counter Ions PF6, Omitted for Clarity)

B


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ACS Applied Materials & Interfaces to the spin-allowed intraligand ππ* transitions and metal-toligand charge transfer (MLCT), respectively (Figure S18, SI). Upon excitation at 405 nm, these complexes exhibited yellow emission (maximum ca. 550 nm) from MLCT excited states (Figure S19, SI). The photophysical data are summarized in Table S5 (SI). Take 2b and 3b as model complexes; their emission intensity and phosphorescence lifetimes exhibited pH insensitivity (Figure S20, SI) but excellent O2 sensitivity (Figure S21, SI). Stern−Volmer plots revealed a good linear relationship: the higher the oxygen concentration, the weaker the phosphorescence and the shorter the lifetime. (Figure S22, SI) 2.2. Cellular Uptake and Localization. Due to the excellent phosphorescence properties of Re(I) complexes, their intracellular distribution can be easily monitored by fluorescence microscopy. Laser scanning confocal microscopic observation showed that complexes 1b−4b effectively penetrated into A549 cells after 20 min incubation, as indicated by their intense emission in the cytoplasm (Figure S23B, SI). However, the cellular uptake of complexes 1a−4a was not efficient under the same conditions; even when the incubation time was elongated, the intracellular emission of 1a−4a was not clearly visualized (Figure S23A, SI). On the other hand, as rhenium is an exogenous element, the cellular uptake efficacies of Re(I) contents can be quantitatively determined by using inductively coupled plasma−mass spectrometry (ICP-MS; Figure 1B). After incubation of A549 cells with 20 μM

smaller or even negative, indicating that 1b−4b were more lipophilic than 1a−4a (Figure 1A). In combination with the cellular uptake results, it is found that, to some degree, the greater the lipophilicity of these Re(I) complex, the larger the cellular uptake efficacy. We also studied the cellular uptake mechanism by using 2b and 3b as examples. Confocal microscope revealed that incubation of A549 cells with 2b and 3b at lower temperature (4 °C) or pretreatment of A549 cells with carbonyl cyanidemchlorophenyl hydrazone (CCCP, a metabolic inhibitor) resulted in reduced cellular uptake efficiency, whereas pretreatment of cells with chloroquine (an endocytosis modulator) displayed negligible effect on the cellular uptake efficiency (Figure S24, SI) These results indicate that the penetration of these Re(I) complexes into A549 cells is dominated by an energy-dependent process rather than endocytic pathways. Then study was conducted to explore the location of the efficiently uptaken complexes 1b−4b in living A549 cells. In confocal microscopic investigations, a commercially available mitochondria-specific stain, MitoTracker Deep Red (MTDR, 100 nM), was used. Figure 2A showed a high degree of colocalization between the visualized green phosphorescence from Re(I) complexes 1b−4b (10 μM) and MTDR in A549 cells, the Pearson’s correlation coefficients of which were determined to be 85%, 87%, 90%, and 86%, respectively. Meanwhile, negligible colocalization was observed between

Figure 1. (A) Lipophilicity of Re(I) complexes. log PO/W is measured as the logarithmic ratio of the concentration of complexes in n-octanol to that in the aqueous phase. (B) Cellular uptake of Re(I) complexes in A549 cells measured by ICP-MS.

complexes for 1 h, the Re(I) contents in the whole cell were found remarkably higher for b series complexes rather than a series complexes, which was consistent with the confocal microscope. It should be noted that both 3a and 3b with the chloromethylpyridyl moiety exhibited the highest cellular uptake among each series. To find out the influencing factors of cellular uptake efficacies for these Re(I) complexes, their lipophilicity referred to as log Po/w (octanol/water partition coefficient) values was determined by the shake-flask method and UV−visible spectroscopy. It is clearly seen that the log Po/w values of 1b−4b, which possessed more extended delocalized aromatic N−N ligand, were positive while those of 1a−4a were relatively

Figure 2. (A) Intercellular colocalization of 1b−4b with MTDR imaged by CLSM. A549 cells were incubated with 20 μM and at 20 min and then stained with MTDR (150 nM, 30 min) at 37 °C (1b− 4b, λex= 405 nm and λem = 550 ± 30 nm; MTDR, λex= 633 nm and λem = 655 ± 20 nm). (B) Distribution of complexes 1b−4b (20 μM, 1 h) in various organelles of A549 cells measured by ICP-MS. C


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extracted from complex 3b-treated cells displayed intensive emission whereas emissive bands were not observed in proteins isolated from 1b-, 2b-, and 4b-treated A549 cells. The emission originated from the irradiation-induced phosphorescence of Re(I) complex. This result indicates that complex 3b containing chloromethylpyridyl moiety, rather than other tested Re(I) compounds, could conjugate to intracellular proteins. Then the immobilization ability of complexes 1b−4b in mitochondria through covalent binding was further ascertained by detecting the changes in the emission intensity of Re(I) complexes upon cell fixation and elution. A commercially available mitochondrial-immobilized probe MTDR, also containing chloromethylpyridyl moiety and possessing reactivity for thiol group, was used as a positive control, which could be retained during cell fixation.41 Another commercially available mitochondrial dye, Rhodamine 123, was used as a negative control, which could be easily washed away once the loss of mitochondrial membrane potential occurred.42 As shown in the confocal microscopy image (Figure 3B), after cell fixation and repeated elution the intracellular emissions of complexes 1b, 2b, 4b, and Rhodamine 123 were barely seen, suggesting that they were easily washed away from fixed cells. Meanwhile, only MTDR and complex 3b were still largely retained in A549 cells under the same conditions, as indicated by the intensive intracellular emission. The combination of these observations with colocalization results indicates that complex 3b targets and selectively accumulates in mitochondria and then conjugates with thiol-containing proteins in mitochondria due to its thiol reactivity, thus being capable of acting as an effective mitochondria-immobilized probe for realtime tracking of mitochondrial morphologies. Moreover, the immobilization ability may be beneficial for prolonging the retention time of 3b in mitochondria, accordingly enhancing mitochondria-mediated cytotoxicity. 2.4. In Vitro Cytotoxicity and Selective Killing Cancer Cells. The in vitro cytotoxicity of the tested eight Re(I) compounds against several different cell lines (HeLa, human pulmonary carcinoma; A549, human lung carcinoma; A549R, cisplatin-resistant cell line; LO2, human normal liver cell line) was determined by MTT assay after 48 h of treatment in the dark. The resulting IC50 values were calculated and listed in Table 1, and cisplatin was adopted as the positive control. It can be seen that complexes 1a−4a with phen ligand only showed

