Mitochondrial Localization of Highly Fluorescent and Photostable

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Article Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX

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Mitochondrial Localization of Highly Fluorescent and Photostable BODIPY-Based Ruthenium(II), Rhodium(III), and Iridium(III) Metal Complexes Gajendra Gupta,*,† Pratibha Kumari,‡ Ji Yeon Ryu,§ Junseong Lee,§ Shaikh M. Mobin,*,‡ and Chang Yeon Lee*,† †

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Department of Energy and Chemical Engineering/Innovation Center for Chemical Engineering, Incheon National University, Yeonsu-gu, Incheon 22012, Republic of Korea ‡ Discipline of Biosciences and Biomedical Engineering, Indian Institute of Technology Indore, Simrol 453552, Madhya Pradesh, India § Department of Chemistry, Chonnam National University, Gwangju 61186, Republic of Korea S Supporting Information *

ABSTRACT: A new N,O-based BODIPY ligand was synthesized and further utilized to develop highly fluorescent and photostable Ru(II), Rh(III), and Ir(III) metal complexes. The complexes were fully characterized by different analytical techniques including single-crystal XRD studies. The photostabilities and live cell imaging capabilities of the complexes were investigated via confocal microscopy. The complexes localized specifically in the mitochondria of live cells and showed negligible cytotoxicities at a concentration used for imaging purposes. They also exhibited high photostabilities, with fluorescence intensities remaining above 50% after 1800 scans.

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and strong fluorescence, but their serious toxicities arising from the release of heavy metals raise concerns regarding live cells imaging.7,8 Therefore, there is a need for new fluorescent mitochondriaselective probes that are photostable and have low cytotoxicities for live cells imaging. Boron dipyrromethene, widely known as BODIPY, is a class of fluorescent dyes exhibiting several interesting photophysical and photochemical properties.9−11 They are highly soluble in common organic solvents and chemically inert, have small Stokes shifts and high fluorescent quantum yields, and exhibit sharp emission and excitation peaks.9−11 These attractive features of BODIPY-based molecules make them important candidates for various applications, including bioimaging experiments for the detection of tagged entities in biological cells.12−14 In the past decade, half-sandwich Ru(II) and Ir(III) organometallic complexes have received considerable interest as anticancer agents.15−17 Two interesting ruthenium complexes,

INTRODUCTION Mitochondria are the energy (ATP) producing organelles present in most eukaryotic cells. They play important roles in the metabolism of lipids and some amino acids, the maintenance of redox balance, the regulation of respiration, and the control of cell death. Dysfunction of mitochondria causes various neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases, cancer, and diabetes.1−4 Thus, tracking mitochondria is essential to further understanding their various cellular functions and subsequently treating mitochondria-related pathologies. Nowadays, fluorescent probes receive worldwide attention for such goals since they enable the imaging of cellular compartments and real-time monitoring of their changing morphology. Commercially available conventional fluorescent dyes like JC-1 and MitoTrackers Green FM and Red FM are commonly used as mitochondria markers. However, the poor photostabilities of these commercial organic dyes limit their use in live cells, since changes in mitochondria morphology are lengthy processes.5,6 Fluorescent semiconductor quantum dots are also widely employed as imaging probes owing to their high photostabilities © XXXX American Chemical Society

Received: March 28, 2019

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DOI: 10.1021/acs.inorgchem.9b00898 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

