Biomaterials 56 (2015) 140e153

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Ruthenium(II) polypyridyl complexes as mitochondria-targeted two-photon photodynamic anticancer agents Jiangping Liu, Yu Chen, Guanying Li, Pingyu Zhang, Chengzhi Jin, Leli Zeng, Liangnian Ji, Hui Chao* MOE 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 12 January 2015 Received in revised form 30 March 2015 Accepted 2 April 2015 Available online

Clinical acceptance of photodynamic therapy is currently hindered by poor depth efficacy and inefficient activation of the cell death machinery in cancer cells during treatment. To address these issues, photoactivation using two-photon absorption (TPA) is currently being examined. Mitochondria-targeted therapy represents a promising approach to target tumors selectively and may overcome the resistance in current anticancer therapies. Herein, four ruthenium(II) polypyridyl complexes (RuL1eRuL4) have been designed and developed to act as mitochondria-targeted two-photon photodynamic anticancer agents. These complexes exhibit very high singlet oxygen quantum yields in methanol (0.74 e0.81), significant TPA cross sections (124e198 GM), remarkable mitochondrial accumulation, and deep penetration depth. Thus, RuL1eRuL4 were utilized as one-photon and two-photon absorbing photosensitizers in both monolayer cells and 3D multicellular spheroids (MCSs). These Ru(II) complexes were almost nontoxic towards cells and 3D MCSs in the dark and generate sufficient singlet oxygen under oneand two-photon irradiation to trigger cell death. Remarkably, RuL4 exhibited an IC50 value as low as 9.6 mM in one-photon PDT (lirr ¼ 450 nm, 12 J cm2) and 1.9 mM in two-photon PDT (lirr ¼ 830 nm, 800 J cm2) of 3D MCSs; moreover, RuL4 is an order of magnitude more toxic than cisplatin in the latter test system. The combination of mitochondria-targeting and two-photon activation provides a valuable paradigm to develop ruthenium(II) complexes for PDT applications. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Ruthenium(II) complex Two-photon Photodynamic therapy Mitochondria 3D multicellular spheroids

1. Introduction As a minimally invasive medical technique to destroy cancer cells without systemic toxicity, photodynamic therapy (PDT) has been received increased attention. PDT involves the use of photosensitizers to transfer energy from light to molecular oxygen to generate reactive oxygen species (ROS), which ultimately cause tissue damage [1]. Singlet oxygen (1O2), a well-known major ROS agent, is substantially liable for photobiological activity [2]. The lifetime and diffusion of 1O2 is quite limited. Thus, following irradiation, subsequent reactions involving 1O2 occur in the immediate vicinity of the accumulated photosensitizers, which is where the biological responses occur that make regioselective cell death possible.

* Corresponding author. Tel.: þ86 20 84110613; fax: þ86 20 84112245. E-mail address: [email protected] (H. Chao). http://dx.doi.org/10.1016/j.biomaterials.2015.04.002 0142-9612/© 2015 Elsevier Ltd. All rights reserved.

By virtue of its role as both the power plant and apoptosis center of cells [3e5], the mitochondrion has become an ideal target for PDT agents. Evidence suggests that cell death is apt to proceed via apoptosis rather than necrosis in PDT when photosensitizers localize in the mitochondria [6]. Mitochondria-targeted anticancer drugs that act through disrupting the redox homeostasis of the cell, which leads to the activation of the mitochondrial-dependent cell death signaling pathway, may overcome cancer resistance [7,8]. Therefore, apoptosis can be triggered efficiently with excess oxidative stress by targeting mitochondria. In this regard, rationally designed photosensitizers that target mitochondria are highly sought. There are many molecular design strategies that seek to target mitochondria [9], among which triphenylphosphine (TPP) salts is an extensively adopted functionality to impart a delocalized charge and lipophilic character to a compound, which increases its affinity for mitochondria [10e12]. Additionally, many PDT agents have been demonstrated to trigger cell death efficiently in vitro. However, these results do not necessarily indicate that an identical

J. Liu et al. / Biomaterials 56 (2015) 140e153

therapeutic outcome may be expected in vivo, for all PDT agents are confronted with barriers to drug penetration and oxygen shortage in tumors, leading to their inability to completely eradicate cancer cells [1]. To address these problems, a more reliable biomodel for drug screening was highly desired. It has been noted [13,14] that 3D multicellular spheroids (MCSs) can bridge the gap between in vitro and in vivo models to serve as a model for drug delivery and to evaluate the efficiency of PDT therapy. Large MCSs (i.e., >200 mm) could form a necrotic core, a quiescent intermediate region and a proliferating periphery region that enable a MCS to mimic the microenvironment of a tumor more closely than a monolayer r cell system [15]. PDT combined with two-photon absorbing photosensitizers in the NIR therapeutic window can offer new perspectives for cancer treatments because of its deeper tissue penetration and unique spatial resolution [16]. Until recently, one-photon PDT molecular agents have been predominant [17e20], and very few two-photon PDT molecular agents were explored [21e24]. Anderson et al. [25] for the first time demonstrated two-photon PDT in a living mammal with conjugated porphyrin dimer. Zou et al. [21] have developed coumarin derivatives as two-photon PDT agents. Furthermore, Arnbjerg et al. [23] have explored a new tetraphenylporphyrin (H2TPP) derivative as a potential molecular twophoton PDT candidate. Unlike conventional porphyrin-based photosensitizers [1,2,23,26e28], metal complexes demonstrate excellent photostability, outstanding photophysical properties, and favorable solubility in aqueous solution. With proper modification, metal complexes are capable of possessing specific affinity for certain organelles [29,30]. For example, Ru(II) complexes exhibit the advantages of rapid systemic clearance [31] and practical excitation wavelengths for cells and have been explored for cancer therapy [20,32e41]. Our previous work on Ru(II) polypyridyl complexes as DNA photocleavage agents [42] and two-photon luminescence cellular imaging probes [43] have stimulated us to further develop Ru(II) polypyridyl complexes as mitochondria-targeted twophoton photodynamic anticancer agents. In this work, a series of Ru(II) polypyridyl complexes (i.e., RuL1eRuL4, Scheme 1) containing 4,7-diphenyl-1,10phenanthroline (DIP) as an ancillary ligand and a TPP scaffold

