Biomaterials 39 (2015) 95e104

Contents lists available at ScienceDirect

Biomaterials journal homepage: www.elsevier.com/locate/biomaterials

Phosphorescent iridium(III)-bis-N-heterocyclic carbene complexes as mitochondria-targeted theranostic and photodynamic anticancer agents Yi Li 1, Cai-Ping Tan 1, Wei Zhang, Liang He, Liang-Nian Ji, Zong-Wan Mao* MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou 510275, PR China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 August 2014 Accepted 20 October 2014 Available online

Mitochondria-targeted compounds represent a promising approach to target tumors selectively and overcome resistance to current anticancer therapies. In this work, three cyclometalated iridium(III) complexes (1e3) containing bis-N-heterocyclic carbene (NHC) ligands have been explored as theranostic and photodynamic agents targeting mitochondria. These complexes display rich photophysical properties, which greatly facilitates the study of their intracellular fate. All three complexes are more cytotoxic than cisplatin against the cancer cells screened. 1e3 can penetrate into human cervical carcinoma (HeLa) cells quickly and efficiently, and they can carry out theranostic functions by simultaneously inducing and monitoring the morphological changes in mitochondria. Mechanism studies show that these complexes exert their anticancer efficacy by initiating a cascade of events related to mitochondrial dysfunction. Additionally, they display up to 3 orders of magnitude higher cytotoxicity upon irradiation at 365 nm, which is so far the highest photocytotoxic responses reported for iridium complexes. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Iridium(III) complexes N-Heterocyclic carbene Theranostic Phosphorescent emission Mitochondrion

1. Introduction The development of multifunctional agents that can realize simultaneous tumor targeting, imaging and treatment has become a major goal in cancer research [1,2]. Multifunctional theranostic agents are expected to contribute significantly to the realization of personalized cancer therapy, and have significant clinical applications by integrating diagnosis and therapy simultaneously [3,4]. Mitochondria are not only the “powerhouse” of the cell, but also reservoirs of a number of apoptosis-promoting proteins that are essential for apoptosis induction [5]. Human cancers are unequivocally associated with alterations in mitochondrial structure and functions [6]. With a diverse range of mitochondria-targeted drugs currently in clinical trials, targeting mitochondria as a cancer therapeutic strategy has gained great success in recent years [5,7,8]. Conventional chemotherapeutic agents cause mitochondrial dysfunction in an indirect fashion by induction of endogenous effectors involved in the physiologic control of apoptosis [9]. In contrast, mitochondria-targeted anticancer drugs act through

* Corresponding author. Tel.: þ86 20 84113788; fax: þ86 20 84112245. E-mail address: [email protected] (Z.-W. Mao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.biomaterials.2014.10.070 0142-9612/© 2014 Elsevier Ltd. All rights reserved.

directly disrupting the energy producing systems of cancer cell mitochondria, producing reactive oxygen species (ROS) and activating mitochondrial-dependent cell death signaling pathways [5]. Such agents may overcome resistance of conventional drugs by inducing apoptosis when endogenous apoptosis induction pathways are disrupted [5,10]. Additionally, based on the fact that most mitochondrial damage can lead to apoptosis, it has been proven that photodynamic therapy targeting mitochondria is a very effective strategy [11e13]. Metal complexes containing N-heterocyclic carbene (NHC) ligands have found wide applications in various fields, particularly as catalysts and anticancer agents [14,15]. The exploration of metaleNHCs as potential anticancer drugs constitutes a very rapidly growing field of research [16e19]. Cancer cells are characterized by abnormally high mitochondrial membrane permeabilization, and it has been shown that metaleNHCs can achieve some degree of selectivity for tumor cells by targeting mitochondria [16,20,21]. On the other hand, iridium complexes are very promising alternatives to platinum-based metallo-anticancer agents [22e24]. Additionally, phosphorescent cyclometalated Ir(III) complexes are widely explored as bioimaging and biosensing agents, which is attributed to their high quantum yields, large Stokes shifts, long-lived luminescence, good photostability and cell permeability [25e29]. Cyclometalated Ir(III) complexes also exhibit strong anticancer

96

Y. Li et al. / Biomaterials 39 (2015) 95e104

potential through mechanisms that are distinct from those of platinum-based anticancer agents [30,31]. Through the combination of their antitumor properties and bioimaging capabilities, phosphorescent Ir(III) complexes have great potential for the construction of novel theranostic platforms. In this work, a series of phosphorescent cyclometalated Ir(III) complexes (1e3, Scheme 1) containing different bis(NHC) ligands were designed, synthesized and characterized. The in vitro antiproliferative activity of 1e3 was investigated against several cancer cell lines and a human normal cell line. The anticancer properties of 1e3, which included cellular localization, impact on mitochondrial damage, initiation of a series of mitochondria-associated events that leads to cellular apoptosis, were explored using a variety of methods. Additionally, the photocytotoxicity of these complexes was also studied. 2. Experimental section 2.1. General materials and methods All starting materials were used as received from commercial sources unless otherwise stated. Solvents were purified and degassed by standard procedures. Iridium chloride hydrate (Alfa Aesar), ppy (2-phenylpyridine, Sigma Aldrich), cisplatin (Sigma Aldrich), CCCP (carbonyl cyanide 3-chlorophenylhydrazone, Sigma Aldrich), DPBF (1,3-diphenylisobenzofuran, Sigma Aldrich), MB (methylene blue, Sigma Aldrich), DMSO (dimethyl sulfoxide, Sigma Aldrich), MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma Aldrich), PI (propidium iodide, Sigma Aldrich), JC-1 (5,50 ,6,60 -tetrachloro-1,10,3,30 -tetraethylbenzimidazolylcarbocyanine iodide, Sigma Aldrich), H2DCFDA (20 ,70 dichlorodihydrofluorescein diacetate, Sigma Aldrich) and MTR (MitoTracker Red, Life Technologies, USA) were used as received. Caspase-3/7 activity assay kit was purchased from Promega (USA). The chloro-bridged dinuclear Ir(III) precursor [Ir(ppy)2]2Cl2 [32] was synthesized according to the literature procedure. The tested compounds were dissolved in DMSO just before the experiments, and the concentration of DMSO was 1% (v/v). The solutions of complexes 1e3 in PBS (phosphate buffered saline) were proved to be stable for at least 48 h at room temperature as monitored by UV/Vis spectroscopy and electrospray mass spectra (ESI-MS). ESI-MS were recorded on a Thermo Finnigan LCQ DECA XP spectrometer (USA). The quoted m/z values represent the major peaks in the isotopic distribution. NMR spectra were recorded on a Varian Mercury Plus 300 MHz spectrometer or a Varian Unity/Inova-500 NB (500 MHz) spectrometer. Shifts were referenced relative to the internal solvent signals. Microanalysis (C, H, and N) was carried out using an Elemental Vario EL CHNS analyzer (Germany). Fluorescence spectra and quantum yields were recorded on a Shimadzu RF5301 spectrofluorophotometer. Emission lifetimes were measured on an FLS 920 combined fluorescence lifetime and steady state spectrometer (Edinburgh). Quantum yields at room temperature were measured by using quinine sulfate in 1 N H2SO4 as a reference standard (FPL ¼ 0.546) [33,34]. UV/Vis spectra were recorded on a Varian Cary 300 spectrophotometer. 2.2. Crystallographic structure determination Crystals of complexes 2 and 3 qualified for X-ray analysis were obtained by slow diffusion of diethyl ether into a concentrated solution of the Ir(III) complex in CH2Cl2. The data were collected at 293(2) K or 150(2) K on a Rigaku Pilatus diffractometer equipped with Mo Ka radiation (l ¼ 0.71073 Å). The empirical absorption corrections were applied using the SADABS program [35]. The crystal structures of 2 and 3 were solved by direct methods with program SHELXS and