1b−4b and the lysosome-specific stain LysoTracker Deep Red (LTDR; Figure S25, SI). ICP-MS was also utilized to further ascertain the mitochondria-specific accumulation of 1b−4b in A549 cells. After incubation with 1b−4b (20 μM) for 1 h, different cellular components such as mitochondria, cytoplasm, and nucleus were isolated from A549 cells, respectively, and Re(I) contents in different cellular components were quantified. Figure 2B revealed that Re(I) contents in mitochondria were much higher than those in cytoplasm and nucleus, indicating that 1b−4b could target mitochondria with high specificity in A549 cells. It is worth mentioning that, as measured by ICP-MS, complex 3b with chloromethylpyridyl moiety exerted the highest cellular uptake efficacies and the greatest mitochondrial accumulation over other Re(I) complexes explored in this work. 2.3. Mitochondrial Immobilization. It has been reported that compounds containing a chloromethyl group are capable of reacting with the thiol groups of cysteine residues within polypeptide and proteins to from stable covalent bonds.22,38 A model experiment was conducted by treating chloromethylcontaining complex 3b with glutathione in a mixed solution of alkaline water and methanol to demonstrate its reactivity with biologically relevant thiols. The product was characterized by ESI-MS, and the formation of a rhenium−glutathione conjugate [Re(CO)3(DIP) (py-3-CH2−GSH)]+ was confirmed by the molecular ion peak and a series of reasonable fragmentation peaks (Figure S26, SI), indicating that complex 3b possesses the reactivity for a biologically relevant thiol group. The intracellular protein conjugation abilities of complexes 1b−4b were investigated using polyacrylamide gel electrophoresis (PAGE). As shown in Figure 3A, proteins isolated from lysed Re(I)-treated (30 μM, 1 h) A549 cells were gel-separated and clearly visualized after CBB (Coomassie Brilliant Blue) staining; however, upon irradiation at 365 nm only the proteins

Table 1. IC50 (μM) Values of Tested Compounds Toward Different Cell Linesa IC50 (μM)

Figure 3. (A) Retaining of emission intensity of Rh123 (150 nM), MTDR (150 nM), and 1b−4b (20 μM) after fixation and washing. A549 cells were incubated with indicated compound for 1 h, fixed by paraformaldehyde, and washed twice with PBS/DMSO (9/1, v/v). The cells were imaged by the confocal instrument after each step. Scale bar: 10 μm. (B) SDS-PAGE analysis of proteins purified from lysed 1b−4b (20 μM, 1 h)-treated A549 cells. The gel was scanned with transmissive ultraviolet (λex =365 nm, top) and then stained with CBB (bottom).

compd

A549

A549R

HeLa

LO2

1a 2a 3a 4a 1b 2b 3b 4b cisplatin

>100 >100 75.8 ± 2.3 39.8 ± 0.7 3.9 ± 0.7 5.5 ± 0.6 3.4 ± 0.6 22.4 ± 1.2 21.5 ± 2.5

>100 >100 37.3 ± 1.1 36.5 ± 1.8 1.2 ± 0.5 2.7 ± 0.5 0.75 ± 0.12 8.5 ± 1.1 65.6 ± 1.6

>100 >100 64.6 ± 2.2 52.5 ± 3.0 0.95 ± 0.11 1.7 ± 0.4 0.52 ± 0.07 5.9 ± 0.9 8.9 ± 1.0

>100 >100 >100 47.9 ± 1.5 3.1 ± 0.5 7.6 ± 0.9 18.7 ± 1.1 6.4 ± 0.7 29.9 ± 2.1

a

IC50 values are drug concentrations necessary for 50% inhibition of cell viability. Data are presented as means ± standard deviations (SDs) obtained in at least three independent experiments, and the drug treatment period was 48 h.

D


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selectively localize in mitochondria, their capability to induce mitochondrial dysfunction was investigated. Mitochondrial dysfunction is usually accompanied by the loss of mitochondrial membrane potential (MMP, ΔΨm); thus the Re(I) complexinduced changes in MMP were initially detected by using flow cytometry with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining, a mitochondriaselective aggregate dye.43 The loss of MMP was characterized by an increase in green fluorescence (JC-1 monomers) and a decrease in red fluorescence (JC-1 aggregates). As shown in Figure 5A, in control cells red fluorescence was mainly observed indicating the high membrane potentials; after treating cells with 1b−4b for 6 h in the dark, a significant dose-dependent red-to-green color shift is observed indicating the remarkable decrease in MMP. Take 3b-treated cells as an example, the percentage of cells with depolarized mitochondrial membranes increased from 3.17 ± 0.5% (control) to 28.4 ± 1.1% (2.5 μM), 77.6 ± 0.5% (5 μM), and 91.7 ± 0.5% (10 μM), respectively. The representative JC-1 red/green ratio signals in Re(I)-treated cells were also compared (Figure S27, SI), showing that the most prominent Re(I) complex-induced MMP loss was observed in 3b-treated cells. The capability of these Re(I) complexes to induce MMP loss is correlated with their cytotoxicity to some extent. Because mitochondrion is known as the bioenergetic center for cell metabolism, we further explored the impact of Re(I) complexes on mitochondrial metabolic and bioenergetic status, mainly the intracellular ATP level and mitochondrial respiration. As shown in Figure 5B, Re(I)-treated A549 cells exhibit a dose-dependent decrease in the intracellular ATP levels compared with the untreated cells. Among all the tested compounds, 3b exhibits the strongest ability to reduce ATP levels, decreasing the ATP level from 120 ± 3.2 nM (control) to 57.0 ± 2.32 nM (1.25 μM), 30.5 ± 2.6 nM (5 μM), and 15.3 ± 3.1 nM (10 μM) per million cells, respectively. Notably, the capability of 1b−4b to reduce the ATP levels in A549 cell is correlated with their cytotoxicity. Further, we chose mitochondria-targeted complex 2b and mitochondrial-immobilized complex 3b as model compounds to explore their impact on mitochondrial respiration. Seahorse XF24 extracellular flux analyzer was utilized to quantify the mitochondrial respiration by measuring the oxygen consumption rate (OCR).44 Panels C and D of Figure 5 compare the OCR changes of untreated and Re(I)-treated A549 cells upon addition of respiration modulators (oligomycin, FCCP, and antimycin A/rotenone) at different time points, which can target different components on the electron transport chain. Initially, treatment with 3b (0.5 and 1.0 μM) causes a dramatic dose-dependent decrease in the basal OCR from 5.51 ± 0.31 (control), 2.29 ± 0.30 (0.5 μM), and 1.01 ± 0.12 (1.0 μM) to 0.45 ± 0.14 (2.0 μM) (Figure 5E). After addition of oligomycin (an ATP synthase inhibitor, 1 μM), coupled respiration for ATP synthesis and uncoupled respiration for driving the proton pumping−leaking cycle across the inner mitochondrial membrane can be affected.44,45 Compared with control cells, 3b-treated cells display a prominent dose-dependent decrease in ATP production from 4.44 ± 0.41 (control) and 0.61 ± 0.11 (0.5 μM) to 0.17 ± 0.05 (1.0 μM) (Figure 5F). Proton leak also changes in 3b-treated cells as compared with control cells: lower dose treatment (0.5 μM) enhances proton leak, and higher dose treatment (1.0 μM) declines proton leak (Figure 5G). Then after addition of FCCP (a potent mitochondrial uncoupling agent, 1 μM), the control ability of ATP synthase to

moderate cytotoxicity or even noncytotoxicity, whereas complexes 1b−4b with DIP ligand showed much higher cytotoxicity than cisplatin against all the human cancer cell lines tested, with IC50 values ranging from 0.52 ± 0.08 to 22.4 ± 0.5 μM. This is in accordance with the cellular uptake level: the higher the cellular uptake level, the greater the cytotoxicity of Re(I) complexes. On the other hand, selectively killing cancer cells with minimum side effect on normal cells is one of the major obstacles for cancer treatment. According to the IC50 values listed in Table 1, complexes 1b−4b also possess some extent of selectivity toward cancer cell lines with less toxicity against normal cells. The high cytotoxicity against the A549R cell line also indicates that they could overcome cisplatin resistance. Among them 3b exhibits the highest antiproliferative activity against cancer cells, which is ca. 7-fold, 17-fold, and 87-fold more potent than cisplatin in killing A549, HeLa, and cisplatinresistant A549R cells, respectively. Notably, 3b also possesses the highest selectivity toward cancer cells A549 (ca. 6-fold) and HeLa cells (ca. 36-fold) over normal cells LO2. A LO2 and A549 cell coculture model was constructed to further elucidate the capability of 3b to selectively kill cancer cells (Figure 4). In this model, Hoechst staining was performed