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were designed and synthesized. The singlet oxygen quantum yield and TPA cross sections of Ru(II) complexes were determined. The cellular uptake and distribution of the complexes were also investigated with inductively coupled plasma mass spectrometry (ICP-MS). Finally, TPA-induced singlet oxygen generation and oneand two-photon PDT evaluation were conducted in monolayer cells and in 3D MCSs. 2. Materials and methods 2.1. Materials and instruments Unless otherwise noted, all chemical reagents and solvents were commercially available and used without further purification. Twice-distilled water was used throughout all experiments. RuCl3$nH2O, 4,7-diphenyl-1,10-phenanthroline, aniline, and triphenylphosphine (TPP) were purchased from SigmaeAldrich and used without further purification. Agarose was purchased from SigmaeAldrich. Dulbecco's modified eagle medium (DMEM), fetal bovine serum (FBS), penicillin, streptomycin and MitoTracker® Green FM were purchased from Invitrogen. PBS buffer was prepared as follows: 11.65 g Na2HPO4, 1.65 g KH2PO4, 9 g NaCl, 1000 mL H2O, pH ¼ 7.4. 1,10-Phenanthroline-5,6-dione [44], cis[Ru(DIP)2Cl2]$2H2O [45], L1 [46] and L2 [47] were prepared according to the previously reported methods. The Ru(II) complexes were dissolved in DMSO preceding the experiments; the calculated quantities of the Ru(II) complex solutions were then added to the appropriate medium to yield a final DMSO concentration of less than 2% (v/v). Microanalysis (C, H, and N) was performed using a PerkineElmer 240Q elemental analyzer. Electrospray ionization mass spectra (ESI-MS) were recorded on a LCQ system (Finnigan MAT, USA). The expected and measured isotope distributions were compared. The 1H NMR and 31P NMR spectra were recorded on a Varian INOVA 500NB Superconducting Fourier Transform Nuclear Magnetic Resonance Spectrometer (500 Hz) at 278 K. The IR spectra were recorded on a EQUINOX 55 Fourier transformation infra-red spectrometer (Bruker, German). The UVeVis spectra were recorded on a Varian Cary 300 spectrophotometer. Emission spectra

Scheme 1. The chemical structures of complexes RuL1eRuL4.

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were recorded on a PerkinElmer LS 55 fluorescence spectrometer at room temperature. Time-resolved emission and two-photon absorption cross section measurements were performed on an FLSP960 (Edinburgh Instruments) combined fluorescence lifetime and steady state spectrometer equipped with an Opolette™ 355II laser source (tuning range 750e1000 nm, Spectra Physics Inc., USA). Luminescence lifetime studies were performed with an Edinburgh FLSP-920 photo-counting system using a hydrogen-filled lamp as the excitation source. Luminescence quantum yields of RuL1eRuL4 in an aerated methanol solution were measured with reference to [Ru(bpy)3]2þ (Ф ¼ 0.042, 10 mM in MeOH, 25  C) [48,49]. The inductively coupled plasma mass spectrometry (ICP-MS) experiments were performed on an Agilent's 7700x instrument. One- and two-photon luminescent imaging was conducted on a LSM 710 (Carl Zeiss, Germany) Laser Scanning Confocal Microscope. Visible one-photon irradiation (lirr ¼ 450 nm) in PDT was provided by a commercially available LED visible area light source (Height LED Instruments, China). All data were processed with the OriginPro 8.0 software package. 2.2. Synthesis 2.2.1. Synthesis of 4-(1-phenyl-1H-imidazo[4,5-f][1,10] phenanthrolin-2-yl)benzaldehyde (1) A mixture of ammonium acetate (9.24 g, 120 mmol), aniline (0.75 g, 8 mmol) and terephthalaldehyde (1.34 g, 10 mmol) was dissolved in glacial acetic acid (100 mL) and stirred under an argon atmosphere at 135  C. A suspension of 1,10phenanthroline-5,6-dione (1.68 g, 8 mmol) in 60 mL of glacial acetic acid was added to the solution over 1 h and was allowed to reflux overnight. After cooling to room temperature, the solution was adjusted to pH 6 using a 25% NH3 solution. The precipitate was allowed to stand overnight at 4  C and then filtered and dried under vacuum. The crude product was purified by silica gel chromatography using CH2Cl2/EtOH (30:1, v/v) as the eluent to afford a pale yellow product (Yield ¼ 56%). Anal. Calcd. for C26H16N4O (%): C, 77.99; H, 4.03; N, 13.99. Found (%): C, 78.07; H, 4.11; N, 13.86. ES-MS: m/z ¼ 400.9 [M þ H]þ. 1H NMR (500 MHz, CDCl3) d 9.31 (s, 1H), 9.07 (dd, J ¼ 4.3, 1.8 Hz, 1H), 8.99e8.91 (m, 2H), 8.37 (s, 1H), 7.97e7.83 (m, 4H), 7.65 (s, 1H), 7.59 (d, J ¼ 4.2 Hz, 2H), 7.56e7.45 (m, 4H). FT-IR (KBr pellet, cm1): n (C]O) 1728.7, n (C]C, C]N) 1668.4. 2.2.2. Synthesis of (4-(1-phenyl-1H-imidazo[4,5-f][1,10] phenanthrolin-2-yl)phenyl)meth-anol (2) 0.18 g (4.7 mmol) of sodium borohydride was dissolved in a 0.2 M sodium hydroxide solution (1.5 mL) and added drop wise to a solution of 0.85 g (2.1 mmol) of 1 dissolved in 60 mL of methanol, with preliminary cooling in an ice bath (0  C). The solution was stirred at room temperature for 1 h. Then, 5.7 mL of a saturated sodium carbonate solution was added and the mixture was stirred for an additional 0.5 h. The suspension was evaporated to dryness, poured into water (10 mL) and then extracted three times with CHCl3 (100 mL). The organic layers were combined, dried with MgSO4 and evaporated to dryness to afford a pale yellow solid (Yield ¼ 90%). The solid can be further purified by silica gel chromatography with CH2Cl2/EtOH (15:1, v/v) as the eluent or used as is. Anal. Calcd. for C26H18N4O (%): C, 77.59; H, 4.51; N, 13.92. Found (%): C, 77.70; H, 4.62; N, 13.81. ES-MS: m/z ¼ 403.2 [M þ H]þ; 425.5 [M þ Na]þ; 806.1 [2M þ H]þ. 1H NMR (500 MHz, CDCl3) d 9.07 (dd, J ¼ 4.3, 1.8 Hz, 1H), 8.99e8.91 (m, 2H), 8.37 (m, 3H), 7.56 (m, 7H), 7.37 (d, J ¼ 7.5 Hz, 2H), 4.59 (s, 2H), 3.25 (s, 1H). FT-IR (KBr pellet, cm1): n (eOH) 3514.5, n (eCH2e) 2895.3, n (C]C, C]N) 1660.1, d (eCH2e) 1465.2.