refined using the full-matrix least-squares program SHELXL [36]. In the final stage of least-squares refinement, non-hydrogen atoms were refined anisotropically. The structural plots were drawn using the xp package in SHELXTL with thermal ellipsoids at the 30% probability level. Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre (CCDC). The deposition numbers for 2 and 3 are CCDC 1008895 and 1008896, respectively. 2.3. Synthesis and characterizations 2.3.1. Synthesis of the ligands General synthetic procedure of the ligands: 1-alkyl-1H-imidazole (60 mmol) and CH2Cl2 (20 mmol) was heated for 24 h at 85  C in a sealed reaction container. The solution was cooled to ambient temperature, and the solvent was removed under reduced pressure. Then the mixture was redissolved in 5 mL CH3OH. Tetrahydrofuran (THF) was added and the mixture was stirred for another 2 h. The suspension was filtered. The precipitate was washed with THF, and dried under vacuum. 1,10 -Methylenebis(3-methyl-1H-imidazol-3-ium) dichloride (L1) [37]. Yield: 2.123 g (42.6%). 1H NMR (300 MHz, DMSO) d 9.86 (s, 2H), 8.27 (s, 2H), 7.79 (s, 2H), 6.89 (s, 2H), 3.88 (s, 6H). 13C NMR (75 MHz, DMSO) d 138.69, 124.60, 122.53, 57.99, 36.66. ESI-MS (CH3OH): m/z 177.1 [M  2Cl  H]þ, 89.0 [M  2Cl]2þ. Elemental analysis: calcd (%) for C9H14Cl2N4: C, 43.39; H, 5.66; N, 22.49; found C, 43.15; H, 5.43; N, 22.66. 1,10 -Methylenebis(3-ethyl-1H-imidazol-3-ium) dichloride (L2). Yield: 2.483 g (44.8%). 1H NMR (300 MHz, DMSO) d 10.02 (s, 2H), 8.30 (d, J ¼ 1.8 Hz, 2H), 7.91 (s, 2H), 6.85 (s, 2H), 4.23 (q, J ¼ 7.3 Hz, 4H), 1.43 (s, 6H). 13C NMR (75 MHz, DMSO) d 137.96, 122.92, 122.44, 58.06, 45.17, 15.10. ESI-MS (CH3OH): m/z 205.6 [M  2Cl  H]þ, 102.7 [M  2Cl]2þ. Elemental analysis: calcd (%) for C11H18Cl2N4: C, 47.66; H, 6.55; N, 20.21; found C, 47.36; H, 6.73; N, 20.04. 1,10 -Methylenebis(3-butyl-1H-imidazol-3-ium) dichloride (L3). Yield: 3.093 g (46.4%). 1H NMR (300 MHz, DMSO) d 10.10 (s, 2H), 8.37 (s, 2H), 7.92 (s, 2H), 6.89 (s, 2H), 4.21 (t, J ¼ 7.2 Hz, 4H), 1.88e1.68 (m, 4H), 1.26 (dq, J ¼ 14.5, 7.4 Hz, 4H), 0.89 (t, J ¼ 7.3 Hz, 6H). 13C NMR (75 MHz, DMSO) d 138.29, 123.41, 122.74, 57.86, 49.49, 31.46, 19.22, 13.72. ESI-MS (CH3OH): m/z 262.4 [M  2Cl  H]þ, 131.2 [M  2Cl]2þ. Elemental analysis: calcd (%) for C15H26Cl2N4: C, 54.05; H, 7.86; N, 16.81; found C, 54.35; H, 7.80; N, 16.65. 2.3.2. Synthesis of the complexes General synthetic procedure of the complexes: A mixture containing L (0.15 mmol), Ag2O (0.15 mmol), [Ir(ppy)2]2Cl2 (0.07 mmol) and CH2Cl2 (8 mL) was heated overnight at 95  C in a sealed reaction container. After cooling to ambient temperature, the mixture was filtered through Celite and washed with CH2Cl2. The filtrate was concentrated to approximately 2 mL under reduced pressure. 20 mL diethyl ether was added to give a light-yellow precipitate. The precipitate was filtered, washed with ether, and then dried under vacuum. [Ir(ppy)2(L1)]Cl (1) [34]. Yield: 85 mg (84.7%). 1H NMR (500 MHz, DMSO) d 8.24 (s, 2H), 8.23 (s, 2H), 8.00e7.93 (m, 2H), 7.80 (d, J ¼ 7.3 Hz, 2H), 7.59 (s, 2H), 7.34 (d, J ¼ 1.9 Hz, 2H), 7.24e7.17 (m, 2H), 6.87e6.81 (m, 2H), 6.74 (td, J ¼ 7.4, 1.1 Hz, 2H), 6.19 (dd, J ¼ 7.5, 0.7 Hz, 2H), 6.12 (s, 2H), 3.49 (dd, J ¼ 13.2, 7.0 Hz, 2H), 3.38e3.33 (m, 2H), 0.23 (s, 6H). 13C NMR (126 MHz, DMSO) d 169.20, 163.23, 153.53, 144.99, 138.01, 131.63, 128.89, 124.99, 124.33, 123.81, 122.06, 121.60, 120.50, 62.29, 37.03. ESI-MS (CH3OH): m/z 677.0 [M  Cl]þ. Elemental analysis: calcd (%) for IrC31H28ClN6$1.5CH2Cl2$0.5H2O: C, 46.00; H, 3.80; N, 9.90; found C, 45.73; H, 3.62; N, 9.95. [Ir(ppy)2(L2)]Cl (2). Yield: 100 mg (96.2%). 1H NMR (500 MHz, DMSO) d 8.24 (s, 2H), 8.23 (s, 2H), 8.01e7.93 (m, 2H), 7.80 (d, J ¼ 7.3 Hz, 2H), 7.59 (s, 2H), 7.34 (d, J ¼ 1.9 Hz, 2H), 7.24e7.18 (m, 2H), 6.88e6.81 (m, 2H), 6.74 (td, J ¼ 7.4, 1.1 Hz, 2H), 6.19 (dd, J ¼ 7.5, 0.7 Hz, 2H), 6.12 (s, 2H), 3.49 (dd, J ¼ 13.2, 7.0 Hz, 2H), 0.23 (t, J ¼ 7.1 Hz, 6H). 13C NMR (126 MHz, DMSO) d 169.12, 163.03, 162.56, 153.50, 144.91, 137.99, 131.21, 129.32, 124.89, 123.80, 123.01, 122.14, 121.56, 120.70, 62.23, 44.03, 15.81. ESIMS (CH3OH): m/z 702.9 [M  Cl]þ. Elemental analysis: calcd (%) for IrC33H32ClN6$CH2Cl2$0.5H2O: C, 48.95; H, 4.23; N, 10.07; found C, 48.64; H, 4.11; N, 10.16. [Ir(ppy)2(L3)]Cl (3). Yield: 108 mg (96.9%). 1H NMR (500 MHz, DMSO) d 8.23 (d, J ¼ 8.1 Hz, 2H), 8.21 (d, J ¼ 5.6 Hz, 2H), 8.01e7.93 (m, 2H), 7.80 (d, J ¼ 7.5 Hz, 2H), 7.59 (d, J ¼ 1.9 Hz, 2H), 7.34 (d, J ¼ 1.9 Hz, 2H), 7.25e7.17 (m, 2H), 6.88e6.81 (m, 2H), 6.72 (td, J ¼ 7.5, 1.0 Hz, 2H), 6.18 (d, J ¼ 7.0 Hz, 2H), 6.12 (s, 2H), 3.37 (dd, J ¼ 12.3, 5.4 Hz, 2H), 3.27e3.18 (m, 2H), 0.94 (ddd, J ¼ 16.5, 11.2, 5.6 Hz, 2H), 0.73e0.59 (m, 4H), 0.54 (t, J ¼ 7.2 Hz, 6H), 0.18e0.07 (m, 2H). 13C NMR (126 MHz, DMSO) d 169.10, 163.16, 162.68, 153.50, 144.86, 137.93, 131.11, 129.35, 124.94, 123.74, 122.85, 122.49, 121.40, 120.69, 62.22, 48.83, 33.32, 19.62, 13.86. ESI-MS (CH3OH): m/z 759.0 [M  Cl]þ. Elemental analysis: calcd (%) for IrC37H40ClN6$CH2Cl2$0.5H2O: C, 51.26; H, 4.87; N, 9.44; found C, 50.96; H, 4.82; N, 9.68. 2.4. Cell lines and culture conditions