Figure 4. Capability of complex 2b and 3b (5 μM, 24 h) to selectively induce cancer cell apoptosis in an A549/LO2 cocultured cell model measured by annexin V/PI double staining. A549 cells were prelabeled by Hoechst (blue). Scale bar: 20 μm.

on A549 nuclei in advance, and then both prestained A549 and LO2 cells were treated with 3b and co-incubated followed by annexin V/PI staining. The confocal microscopic images clearly showed that most of the A549 cells (blue nuclei) were also positively stained by annexin V (green color) and PI (red color), whereas LO2 cells were not successfully stained by annexin V/PI (Figure 4 lower panel). This indicates that apoptotic cell death was selectively induced in A549 cells while LO2 cells were still vigorous after the same treatment with 3b. Compound 2b was also conducted under the same conditions as a comparison, showing that the Hoechst staining (blue nuclei) and annexin V/PI staining (green and red color) were not perfectly localized in the same cells (Figure 4 upper panel). These observations further confirmed that complex 3b could selectively kill cancer cells rather than normal cells while the selectivity of 2b for cancer cells was not good. The relatively higher cytotoxicity and better cancer cell selectivity of complex 3b among all the tested Re(I) compounds may be partially attributed to its immobilization capability and prolonged retention time in mitochondria. 2.5. Induction of Mitochondrial Dysfunction and Metabolic Repression. Because complexes 1b−4b could E


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Figure 5. Induction of mitochondrial dysfunction by rhenium complexes 1b−4b. (A) MMP of Re(I)-treated (6 h) A549 cells analyzed by flow cytometry at indicated concentrations (JC-1 staining, λex = 488 nm and λem = 530 ± 30 nm (green) and 585 ± 30 nm (red)). (B) Intracellular ATP levels in Re(I)-treated (6 h) A549 cells. (C−D) Respiratory profiles of untreated and Re(I)-treated A549 cells at the indicated concentrations measured by a Seahorse XF24 extracellular flux analyzer. Respiration modulators oligomycin (1 μM), FCCP (1 μM), and the mixture of rotenone (1 μM) and antimycin A (1 μM) were added at different time points. The OCR values were normalized to 1 μg protein determination by the BCA assay. (E) Basal respiration calculated by subtracting OCR values after the addition of the mixture of rotenone (1 μM) and antimycin A (1 μM) from basal OCR. (F) ATP production calculated by subtracting OCR values after the addition of oligomycin from basal OCR. (G) Proton leak calculated by subtracting OCR values after the addition of the mixture of rotenone (1 μM) and antimycin A (1 μM) from OCR values obtained after the addition of FCCP. (H) Nonmitochondrial respiration was the OCR value after the addition of the mixture of rotenone (1 μM) and antimycin A (1 μM).

respiration and the proton gradient is eliminated, shown as the rebound of the OCR peak curve in control cells. However, a dramatic decrease in the OCR peak rather than the rebound is observed in 3b-treated cells under the same conditions (Figure 5D). This indicates the loss of spare respiratory capacity of 3btreated cells.44,45 Finally, a mixture of antimycin A (a mitochondrial complex III inhibitor, 1 μM) and rotenone (a mitochondrial complex I inhibitor, 1 μM) is added to shut down the mitochondrial respiratory chain thoroughly, resulting in substantial decline of OCR peak both in control and 3btreated cells (Figure 5D). The remaining OCR values represent nonmitochondrial O2 consumption, including substrate oxidation and cell surface O2 consumption.44,45 It is observed that nonmitochondrial O2 consumption in 3b-treated cells is much less (0.18 ± 0.05, 2.0 μM) than that in control cells (1.08 ± 0.11) (Figure 5H). These results indicate that both the mitochondrial respiration and nonmitochondrial respiration are effectively inhibited, contributing to the relatively superior cytotoxicity of 3b. As a comparison, treatment with 2b at the same concentrations under the same conditions (Figure 5C)

also causes metabolic repression, shown as decreased basal OCR, reduced ATP production, enhanced proton leak, and inhibited nonmitochondrial respiration compared with control cells (Figure 5E−H). However, it is worth noting that 2b at the same concentrations displays a much less prominent impact on the OCR than 3b. All these results combined with the MMP and ATP assay indicate that the mitochondrial-immobilized complex 3b possesses stronger capability to induce mitochondrial dysfunction and metabolic inhibition than the nonimmobilized complexes. This could be explained partially by the mitochondrial immobilization capability of 3b and the prolonged retention time. 2.6. Real-Time Tracking of Mitochondria Dysfunction. Because of the high quantum yields and the capability of targeting and inducing mitochondrial dysfunction, Re(I) complexes may possess great potential acting as theranostic agents for real-time tracking of Re(I)-induced mitochondrial dysfunction. As the lifetimes of 2b and 3b are very sensitive to oxygen concentrations, the intracellular oxygen consumption in F


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3b-treated cells. This is consistent with the Seahorse results that 3b can inhibit mitochondrial metabolism and reduce oxygen consumption rate more effectively. PLIM results prove the promising application of 2b and 3b as theranostic agents capable of inducing and monitoring the mitochondrial metabolic inhibition simultaneously. In addition, mitochondria are dynamic organelles and mitochondrial dysfunction is usually accompanied by the abnormal mitochondrial morphology.46 Complexes 2b and 3b were chosen as representatives of mitochondria- nonimmobilized and -immobilized Re(I) compound, respectively, and their emission can be monitored by confocal microscopy thus displaying the real-time changes in the mitochondrial morphology. As shown in Figure 7 and Figure S29A (SI),

Re(I)-treated cells can be monitored by phosphorescence lifetime imaging (PLIM), accordingly reflecting the extent of Re(I)-induced inhibition of mitochondrial respiration and the mitochondrial metabolic status. The higher oxygen concentration in living cells represents less oxygen consumption, thus the slower oxygen consumption rate and the more inhibited mitochondrial metabolism. The upper panel of Figure 6A shows the PLIM imaging of 3b-treated fixed A549 cells under different oxygen concen-

Figure 7. Real-time tracking of mitochondrial morphology in 3btreated A549 cells (20 μM) by CLSM. Scale bar: 5 μm.