Htereph), 7.42 (d, 2H, J = 8, Htereph), 6.00 (s, 2H, Hpyrrole), 5.89 (s, 1H, Hpyrrole), 2.56 (s, 6H, HCH3), 2.32 (s, 3H, HCH3), 1.91 (s, 3H, HCH3), 1.43 (s, 6H, HCH3). 13C{1H} NMR (100 MHz, CDCl3): δ = 184.74, 155.76, 142.87, 140.86, 140.57, 137.66, 136.12, 131.10, 130.28, 129.04, 128.10, 127.70, 121.39, 113.27, 14.71, 14.57, 14.17, 13.15. 19F NMR (376 MHz, CDCl3): δ = 146.28 (q, 2F, FB−F). Anal. Calcd (%) for C26H26BF2N3O: C, 70.13; H, 5.89; N, 9.44. Found: C, 69.98; H, 5.85; N, 9.71. Synthesis of Ru−BODIPY Complex 1. BODIPY ligand L (25 mg, 0.056 mmol) and BuOK (6.3 mg, 0.056 mmol) were dissolved in dry methanol (15 mL) and nitrogen was bubbled for few minutes. The reaction mixture was stirred for 1 h. After this, ruthenium p-cymene dimer (17.1 mg, 0.028 mmol) was added into the reaction and stirred for 24 h. The reaction mixture was filtered through Celite to remove any undissolved particles. DEE was slowly added to the solution and kept at room temperature for slow evaporation. After 2−3 days, X-ray quality crystals were formed, which were collected and dried under vacuum. Yield: 34 mg (85%). ESI MS (CHCl3): m/z = 680.23 [M − Cl]+. 1H NMR (400 MHz, CDCl3): δ = 7.60 (d, 2H, J = 8, Htereph), 7.33 (d, 2H, J = 8, Htereph), 6.03 (s, 1H, Hpyrrole), 5.98 (s, 2H, Hpyrrole), 5.72 (d, 1H, J = 8, Hp‑cym), 5.63 (dd, 2H, J = 8, Hp‑cym), 5.35 (d, 1H, J = 8, Hp‑cym), 2.75 (sept, 1H, J = 8, CH(CH3)2), 2.59 (s, 3H, CH3), 2.55 (s, 6H, CH3), 2.34 (s, 3H, CH3Ccym), 1.85 (s, 3H, CH3), 1.41 (s, 6H, CH3), 1.24 (d, 3H, J = 8, CH(CH3)2), 1.18 (d, 3H, J = 8, CH(CH3)2). 13C{1H} NMR (100 MHz, CDCl3): δ = 183.06, 155.68, 154.34, 142.95, 140.83, 138.24, 137.59, 136.68, 136.26, 131.16, 129.21, 127.58, 121.29, 119.59, 100.79, 99.26, 83.58, 82.59, 81.08, 31.08, 22.26, 22.20, 18.97, 17.73, 14.79, 14.58, 14.26. 19F NMR (376 MHz, CDCl3): δ = 146.29 (q, 2F, FB−F). Anal. Calcd (%) for C36H39BClF2N3ORu: C, 60.47; H, 5.50; N, 5.88. Found: C, 60.28; H, 5.42; N, 5.94. Synthesis of Ir−BODIPY Complex 2. BODIPY ligand L (25 mg, 0.056 mmol) and BuOK (6.3 mg, 0.056 mmol) were dissolved in dry methanol (15 mL) and nitrogen was bubbled for few minutes. The reaction mixture was stirred for 1 h. After this, pentamethylcyclopentadienyl iridium dimer (22.3 mg, 0.028 mmol) was added into the reaction and stirred for 24 h. The red precipitate formed was collected by filtration and washed with a small amount of diethyl ether. DEE was added into the filtrate and left for slow evaporation. After 2−3 days, X-ray quality crystals were observed, which were also collected and dried under vacuum. Yield: 38 mg (84%). ESI MS (CHCl3): m/z = 772.27 [M − Cl]+. 1H NMR (400 MHz, CDCl3): δ = 7.70 (d, 2H, J = 8, Htereph), 7.36 (d, 2H, J = 8, Htereph), 6.11 (s, 1H, Hpyrrole), 5.99 (s, 2H, Hpyrrole), 2.56 (s, 6H, CH3), 2.55 (s, 3H, CH3), 2.01 (s, 3H, CH3), 1.76 (s, 15H, Cp*), 1.44 (s, 6H, CH3). 13C{1H} NMR (100 MHz, CDCl3): δ = 183.64, 155.74, 152.20, 143.02, 140.78, 139.44, 137.61, 136.77, 136.61, 131.10, 129.47, 127.55, 121.29, 118.88, 84.64, 17.21, 14.79, 14.58, 14.30, 9.41. 19F NMR (376 MHz, CDCl3): δ = 146.29 (q, 2F, FB−F). Anal. Calcd (%) for C36H40BClF2N3OIr: C, 53.57; H, 4.99; N, 5.21. Found: C, 53.59; H, 4.61; N, 5.26. Synthesis of Rh−BODIPY Complex 3. A similar procedure was followed as that for 2, replacing pentamethylcyclopentadienyl iridium dimer with pentamethylcyclopentadienyl rhodium dimer (17.3 mg, 0.028 mmol). Yield: 33 mg (82%). ESI MS (CHCl3): m/z = 682.14 [M - Cl]+. 1H NMR (400 MHz, CDCl3): δ = 7.67 (d, 2H, J = 8, Htereph), 7.34 (d, 2H, J = 8, Htereph), 6.04 (s, 1H, Hpyrrole), 5.99 (s, 2H, Hpyrrole), 2.56 (s, 6H, CH3), 2.48 (s, 3H, CH3), 1.88 (s, 3H, CH3), 1.76 (s, 15H, Cp*), 1.43 (s, 6H, CH3). 13C{1H} NMR (100 MHz, CDCl3): δ = 182.63, 155.63, 152.75, 142.99, 140.95, 138.82, 136.50, 136.48, 131.21, 129.23, 127.54, 121.27, 119.09, 93.19, 93.10, 17.21, 14.75, 14.60, 14.58, 9.26. 19F NMR (376 MHz, CDCl3): δ = 146.28 (q, 2F, FB−F). Anal. Calcd (%) for C36H40BClF2N3ORh: C, 60.23; H, 5.62; N, 5.85. Found: C, 59.80; H, 5.57; N, 5.82.