Scheme 1. Chemical structures of Ir(III) complexes studied in this work.

HeLa (human cervical cancer), A549 (human pulmonary carcinoma), A549R (cisplatin-resistant A549), MCF-7 (human breast adenocarcinoma), HepG2 (human hepatocellular liver carcinoma) and LO2 (human normal liver) cells were obtained from Experimental Animal Centre of Sun Yat-sen University (Guangzhou, China).

Y. Li et al. / Biomaterials 39 (2015) 95e104 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 mg/mL streptomycin, 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 a constant temperature of 37  C. A549R cells were cultured in medium containing cisplatin to maintain the resistance. 2.5. Cellular uptake and localization 2.5.1. Determination of log Po/w The lipophilicity of the complexes, which was referred to as the n-octan-1-ol/ water partition coefficient (log Po/w), was determined following a reported procedure [38]. log Po/w was calculated as the logarithmic ratio of Ir(III) concentration in n-octanol to that in aqueous phase. 2.5.2. Inductively coupled plasma mass spectrometry (ICP-MS) The intracellular iridium contents of 1, 2 and 3 were determined by a method reported in literature with slight modifications [39]. Briefly, HeLa cells were grown in 60 mm tissue culture dishes and incubated for 24 h. The medium was removed and replaced with medium-DMSO (99:1 v/v) containing 1, 2 or 3 (10 mM). After 1 h incubation, the cells were trypsinised and collected in PBS (1 mL). The cells were counted, and digested with HNO3 (65%, 2 mL) at 60  C for 1 h. The solution was then diluted to a final volume of 10 mL with Milli-Q water. The concentration of iridium was measured using the XSERIES 2 ICP-MS. 2.5.3. Colocalization assay HeLa/A549 cells were seeded in 35 mm dishes for 24 h and then incubated with MTR (100 nM) at 37  C for 20 min. The cells were further co-incubated with 1, 2 or 3 (20 mM) at 37  C for 10 min. Cells were washed three times with ice-cold PBS and €ttingen, Germany) visualized by confocal microscopy (LSM 710, Carl Zeiss, Go immediately. Ir(III) complexes were excited at 405 nm and MTR was excited at 543 nm. Emission was collected at 520 ± 20 nm (1e3) or 600 ± 20 nm (MTR). 2.5.4. Live cell imaging after treatment with metabolic or endocytic inhibitors HeLa cells were seeded in 35 mm dishes for 24 h and preincubated with CCCP (10 mM) or chloroquine (50 mM) for 1 h. The medium was removed and the cells were then incubated with 3 (10 mM) for 10 min. The cells were washed three times with €ttingen, ice-cold PBS and visualize by confocal microscopy (LSM 710, Carl Zeiss, Go Germany) immediately. 2.6. Cytotoxicity assay Cell growth inhibitory effects of the tested compounds were determined by MTT assay as previously described [21]. 2.7. Mitochondrial damage and real-time tracking 2.7.1. Analysis of mitochondrial membrane potential (MMP) The impact of the tested compounds on MMP was determined as previously described [21]. Briefly, HeLa cells were treated with the tested compounds at the indicated concentrations for 6 h. The cells were then collected and stained with JC-1 (5 mg/mL) and analyzed immediately by flow cytometry (FACSCalibur™, Becton Dickinson, NJ, USA). Data were analyzed by FlowJo Software (Tree Star, OR, USA). 2.7.2. Real-time tracking of mitochondria HeLa cells seeded in 35 mm dishes were incubated with 3 (20 mM) in a humidified incubator connected with the confocal microscope. The incubator provided an atmosphere of 5% CO2 and 95% air at a constant temperature of 37  C Cell imaging was then carried out immediately by confocal microscopy (LSM 710, Carl Zeiss, € ttingen, Germany). Emission was collected at 520 ± 20 nm upon excitation at Go 405 nm. 2.8. Cell cycle analysis Cell cycle distribution was analyzed by flow cytometry and PI staining as previously described [40]. Briefly, HeLa cells were exposed to Ir(III) at the indicated concentrations for 24 h. After an overnight storage at 20  C in 70% ethanol, the cells were washed twice with PBS, then resuspended in 500 mL staining solution containing PI (10 mg/mL) and DNAse-free RNase (100 mg/mL) and analyzed by flow cytometry (FACSCalibur™, Becton Dickinson, NJ, USA). Data were analyzed by ModFit LT 2.0 software (Variety Software House, Inc., Topsham, ME, USA). 2.9. Induction of apoptosis 2.9.1. Western blot analysis Western blotting was performed as previously described [21]. Briefly, HeLa cells seeded in 6 cm culture dishes were exposed to 3 at 1 mM, 5 mM or 10 mM for 12 h. Cells were lysed in radio-immunoprecipitation assay (RIPA) buffer. The proteins were separated on SDS-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes (Millipore, MA, USA). The membrane was