Figure 6. (A) PLIM images of 3b-treated (10 μM, 30 min) fixed and living A549 cells under different oxygen partial pressures at 37 °C (λex = 405 nm and f = 0.5 MHz). Scale bar: 20 μm. (B) Luminescence lifetime distributions of PLIM imaging of 3b-treated fixed cells. (C) Fitted Stern−Volmer plots of 3b. Red dots and green triangles represent the corresponding data collected from 3b-treated fixed and living cells, respectively.

both 2b and 3b (10 μM) could effectively penetrate into A549 cells in 5 min, the intensive emission of which ascertained the healthy mitochondria with normal filamentous network. Prolonged treatment of A549 cells with 2b and 3b caused gradual mitochondrial swelling. After treatment for 40 min, the hollow spheres and solid granules can be clearly seen in most cells, representing the extremely swollen and damaged mitochondria. Notably, with the continuous extension of incubation time the capability of 2b and 3b to specifically localize in mitochondria may be affected by the loss of membrane potential and mitochondrial damage. In Figure S29B (SI), after treatment for 2 and 4.5 h, 3b was still colocalized excellently with MTDR (Pearson’s correlation coefficients of 85−91%), but the colocalization degree of 2b with MTDR was reduced with lower Pearson’s correlation coefficients of 53− 56%. This can be explained by the fixation of 3b in mitochondria and the escape of nonfixable 2b from mitochondria when the membrane potential was lost. These findings indicate that mitochondrial-immobilized complex is more advantageous for real-time tracking of morphological changes during mitochondrial damage. 2.7. Intracellular ROS Levels. Intracellular ROS is one of the most important mediators of cell death, and mitochondria are known as a major source of ROS. Hence the capability of mitochondria-targeted compounds 1b−4b to induce intracellular ROS elevation was examined by using flow cytometry with 2′,7′-dichlorofluorescein diacetate (H2DCFDA) staining. H2DCFDA is naturally nonfluorescent and can be converted into highly fluorescent 2′,7′-dichlorofluorescein (DCF) by intracellular ROS.47 After 6 h treatment, 1b−4b caused a dramatically dosedependent increase in intracellular ROS levels (Figure 8A). At a concentration of 10 μM, the mean fluorescent intensity of DCF

trations, and elongated lifetimes are observed upon decreasing O2 concentration from 21% to 2%. The corresponding lifetime distribution histogram is shown in Figure 6B. Because it is generally supposed that the O2 concentration in fixed cells is the same as the extracellular environment, a calibration curve of intracellular O2 concentrations as a function of phosphorescence lifetimes of 3b is constructed (Figure 6C), and the red dots are collected from the data of Figure 6B. Under the same conditions, the calibration curve of 2b is also obtained using PLIM imaging of 2b-treated fixed A549 cells (Figure S28, SI). Then the PLIM imaging of 2b- and 3b-treated living cells is collected (Figure 6A and Figure S28A, bottom panel, SI). Under different incubation conditions, the lifetime changes and the intracellular O2 concentrations in Re(I)-treated living cells can be calculated according to the calibration curves, which are represented as green triangles in Figure 6C and summarized in Tables S8 and S9 (SI). For example, under ambient conditions, the living A549 cells treated with 2b and 3b exhibit a phosphorescent lifetime of ca. 470 and 410 ns, respectively, indicating the mitochondrial oxygen concentrations of 24% and 29%. When the extracellular oxygen concentration is reduced to 2%, the phosphorescent lifetimes of 2b and 3b were elongated to 650 and 620 ns and the mitochondrial oxygen concentrations were calculated to be 8% and 10%, respectively. The higher oxygen concentration in 3b-treated rather than 2b-treated cells is likely the result of less oxygen consumption and thus a slower respiration rate, indicating that mitochondrial metabolism is more inhibited in G


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c, from the membrane between the space of mitochondria to cytosol, thus initiating the death signaling cascade.48 Complexes 2b and 3b were chosen as representative of mitochondrialnonimmobilized and -immobilized Re(I) compound, respectively, to explore their capability to induce apoptosis. First, confocal microscopy was utilized to monitor the nuclear morphology of Re(I)-treated A549 cells stained with Hoechst 3342 (Figure S30, SI). Control cells show normal morphology and exhibit homogeneous nuclear staining, whereas treatment with 2b and 3b (8 μM, 24 h) both increase the percentage of apoptotic cells with morphological characteristics, e.g., membrane blebbing, cell shrinkage, and nuclear fragmentation.49 Next, phosphatidylserine externalization50 was detected as a hallmark of early apoptosis using Annexin V/PI labeling. As shown in flow cytometry analysis (Figure 9A), annexin Vpositive/PI-negative cells are considered as early apoptotic

Figure 8. (A) Intracellular ROS generation in Re(I)-treated (2.5−10 μM, 6 h) A549 cells measured by flow cytometry (λex = 488 nm and λem = 525 nm). (B) Dose-dependent inhibition of cell death upon the incubation of A549 cells with ROS inhibitor NAC. Data are represented as means ± SD of three independent experiments.

in 1b−4b-treated cells was approximate 4−7-fold higher than that in vehicle-treated cells. Upon the treatment of ROS inhibitor NAC, the cell viability was increased dramatically depending on the NAC concentration (Figure 8B). These findings indicate that complexes 1b−4b can induce intracellular ROS elevation and ROS-dependent cell death. Because of the inefficient cellular uptake and low cytotoxicity of 1a−4a, they were excluded from this and the following intracellular investigations. 2.8. Induction of Apoptosis. Mitochondria are essential components of the intrinsic pathway of apoptosis, which regulates the release of pro-apoptotic proteins, e.g., cytochrome

Figure 9. (A) Annexin V/PI double staining analyzed by flow cytometry. A549 cells were incubated with 2b and 3b for 6 h. (B) Representative TEM images showing the morphological features of A549 cells treated with 3b (1 and 3 μM) for 24 h. Panels d and e are enlarged views of panel b, and panel f is the enlarged view of panel c, respectively. N, nuclear. Scale bars: 2 μm. H


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consumption, thus reflecting the Re(I)-induced mitochondrial respiration repression. The excellent phosphorescence of 2b and 3b can also be utilized for real-time tracking of Re(I)induced mitochondrial morphological changes. By comparison, complex 3b functions as a better theranostic agent for in situ tracking of therapeutic effect because it is still well-fixed within mitochondria even when the mitochondria are already damaged. Overall, our work shows that targeting mitochondria and mitochondrial metabolism is an effective strategy for cancer therapy. Rational construction of multifunctional theranostic anticancer agents with mitochondrial-immobilization property can enhance cytotoxicity and selectivity against cancer cells, as well as the mitochondrial tracking function.

while annexin V/PI double positive cells are assigned to be late apoptotic and necrotic. It can be seen that treatment with 3b caused a dose-dependent increase in the percentage of cells in both early apoptotic and late/necrotic phases and mainly induced early apoptosis. After 24 h treatment with 3b, the percentages of early apoptotic cells increased from 5.2 ± 0.5% to 17.5 ± 1.12% (10 μM) and 63.3 ± 0.82% (20 μM). In contrast, the capability of complex 2b to induce apoptosis was much weaker, and the percentages of cells in early apoptotic phase was only 6.7 ± 0.03% (10 μM) and 11.6 ± 0.01% (20 μM) under the same conditions. The percentages of late apoptotic cells among 3b-treated cells were also much higher than those among untreated and 2b-treated cells. Transmission electron microscope (TEM) was also utilized to investigate the mitochondrial morphology of 3b-treated A549 cells (1 and 5 μM, 24h). Compared with vehicle-treated cells, 3b induced substantial morphological changes including swollen mitochondria with disrupted cristae at lower dose, a remarkable increase in the numbers of damaged mitochondria, and cytoplasmic vacuolation at higher dose. These morphological changes are the typical features of mitochondria dysfunction-mediated apoptosis (Figure 9B). During apoptosis the release of pro-apoptotic proteins from mitochondria can activate the death-driving proteolytic proteins caspases.51 A homogeneous luminescent Caspase-Glo assay was operated to elucidate the effects of 2b and 3b on the caspase-3/ 7 activity. As shown in Figure S31, treatment of 2b and 3b moderately induced the activation of caspase-3/7 in a dosedependent manner, indicating that both 2b and 3b can induce caspase-dependent apoptotic pathways. This can be considered as a consequence of Re(I)-induced mitochondrial dysfunction.