NAMI-A and KP1019, developed by Sava and Keppler, respectively, are being tested as potential drug candidates.18 Ruthenium complexes are generally less toxic than platinum-based complexes and appear to follow a different mode of action.19,20 Detailed biological studies on several fluorescence metal complexes have also been explored.21−25 Although a few platinum-based26−28 BODIPY complexes (Figure 1) have recently been used in

Figure 1. Known Pt−BODIPY complexes used for mitochondrial imaging.26−28

mitochondrial imaging experiments, to the best of our knowledge, half-sandwich ruthenium, iridium, or rhodium BODIPY complexes have not yet been studied for such imaging purposes. Therefore, to combine the important properties of BODIPY and half-sandwich metal complexes, herein, we designed a new series of Ru(II)-, Ir(III)-, and Rh(III)-based BODIPY complexes that showed superior photostabilities and were specifically localized in the mitochondria of living cells.

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EXPERIMENTAL SECTION

Materials and Instruments. 2,4-Dimethylpyrrole was purchased from Alfa-Aesar, Korea. Terephthalolyl chloride was obtained from TCI, Korea. Dry dichloromethane was obtained from Acros Organics, Korea. All other reagents and solvents were commercially available and used without further purification. 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) was purchased from SigmaAldrich Chemical Co., USA. Corning 35 mm confocal dishes were used. Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco. MitoTracker Red CMXRos and Hoechst 33342 were purchased from thermo Fisher. Cervical cancer cell line (HeLa cells) and lung cancer cell line A549 cells were obtained from the National Centre for Cell Science, Pune. The 1H and 13C{1H} NMR spectra were recorded on an Agilent 400-MR spectrometer using the residual protonated solvent as internal standard. UV−visible absorption spectra were recorded on a Thermo Scientific Genesys 10S UV−vis spectrophotometer. Fluorescence spectra were recorded on a Scinco FluoroMate FS-2 fluorescence spectrometer. HRESI-MS were recorded on a Waters SynaptG2Si spectrometer at KBSI, South Korea. The CHN analysis were measured on an elemental analyzer (Elementar Analysensysteme GmbH, Germany), KBSI, Busan Center, South Korea Synthesis. Synthesis of BODIPY Ligand L. Terephthaloyl chloride (1.06 g, 0.005 mmol) was dissolved in dry dichloromethane (100 mL) under nitrogen. Then 2,4-dimethylpyrrole (2 mL, 0.0194 mmol) was added into the solution, which was stirred at room temperature for 12 h. Triethylamine (10 mL) and BF3·OEt3 (10 mL) were added dropwise simultaneously at 0 °C and stirred at room temperature for 1−1.5 h. The reaction mixture was extracted with dichloromethane and water. The dichloromethane layer was collected and dried over Na2SO4 and evaporated under vacuum. The crude residue was purified by column chromatography on silica gel using a mixture of CH2Cl2:PE as eluent. The light red product was collected and dried. It also gives a dual BODIPY in less than 5% yield, which is a subject of other studies, and therefore, the details are not described here. Yield: 780 mg (33%). ESI MS (CHCl3): m/z = 446.32 [M + H]+. 1H NMR (400 MHz, CDCl3): δ = 9.08 (s, 1H, NH), 7.78 (d, 2H, J = 8,