2.2.3. Synthesis of 2-(4-(bromomethyl)phenyl)-1-phenyl-1Himidazo[4,5-f][1,10]phenan-throline (3) 0.8 g (2.0 mmol) of 2 was dissolved into 48% hydrobromic acid (25 mL), stirred, heated to 125  C and added to concentrated sulfuric acid (2 mL) in a drop wise manner. The solution was refluxed for 6 h, cooled to room temperature and then poured into 30 mL water, and then treated with saturated sodium carbonate solution to obtain pH ¼ 8. The product was subsequently extracted with CH2Cl2 and dried with MgSO4. The solvent was removed to give a yellow solid. The crude product was purified by silica gel chromatography using CH2Cl2/EtOH (50: 1, v/v) as the eluent, affording a pale yellow powder (Yield ¼ 45%). Anal. Calcd. for C26H17BrN4 (%): C, 67.11; H, 3.68; N, 12.04. Found (%): C, 67.01; H, 3.60; N, 12.17. ESMS: m/z ¼ 385.3 [M  Br]þ; 464.1 [M þ H]þ; 488.3 [M þ Na]þ. 1H NMR (500 MHz, CDCl3) d 9.07 (dd, J ¼ 4.3, 1.8 Hz, 1H), 8.99e8.91 (m, 2H), 8.37 (m, 3H), 7.64 (s, 1H), 7.59 (d, J ¼ 7.0 Hz, 2H), 7.49 (m, 4H), 7.39 (m, 2H), 4.63 (s, 2H). FT-IR (KBr pellet, cm1): n (eCH2e) 2921.5, n (C]C, C]N) 1663.6, d (eCH2e) 1471.5. 2.2.4. Synthesis of L3 A mixture of 3 (0.23 g, 0.5 mmol) and TPP (0.5 g, 2 mmol) were dissolved in DMF (6 mL) and refluxed for 24 h at 110  C. The mixture was allowed to cool to room temperature and then it was poured into 200 mL of toluene. The mixture stood overnight at 4  C to give a light yellow precipitate. The product was filtered and washed with a small amount of toluene and ether and dried under vacuum (Yield ¼ 75%). Anal. Calcd. for C44H32BrN4P (%): C, 72.63; H, 4.43; N, 7.70. Found (%): C, 72.55; H, 4.51; N, 7.61. ES-MS: m/z ¼ 647.7 [M  Br]þ. 1H NMR (300 MHz, DMSO-d6) d 9.07 (dd, J ¼ 4.3, 1.8 Hz, 1H), 8.99e8.91 (m, 2H), 7.94e7.83 (m, 4H), 7.77e7.58 (m, 17H), 7.47 (dd, J ¼ 8.5, 4.3 Hz, 1H), 7.36 (dd, J ¼ 13.6, 5.0 Hz, 3H), 6.92 (dd, J ¼ 8.4, 2.4 Hz, 2H), 5.17 (d, J ¼ 15.7 Hz, 2H). 2.2.5. Synthesis of 4-(1-phenyl-1H-imidazo[4,5-f][1,10] phenanthrolin-2-yl)phenol (4) A mixture of 1,10-phenanthroline-5,6-dione (1.05 g, 5 mmol), ammonium acetate (4.63 g, 60 mmol), aniline (0.56 g, 6 mmol) and p-hydroxybenzaldehyde (0.61 g, 5 mmol) were dissolved in 35 mL of glacial acetic acid and refluxed overnight under an argon atmosphere. The reaction mixture was cooled to room temperature, poured into water (50 mL), treated with a 25% NH3 solution until the pH ¼ 6, giving rise to a thick dark green suspension. The suspension was added to 50 mL of CHCl3, stirred and then filtered to yield a dark gray crude product and a dark green filtrate, which was then extracted three times with CHCl3 (200 mL). The organic layers were combined, washed with brine (40 mL), dried with MgSO4 and evaporated under vacuum, giving a dark green solid. The solids were combined and purified by silica gel chromatography with CH2Cl2/EtOH (40:1, v/v), affording a white powder (Yield ¼ 75%). Further purification was obtained via re-crystallized with CHCl3/ toluene to give a white crystal. Anal. Calcd. for C25H16N4O (%): C, 77.30; H, 4.15; N, 14.42. Found (%): C, 77.18; H, 4.25; N, 14.53. ES-MS: m/z ¼ 338.1 [M þ 1]þ, 777.8 [2M þ H]þ. 1H NMR (500 MHz, CDCl3) d 9.08 (dd, J ¼ 4.3, 1.8 Hz, 1H), 9.02 (d, J ¼ 1.8 Hz, 1H), 8.80 (s, 1H), 8.38 (s, 1H), 7.96 (m, 2H), 7.65 (s, 1H), 7.60 (d, J ¼ 4.4 Hz, 2H), 7.49 (m, 4H), 7.02 (m, 2H), 6.35 (s, 1H). 2.2.6. Synthesis of 2-(4-(4-bromobutoxy)phenyl)-1-phenyl-1Himidazo[4,5-f][1,10]phena-nthroline (5) 0.58 g (1.5 mmol) of 4, 4.0 g (18.5 mmol) of 1,4-dibromobutane and 0.42 g (3 mmol) of potassium carbonate were stirred in a mixture of DMF (100 mL) and CHCl3 (50 mL) at 70  C for 6 h. After the reaction was complete, the hot mixture was filtered to give a light yellow solution, which was evaporated to dryness. The residue was purified by silica gel chromatography using CH2Cl2/EtOH (20:1,