4. MATERIALS AND METHODS Re(CO)5Cl (Sigma-Aldrich), 4,7-diphenyl-1,10-phenanthroline (DIP, Sigma-Aldrich), NH4PF6 (Alfa Aesar), pyridine (J&K Scientific Ltd.), 3-(hydroxymethyl)pyridine (J&K), 3-(chloromethyl)pyridine (J&K), 3-pyridinylmethanamine (J&K), 1-ethlimidazole (J&K), silver trifluoromethanesulfonate (Sigma-Aldrich), cisplatin (Sigma-Aldrich), DMSO (dimethyl sulfoxide; Sigma-Aldrich), MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; Sigma-Aldrich), MTDR (MitoTracker Deep Red FM; Life Technologies, USA), LTDR (LysoTracker Deep Red FM; Life Technologies), Hoechst 33342 (SigmaAldrich), JC-1 (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide; Sigma-Aldrich), and H2DCFDA (2′,7′-dichlorodihydrofluorescein diacetate, Sigma-Aldrich) were used as received. Caspase-3/7 activity assay kit, Cell Titer-Glo luminescent Cell Viability Assay kit, and ribonucleotide triphosphates (rRTPs) kit were purchased from Promega (USA). All the tested compounds were dissolved in DMSO and diluted by PBS just before the cellular experiments, and the concentration of DMSO was less than 1% (v/v). NMR spectra were recorded on a Bruker Avance 400 spectrometer (Germany). ESI-MS were recorded on a Thermo Finnigan LCQ DECA XP spectrometer (USA). Microanalysis (C, H, and N) was carried out using an Elemental Vario EL CHNS analyzer (Germany). UV/vis spectra were recorded on a Varian Cary 300 spectrophotometer (USA). Emission measurements were conducted on an FLS 920 combined fluorescence lifetime and steady state spectrometer (Japan). Quantum yields of luminescence at room temperature were calculated using [Ru(bpy)3 ](PF6)2 as the reference. Oxygen concentration was controlled by flow counters (HORIBA STEC, SEC-E40JS, 60 cm3(STP) min−1) or oxygen concentration-changeable multigas incubator (Thermo Scientific, SERIES II WATER JACKET CO2 Incubator, Model: 3131, S/N: 112620-1988) during the spectra, lifetime measurements, and cell imaging. The PLIM setup is integrated with an Olympus FV1000 laser scanning confocal microscope equipped with a 40 immersion objective lens. The lifetime values were calculated with professional software provided by PicoQuant Company. 4.1. Synthesis and Characterizations. General Synthetic Procedure of [Re(CO)3(N−N)L](PF6). A mixture of precursor [Re(CO)3(N−N)]Cl (0.20 mmol) and AgCF3SO3(0.30 mmol) in CH3CN (50 mL) was refluxed overnight under N2 protection. After removing off-white AgCl precipitate, the remaining solution was evaporated to obtain yellow solids of [Re(CO)3(N−N) (CH3CN)](CF3SO3), which was used directly for further reaction without purification. L (2 mmol) was added into 50 mL THF solution of [Re(CO)3(N−N) (CH3CN)](CF3SO3) and refluxed for 20 h under N2 protection. The solvent was evaporated, and the resulting solids were resolved in 3 mL of CH3CN, and then aqueous solution of 10fold excess of NH4PF6 was added and stirred for another 1 h. Finally, the precipitates were washed with diethyl ether and further recrystallized from CH3CN/diethyl ether. However, the synthesis route of 3a and 3b was an exception and presented in Scheme S1 (SI): 2a or 2b was resolved in minimum CH2Cl2 and 5 mL of SO2Cl2 was added, the mixture was refluxed for 5 h under N2 protection, and

3. CONCLUSIONS In summary, eight organometallic Re(I) tricarbonyl polypyridyl complexes with different lipophilicities have been synthesized as mitochondria-targeted anticancer agents. Among them, complexes 1b−4b can quickly and effectively penetrate into A549 cells and specifically localize in mitochondria while the cellular uptake of 1a−4a is not efficient. The cytotoxicities of 1b−4b are superior to cisplatin against various cancer cells, including cisplatin-resistant A549 cells, whereas the cytotoxicities of 1a− 4a are much lower. This is highly related to their lipophilicity and cellular uptake efficacy. Notably, complexes 3b containing a reactive chloromethylpyridyl moiety can be immobilized within mitochondria probably through nucleophilic substitution with reactive thiol groups within mitochondrial proteins. The mitochondrial immobilization of 3b results in relatively higher cytotoxicity against cancer cells than other nonimmobilized Re(I) complexes. Moreover, 3b can selectively kill cancer cells among the co-incubated A549 and LO2 cells, whereas the cancer cell selectivity was not good for the nonimmobilized complex 2b under the same condition. This may be attributed to the higher cellular uptake efficacy as well as longer retention time in mitochondria. Mechanism studies show that mitochondria-targeted 1b−4b mainly induce a series of mitochondria-dependent events, including mitochondrial damage, mitochondrial respiration inhibition, ATP production depletion, ROS level elevation, and caspase-dependent cellular apoptosis. By comparison, mitochondria-immobilized 3b causes more effective repression of mitochondrial metabolism than nonimmobilized complexes. Simultaneously, the O2-sensitive phosphorescent lifetimes of 2b and 3b can be utilized for PLIM imaging of intracellular oxygen I