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RESULTS AND DISCUSSION

The new N,O-based BODIPY ligand, L, was synthesized and characterized via different analytical techniques, including singlecrystal XRD structural analysis (Scheme 1 and Experimental Section). The single-crystal structure of L, obtained by slow B

DOI: 10.1021/acs.inorgchem.9b00898 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry evaporation of a dichloromethane/hexane mixture, confirmed the formation of the desired molecule. The analysis indicated the presence of a BODIPY moiety on one side of the ligand and

dimethylpyrrole methanone on the other, which rendered free N and O atom sites for metal coordination. The reaction of BODIPY L under nitrogen with potassium tert-butoxide followed by addition of the desired metal (Ru, Rh, or Ir) dimer resulted in the formation of the three new neutral metal complexes 1−3 (Scheme 2 and Supporting Information). Complexes 1−3 were fully characterized, and their formations were confirmed with different analytical techniques. They are highly soluble in common organic solvents and are stable in air. In addition to aromatic and methyl proton peaks, the 1H NMR

Scheme 1. Synthesis of BODIPY Ligand L and Its SingleCrystal XRD Structure

Scheme 2. Synthesis of Ru, Ir, and Rh Metal Complexes 1−3a

a

Ru P-cym = Ru2Cl2(C10H14)2Cl2 and MC5Me5 = M2Cl2(C10H15)2Cl2, where M = Ir and Rh.

Figure 3. Single-crystal X-ray structures of Ru−BODIPY complex 1 and Ir−BODIPY complex 2.

Figure 2. Comparison of 1H NMR spectra of free BODIPY ligand L and its metal complexes 1−3. Detailed assignment of the peaks is described in the Experimental Section. C

DOI: 10.1021/acs.inorgchem.9b00898 Inorg. Chem. XXXX, XXX, XXX−XXX

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Inorganic Chemistry

in accordance with the calculated m/z values, further confirmed the formation of the desired products 1−3 (Figure S2, Supporting Information). Although multinuclear NMR and ESI-MS initially indicated the formation of the metal complexes, final structural confirmation was achieved via a single-crystal XRD study. Single crystals of 1 and 2 were obtained by slow evaporation of the reaction methanol solution, yielding dark-colored crystals suitable for X-ray analysis. The molecular structures of 1 and 2 are shown in Figure 3. Ruthenium complex 1 and iridium complex 2 crystallized in a monoclinic system with P21/n and P21/c space groups, respectively. They showed typical piano-stool geometries with the metals binding to the O1 and N1 binding

spectra of the free BODIPY ligand L in CDCl3 showed a sharp, broad peak at δ 9.08 ppm, which was attributed to the NH proton of the dimethyl pyrrole moiety (Figure S1, Supporting Information). The disappearance of the NH peak in the 1H NMR spectra upon reaction of free BODIPY ligand L with the metal dimers initially confirmed the formation of metal− nitrogen bonds to yield complexes 1−3 (Figure 2). Two doublets from the terephthaloyl benzene were observed between δ 7.70 and 7.33 ppm, which were shifted upfield compared with those of the free ligand L. The complexes also showed a single quartet in the 19F NMR at approximately δ 146.29 ppm attributed to the F atoms in the BODIPY groups. The ESI-MS spectra measured in chloroform, which showed M−Cl peaks that were

Figure 4. Quantitative cellular uptake analysis of L and 1−3 via flow cytometry. HeLa cells were stained with L and 1−3 (5 μM, 30 min), and unstained cells were used as a control sample.