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v/v) as the eluent to afford a pale yellow product (Yield ¼ 61%). Anal. Calcd. for C29H23BrN4O (%): C, 66.54; H, 4.43; N, 10.70. Found (%): C, 66.68; H, 4.45; N, 10.56. ES-MS: m/z ¼ 443.5 [M  Br]þ; 523.4 [M þ H]þ. 1H NMR (500 MHz, CDCl3) d 9.08 (dd, J ¼ 4.3, 1.8 Hz, 1H), 9.02 (d, J ¼ 1.8 Hz, 1H), 8.81 (s, 1H), 8.39 (s, 1H), 8.09 (m, 2H), 7.64 (s, 1H), 7.58 (d, J ¼ 9.9 Hz, 2H), 7.48 (m, 4H), 7.02 (d, J ¼ 8.1 Hz, 2H), 4.08 (t, J ¼ 6.2 Hz, 2H), 3.59 (s, 2H), 1.94 (d, J ¼ 6.6 Hz, 2H), 1.72 (s, 2H). 2.2.7. Synthesis of L4 A mixture of 5 (0.45 g, 0.86 mmol) and TPP (0.90 g, 3.4 mmol) were dissolved in DMF (10 mL) and refluxed for 24 h at 110  C. The mixture was allowed to cool to room temperature, poured into 250 mL of toluene and allowed to stand overnight at 4  C, which gave a light yellow precipitate. The product was filtered and washed with a small amount of toluene and ether and dried under vacuum (Yield ¼ 56%). Anal. Calcd. for C47H38BrN4OP (%): C, 71.85; H, 4.87; N, 7.13. Found (%): C, 71.70; H, 4.89; N, 7.20. ES-MS: m/ z ¼ 705. 3 [M  Br]þ. 1H NMR (300 MHz, DMSO-d6) d 9.06 (dd, J ¼ 4.3, 1.8 Hz, 1H), 9.00 (d, J ¼ 1.8 Hz, 1H), 8.92 (dd, J ¼ 4.3, 1.7 Hz, 1H), 8.02e7.63 (m, 21H), 7.58e7.40 (m, 3H), 7.31 (dd, J ¼ 8.5, 1.6 Hz, 1H), 6.85 (d, J ¼ 8.9 Hz, 2H), 4.04 (t, J ¼ 6.0 Hz, 2H), 3.65 (s, 2H), 1.89 (d, J ¼ 6.8 Hz, 2H), 1.72 (s, 2H). 2.2.8. Synthesis of compounds RuL1eRuL4 A mixture of L1eL4 (0.1 mmol) and cis-[Ru(DIP)2Cl2]$2H2O (0.1 mmol) in EtOH/H2O (10 mL/5 mL) was refluxed under an argon atmosphere at 80  C for 24 h to give a clear red solution. After the reaction, the solution was cooled and evaporated to dryness. The resultant extract was purified by column chromatography on alumina with CH3CN/EtOH to afford a red solid. The solid was then dissolved in water and added drop wise to a saturated NaClO4 solution to give a red precipitate. The precipitate was collected, washed with a small amount of ether and dried to afford the perchlorate complex. To promote their solubility in water, the product was further converted to chloride for the biological activity test as follows: the perchlorate complex was dissolved in acetone (as little as possible), filtered and excess (n-CH3CH2CH2CH2)4NCl was added to give a red precipitate. The resulting precipitate was collected, washed with acetone and dried under vacuum to give the pure chloride product. The yield, 1H NMR, 31P NMR, ESI-MS and element analysis data are listed as follows. RuL1: Yield 72%. Anal. Calcd. for C67H44Cl2N8 Ru (%): C, 71.02; H, 3.91; N, 9.89. Found (%): C, 71.13; H, 3.89; N, 9.81. ES-MS (CH3OH): m/z ¼ 530.8 [M  2Cl]2þ. 1H NMR (500 MHz, DMSO) d 9.10 (d, J ¼ 8.0 Hz, 2H), 8.36 (t, J ¼ 6.0 Hz, 4H), 8.27 (d, J ¼ 10.7 Hz, 4H), 8.23 (d, J ¼ 5.5 Hz, 2H), 8.12 (s, 2H), 7.88 (s, 2H), 7.83 (d, J ¼ 5.5 Hz, 2H), 7.78 (d, J ¼ 5.6 Hz, 2H), 7.73e7.57 (m, 24H), 7.53 (s, 1H). RuL2: Yield 69%. Anal. Calcd. for C73H48Cl2N8Ru (%): C, 72.51; H, 4.00; N, 9.27. Found (%): C, 72.60; H, 4.11; N, 9.18. ES-MS (CH3OH): m/z ¼ 569.3 [M  2Cl]2þ. 1H NMR (500 MHz, DMSO) d 9.26 (d, J ¼ 7.1 Hz, 1H), 8.35 (d, J ¼ 5.5 Hz, 1H), 8.31 (t, J ¼ 4.1 Hz, 1H), 8.30e8.24 (m, 5H), 8.24e8.20 (m, 2H), 8.18e8.14 (m, 1H), 7.98 (dd, J ¼ 8.3, 5.3 Hz, 1H), 7.83 (dd, J ¼ 5.2, 2.4 Hz, 2H), 7.78 (dt, J ¼ 5.7, 4.4 Hz, 6H), 7.75e7.58 (m, 26H), 7.50e7.41 (m, 4H). RuL3: Yield 58%. Anal. Calcd. for C92H64Cl3N8PRu (%): C, 72.70; H, 4.24; N, 7.37. Found (%): C, 72.79; H, 4.17; N, 7.31. ES-MS (CH3OH): m/z ¼ 470.9 [M  3Cl]3þ, 705.2 [M-3CleH]2þ. 1H NMR (500 MHz, DMSO) d 9.20 (d, J ¼ 8.2 Hz, 1H), 8.35 (d, J ¼ 5.5 Hz, 1H), 8.30 (d, J ¼ 5.5 Hz, 1H), 8.26 (dd, J ¼ 13.0, 2.8 Hz, 5H), 8.22e8.19 (m, 2H), 8.17e8.14 (m, 1H), 7.95 (ddd, J ¼ 15.2, 8.4, 6.1 Hz, 4H), 7.83 (d, J ¼ 5.5 Hz, 1H), 7.80e7.72 (m, 13H), 7.71e7.61 (m, 30H), 7.50 (d, J ¼ 8.8 Hz, 1H), 7.45 (d, J ¼ 8.0 Hz, 2H), 6.98 (dd, J ¼ 8.4, 2.1 Hz, 2H), 5.18 (d, J ¼ 16.0 Hz, 2H). 31P NMR (500 MHz, DMSO) d 28.05.

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RuL4: Yield 56%. Anal. Calcd. for C95H70Cl3N8OPRu (%): C, 72.31; H, 4.47; N, 7.10. Found (%): C, 72.28; H, 4.40; N, 7.01. ES-MS (CH3OH): m/z ¼ 490.3 [M  3Cl]3þ, 753.7 [M  2Cl]2þ. 1H NMR (500 MHz, DMSO) d 9.23 (d, J ¼ 8.5 Hz, 1H), 8.35 (d, J ¼ 5.5 Hz, 1H), 8.31 (d, J ¼ 5.5 Hz, 1H), 8.29e8.23 (m, 5H), 8.21 (dd, J ¼ 5.4, 4.0 Hz, 2H), 8.16e8.12 (m, 1H), 7.97 (dd, J ¼ 8.2, 5.3 Hz, 1H), 7.92e7.88 (m, 3H), 7.83e7.74 (m, 20H), 7.70e7.62 (m, 20H), 7.56 (d, J ¼ 8.8 Hz, 2H), 7.45 (d, J ¼ 8.9 Hz, 1H), 6.92 (d, J ¼ 9.0 Hz, 2H), 4.06 (t, J ¼ 6.0 Hz, 2H), 3.65 (td, J ¼ 14.1, 8.7 Hz, 2H), 1.91 (t, J ¼ 5.6 Hz, 2H), 1.77e1.65 (m, 2H). 31P NMR (500 MHz, DMSO) d 24.05. 2.3. Two-photon absorption cross section measurements The theoretical framework and experimental protocol for the two-photon absorption cross section measurements have already been outlined by Webb and Xu [50]. The two-photon luminescence of RuL1eRuL4 was measured through quartz cuvettes by the combined spectrometer at a concentration of 4  104 M in methanol at 298 K. The two-photon excitation ratios of the reference and sample systems are given below:

sS ¼ sR

fR CR IS nS fS CS IR nR

(1)

where s is the TPA cross section, F is the quantum yield, C is the concentration, n is the refractive index, and I is the integrated photoluminescent spectrum. The superscript S and R represent sample and reference, respectively. In our experiment, we have kept an identical excitation intensity and wavelength for both the sample and reference. Rhodamine B was utilized as the reference to measure the cross section values s. 2.4. Quantification of singlet oxygen formation The quantification of 1O2 was conducted according to reported protocols [51,52]. 1,3-Diphenylisobenzofuran (DPBF) was used to measure the singlet oxygen quantum yield for RuL1eRuL4. DPBF was luminescent (lex ¼ 411 nm, lem ¼ 479 nm), but its corresponding photo-oxidative product was not. A series of 3 mL air-saturated methanol solutions containing DPBF (30 mM) and the complexes, whose absorbance at 479 nm were adjusted to the same value (OD479nm ¼ 0.08), were separately charged into a 1 cm path fluorescence cuvette (obtained from a PerkineElmer LS 55 fluorescence spectrometer). The consumption of DPBF under irradiation at 450 nm was followed by monitoring the decrease in its fluorescence intensity at the emission maximum. [Ru(bpy)3]2þ was used as the standard, whose 1O2 formation quantum yield was determined to be 0.81 [53] in air-saturated methanol. The emission maxima of DPBF with different irradiation times were obtained, and the singlet oxygen quantum yields were determined using the following equations:

D½DPBF I0  It ¼ ¼ Iin Fab FD Fr  t t

(2)

k F FD ¼ ab ¼ ks Fsab FsD

(3)

where Iin is the incident monochromatic light intensity, Fab is the light absorbing efficiency, Fr is the reaction quantum yield of DPBF, t is the reaction time, I0/It is the fluorescence intensity before/after irradiation of the complexes, and k is the slope of plots. The superscript s indicates the reference.