97

blocked and incubated with the primary antibodies (Cell Signaling Technology, MA, USA) at 4  C overnight. After a subsequent washing step, the membrane was incubated with the secondary antibody for 2 h. The immunoreactivity was detected using the enhanced chemiluminescence detection kit (Amersham Inc, IL, USA). Images were captured using a FluorChem M imaging station and analyzed manually with AlphaView software (ProteinSimple, CA, USA). 2.9.2. Caspase-3/7 activity assay Caspase-3/7 activity was measured using Caspase-Glo® Assay kit (Promega, Madison, WI, USA) according to the manufacturer's instructions. Cells cultured in 96-well plates were treated with the tested compounds for 12 h. 100 mL of CaspaseGlo® 3/7 reagent was added to each well containing 100 mL culture medium. The mixture was incubated at room temperature for 1 h. The luminescence was €nnedorf, measured using a micro-plate reader (Infinite M200 Pro, Tecan, Ma Switzerland). 2.9.3. Morphological observations HeLa cells were seeded in 35 mm culture dishes and incubated for 24 h. The growth medium was removed, and the cells were exposed to 3 (0.5, 1 or 2 mM) for 12 h. Cell images were acquired by a confocal microscope (LSM 710, Carl Zeiss, €ttingen, Germany). Go 2.9.4. Annexin V/PI assay The assay was performed according to the manufacturer's protocol. HeLa cells cultured in 6-well plates were exposed to the tested compounds at the indicated concentrations for 24 h. The cell suspension was stained with 5 mL annexin V and 10 mL PI at room temperature for 10 min in the dark, and analyzed immediately by flow cytometry (FACSCalibur™, Becton Dickinson, NJ, USA). Data were analyzed by FlowJo Software (Tree Star, OR, USA). 2.10. ROS detection The impact of 1e3 on ROS levels was determined as previously described [21]. Briefly, cells were treated with Ir(III) for 6 h and then incubated with H2DCFDA (10 mM) at 37  C for 20 min. The fluorescence intensity of the cells was measured by flow cytometry (FACSCalibur™, Becton Dickinson, NJ, USA) with excitation at 488 nm and emission at 530 nm. Data were analyzed by FlowJo Software (Tree Star, OR, USA). 2.11. Photodynamic activity 2.11.1. Quantification of singlet oxygen (1O2) generation The 1O2 quantum yields (ФD) were detected by monitoring the photooxidation of DPBF sensitized by 1e3 in DMSO [41]. DMSO solutions containing Ir(III) and DPBF (50 mM) were aerated for 10 min, and then excited at 365 nm (20 mW cm2). The absorbance of DPBF at 418 nm was recorded every 2 s. MB was used as the reference of 1O2 sensitization (ФD ¼ 0.52). The absorbance at 365 nm of Ir(III) complexes and MB was kept at 0.15. The 1O2 quantum yields of Ir(III) complexes were calculated according the following equation. IrðIIIÞ

FD


.
 IrðIIIÞ ¼ FMB  F MB sMB  F IrðIIIÞ D  s

where s is the slope of a linear fit of the change in absorbance of DPBF (at 418 nm) with the irradiation time, and F is the absorption correction factor, which is given by F ¼ 1e10OD (OD is the optical density at the irradiation wavelength). 2.11.2. Determination of phototoxicity Cells seeded in 96-well plates were incubated with the tested compounds in the dark for 12 h and then exposed to 365 nm light (20 mW cm2) for 10 min. After further incubation for 32 h in the dark, 20 mL MTT solution (5 mg/mL) was added to each well, and the plates were incubated for an additional 4 h. The media was removed and 150 mL DMSO was added per well. Cell viability was determined by MTT assay described previously [21]. 2.12. Statistical analysis All biological experiments were performed at least twice with triplicates in each experiment. Representative results were depicted in this report. Data were presented as means ± standard deviations.

3. Results and discussion 3.1. Synthesis and photophysical properties The biscarbene ligand L1 was prepared and isolated from onepot reaction of 1-methyl-1H-imidazole with dichloromethane according to the literature method with slight modifications [37]. L2 and L3 were synthesized similarly. Complexes 1 was synthesized by

98

Y. Li et al. / Biomaterials 39 (2015) 95e104

direct reaction of L1, [Ir(ppy)2]2Cl2 and Ag2O in dichloromethane according to the method previously reported (Scheme S1) [34]. Complexes 2 and 3 were synthesized adopting the similar method. The ligands and complexes were characterized by ESI-MS, 1H NMR, 13C NMR and elemental analysis (Figs. S1eS6). The structures of complexes 2 and 3 were determined by X-ray crystallography. The perspective views of 2 and 3 are shown in Fig. 1. The crystallographic parameters and selected bond angles/distances are shown in Tables S1 and S2, respectively. Both complexes adopt a distorted octahedral structure. In both cases the pyridine groups of the C^N ligands are in the trans configuration. The :C^C: ligands are significantly distorted because of the methylene bridge, while the N^C: ancillary ligands are almost flat. The bite angles of the :C^C: ligands (2: 85.2(2); 3: 84.6(2)) are wider than the average bit angles of the N^C: ligands (2: 79.3(2); 3: 79.8(7)), which is also observed for other complexes with similar structures [34,42]. The electronic absorption spectra of the 1e3 in PBS, CH2Cl2 and CH3CN at 298 K are shown in Fig. 2A. The strong absorption bands below 300 nm are originated from the spin-allowed 1pep* electronic ligand-centered (LC) transitions [34]. The structureless bands at 300e360 nm can be assigned to phenyl-to-pyridine pep* ligand-centered charge transfer (LCCT) and metal-to-ligand charge-

Fig. 1. Perspective views of the cations in 2 and 3. The hydrogen atoms and counter ions are omitted for clarity.

transfer (MLCT) (dp(Ir)ep*(phenylpyridine)) transitions [34,42]. The lower-lying bands in the visible region (>360 nm) are attributed to both singlet and triplet MLCT transitions [34,42,43]. Delicate difference in the absorption spectra of 1, 2 and 3 is observed in solvents of different polarity. The room temperature phosphorescence spectra of complexes 1e3 are shown in Fig. 2B. The photophysical data are summarized in Table S3. Under ambient conditions, 1e3 display blue-green emission with a maximum wavelength around 500 nm upon excitation at 380 nm. The phosphorescence spectra of 1e3 are vibronically structured in PBS, CH2Cl2 and CH3CN, which indicates that the emissive excited states have a pronounced LC pp* character in addition to MLCT character [42]. The emission intensities and lifetimes of 1e3 show a dependence on the polarity of the solvents with no spectral shift observed. The emission properties of the complexes are also oxygen-sensitive, as they show higher quantum yields and longer emission lifetimes in degassed solvents as compared with those obtained in air-saturated solutions. 3.2. Cellular localization Due to their rich photophysical properties, the intracellular distribution of phosphorescent iridium complexes can be easily monitored by fluorescent microscopy [25e27]. Confocal microscopic observation shows that 1, 2 and 3 can effectively penetrate into HeLa cells after 10 min incubation, as indicated by the intense and punctate green fluorescence in the cytoplasm (Fig. 3). Colocalization analysis with the organelle-specific stain for mitochondria shows high degree of colocalization between Ir(III) (20 mM) and conventional MTR (100 nM) in HeLa cells. Pearson's correlation coefficients for Ir(III) and MTR are 84%, 85% and 88% for 1, 2 and 3, respectively. Similar results are obtained in A549 cells (Fig. S7).

Fig. 2. (A) UV/Vis spectra of 1, 2 and 3 (2  105 M) in PBS, CH2Cl2, and CH3CN at 298 K. (B) Emission spectra of 1, 2 and 3 (2  105 M) in air-saturated PBS, CH2Cl2, and CH3CN at 298 K (lex ¼ 380 nm).

Y. Li et al. / Biomaterials 39 (2015) 95e104

99

Fig. 3. Determination of intercellular localization of complexes 1e3 by confocal microscopy. HeLa cells were incubated with MTR (100 nM) for 20 min and then co-incubated with 1 (20 mM), 2 (20 mM) and 3 (20 mM) for 10 min at 37  C. The Ir(III) complexes were excited at 405 nm and the emission was collected at 520 ± 20 nm. MTR was excited at 543 nm and the emission was collected at 600 ± 20 nm. Scale bar: 20 mm.