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7.69 (s, 10H; H3−H7, H10−H14 of Ph2-phen), 7.40−7.44 (m, J = 7.8, 5.8 Hz, 1H; H5 of pyridine), 4.70 (s, 2H; 2H; CH2). ESI-MS: m/z 729.9 [M − PF6−]+. [Re(CO)3(DIP)(L4)](PF6) (4b). Yield: 122.7 mg (70%). Elem. Anal. (%). Calcd for C33H24F6N4O3PRe·4H2O (928.15): C, 44.72; H, 3.48; N, 6.04; C/N, 7.40. Found: C, 44.63; H, 3.51; N, 5.89; C/N, 7.57. 1H NMR (400 MHz, DMSO): δ 9.48 (d, J = 5.4 Hz, 2H; H1 and H16 of Ph2-phen), 8.42−8.44 (m, 2H; H1, H6 of pyridine), 8.15−8.20 (m, J = 5.4 Hz, 4H; H8, H9, H2, H15 of Ph2-phen), 7.72 (s, 11H; H3−H7, H10−H14 of Ph2-phen, H4 of pyridine), 7.40−7.44 (dd, J = 7.8, 4.8 Hz, 1H; H5 of pyridine), 4.68−4.71 (m, 2H, 2H; CH2). 3.97−4.00 (m, 2H, 2H; NH2). ESI-MS: m/z 711.0 [M − PF6−]+; 752.0 [M − PF6−]+ + CH3CN. 4.2. Crystallographic Structure Determination. Crystals of 2a and 3a qualified for X-ray analysis were obtained by the slow diffusion of diethyl ether into the acetonitrile solution of complexes. X-ray diffraction measurements were performed on a Bruker Smart 1000 CCD diffractometer with Mo Kα radiation (λ = 0.71073 Å) at 173 or 150 K. The crystal structures were solved by direct methods with program SHELXS and refined using the full matrix least-squares program SHELXL.40 The CCDC deposit numbers for 2a and 3a are 1520808 and 1520690, respectively. Crystallographic data, details of data collection, and structure refinements are listed in Tables S1 and S3. Selected bond distances and angles are listed in Tables S2 and S4. The structural plots were drawn using the xp package in SHELXTL at a 30% thermal ellipsoids probability level. 4.3. Cell Lines and Culture Conditions. A549, A549cisR, HeLa, and LO2 cells were obtained from Experimental Animal Center of Sun Yat-sen University (Guangzhou, China). Cells were maintained in DMEM (Dulbecco’s modified Eagle’s medium; Gibco BRL) or RPMI 1640 (Roswell Park Memorial Institute 1640, Gibco BRL) medium, which contained 10% FBS (fetal bovine serum; Gibco BRL), 100 μg/ mL streptomycin (Gibco BRL), and 100 U/ml penicillin (Gibco BRL). The cells were cultured in a humidified incubator, which provided an atmosphere of 5% CO2 and 95% air at 37 °C. In each experiment, cells treated with vehicle control (1% DMSO) were used as the reference group. 4.4. Cellular Uptake. A549 cells were treated with 1a−4a (200 μM) for 2 h and 1b−4b (20 μM) for 30 min at 37 °C, then washed three times with ice-cold PBS, and visualized by confocal microscopy (LSM 710, Carl Zeiss, Göttingen, Germany). Emission was collected at 550 ± 20 nm upon excitation at 405 nm. Colocalization Assay. A549 cells were co-incubated with 1b−4b (20 μM) and MTDR (200 nM) or LTDR (150 nM) at 37 °C for 30 min. Cells were washed three times with PBS and visualized by confocal microscopy immediately. The excitation wavelengths of Re(I) complexes, MTDR and LTDR, are 405 and 633 nm, respectively. Emission was collected at 540 ± 20 nm for 1b−4b and 665 ± 20 nm for MTDR and LTDR. ICP-MS Measurement. A549 cells were seeded in 10 cm tissue culture dishes at a density of 1 × 105 cells/mL in 5 mL of RPMI 1640 medium (Invitrogen). Cells were incubated at 37 °C with 1a−4a (100 μM) for 4 h and 1b−4b (20 μM) for 1 h, respectively. After digestion in trypsin−EDTA solution, A549 cells were counted and digested in 60% HNO3 at room temperature overnight and then diluted with Milli-Q H2O to obtain 2% HNO3 solutions for ICP-MS (ThermoElemental, USA) measurement of the whole cell rhenium contents. For the measurements of rhenium contents in various organelles, nuclear and cytoplasm fractions were separated by a nuclear and cytoplasmic protein extraction kit (Shanghai Sangon Biological Engineering Technology & Services Co. Ltd.) and mitochondria fractions were separated by a cell Mitochondria Isolation Kit (Beyotime Biotechnology), respectively, just before the 60% HNO3 digestion and Milli-Q H2O dilution. The standards for rhenium calibration were freshly prepared by diluting a ReNO3 stock solution with 2% HNO3 in Milli-Q H2O. 4.5. Mitochondrial Immobilization. Confocal Microscopy before and after Cell Fixation. A549 cells were cultured and incubated with 1b−4b (10 μM) and MTDR and Rhodamine 123 for 2 h. The cells were then washed twice by PBS, fixed in 2 mL of 4%