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2.5. Cell line and culture conditions HeLa cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cell lines were maintained in either RPMI-1640 or DMEM media supplemented with fetal bovine serum (10%), penicillin (100 units/mL) and streptomycin (50 units/ mL) at 37  C in a CO2 incubator (95% relative humidity, 5% CO2). 2.6. Cellular uptake and distribution (ICP-MS) For the cellular uptake studies, exponentially growing HeLa cells were harvested, and the resulting single-cell suspension was plated into 100 mm tissue culture plates (Costar). HeLa cells were cultured to a density of 5  105 cells per mL in a volume of 5 mL of DMEM, treated with 10 mM Ru(II) complexes and incubated for 2 h at 37  C. After digestion, HeLa cells were collected (preserved in an ice bath), counted and subsequently divided into two equal parts for the extraction of the nucleus and cytoplasm. The nucleus portion was treated with a nucleus extraction kit (Pierce, Thermo) to give the nucleus extract and the cytoplasm was treated with a cytoplasm extraction kit (Pierce, Thermo) to give the mitochondrial portion. The procedures above were conducted in strict dark conditions. All of the final extracts were treated with 60% HNO3 and stood for over 24 h at room temperature to ensure complete digestion. Each sample was then diluted with twice-distilled water to achieve a final volume of 10 mL containing 2% HNO3. In addition, a standard curve was made for the quantitative determination. The concentration of Ru in three domains was determined by an inductively coupled plasma mass spectrometer, associating with cell number to afford the absolute Ru content per cell. Data were reported as the means ± standard deviation (n ¼ 3). 2.7. Monolayer cells confocal luminescence imaging Cells were plated onto 35 mm glass bottom dishes (Corning) and allowed to adhere for 24 h. The cells were washed with PBS, incubated with 10 mM of the Ru(II) complexes in DMSO/PBS (pH ¼ 7.4, 2:98, v/v) for 2 h in the dark at 37  C. For mitochondrial co-localization, the cells were further incubated with Mitotracker Green for 30 min in the incubator. For singlet oxygen detection, the cells were further incubated with 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) [54] for 20 min. Cell imaging was performed after the cells were washed with PBS. 2.8. (Photo)cytotoxicity in monolayer HeLa cells Exponentially grown HeLa cells were seeded in triplicate into 96-well plates at 1  104 cells/well. After incubation for 24 h, the cells were treated with increasing concentrations of the tested complexes. Control wells were prepared by the addition of culture medium (100 mL). Wells containing the culture medium only (i.e., without cells) were used as the blanks. The plates were incubated in the dark for 24 h. Then, all of the culture media were refreshed. For the cell cultures exposed to light, all of the experiment, control and blank wells were irradiated by an LED area light source (20 mW cm2) for 10 min. Both of the cell cultures from the dark and light groups were incubated for an additional 48 h. Upon completion of the incubation period, the viability and IC50 values of the compounds in the two groups were measured by ATP concentration with the CellTiter-Glo® Luminescent Cell Viability kit (Promega) according to the published protocol. The cell survival rate in the control wells of the dark group was considered to represent 100% cell survival. Each experiment was repeated at least three times to obtain the mean survival rates and standard

deviation. The IC50 values were determined by plotting the percentage of viability versus concentration on a logarithmic graph. 2.9. Formation and Z-axis scanning of 3D MCSs A suspension of 1% agarose in fresh PBS was submitted to a highpressure steam sterilization pot and sterilized in liquid mode for 20 min. Upon completion, the resulting clear solution was charged into a hot 96-well microassay culture plate (50 mL per well). After cooling, the plates were exposed to UV light for 3 h to ensure that they were sterile. HeLa single-cell suspensions at a density of 1  105 cells per mL were then charged into the preprocessed 96well microassay culture plates with a volume of 200 mL per well. The cells were incubated in an incubator and the culture medium was replaced every two days. HeLa MCSs formed spontaneously in 3 days with a diameter of approximately 800 mm. Each MCS in the 96-well plates was imaged with a phase-contrast microscope 10 object to monitor MCS integrity, diameter and volume. The volume (mm3) of each tumor spheroid was calculated by the formula: V ¼ 4/ 3 pr3. 3D MCSs pretreated with 10 mM Ru(II) complexes and H2TPP, respectively, for 8 h in the dark were carefully washed three times with PBS and subjected to confocal microscopy. The one-/twophoton excited luminescent images of sections along the z-axis were captured and stacked in the z-stack mode to give a final one-/ two-photon z-axis stack imaging. 2.10. Two-photon induced singlet oxygen generation 3D MCSs were treated with 10 mM Ru(II) complexes and H2TPP for 8 h in the dark. The culture medium was removed and the cells were allowed to incubate for an additional 30 min with 2 mL PBS containing 10 mM DHFA-DA. The culture medium was refreshed with fresh PBS after this period and subjected to two-photon irradiation (100 mW, 80 MHz, 100 fs) at 810(RuL2)/820(RuL1,RuL3)/ 830(RuL4) nm for 3 min/section (section interval: 3 mm) using a laser source equipped in an LSM 710 Carl Zeiss Laser Scanning Confocal Microscope. Fluorescent images were captured using an excitation wavelength at 488 nm, and the emission was recorded between 510 and 550 nm. 2.11. 3D MCSs growth inhibition and viability assay 3D MCSs were treated with DMEM, 10 mM of the Ru(II) complexes, 10 mM of H2TPP, 10 mM and 30 mM cisplatin, respectively, and incubated in the dark for 3 days. Then, the HeLa MCSs were subjected to two-photon irradiation (100 mW, 80 MHz, 100 fs) at 800(H2TPP,Control, cisplatin)/810(RuL2)/820(RuL1,RuL3)/ 830(RuL4) nm for 3 min/section (section interval: 3 mm) using the same equipment noted above, and the samples were incubated in the dark for an additional 3 days. The culture media were refreshed every two days without altering the drug concentration. All of the procedures were conducted strictly in the dark. Images and diameter data of 3D MCSs were collected every 24 h. MCSs volume changes were recorded over 6 days. During this procedure, the viability assay of 3D MCSs before irradiation (Day 3) and 48 h after irradiation (Day 5) was conducted according to the protocol of the Viability/Cytotoxicity Kit for mammalian cells (Life Technologies) to offer a visualized analysis of the survival/death rate in the MCSs [55]. Live cells were distinguished by the presence of ubiquitous intracellular esterase activity, as determined by the enzymatic conversion of the virtually non-fluorescent cell permeant calcein AM to the intensely fluorescent calcein (lex ¼ 495 nm, lem ¼ 515 nm). EthD-1 would enter cells with damaged membranes and undergo a 40-fold enhancement of fluorescence upon binding

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to nucleic acids, producing a bright red fluorescence in dead cells (lex ¼ 495 nm, lem ¼ 635 nm). As EthD-1 overlaps with the Ru(II) complex channel, only calcein AM was used in this experiment. The determination of cell viability depends on these physical and biochemical cell properties. After refreshing the culture medium, the 3D MCSs were incubated with the calcein AM (2 mM) solution for 30 min and imaged directly using an inverted fluorescence microscope.