Figure 5. Comparisons of L, 1−3, and Mitotracker Red CMXRos photobleacking results. (a) Fluorescent images of live HeLa cells stained with 5 μM L and 1−3 in separate dishes with an increasing number of scans. (b) Photobleaching relative intensities of L, 1−3, and Mitotracker Red CMXRos. Complex λex = 488 nm, λem = 500−550 nm; Mitotracker Red CMXRos λex = 559 nm, λem = 575−675 nm. D

DOI: 10.1021/acs.inorgchem.9b00898 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 6. Intracellular compound localization study using confocal microscopy. Live HeLa cells were treated with 5 μM L and 1−3 (complexes were dissolved in 0.01% DMSO) for 30 min followed by counterstaining with Hoechst (5 μg/mL for 30 min, stain nucleus). Hoechst, λex = 405 nm, λem = 420−470 nm; complex, λex = 488 nm, λem = 500−550 nm. Inset scale bars: 20 μm.

sites. The p-cymene or pentamethylcyclopentadienyl ring occupied the remaining three coordination sites to complete the octahedral geometry around the ruthenium and iridium metal centers. The chlorine atom was attached to the metal centers, with M−Cl distances of approximately 2.402 and 2.386 Å for 1 and 2, respectively. A detailed summary of the crystallographic data is given in Table S1 (Supporting Information). The UV−visible and fluorescence properties of bodipy ligand L and complexes 1−3 were studied in dichloromethane at room temperature (Figure S3, Supporting Information). Free BODIPY ligand L displayed an intense, sharp absorption peak at 502 nm arising from the S0−S1 transitions and a broad peak at approximately 318 nm from the S0−S2 transitions. The metal complexes 1−3 showed similar sharp peaks at approximately 503 nm and broad peaks at approximately 340 nm. Upon excitation at 490 nm, the ligand L and complexes 1−3 displayed strong emissions at 512 and 515 nm, respectively. These emission peaks were attributed to the BODIPY chromophore present in the complexes, which can be effectively further used for bioimaging studies. The quantum yields of free BODIPY ligand L and metal complexes 1−3 were measured in different solvents. In dichloromethane, the quantum yields of ligand L is 0.49 and that of the metal complexes 1−3 are 0.11, 0.08 and 0.15, respectively. In acetonitrile the quantum yields were found to be 0.39, 0.08,

0.12, and 0.04, respectively. On the other hand, in DMSO, the quantum yields were 0.36, 0.10, 0.20, and 0.06 for L and 1−3, respectively (Table S2, Supporting Information). In general, the quantum yields29 of the complexes decreased in the order L > 2 > 1 > 3, and no significant variation is observed with increase in solvent polarity. However, in the case of water, fluorescence intensities decreased in the order 2 > 1 > 3 > L. This change in behavior may be due to the higher solubility of the metal complexes in water than that of the free BODIPY ligand L, where the iridium complex 2 had the highest solubility in aqueous medium (Figure S4, Supporting Information). Cytotoxicity Measurements. Biological probes for live-cell imaging should negligibly perturb live cell proliferation at the concentration used for imaging. Therefore, MTT assays were performed to determine the cytotoxicities of free BODIPY ligand L and metal complexes 1−3 against HeLa cells.30 The concentration-dependent effects of L and 1−3 after 12 h of incubation are shown in Figure S5 (Supporting Information). No significant cytotoxic responses were observed at 5 μM, as more than 80% cells were viable. This concentration was thus further used for live-cell imaging. These results indicated that the complexes have negligible toxicities for cell imaging at a concentration of 5 μM and an incubation time of 30 min. Flow Cytometry Study. To more comprehensively understand the bioimaging behavior of the synthesized complexes, E

DOI: 10.1021/acs.inorgchem.9b00898 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 7. Mitochondria imaging study via confocal microscopy. Live HeLa cells were treated with 5 μM L and 1−3 (complexes were dissolved in 0.01% DMSO) for 30 min followed by counterstaining with MitoTracker Red (80 nM for 30 min, stain mitochondria). MitoTracker Red, λex = 559 nm, λem = 575−675 nm; complex, λex= 488 nm; λem = 500−550 nm. Inset scale bars: 20 μm.