As iridium is an exogenous element, the cellular uptake levels of Ir(III) can be quantitatively determined by ICP-MS [39]. After incubation with 10 mM complexes for 1 h, the intracellular iridium contents of 1, 2 and 3 are in the following order: 3 (4.1 ± 0.3 pmol per cell) > 2 (2.2 ± 0.2 pmol per cell) > 1 (0.6 ± 0.1 pmol per cell). It has been reported that many factors, e.g., lipophilicity and molecular size, can influence the cellular uptake efficiencies of iridium complexes, depending on the molecular structures [44,45]. The lipophilicity of 1e3 was determined by the shake-flask method [38]. As estimated, the log Po/w values obtained for 1 (0.074), 2 (0.49) and 3 (1.70) are increased with the length of the alkyl substitutions. The lipophilicity of 1e3 is correlated with their cellular uptake level. The cellular uptake mechanisms of small molecules include energy-dependent (endocytosis and active transport) or energyindependent (facilitated diffusion and passive diffusion) pathways [46,47]. Microscopic observations indicate that incubation of HeLa

cells with complex 3 at lower temperature (4  C) or upon treatment with the metabolic inhibitor CCCP can lead to reduced cellular uptake efficiency (Fig. S8). Chloroquine, an endocytosis modulator inhibiting the acidification of endosomes, shows no effect on the ability of 3 to cross the plasma membrane. The results indicate that complex 3 enters HeLa cells possibly through an energy-dependent pathway, e.g., active transport, and endocytosis is not responsible for the cell-membrane penetration process [45,46]. 3.3. In vitro cytotoxicity The in vitro cytotoxicity of the tested compounds was determined against HeLa, MCF-7, A549, A549R, HepG2 and LO2 cells by MTT assay after 48 h of treatment. The resulting IC50 values are listed in Table 1. Complexes 1e3 display higher cytotoxicity than cisplatin against all the human cancer cell lines tested. Based on the IC50 values, the in vitro antiproliferative activity of these complexes

Table 1 IC50 (mM) values of the tested compounds towards different cell lines.a Compounds

HeLa

A549

A549R

HepG2

MCF-7

LO2

1 2 3 L1 L2 L3 Cisplatin [Ir(ppy)2]2Cl2

1.5 ± 0.1 1.2 ± 0.1 1.0 ± 0.1 >100 >100 >100 11.4 ± 0.7 13.5 ± 1.6

5.7 ± 0.8 3.4 ± 0.2 3.1 ± 0.4 >100 >100 >100 16.4 ± 0.2 19.6 ± 2.6

5.3 ± 0.7 3.8 ± 0.4 3.0 ± 0.2 >100 >100 >100 91.2 ± 5.2 20.2 ± 2.3

9.8 ± 0.8 6.2 ± 0.2 3.3 ± 0.2 >100 >100 >100 12.0 ± 1.3 22.0 ± 1.8

10.4 ± 0.7 6.0 ± 0.4 2.7 ± 0.2 >100 >100 >100 10.9 ± 0.9 15.4 ± 1.3

17.3 ± 2.0 10.7 ± 0.3 8.0 ± 0.7 >100 >100 >100 20.7 ± 0.3 23.2 ± 2.8

a IC50 values are drug concentrations necessary for 50% inhibition of cell viability. Data are presented as means ± standard deviations obtained in at least three independent experiments. Cells are treated with the compounds for 48 h.

100

Y. Li et al. / Biomaterials 39 (2015) 95e104

is in the following order: 3 > 2 > 1 > cisplatin. An exceptionally high antiproliferative activity is observed for complex 3, whose cytotoxicity is more than 10-fold higher than that of cisplatin against HeLa cells. The in vitro inhibition potency of Ir(III) complexes is in accordance with their relative lipophilicity. Among the cancer cells tested, a selectivity for HeLa cells is observed for 1e3. 1e3 also show some degree of selectivity for cancer cells, as they are less cytotoxic against normal LO2 cells than cancer cells screened. The chloro-bridged Ir(III) dimer is less cytotoxic than 1e3 against all the cell lines screened. It should also be noted that ligands L1eL3 are inactive against all the cell lines tested (IC50 > 100 mM). In addition, complexes 1e3 are approximately 20e30 fold more potent than cisplatin in killing cisplatin-resistant A549R cells, which indicates that they can overcome cisplatin resistance. 3.4. Mitochondrial damage and real-time tracking Mitochondrion is the bioenergetic center of the cell, and it is also an essential component of the intrinsic apoptotic signaling pathway [48]. Once the membrane integrity of mitochondria is affected, the pro-death factors are released from mitochondria and initiate the death signaling cascade [49]. In order to investigate whether mitochondrial dysfunction was involved in cell death induced by 1e3, the changes in MMP were detected by JC-1 staining and flow cytometry. Depolarization of mitochondria is characterized by a decrease in red fluorescence (JC-1 aggregates) and an increase in green fluorescence (JC-1 monomers) [50]. As can be seen from Fig. 4, treatment of HeLa cells with complexes 1, 2 and 3 causes a concentration-dependent red to green color shift, indicating loss of MMP. Treatment of 3 (10 mM) for 6 h increases the percentage of cells with depolarized mitochondrial membranes from 12.2 ± 0.3% to 99.5 ± 0.4%. In healthy cells, mitochondria exist as precisely controlled dynamic networks, and changes in number and morphology of mitochondria are often observed when they are damaged [51]. As complexes 1e3 could localize to mitochondria and damage mitochondrial integrity, the possibility of them to be used as bifunctional theranostic agents was explored. As complex 3 is most potent in damaging mitochondria, it is used as a model complex for realtime monitoring of changes in mitochondrial morphology caused by Ir(III) treatment. As can be seen in Fig. 5 and Video S1, complex 3 can be effectively taken up by HeLa cells after 1 min incubation, and the green fluorescence shows the normally filamentous mitochondrial morphology. Prolonged incubation of HeLa cells with 3 causes morphological signs of gradually damaged mitochondria.

After 15 min incubation, most mitochondria appear as solid granules. Hollow spheres representing extremely swollen mitochondria can be observed after 25 min treatment. Co-localization analysis of 3 and MTR gives further evidence that the bubbles are swelling mitochondria (Fig. S9). Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.10.070. 3.5. Cell cycle arrest The cytotoxicity of many anticancer drugs is often associated with genomic DNA damage and cell cycle perturbation [52,53]. The effects of complexes 1e3 on cell cycle distribution were investigated by flow cytometry and PI staining (Fig. S10). HeLa cells were treated with 1, 2 and 3 at a concentration of 1 mM for 12, 24, 36 and 48 h. No obvious effects on cell cycle distribution are observed for 1e3. The phenomenon is consistent with the results reported for other mitochondria-targeted compounds, which further confirms that the action mechanisms of these complexes are very different from those of traditional metal-based anticancer agents [53,54]. 3.6. Induction of apoptosis Mitochondria control the intrinsic pathway of apoptosis by regulating the translocation of pro-apoptotic proteins, e.g., cytochrome c, from mitochondrial intermembrane space to cytosol [48]. The release of proapoptotic proteins from mitochondria can activate death-driving proteolytic proteins known as caspases [55]. The Bcl-2 family of proteins, consisting of anti-apoptotic and proapoptotic members, regulate MMP and mitochondrial permeability during apoptosis [56]. Western blotting analysis of several key proteins involved in mitochondrial damage was performed by choosing the most active complex 3 as a model compound. As shown in Fig. 6, Bcl-2 expression is suppressed after treatment of HeLa cells with complex 3, accompanied by a concentration-dependent increase in protein expression of Bax and cytochrome c. The cleavage of full length caspase-3 (35 kDa) to large fragment (17/19 kDa) of activated caspase-3 is also observed. Accordingly, the well-known substrate of activated caspase-3 involved in apoptotic signaling, poly (ADP-ribose) polymerase (PARP), is cleaved to an 85-kD signature peptide in 3-treated HeLa cells in a dose-dependent manner (Fig. 6). The results show that mitochondrial damage caused by 3 initiates a cascade of events that can lead to execution of cell death.