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Table 1 Photophysical properties of RuL1eRuL4.a Complex

labs/nm (log ε)

lem/nm

Fem

t/ns

s2b/GM

RuL1 RuL2 RuL3 RuL4

467 461 464 464

617 606 608 605

0.031 0.036 0.036 0.037

253 255 264 225

124 155 170 198

a b

(4.41) (4.46) (4.40) (4.45)

All data were obtained in methanol at 298 K. Maximum two-photon absorption cross section, s2, at 810e830 nm.

2.12. Evaluation of (photo)toxicity towards 3D MCSs 3D MCSs were treated with increasing concentrations of the tested compounds (RuL1eRuL4, H2TPP and cisplatin). Controls were prepared by the addition of the culture medium. After incubating in the dark for 24 h, they were divided into two identical groups: one remained unchanged, one was subjected to irradiation. Both groups were incubated for another 48 h. The viability and IC50 values of the compounds in these two groups were measured via their ATP concentration with the CellTiter-Glo® 3D Cell Viability kit (Promega). Both OPA- and TPA-PDT treatments were conducted in this experiment. 3. Results and discussion 3.1. Design and synthesis We synthesized two Ru(II) polypyridyl complexes RuL1eRuL2 at the beginning and found that they showed considerably high 1O2 quantum yields and low cytotoxicity in the dark. In addition, these two complexes demonstrated significant two-photon absorption cross sections. We found that the latter complex manifested better properties for PDT. For this reason, we made some modifications to RuL2 with a view to improve therapy efficacy by targeting the mitochondria more efficiently. We presumed that subtle changes of the charge and lipophilic distribution in the molecular structure might make an obvious difference in mitochondrial affinity. In this respect, we tried to introduce the TPP scaffold into RuL2 in two distinct ways: i) with the TPP moiety conjugated directly to L2 to give L3, and ii) with TPP linked to L2 like a pendant with a flexible saturated carbon spacer to give L4. The results of these efforts afforded the complexes RuL3 and RuL4, respectively. The synthetic routes of the ligands are presented in Fig. S1. The ligands were synthesized based on the method for imidazole ring preparation established by Steck and Day [56]. L3 was synthesized by the reduction of 4-(1-phenyl-1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)benzaldehyde with NaBH4 at room temperature, followed by a bromination reaction with hydrobromic acid. Finally, TPP was introduced in refluxed DMF [10]. L4 was obtained via a substitution reaction of 4-(1-phenyl-1H-imidazo[4,5-f][1,10]phenanthrolin-2-yl)phenol with excess 1,4-dibromobutane, followed by the introduction of TPP. In each step of these reactions, the product was obtained at a reasonable yield, as the L2 scaffold is sufficiently stable to minimize undesired side reactions. The complexes were obtained by coordination of Ru(DIP)2Cl2 with the ligands in a refluxed EtOH/H2O solvent mixture at a molar ratio of 1:1. The complexes were then purified by aluminum oxide chromatography and crystallization. The TPP scaffold proved to elevate the solubility of complexes without hindering their cellular uptake ability. These complexes were characterized by elemental analysis, ES-MS, IR, 1H NMR and 31P NMR spectroscopy. 3.2. Photophysical properties The electronic absorption and emission spectra of complexes RuL1eRuL4 in methanol are shown in Fig. S6 and the

corresponding data are summarized in Table 1. All of these complexes showed intense and similar absorption features (with ε on the order of 104 M1 cm1) at approximately 460 nm (i.e., the MLCT absorption band). The emission spectra exhibited lem values at approximately 610 nm, with moderate quantum yields (F) of 0.031e0.037, and with [Ru(bpy)3]2þ as the reference. To determine their lifetimes, a luminescent decay experiment was performed at 298 K, which demonstrated that the lifetimes of the RuL1eRuL4 complexes were 225e264 ns. These lifetimes were obtained through fitting the data to a single exponential decay function. As with many other Ru(II) complexes, these complexes also showed a very large stokes shift (of over 140 nm). The two-photon absorption cross section s2 values were measured in methanol from 760 to 900 nm in 10 nm intervals by the TPEF method [50] with rhodamine B (4  104 M) as the reference [57]. The results illustrated in Fig. 1 indicated that the largest s2 values of RuL1eRuL4 appeared with excitation wavelengths at 810e830 nm. The s2 values ranged from 124 to 198 GM (1 GM ¼ 1  1050 cm s4 photon1 molecule1), which is much larger than that of H2TPP (s ¼ 2.2 GM) [58] and many recently reported two-photon bioactive organometallic molecular probes [29,59e61]. The substitution of the hydrogen atom by a phenyl group in the imidazole moiety results in an obvious enhancement in the TPA cross sections. TPP made contributions to the TPA cross section values as well. The two-photon induced emission spectra (Figs. S7e10) are similar to linear emission spectra. The quadratic dependence of the two-photon induced fluorescence intensity on the excitation power is depicted by the logarithmic figure in the inset. The slopes of the logarithmic plots are approximately 2, confirming that the RuL1eRuL4 complexes are two-photon active. 3.3. Singlet oxygen (1O2) quantum yields (FD) To investigate the efficiency of singlet oxygen generation from the complexes, the 1O2 quantum yields (FD) were determined using 1,3-diphenyl-isobenzofuran (DPBF) as a singlet oxygen scavenger [62]. The mixture of RuL1eRuL4 and DPBF was irradiated with 450 nm monochromatic light. The reduction of the fluorescent intensity of DPBF versus time is illustrated in Fig. 2. The FD values of RuL1eRuL4 in methanol were calculated to be 0.74e0.81 (Table 2) by the slopes derived from the graph associated with the slope of [Ru(bpy)3]2þ, whose quantum yield was 0.81 in methanol [53]. The singlet oxygen yields are significantly higher than most organic and organometallic photosensitizers, including many porphyrin derivatives [17,19,27,63e65], which indicates that RuL1eRuL4 can generate 1O2 more efficiently than these compounds. Simultaneously, a direct way to detect 1O2 generation is to trace 1O2 phosphorescence [1Dg / 3S g (0, 0)] at 1273 nm [66]. Significant phosphorous signals were detected in the presence of RuL1eRuL4 at approximately 1273 nm in CD3CN (Fig. S11). The emission intensities were strictly in accordance with the determined 1O2 quantum yields above. Moreover, the s2  FD values of RuL1eRuL4 (92e160) are much larger than that of H2TPP (1.5). A high s2  FD value expresses the merit of singlet oxygen generation by twophoton excitation [27].

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Fig. 1. Two-photon absorption cross sections of RuL1eRuL4 at different excitation wavelengths from 760 to 900 nm in methanol.

Fig. 2. 1O2 production via changes in the absorbance by DPBF at 411 nm versus irradiation time (lirr ¼ 450 nm) in the presence of adjusted concentrations of RuL1eRuL4 in aerated methanol vs. [Ru(bpy)3]2þ as the standard. Inset: Emission spectra of the mixture of RuL4 and DPBF upon irradiation. The arrow indicates the direction of the changes.