both flow cytometry and fluorescence microscopic techniques were employed for analysis. Flow cytometry provides fluorescent signals from large populations of cells in a flow (suspension cells), whereas confocal microscopy provides morphological details regarding surface-attached cells. Here, a flow cytometry study was performed to quantitatively examine the cellular uptake of the BODIPY-based complexes via a large population of cells. HeLa cells were treated with L and 1−3 (5 μM, 30 min), and untreated cells were used as a control sample. Almost all cells were able to uptake L and 1−3 with high efficiencies, indicated by the increased fluorescence intensities of nearly 100% of the cells, resulting in a right-shift in the histogram with respect to the control sample (Figure 4). Although L shows the least solubility and least quantum yield in aqueous medium, the cellular uptake is the highest. This could be due to the fact that cell membranes as well as mitochondrial double layered membranes are made up of a phospholipid bilayer, which can allow diffusion of strong lipophilic molecules more easily as compare to less lipophilic metal complexes. Also, the quantum yield of L is more than twice that of the metal complex in organic solvents (Table S2, Supporting Information). Photostabilities of BODIPY-Based Complexes. The photostability of any new fluorescent imaging probe is one of the most significant parameters for establishing its feasibility. Photobleaching is the most common issue with most organic

probes (dyes), as it compromises the long-time monitoring of any dynamic events inside living cells.5,31−33 Here, photobleaching experiments on L and 1−3 were performed with a confocal laser scanning microscope. After 1800 scans, the fluorescence intensities of L and 1−3 remained at 68.8%, 52.7%, 53.5%, and 96.8%, respectively, whereas the fluorescence intensity of MitoTracker Red CMXRos remained at 68.8%. The photostability properties of these complexes decreased in the order 3 > L > 2 > 1, where rhodium complex 3 was the most photostable among the metal complexes. All the investigated complexes can be considered as photostable, since they retained more than 50% fluorescence intensities after 1800 scans (Figure 5, Videos S1−S5, Supporting Information). Therefore, these complexes can be used for long-term live cells imaging. Cellular Uptake and Bioimaging. The uptake of fluorescent complexes by cells is an important factor for cellular imaging. To explore the subcellular localization of the synthesized complexes, a commercially available nucleus staining probe (Hoechst 33342) and a mitochondria tracking probe (MitoTracker Red CMXRos) were used for a colocalization study. The colocalization effects of L and 1−3 with Hoechst 33342 and MitoTracker Red CMXRos were quantified using Pearson’s correlation coefficient (Rr from +1 to −1).34 The standard fluorescent dye Hoechst 33342, which is blue-regionemitting fluorescent and localizes in the nucleus, was employed F

DOI: 10.1021/acs.inorgchem.9b00898 Inorg. Chem. XXXX, XXX, XXX−XXX

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Figure 8. Living HeLa cells stained with L and 1−3 (5 μM) and costained with MitoTracker Red CMXRos, before imaging cells treated with CCCP (10 μM, 50 min).

stress from drug treatments, metabolic stress or autophagy can lead to the selective elimination of damaged mitochondria. Under stress conditions, Rhodamine 123 is removed from the live cells. Hence, to assess the tolerance of ligand L and complexes 1−3 during changes in membrane potential, the mitochondria membrane-potential uncoupler agent carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used, which abolishes membrane potentials.36,37 When the living cells were treated with 10 μM CCCP for 50 min before imaging, the fluorescence intensities decreased slightly. The sensitivities and specificities of complexes L and 1−3 remained, but the standard MitoTracker Red CMXRos lost its specificity to mitochondria (Figure 8).