Fig. 4. Effects of 1, 2 and 3 on MMP analyzed by JC-1 staining and flow cytometry. HeLa cells were treated with vehicle or Ir(III) at the indicated concentrations for 6 h. JC-1 was excited at 488 nm and monitored simultaneously at 530 ± 15 nm (Green) and 590 ± 15 nm (Red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Y. Li et al. / Biomaterials 39 (2015) 95e104

101

Fig. 6. Western blot analysis of the dose-dependent effect of complex 3 on expression of cytochrome c, Bcl-2, Bax, caspase 3 and PARP. HeLa cells were exposed to 3 at the indicated concentrations for 12 h.

but PI-negative because plasmic membrane is intact while phosphatidylserine is externalized. Annexin V/PI double positive cells can be both late apoptotic and necrotic [58]. Flow cytometric analysis shows that treatment of HeLa cells with 1e3 leads to a dose-dependent increase in the percentage of apoptotic cells (Fig. 8). After treated with 3 (10 mM) for 12 h, the percentages of cells in early apoptotic phase and late-apoptotic/necrotic phase are 28.4 ± 4.1% and 57.7 ± 5.2%, respectively. The ability of 1e3 to induce apoptosis is in accordance with their antitumor activity. 3.7. Elevation of intracellular ROS levels

Fig. 5. Real-time tracking of mitochondria in HeLa cells stained with 3 (20 mM) at 37  C for different time intervals. lex ¼ 405 nm, lem ¼ 520 ± 20 nm. Scale bar: 20 mm.

Mitochondria are the major source of intracellular ROS, and mitochondrial dysfunction is closely related to ROS accumulation in the process of apoptosis [59]. To elucidate whether depolarization of mitochondria caused by 1e3 treatment led to ROS production, the effects of 1e3 on intracellular ROS levels were examined by 20 ,70 -dichlorofluorescein (DCF) fluorescence assay measured by flow cytometry (Fig. 9). After 6 h treatment, HeLa cells are stained with H2DCFDA, which is nonfluorescent and can be converted into highly fluorescent DCF species in the presence of intracellular ROS. The results show that 1e3 (10 mM) cause an approximately 2~3-fold concentration-dependent increase in DCF fluorescence signals as compared with vehicle-treated cells, and the capabilities of 1e3 to elevate ROS levels are consistent with their in vitro cytotoxic potencies.

In addition, activation of caspase-3/7 by 1e3 was measured by a homogeneous luminescent assay. As shown in Fig. 7, treatment of 1e3 markedly stimulates activation of caspase-3/7 in a dosedependent manner, which is similar to that observed for cisplatin. Apoptosis is characterized by some biochemical and morphological features [57]. The capability of 1e3 to induce apoptosis was further studied by monitoring morphological changes and phosphatidylserine externalization. Representative images of microscopic analysis of morphological changes in cells treated with 3 are shown in Fig. S11. It can be seen that treatment of 3 impairs the proliferation capacity of HeLa cells as compared with the vehicle-treated control cells. After treatment of 3 (2 mM) for 12 h, most of the cells have lost their normal morphology and show marked apoptotic morphological characteristics, e.g., membrane blebbing and cell shrinkage. Phosphatidylserine externalization is considered to be a hallmark of early apoptosis. Early apoptotic cells are annexin V-positive

Fig. 7. Activation of caspases-3/7 by Ir(III) treatment. HeLa cells were exposed to cisplatin, 1, 2 and 3 at the indicated concentrations for 12 h.

102

Y. Li et al. / Biomaterials 39 (2015) 95e104

Fig. 8. Flow cytometric quantification of annexin V and PI double labeled HeLa cells after treatment with cisplatin, 1, 2 and 3 for 24 h at the indicated concentrations.

3.8. Photodynamic activity Iridium complexes with long-lived triplet excited states can be used as effective 1O2 sensitizers [60e62]. 1O2 is considered to be the main cytotoxic species in photodynamic therapy [63]. The efficiency of 1e3 to generate photosensitized 1O2 was determined by monitoring the dye-sensitized photooxidation of DPBF [41]. 1O2 quantum yields (ФD) were calculated using MB (ФD ¼ 0.52) as the standard (Fig. 10). The results show that 1O2 generation capability of 1e3 is in the following order: 3 (FD ¼ 0.62) > 2 (FD ¼ 0.59) > 1 (FD ¼ 0.58). Subsequently, the phototoxicity of complexes 1, 2 and 3 was examined against HeLa, A549, and A549R cells. Cells were exposed to the tested compounds at various concentrations for 12 h, and then irradiated at 365 nm light (20 mW cm2) for 10 min. The optimal total light dose was determined in a preliminary series of experiments using 3 as the representative compound. Cell cytotoxicity was determined by MTT assay 36 h after the end of irradiation. The phototoxic indexes are calculated as ratios of the IC50 values obtained in the dark to those obtained under light

Fig. 9. Intracellular ROS production measured by DCF fluorescence (Ex ¼ 488 nm; Em ¼ 530 nm) in HeLa cells after treatment with complexes 1, 2 and 3 at the indicated concentrations for 6 h.

irradiation. The results show that the IC50 values of 1, 2 and 3 under light are markedly decreased by 9- to 3488-fold as compared with the values obtained in the dark (Table 2). No significant difference in cytotoxicity is found for cancer cells treated with cisplatin in the presence and absence of light. Notably, 3 shows the most potent photodynamic activity. The phototoxic indexes of 3 against HeLa, A549 and A549R are 49, 239 and 3488, respectively. To the best of our knowledge, these are among the highest phototoxic index values ever reported for iridium complexes [41,64,65]. The result suggests that these Ir(III)eNHCs complexes may be used as potent photodynamic therapeutic agents.

4. Conclusions In conclusion, three cyclometalated iridium(III) complexes bearing bis(NHC) ligands with different lipophilicity have been prepared and characterized. All of the newly synthesized complexes show higher antiproliferative activities than cisplatin against various cancer cells including cisplatin-resistant A549 cells. The cytotoxicities of the complexes are found to be correlated with their lipophilicities and cellular uptake efficacies. Notably, 1e3 can be quickly and effectively taken into HeLa cells and specifically localize

Fig. 10. Photooxidation of DPBF (50 mM) in aerated DMSO by complexes 1, 2 and 3 under light irradiation. Changes in absorption spectra of DPBF at 418 nm upon irradiation at 365 nm in the presence of 1e3 were monitored, and MB was used as a standard.