Table 2 The efficiency of singlet oxygen generation of compounds RuL1eRuL4 by onephoton (450 nm) and two-photon (810e830 nm) excitation. a

Complex

One-photon FD

s2/GM

s2  FD intensity

RuL1 RuL2 RuL3 RuL4 H2TPP

0.74 0.77 0.77 0.81 0.70b

124 155 170 198 2.2b

92 119 131 160 1.5b

a b

Quantum yield of singlet oxygen generation FD. Value taken from Ref. [27].

3.4. Cellular uptake and imaging To determine the location of the complexes within the cells, colocalization studies of RuL1eRuL4 with MitoTracker Green (MTG), which is a commercially available mitochondria imaging agent, were conducted in HeLa cells. As shown in Fig. 3a, an excellent superimposition pattern between the MTG and RuL4 could be observed. The observed red phosphorescence from RuL4 is localized in the mitochondria of living HeLa cells with a very high correlation coefficient (R ¼ 0.88). However, these two channels are not completely overlapped for the other three complexes (Fig. S12),

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Fig. 3. a) One- and two-photon luminescent imaging of HeLa cells incubated with 10 mM of RuL4 (lex ¼ 458 nm/830 nm, lem ¼ 610 ± 30 nm) in DMSO and PBS (pH ¼ 7.4, 2:98, v/v) for 2 h at 37  C, followed by 50 nM of Mito-tracker Green (MTG) (lex ¼ 488 nm, lem ¼ 510e530 nm). The inset scale bar represents 20 mm. The co-localization coefficient of RuL4 and MTG is 0.88. b) ICP-MS quantification of the internalized Ru by the HeLa cells. HeLa cells were treated with RuL1eRuL4 (10 mM) at 37  C for 2 h in the dark. Nuclei (Nuc.), mitochondria (Mito.) and cytoplasm (Cyto.) were extracted using mitochondrial and nuclear isolation kits. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

implying that complexes RuL1eRuL3 only partly accumulated in the mitochondria. The uptake of photosensitizers by cancer cells is a crucial factor in determining cellular imaging and PDT treatment efficacy. In order to quantify the cellular uptake and location degree of Ru(II) complexes within HeLa cells more precisely, the cellular ruthenium content and distribution (Fig. 3b, Table S1) were studied by ICP-MS. The analysis revealed the Ru contents in HeLa as high as 31e39 ng per million cells after 2 h incubation. All complexes could pass through the membrane of HeLa cell and the majority accumulated in the cytoplasm and only a small proportion entered the nuclei within 2 h. These results agree with the previous work reported by Puckett et al. [45]. The mitochondrial Ru uptake was further ascertained. The result indicated that Ru(II) complexes accumulated in mitochondria to different degrees among which RuL4 exerts the most salient affinity for mitochondria. In sharp contrast,

there is a wide gap in mitochondrial uptake between RuL4 and RuL3. Of particular interest is the observation that the mitochondrial uptakes for RuL1-RuL2 are also higher than RuL3. 3.5. Two-photon induced 1O2 generation and (photo)cytotoxicity in monolayer HeLa cells The mechanism for two-photon induced singlet oxygen generation has been shown to be identical to the one-photon case [67], and its 1O2 quantum yield is exactly half of the one-photon induced mechanism [22]. To demonstrate the ability of RuL1eRuL4 to produce singlet oxygen in vitro with two-photon laser irradiation, we incubated HeLa cells with RuL1eRuL4 (10 mM) and the 2,7-dichlorodihydro-fluorescein diacetate (DCFH-DA). Once in the cells, DCFH-DA is hydrolyzed by esterase enzymes to DCFH, which is vulnerable to singlet oxygen and can be oxidized to the

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fluorescent compound 2,7-dichlorofluorescein (DCF). Thus, the fluorescent intensity can reflect the intracellular singlet oxygen level [54,68]. We recorded the confocal fluorescence images of the cells before and after two-photon irradiation at 810e830 nm for 3 min (light dose: 800 J cm1) (Fig. S13). Cells treated with only DCFH-DA as the control showed no obvious fluorescence enhancement following irradiation. By contrast, a significant fluorescence increase was observed following irradiation in the cells treated with both the complexes and DCFH-DA, suggesting that RuL1eRuL4 are efficient two-photon absorbing photosensitizers in the cells. The one-photon (photo)cytotoxicity of RuL1eRuL4 in monolayer HeLa cells was also investigated. HeLa cells incubated with RuL1eRuL4 in the presence or absence of light were treated with the CellTiter-Glo® Luminescent Cell Viability kit (Promega). The CellTiter-Glo® Luminescent Cell Viability Assay is a homogeneous method to determine the number of viable cells in culture and is based on quantifying the ATP present, which indicates the presence of metabolically active cells. The dark and light cytotoxicity profile for HeLa cells are summarized in Table 3, revealing that RuL1eRuL4 showed essentially nontoxicity towards HeLa cells in the dark (IC50 values were all above 100 mM). Nevertheless, their cytotoxicity can be very high when exposed to visible light (lirr ¼ 450 nm, light dose: 12 J cm2). The IC50 values of RuL1eRuL4 in the presence of light were calculated and cisplatin was adopted as the positive control. Cisplatin showed a photocytotoxicity index (PI) value of approximately 1 because of its small photosensitization activity [20]. The complexes RuL1eRuL3 demonstrated similar photodynamic activities, with IC50 values ranging from 12.4 to 15.5 mM. Notably, RuL4 exhibited the lowest IC50 value (at 3.5 mM) and a 28fold enhancement (at least) in cytotoxicity when exposed to a very small dose of visible light (12 J cm2), which is a result of its strongest photosensitization activity and high mitochondrial uptake. Compared with organic and many organometallic photosensitizers, RuL4 exhibited a better therapeutic outcome with less light dose [19,21,65,69,70]. Moreover, unlike many Ir(III) PDT agents [18,19], the irradiation light lies in the NIR area instead of the cytotoxic UV area, which avoids additional damage to normal cells. As depicted in Fig. S14, both of the control group cells in the presence or absence of irradiation exhibited approximate viability. In addition, visible light dose-dependent cytotoxicity of RuL4 in HeLa cells was examined (Fig. 3). Almost 60% of the HeLa cells were killed at a light dose of 2 J cm2 when the cells were incubated with 10 mM of RuL4 for 2 h. Additionally, nearly 50% of the cells were killed with a light dose of ca. 1.7 J cm2 at the same concentration. This remarkable photocytotoxicity can be explained by its high singlet quantum yield and mitochondrial-selective accumulation.