in a colocalization study. The obtained Pearson’s correlation coefficients (Rr) of the blue (Hoechst 33342) and green fluorescent signals from L and 1−3 were very poor (−0.04, 0.32, 0.41, and 0.34, respectively) on live HeLa cells (Figure 6 and Figure S6, Supporting Information). A similar experiment was performed on a lung cancer cell line (A549), where similarly low Pearson’s correlation coefficients of L and 1−3 with Hoechst 33342 were found (0.07, 0.09, 0.14, and −0.25, respectively (Figures S7 and Figure S8, Supporting Information). These results thus negated any chance of significant colocalization/ nuclear localization. A further colocalization experiment was performed with standard commercially available MitoTracker Red CMXRos (stain mitochondria). The green fluorescence signals of L and 1−3 overlapped with the red fluorescence signals of MitoTracker Red, yielding the high Pearson’s correlation coefficients of 0.85, 0.84, 0.85, and 0.79, respectively, on HeLa cells. This confirmed that the complexes predominantly localized in mitochondria (Figure 7 and Figure S9, Supporting Information). To investigate the universal staining abilities of the complexes, a similar experiment was performed on another cell line, i.e., A549 cells. Similar results were obtained on A549 cells, showing high Pearson’s correlation coefficients for L and 1−3 with MitoTracker Red of 0.87, 0.79, 0.89, and 0.80, respectively (Figure S10 and Figure S11, Supporting Information). The fluorescence confocal microscopic images and colocalization results with MitoTracker Red CMXRos confirmed that the complexes specifically localized in the mitochondria of the different cell lines. Among the complexes, iridium metal complex 2 exhibited the highest colocalization coefficient. Mitochondria Change Analysis. Mitochondria play a vital role in maintaining the proton gradient across membranes with a very high membrane potential (ΔΨm) of approximately 180 mV.34,35 This large mitochondrial membrane potential gradient attracts positively charged species such as Rhodamine 123, which specifically stains mitochondria. When cells undergo

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CONCLUSION In summary, a new bidentate N,O-containing BODIPY ligand was designed to develop three new half-sandwich Ru(II), Ir(III), and Rh(III) metal complexes. The highly fluorescent BODIPYbased complexes exhibited highly efficient uptakes by live cells and target mitochondria. The ligand L and the metal complexes 1−3 displayed high mitochondria specificities, low cytotoxicities at an imaging concentration, high photostabilities, and greater resistances to the loss of mitochondrial membrane potential. Our interesting findings suggest that more stable BODIPY-based transition metal complexes can be developed and utilized for mitochondrial localization and other biomedical applications.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.inorgchem.9b00898. Experimental section, 1H NMR, 13C NMR, 19F NMR, ESI-MS, UV−vis and fluorescence spectra, tables for crystallographic data and quantum yield measurments, confocal microscopy images, and complete captions for the photobleaching videos (PDF) G

DOI: 10.1021/acs.inorgchem.9b00898 Inorg. Chem. XXXX, XXX, XXX−XXX

Article

Inorganic Chemistry

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Video S1: Photobleaching of L (AVI) Video S2: Photobleaching of 1 (AVI) Video S3: Photobleaching of 2 (AVI) Video S4: Photobleaching of 3 (AVI) Video S5: Photobleaching of MitoTracker Red CMXRos (AVI) Accession Codes

CCDC 1896467−1896469 contain the supplementary crystallographic data for this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/data_request/cif, or by emailing [email protected], or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.

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AUTHOR INFORMATION

Corresponding Authors

*(G.G.) E-mail: [email protected]. *(S.M.M.) E-mail: [email protected]. *(C.Y.L.) E-mail: [email protected]. ORCID

Gajendra Gupta: 0000-0002-0098-8288 Ji Yeon Ryu: 0000-0001-6321-5576 Junseong Lee: 0000-0002-5004-7865 Shaikh M. Mobin: 0000-0003-1940-3822 Chang Yeon Lee: 0000-0002-1131-9071 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the National Research Foundation of Korea and funded by the Ministry of Science, ICT & Future Planning (NRF2018R1D1A1B07045863) and Post-Doctoral Research Program (2018) for G.G. through Incheon National University. P.K. and S.M.M. acknowledge the Discipline of Biosciences and Biomedical Engineering (BSBE) at IIT Indore for facilitating certain parts of this work, as well as MHRD, New Delhi, for research funding.

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REFERENCES

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DOI: 10.1021/acs.inorgchem.9b00898 Inorg. Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.inorgchem.9b00898 Inorg. Chem. XXXX, XXX, XXX−XXX