Y. Li et al. / Biomaterials 39 (2015) 95e104

103

Table 2 IC50 values and phototoxic indexes of 1, 2, 3 and cisplatin. Compounds

IC50 (mM)a HeLa

1 2 3 Cisplatin

0.15 0.13 0.021 10.7

Phototoxic indexb A549

± ± ± ±

0.02 0.01 0.003 0.7

0.16 0.098 0.013 17.2

A549R ± ± ± ±

0.02 0.006 0.002 0.7

0.019 0.0028 0.00086 81.4

± ± ± ±

0.002 0.0003 0.00002 3.6

HeLa

A549

A549R

10 9 49 1

36 35 239 1

279 1357 3488 1

a IC50 values are drug concentrations necessary for 50% inhibition of cell viability. Data are presented as means ± standard deviations obtained in three independent experiments. b Phototoxicity index is the ratio of the IC50 value in dark to that obtained upon light irradiation. Cells were treated with the compounds for 12 h and then exposed to 365 nm UV light (20 mW cm2) for 10 min.

to mitochondria. Complex 3 can function as a novel theranostic agent by inducing and real-time tracking the changes in mitochondrial morphology. Mechanism studies show that 13 induce a series of events associated with mitochondrial damage in HeLa cells including ROS production, cytochrome c release, caspases activation and apoptosis. However, no cell cycle disturbance is observed, which indicates that 1e3 are not genotoxic and exert their activities through mechanisms distinct from those of cisplatin. Interestingly, these complexes can act as efficient photosensitizers. Notably, the cytotoxicity of 1e3 against A549R cells is increased by 279- to 3488-fold upon irradiation at 365 nm. Our study demonstrates that these Ir(III)eNHC complexes have high potential to be utilized as mitochondria-targeted theranostic and photodynamic agents. Funding This work is supported by National Natural Science Foundation of China (Nos. 21172274, 21231007, 21121061 and 21201183), State High-Tech Development Program (863 Program: 2012AA020305), National Basic Research Program (973 Program No. 2014CB 845604), the Ministry of Education of China (Nos., IRT1298 and 313058), and the Fundamental Research Funds for the Central Universities. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.biomaterials.2014.10.070 References [1] Kelkar SS, Reineke TM. Theranostics: combining imaging and therapy. Bioconjug Chem 2011;22:1879e903. [2] Cole AJ, Yang VC, David AE. Cancer theranostics: the rise of targeted magnetic nanoparticles. Trends Biotechnol 2011;29:323e32. [3] Santra S, Kaittanis C, Santiesteban OJ, Perez JM. Cell-specific, activatable, and theranostic prodrug for dual-targeted cancer imaging and therapy. J Am Chem Soc 2011;133:16680e8. [4] Yuan Y, Kwok RT, Tang BZ, Liu B. Targeted theranostic platinum(IV) prodrug with a built-in aggregation-induced emission light-up apoptosis sensor for noninvasive early evaluation of its therapeutic responses in situ. J Am Chem Soc 2014;136:2546e54. [5] Fulda S, Galluzzi L, Kroemer G. Targeting mitochondria for cancer therapy. Nat Rev Drug Discov 2010;9:447e64. [6] Wallace DC. Mitochondria and cancer. Nat Rev Cancer 2012;12:685e98. [7] Gogvadze V, Orrenius S, Zhivotovsky B. Mitochondria as targets for chemotherapy. Apoptosis 2009;14:624e40. [8] Wang F, Ogasawara MA, Huang P. Small mitochondria-targeting molecules as anti-cancer agents. Mol Aspects Med 2010;31:75e92. [9] Hu W, Kavanagh JJ. Anticancer therapy targeting the apoptotic pathway. Lancet Oncol 2003;4:721e9. [10] Smith RA, Hartley RC, Murphy MP. Mitochondria-targeted small molecule therapeutics and probes. Antioxid Redox Signal 2011;15:3021e38. [11] Lim SH, Thivierge C, Nowak-Sliwinska P, Han J, van den Bergh H, Wagnieres G, et al. In vitro and in vivo photocytotoxicity of boron dipyrromethene derivatives for photodynamic therapy. J Med Chem 2010;53:2865e74.

[12] Hamblin MR, Miller JL, Rizvi I, Ortel B, Maytin EV, Hasan T. Pegylation of a chlorin(e6) polymer conjugate increases tumor targeting of photosensitizer. Cancer Res 2001;61:7155e62. [13] Park SY, Oh KT, Oh YT, Oh NM, Youn YS, Lee ES. An artificial photosensitizer drug network for mitochondria-selective photodynamic therapy. Chem Commun 2012;48:2522e4. [14] Herrmann WA. N-heterocyclic carbenes: a new concept in organometallic catalysis. Angew Chem Int Ed 2002;41:1290e309. [15] Diez-Gonzalez S, Marion N, Nolan SP. N-heterocyclic carbenes in late transition metal catalysis. Chem Rev 2009;109:3612e76. [16] Hickey JL, Ruhayel RA, Barnard PJ, Baker MV, Berners-Price SJ, Filipovska A. Mitochondria-targeted chemotherapeutics: the rational design of gold(I) Nheterocyclic carbene complexes that are selectively toxic to cancer cells and target protein selenols in preference to thiols. J Am Chem Soc 2008;130: 12570e1. [17] Cisnetti F, Gautier A. Metal/N-heterocyclic carbene complexes: opportunities for the development of anticancer metallodrugs. Angew Chem Int Ed 2013;52: 11976e8. [18] Hindi KM, Panzner MJ, Tessier CA, Cannon CL, Youngs WJ. The medicinal applications of imidazolium carbene-metal complexes. Chem Rev 2009;109: 3859e84. [19] Liu WK, Gust R. Metal N-heterocyclic carbene complexes as potential antitumor metallodrugs. Chem Soc Rev 2013;42:755e73. [20] Zou T, Lum CT, Lok CN, To WP, Low KH, Che CM. A binuclear gold(I) complex with mixed bridging diphosphine and bis(N-heterocyclic carbene) ligands shows favorable thiol reactivity and inhibits tumor growth and angiogenesis in vivo. Angew Chem Int Ed 2014;53:5810e4. [21] Li Y, Liu GF, Tan CP, Ji LN, Mao ZW. Antitumor properties and mechanisms of mitochondria-targeted Ag(I) and Au(I) complexes containing N-heterocyclic carbenes derived from cyclophanes. Metallomics 2014;6:1460e8. [22] Wilbuer A, Vlecken DH, Schmitz DJ, Kraling K, Harms K, Bagowski CP, et al. Iridium complex with antiangiogenic properties. Angew Chem Int Ed 2010;49:3839e42. [23] Feng L, Geisselbrecht Y, Blanck S, Wilbuer A, Atilla-Gokcumen GE, Filippakopoulos P, et al. Structurally sophisticated octahedral metal complexes as highly selective protein kinase inhibitors. J Am Chem Soc 2011;133: 5976e86. [24] Liu Z, Romero-Canelon I, Qamar B, Hearn JM, Habtemariam A, Barry NPE, et al. The potent oxidant anticancer activity of organoiridium catalysts. Angew Chem Int Ed 2014;53:3941e6. [25] Lo KKW, Zhang KY. Iridium(III) complexes as therapeutic and bioimaging reagents for cellular applications. RSC Adv 2012;2:12069e83. [26] You Y. Phosphorescence bioimaging using cyclometalated Ir(III) complexes. Curr Opin Chem Biol 2013;17:699e707. [27] Zhao Q, Huang C, Li F. Phosphorescent heavy-metal complexes for bioimaging. Chem Soc Rev 2011;40:2508e24. [28] You Y, Han Y, Lee YM, Park SY, Nam W, Lippard SJ. Phosphorescent sensor for robust quantification of copper(II) ion. J Am Chem Soc 2011;133: 11488e91. [29] Woo H, Cho S, Han Y, Chae WS, Ahn DR, You Y, et al. Synthetic control over photoinduced electron transfer in phosphorescence zinc sensors. J Am Chem Soc 2013;135:4771e87. [30] Cao R, Jia J, Ma X, Zhou M, Fei H. Membrane localized iridium(III) complex induces endoplasmic reticulum stress and mitochondria-mediated apoptosis in human cancer cells. J Med Chem 2013;56:3636e44. [31] He L, Liao SY, Tan CP, Lu YY, Xu CX, Ji LN, et al. Cyclometalated iridium(III)-bcarboline complexes as potent autophagy-inducing agents. Chem Commun 2014;50:5611e4. [32] King KA, Watts RJ. Dual emission from an ortho-metalated iridium(III) complex. J Am Chem Soc 1987;109:1589e90. [33] Meech SR, Phillips D. Photophysics of some common fluorescence standards. J Photochem 1983;23:193e217. [34] Monti F, Kessler F, Delgado M, Frey J, Bazzanini F, Accorsi G, et al. Charged biscyclometalated iridium(III) complexes with carbene-based ancillary ligands. Inorg Chem 2013;52:10292e305. [35] Blessing R. An empirical correction for absorption anisotropy. Acta Crystallogr Sect A 1995;51:33e8.