NaOH solution was simultaneously used as the tail gas absorber. The solvent was evaporated, and the resulting solids were resolved in 3 mL of CH3CN; aqueous solution of 10-fold excess of NH4PF6 was added and washed several times with diethyl ether and recrystallized from CH3CN/diethyl ether. [Re(CO)3(phen)(L1)](PF6) (1a). Yield: 113 mg (82%). Elem. Anal (%). Calcd for C20H13F6N3O3PRe·0.05ether (675.02): C, 37.05; H, 2.97; N, 6.11; C/N, 6.06. Found: C, 36.92; H, 2.94; N, 6.20; C/N, 5.95. 1H NMR (400 MHz, DMSO); δ 9.78 (dd, J = 5.1, 1.3 Hz, 2H; H1 and H8 of phen), 8.98 (m, 2H; H3 and H6 of phen), 8.46 (dd, J = 6.5, 1.4 Hz, 2H; H1 and H5 of pyridine), 8.32 (s, 2H; H4 and H5 of phen), 8.30−8.20 (m, 2H; H2and H7 of phen), 7.87 (tt, J = 7.7, 1.5 Hz, 1H; H3 of pyridine), 7.33 (dd, J = 7.6, 6.6 Hz, 2H; H2 and H4 of pyridine). ESI-MS: m/z 529.9 [M − PF6−]+. [Re(CO)3(phen)(L2)](PF6) (2a). Yield: 109.7 mg (76%). Elem. Anal. (%). Calcd for C21H15F6N3O4PRe (705.03): C, 36.05; H, 2.35; N, 5.96; C/N, 6.04. Found: C, 35.65; H, 2.56; N, 5.94; C/N, 6.00. 1H NMR (400 MHz, DMSO): δ 9.78 (dd, J = 5.1, 1.2 Hz, 2H; H1 and H8 of phen), 9.05 (dd, J = 8.3, 1.2 Hz, 2H; H3 and H6 of phen), 8.36 (t, J = 7.8 Hz, 1H; H1 of pyridine), 8.32 (s, 2H; H4, H5 of phen), 8.30 (d, J = 5.5 Hz, 1H; H4 of pyridine), 8.26 (dd, J = 8.3, 5.1 Hz, 2H; H2, H7of phen), 7.77 (d, J = 7.9 Hz, 1H; H2 of pyridine), 7.28 (dd, J = 7.8, 5.7 Hz, 1H, H3 of pyridine), 5.34 (t, J = 5.7 Hz, 1H; OH), 4.32 (d, J = 5.7 Hz, 2H; CH2). ESI-MS: m/z 559.9 [M − PF6−]+. [Re(CO)3(phen)(L3)](PF6) (3a). Yield: 103.6 mg (70%). Elem. Anal. (%). Calcd for C20H14ClF6N3O3PRe (722.99): C, 34.26; H, 2.38; N, 5.43; C/N, 6.30. Found: C, 34.74; H, 2.36; N,5.79; C/N, 6.00. 1H NMR (400 MHz, DMSO): δ 9.79 (dd, J = 5.1, 1.1 Hz, 2H; H1 and H8 of phen), 9.05 (dd, J = 8.3, 1.1 Hz, 2H; H3 and H6 of phen), 8.56 (s, 1H; H1 of pyridine), 8.40 (d, J = 5.2 Hz, 1H; H4 of pyridine), 8.31 (s, 2H; H4 and H5 of phen), 8.26 (dd, J = 8.3, 5.1 Hz, 2H; H2 and H7 of phen), 7.92 (d, J = 8.0 Hz, 1H; H2 of pyridine), 7.33 (dd, J = 7.9, 5.7 Hz, 1H; H3 of pyridine), 4.63 (s, 2H; CH2). ESI-MS: m/z 577.9 [M − PF6−]+. [Re(CO)3(phen)(L4)](PF6) (4a). Yield: 96.6 mg (67%). Elem. Anal. (%). Calcd for C21H16F6N4O3PRe·3H2O (757.59): C, 33.29; H, 2.93; N, 7.40; C/N, 4.49. Found: C, 33.13; H, 2.89; N, 7.48; C/N,4.43. 1H NMR (400 MHz, DMSO): δ 9.42 (d, J = 4.8 Hz, 2H; H1 and H8 of phen), 8.97 (d, J = 8.1 Hz, 2H; H3 and H6 of phen), 8.45 (d, J = 47.1 Hz, 2H; H1, H4 of pyridine), 8.30 (s, 2H; H4, H5 of phen), 8.18 (d, J = 3.9 Hz, 1H; H2 of pyridine), 8.09 (dd, J = 8.1, 5.1 Hz, 2H; H2 and H7 of phen), 7.67 (s, 1H; H3 of pyridine), 6.89−6.66 (m, 2H; NH2), 3.95 (d, J = 5.7 Hz, 2H; CH2). ESI-MS: m/z 559.0 [M − PF6−]+; 600.0 [M − PF6−]+ + CH3CN. [Re(CO)3(DIP)(L1)](PF6) (1b). Yield: 132.1 mg (78%).; Elem. Anal. (%). Calcd for C32H21F6N3O3PRe·11H2O (827.08): C, 37.50; H, 4.23; N, 4.10; C/N, 9.14. Found: C, 37.27; H, 4.27; N, 4.02; C/N, 9.27. 1H NMR (400 MHz, DMSO): δ 9.84 (d, J = 5.4 Hz, 2H; H1 and H16 of Ph2-phen), 8.62 (d, J = 6.5 Hz, 2H; H1 and H5 of pyridine), 8.23 (d, J = 5.4 Hz, 2H; H8 and H9 of Ph2-phen), 8.17 (s, 2H; H2, H15 of Ph2phen), 7.99−7.91 (m, 1H; H3 of pyridine), 7.70 (dd, J = 10.4, 4.9 Hz, 10H; C6H5 at Ph2-phen), 7.43 (t, J = 7.9 Hz, 2H; H4, H2 of pyridine). ESI-MS: m/z 681.9 [M − PF6−]+. [Re(CO)3(DIP)(L2)](PF6) (2b). Yield: 142.1 mg (81%). Elem. Anal. (%). Calcd for C33H23F6N3O4PRe·10H2O (1037.19): C, 46.26; H, 2.71; N, 4.90; C/N, 9.44. Found: C, 46.17; H, 2.72; N, 4.78; C/N, 9.66. 1H NMR (400 MHz, DMSO): δ 9.85 (d, J = 5.4 Hz, 2H; H1 and H16 of Ph2-phen), 8.52 (s, 1H; H1 of pyridine), 8.44 (d, J = 5.5 Hz, 1H; H6 of pyridine), 8.22 (d, J = 5.4 Hz, 2H; H8 and H9 of Ph2phen), 8.16 (s, 2H; H2 and H15 of Ph2-phen), 7.85 (d, J = 8.1 Hz, 1H; H4 of pyridine), 7.76−7.65 (m, 10H; C6H5 at Ph2-phen), 7.33 (m, 1H; H5 of pyridine), 5.40 (t, J = 5.7 Hz, 1H; OH), 4.41 (d, 2H; CH2). ESIMS: m/z 711.9 [M − PF6−]+. [Re(CO)3(DIP)(L3)](PF6) (3b). Yield: 137.8 mg (76%). Elem. Anal. (%). Calcd for C33H22ClF6N3O3PRe·3H2O (929.22): C, 42.65; H, 3.04; N,4.52; C/N, 9.44. Found: C, 42.52; H, 3.03; N, 4.47 C/N, 9.51. 1 H NMR (400 MHz, DMSO): δ 9.84 (d, J = 5.4 Hz, 2H; H1 and H16 of Ph2-phen), 8.69 (s, 1H; H1 of pyridine), 8.52 (d, J = 5.6 Hz, 1H; H6 of pyridine), 8.22 (d, J = 5.4 Hz, 2H; H8, H9 of Ph2-phen), 8.14 (s, 2H; H2, H15 of Ph2-phen), 7.98 (d, J = 8.0 Hz, 1H; H4 of pyridine), J


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indicated concentrations for 5 h at 37 °C under a 5% CO2 atmosphere. Then cell culture medium was removed and replaced by assay medium, and the Microplates were placed into a 37 °C non-CO2 incubator to equilibrate for 1 h. After the completion of cellular basic respiratory measurements, the repiration modulators at indicated concentrations were loaded consecutively in sequence to affect the electron transport chain, and the oxygen consumption in different stages was measured directly. Wave 2.0.0 Software was utilized to calculate the key metabolic parameters of mitochondria. Real-Time Tracking of Changes in Mitochondrial Morphology. A549 cells were cultured in 60 mm dishes (Corning) and treated with complexes 2b and 3b (10 μM). Cell imaging was collected every 5 min by confocal microscopy (λex = 405 nm, λem = 540 ± 20 nm). PLIM Imaging of Mitochondrial Respiration. A549 cells were cultured in 60 mm dishes and treated with complexes 2b and 3b (20 μM) for 30 min at 37 °C followed by twice PBS wash. Then 1 mL of PBS was added for imaging of living cells. For imaging of fixed cells, the cells should be fixed by 4% paraformaldehyde at 4 °C overnight in advance. Then the phosphorescent lifetime imaging of living cells and fixed cells under different O2 concentrations was recorded by PLIM (setup is integrated with an Olympus FV1000 laser scanning confocal microscope; λ ex = 405 nm, λ em = 540 ± 20 nm). The O 2 concentrations and O2/N2 ratios were controlled by adjusting the flow counters in the live cell station. The cell images were taken after the cell plates were equilibrated under a certain oxygen concentration for more than 30 min. The lifetime values were calculated with professional software provided by PicoQuant Co. 4.8. Measurement of Intracellular ROS. Cells were treated with 1b−4b at the indicated concentrations for 6 h and then incubated with 10 μM H2DCFDA in serum-free DMEM for 15 min at 37 °C in the dark. After the cells were washed twice with serum-free DMEM, the fluorescence intensity of the cells was measured immediately by flow cytometry with excitation at 488 nm and emission at 530 nm. Green MFI were analyzed using Flow Jo 7.6 software (Tree Star) 4.9. Induction of Apoptosis. Transmission Electron Microscopy. A549 cells were cultured in 60 mm dishes (Corning) and treated with 2b and 3b at indicated concentrations for 24 h. The cells were washed by PBS and fixed overnight at 4 °C in phosphate buffer (pH 7.4) containing 2.5% glutaraldehyde. After treatment with osmium tetroxide as postfixative, the cells were stained with uranyl acetate and lead citrate, observed by a transmission electron microscope (JEM 100 CX, JEOL, Tokyo, Japan). Images were photographed using the Eversmart Jazz program (Scitex). Hoechst Staining. A549 cells were seeded into 35 mm dishes (Corning) and treated with 2b and 3b for 24 h. The cells were then washed once with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min. After that, cells were labeled with Hoechst 33342 (5 μg/mL in PBS) for 5 min. The cells were analyzed with a confocal microscope immediately. Annexin V/PI Assay. The assay was carried out according to the manufacturer’s protocol. A549 cells were seeded in 6-well plates and treated with indicated concentrations of 2b and 3b for 24 h. The cells were harvested and stained with Annexin V and PI as described above at room temperature for 15 min in the dark and analyzed immediately by flow cytometry (λex = 488 nm), and the absorbance at 488 nm of 1b−4b can be ignored. Data were analyzed by FlowJo Software (TreeStar). Caspase-3/7 Activity Assay. Caspase-3/7 activity was conducted by the Caspase-Glo Assay kit (Promega) according to the manufacturer’s instructions. Briefly, cells were cultured and treated with different concentrations of 2b and 3b for 6 h, and then 50 μL of cell lysate was added to each well, followed by the addition of 50 μL of Caspase-Glo reagents. The mixture was incubated at room temperature for 30 min, and then the luminescence was measured using a TECAN Infinite M200 station. 4.10. Statistical Analysis. All biological experiments were performed at least twice with triplicates in each experiment. Representative results were depicted in this report, and data were presented as means ± standard deviations (SDs).