104

Y. Li et al. / Biomaterials 39 (2015) 95e104

[36] Sheldrick GM. SHELXS 97, program for X-ray crystal structure determination. €ttingen; 1997. SHELXL-97, program for X-ray crystal structure University of Go €ttingen; 1997. refinement, University of Go [37] Ahrens S, Zeller A, Taige M, Strassner T. Extension of the alkane bridge in bisNHCepalladiumechloride complexes. Synthesis, structure, and catalytic activity. Organometallics 2006;25:5409e15. [38] McKeage MJ, Berners-Price SJ, Galettis P, Bowen RJ, Brouwer W, Ding L, et al. Role of lipophilicity in determining cellular uptake and antitumour activity of gold phosphine complexes. Cancer Chemother Pharmacol 2000;46:343e50. [39] Leung SK, Liu HW, Lo KK. Functionalization of luminescent cyclometalated iridium(III) polypyridine complexes with a fluorous moiety: photophysics, protein-binding, bioconjugation, and cellular uptake properties. Chem Commun 2011;47:10548e50. [40] Ye RR, Ke ZF, Tan CP, He L, Ji LN, Mao ZW. Histone-deacetylase-targeted fluorescent ruthenium(II) polypyridyl complexes as potent anticancer agents. Chem Eur J 2013;19:10160e9. [41] Li SP, Lau CT, Louie MW, Lam YW, Cheng SH, Lo KK. Mitochondria-targeting cyclometalated iridium(III)-PEG complexes with tunable photodynamic activity. Biomaterials 2013;34:7519e32. [42] Yang CH, Beltran J, Lemaur V, Cornil J, Hartmann D, Sarfert W, et al. Iridium metal complexes containing N-heterocyclic carbene ligands for blue-lightemitting electrochemical cells. Inorg Chem 2010;49:9891e901. [43] Costa RD, Monti F, Accorsi G, Barbieri A, Bolink HJ, Orti E, et al. Photophysical properties of charged cyclometalated Ir(III) complexes: a joint theoretical and experimental study. Inorg Chem 2011;50:7229e38. [44] Zhang GL, Zhang HY, Gao Y, Tao R, Xin LJ, Yi JY, et al. Near-infrared-emitting iridium(III) complexes as phosphorescent dyes for live cell imaging. Organometallics 2014;33:61e8. [45] Lo KK, Chan BT, Liu HW, Zhang KY, Li SP, Tang TS. Cyclometalated iridium(III) polypyridine dibenzocyclooctyne complexes as the first phosphorescent bioorthogonal probes. Chem Commun 2013;49:4271e3. [46] Li CY, Yu MX, Sun Y, Wu YQ, Huang CH, Li FY. A nonemissive iridium(III) complex that specifically lights-up the nuclei of living cells. J Am Chem Soc 2011;133:11231e9. [47] Puckett CA, Barton JK. Mechanism of cellular uptake of a ruthenium polypyridyl complex. Biochemistry 2008;47:11711e6. [48] Wang C, Youle RJ. The role of mitochondria in apoptosis. Annu Rev Genet 2009;43:95e118. [49] Green DR, Kroemer G. The pathophysiology of mitochondrial cell death. Science 2004;305:626e9. [50] Salvioli S, Ardizzoni A, Franceschi C, Cossarizza A. JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess DJ changes in intact

[51] [52] [53] [54]

[55] [56] [57] [58] [59]

[60]

[61]

[62]

[63] [64]

[65]

cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett 1997;411:77e82. Karbowski M, Youle RJ. Dynamics of mitochondrial morphology in healthy cells and during apoptosis. Cell Death Differ 2003;10:870e80. Shapiro GI, Harper JW. Anticancer drug targets: cell cycle and checkpoint control. J Clin Invest 1999;104:1645e53. Wang D, Lippard SJ. Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov 2005;4:307e20. Wisnovsky SP, Wilson JJ, Radford RJ, Pereira MP, Chan MR, Laposa RR, et al. Targeting mitochondrial DNA with a platinum-based anticancer agent. Chem Biol 2013;20:1323e8. Henry-Mowatt J, Dive C, Martinou JC, James D. Role of mitochondrial membrane permeabilization in apoptosis and cancer. Oncogene 2004;23:2850e60. Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev 1999;13:1899e911. Taylor RC, Cullen SP, Martin SJ. Apoptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol 2008;9:231e41. Vermes I, Haanen C, Reutelingsperger C. Flow cytometry of apoptotic cell death. J Immunol Methods 2000;243:167e90. Trachootham D, Alexandre J, Huang P. Targeting cancer cells by ROS-mediated mechanisms: a radical therapeutic approach? Nat Rev Drug Discov 2009;8: 579e91. Djurovich PI, Murphy D, Thompson ME, Hernandez B, Gao R, Hunt PL, et al. Cyclometalated iridium and platinum complexes as singlet oxygen photosensitizers: quantum yields, quenching rates and correlation with electronic structures. Dalton Trans 2007:3763e70. Sun JF, Zhao JZ, Guo HM, Wu WH. Visible-light harvesting iridium complexes as singlet oxygen sensitizers for photooxidation of 1,5-dihydroxynaphthalene. Chem Commun 2012;48:4169e71. Gao RM, Ho DG, Hernandez B, Selke M, Murphy D, Djurovich PI, et al. Biscyclometalated Ir(III) complexes as efficient singlet oxygen sensitizers. J Am Chem Soc 2002;124:14828e9. Ethirajan M, Chen Y, Joshi P, Pandey RK. The role of porphyrin chemistry in tumor imaging and photodynamic therapy. Chem Soc Rev 2011;40:340e62. Lo KK, Law WH, Chan JC, Liu HW, Zhang KY. Photophysical and cellular uptake properties of novel phosphorescent cyclometalated iridium(III) bipyridine Dfructose complexes. Metallomics 2013;5:808e12. Kastl A, Wilbuer A, Merkel AL, Feng L, Di Fazio P, Ocker M, et al. Dual anticancer activity in a single compound: visible-light-induced apoptosis by an antiangiogenic iridium complex. Chem Commun 2012;48:1863e5.