paraformaldehyde (v/v), washed with PBS(10% DMSO, v/v) at least 3 times, and visualized by confocal microscopy before and after fixation. Covalent Binding with Mitochondrial Proteins in SDS-PAGE. A549 cells were incubated with complexes 1b−4b (30 μM) for 1 h. Digestive cells were washed with PBS, lysed using RIPA buffer, and centrifuged to collect protein supernatant. After determination of protein concentration by BCA assay (Novagen Inc., USA), the same amount of cellular total proteins was denatured by being boiled in sample loading SDS-PAGE (polyacrylamide gel electrophoresis) buffer for 10 min. The denatured proteins were loaded and separated by SDS-PAGE. Then the gel was scanned by transmission ultraviolet (λex = 365 nm) and analyzed by a FluorChem M imaging station AlphaView Software (ProteinSimple, CA, USA). The same gel was then stained with Commassie Brilliant Blue-250 dye, and the imaging was also captured and analyzed. 4.6. Cytotoxicity and Selectivity against Cancer Cells. MTT Assay. Cells were cultured in 96-well plates and grown to confluence. The compounds were dissolved in DMSO (1%, v/v), and diluted with fresh media immediately. The cells were incubated with a series of concentrations of the tested compounds for 44 h at 37 °C. A 20 μL aliquot of MTT solution was then added to each well, and the plates were incubated for an additional 4 h. The medium was carefully removed, and DMSO was added (150 μL per well) and incubated for 10 min with shaking. The absorbance at 595 nm was measured using a microplate reader (Infinite M200 Pro, Tecan, Männedorf, Switzerland). Selective Killing Cancer Cells over Normal Cells. A549 cells were prestained with Hoechst3342 for 10 min, then washed with PBS three times, suspended in fresh medium, then seeded into 35 mm dishes with the same amount of LO2 cells, and incubated for 24 h for cell attachment. The cell mixtures were incubated with complexes 2b and 3b (10 μM) for 24 h, stained with 5 μL of Annexin-V and 10 μL of propidium iodide, and immediately visualized by confocal microscopy. 4.7. Mitochondrial Dysfunction and Real-Time Tracking. MMP Assay. A549 cells were cultured in 60 mm dishes (Corning) and treated with complexes 1b−4b for 6 h. The cells were then collected, resuspended at 1 × 106/mL in prewarmed PBS containing JC-1 5 μg/ mL, and incubated for 30 min at 37 °C. Subsequently, the cells were washed twice with PBS and immediately analyzed in a flow cytometer. Fluorescence was monitored by measuring both the monomer (527 nm emission; green) and the aggregate (590 nm emission; red) forms of JC-1 following excitation at 488 nm. Red and green MFI were analyzed using Flow Jo 7.6 software (TreeStar, USA). For each sample, 10, 000 events were acquired. Assay of ATP Concentration in Cells. ATP concentration of A549 was conducted by the CellTiter-Glo luminescent Cell Viability Assay (Promega) according to the manufacturer’s instructions. Cells were cultured in a 96 round black well plate for 24 h to welt. A sample of complexes 1b−4b at the indicated concentration was added into the cell to co-incubate for 6 h. The cell was washed by PBS once and balanced in PBS for 30 min and 100 μL CellTiter-Glo luminescent cell viability reagent was added into each well. The mixture was lysed for 2 min by a shaken machine and then incubated at room temperature for 10 min. The luminescence was measured using a TECAN Infinite M200 station. On the same condition, a standard curve was obtained; by the known concentration of standard ATP sample, ribonucleotide triphosphates (10 mM), we can obtain the ATP concentration of the cell. Mitochondrial Bioenergetics Analysis. The Seahorse XF24 Extracellular Flux analyzer (Seahorse Bioscience, Billerica, MA, USA) and XF Cell Mito Stress Test kit was utilized to quantify the mitochondrial respiration by measuring the OCR (oxygen consumption rate). According to the manufacturer’s instruction, after a series of preliminary experiments, the final concentrations of the respiration modulators were determined: oligomycin, 1 μM; FCCP, 1 μM; a mixture of antimycin A and rotenone, 1 μM and 1 μM, respectively. A549 cells were seeded in Seahorse 24-well XF Cell Culture Microplates at a density of 3 × 104 cells per well (0.275 cm2), cultured for 24 h, and then treated with complex 2b or 3b at the K


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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/acsami.7b01764. ESI-MS and 1H NMR spectra; stability of rhenium compounds; UV−visible and emission spectra; luminescence intensities and lifetimes; cellular uptake and subcellular localization; MMP assay; PLIM images; 2binduced mitochondrial morphological changes; Hoechst staining; Caspase-3/7 assay; crystallographic data; photophysical data; ICP-MS data; and lifetime data (PDF) Crystallographic information for 2a (CIF) Crystallographic information for 3a (CIF)

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AUTHOR INFORMATION

Corresponding Authors

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

Zong-Wan Mao: 0000-0001-7131-1154 Author Contributions §

J.Y. and J.-X.Z. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Thanks very much to Prof. Qiang Zhao from Nanjing University of Posts and Telecommunications (NUPT) for the utilization of PLIM equipment. We are grateful for financial support from the National Natural Science Foundation of China [Grants 21231007, 21572282, and 21401217], the 973 Program [Grants 2014CB845604 and 2015CB856301], the Science and Technology Program of Guangzhou [Grant 201607010379], the Natural Science Foundation of Jiangsu Province [Grant BK20140600 (to Z.G.)], and Fundamental Research Funds for the Central Universities.

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