3.6. Penetration ability and two-photon induced 1O2 generation in 3D MCSs It has been noted [71,72] that many anticancer agents failed to transit from successful results in monolayer cells to in vivo tests, partly because of the limitations of extracellular barriers, such as the extracellular matrix (ECM), present in vivo that may hinder drug delivery. 3D MCS is the most widely exploited tissue model for the assessment of drug delivery because it is much closer to clinical expression profiles than those seen in monolayer cells and because it is relatively easy to handle [73]. Moreover, limited by the bottleneck of the femtosecond laser scanning area, two-photon PDT in the present work can only be evaluated for small numbers of monolayer cells [16,21,24]. For this reason, 3D MCSs are the optimal choice to evaluate two-photon PDT. Small MCSs with diameters over 200 mm are sufficient to mimic in vivo-like intercellular interactions. However, MCSs with larger diameters were suggested to be a better choice because they can better simulate the pathophysiological conditions of solid tumors, such as the specific hypoxic areas in the tumor's center and proliferation gradients [14]. Hence, we subsequently utilized 800 mm 3D MCSs as the in vivo model to evaluate the PDT therapeutic outcome of our Ru(II) complexes Fig. 4. To probe the penetration ability of the complexes, 800 mm HeLa MCSs were incubated with 10 mM of the RuL1eRuL4 complexes for 8 h, followed by one-photon (OPM) and two-photon (TPM) Z-stack imaging microscopy (Fig. 5 and Fig. S15e18). H2TPP, which is a wellknown photosensitizer in PDT, was used for comparison. The results showed that MCSs incubated with the complexes for 8 h exhibited considerable luminescent intensity at every section of depth, indicating that they could overcome the ECM barrier and accumulate in MCSs within this time period. However, the MCSs showed little luminescence with H2TPP in either the core or shell, partly because of its poor solubility and slow cellular uptake. The strong tissue absorption of visible light accounts for the weaker luminescence in the core than the shell at a depth of over 100 mm in OPM. Moreover, TPM Z-stack scanning showed a larger penetration depth than OPM, owing to the location of the two-photon excitation wavelength in the therapeutic window for the tissue at 700e900 nm. We further examined the two-photon induced singlet oxygen generation in these MCSs with DCFH-DA. As shown in Fig. 6, the control MCSs were incubated without any photosensitizers, and no fluorescence was detected after irradiation. The very low levels of ROS in the HeLa cells can explain this result. Similarly, MCSs treated with H2TPP did not show any obvious fluorescence change, which is likely because of the low cellular concentration of H2TPP and its

Table 3 (Photo)cytotoxicity (IC50 [mM]) towards HeLa monolayer cells and MCSs.a Compound

Cisplatin H2TPP RuL1 RuL2 RuL3 RuL4

Monolayer cells

3D HeLa MCSs

Dark

Lightb

PIc

Dark

Lightb

PIc

Lightd

9.0 ± 0.8 nd >100 >100 >100 >100

8.8 ± 0.8 nd 12.4 ± 1.4 13.1 ± 2.2 15.5 ± 2.3 3.5 ± 1.1

1.02 nd >8.0 >7.6 >6.4 >28

17.4 ± 1.1 >100 >100 >100 >100 >100

17.0 ± 1.9 >100 22.7 ± 1.8 25.4 ± 2.2 27.5 ± 1.3 9.6 ± 1.1

1.02 nd >4.4 >3.9 >3.6 >10

17.2 97.5 6.5 7.6 9.1 1.9

± ± ± ± ± ±

PI 3.0 2.2 0.5 1.1 1.0 0.3

1.01 >1.0 >15 >12 >11 >52

a Monolayer cells/MCSs (diameter ~800 mm) were incubated with the indicated compounds for 72 h in total; that is, irradiations were given after 24 h incubation, and the photocytotoxicity were measured 48 h after PDT treatment. b Irradiated at 450 nm by an LED area light (20 mW cm2) for 10 min. c PI (photocytotoxicity index) is the ratio of dark-to-light toxicity and reflects the effective PDT range of the PSs. d Irradiated at 800 nm (H2TPP, Cisplatin)/810 nm (RuL2)/820 nm (RuL1, RuL3)/830 nm (RuL4) using a confocal microscope equipped with a mode-locked Ti:sapphire laser source (100 mW, 80 MHz, 100 fs) for 3 min per section of 3D HeLa MCSs (section interval: 3 mm, scanned area for every section was 1.5  1.5 mm). Data shown are values from three replicate trials.

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3.7. 3D MCSs growth inhibition and viability assay

Fig. 4. Visible light (lirr ¼ 450 nm) dose-dependent cytotoxicity curve of RuL4 (10 mM) on monolayer HeLa cells after 2 h incubation in the dark. The MTT assay was performed 48 h after PDT treatment. The results were expressed as the mean ± S.D. of three replicate trials.

small TPA cross section. In contrast, a significant fluorescence enhancement was observed by treating the MCSs with RuL1eRuL4. Notably, unlike monolayer cells, the fluorescence in the MCSs showed uneven distributions. Fluorescence in the shell was apparently stronger than that of the MCS core, which is in agreement with the notion that the reduced oxygen concentration moves progressively from the shell to the core. Clearly, the MCSs incubated with RuL4 showed the strongest luminescence, which is consistent with its high TPA photosensitization activity and cellular uptake.

To study the ability of these complexes to inhibit growth, MCSs with diameters of approximately 800 mm were exposed to DMEM, 10 mM H2TPP, 10 or 30 mM cisplatin and 10 mM RuL1eRuL4 and then incubated for three days. The samples were then subjected to two-photon irradiation and were incubated for an additional three days. The incubation environment was entirely dark and the DMEM were changed every two days without altering the drug concentration. H2TPP was used for comparison and cisplatin was used as the positive control. Fig. S19 depicts representative optical images of MCSs with various treatments, and the trend in the volume change of MCSs was summarized in Fig. 7a. It is noted that MCSs treated with culture medium did not show any growth inhibition. The volume in the control group increased over time during this experiment. In addition, the MCSs grew compact and the layering became more apparent as a result of the interaction between the cells and the ECM. Similarly, the treatment of RuL1eRuL4 and H2TPP in the samples exposed to dark showed no obvious MCS growth inhibition in the first three days, whereas cisplatin gave a significant inhibition in the first 24 h at a concentration of 30 mM. We note that a weak inhibition at the relatively low concentration of 10 mM was also observed. Because tumor cells were generally less sensitive to chemo therapeutics in vivo, as predicted, low concentrations of cisplatin failed to achieve an effective therapeutic outcome where the MCSs stopped growing. After laser irradiation, RuL1eRuL4 demonstrated a very effective inhibition of MCSs growth. The volume of the MCSs gradually decreased after irradiation, with complexes RuL4 demonstrating the strongest inhibition. We note that MCSs treated with H2TPP showed only a weak growth delay. In contrast, the other test groups showed no obvious changes following irradiation because of their non-photosensitization activities.

Fig. 5. a) One- and two-photon excited Z-stack images of 3D HeLa MCSs after incubation with RuL4 for 8 h (concentration of 10 mM); from left to right: Brightfield, Ru complex luminescence, Overlay. b) Substrate of OPM and TPM Z-axis scanning images captured every 5.1 mm from the top to bottom of an intact 800 mm spheroid. c) The one- and twophoton 3D Z-stack images of an intact spheroid. The excitation wavelengths of OPM and TPM were 458 and 830 nm, respectively.

Fig. 6. DCFH-DA staining on pre-treated MCSs. The MCSs were incubated with 10 mM RuL1eRuL4 and H2TPP, respectively, for 8 h in the dark and subjected to 800 nm (H2TPP, Control)/810 nm (RuL2)/820 nm (RuL1, RuL3)/830 nm (RuL4) two-photon laser irradiations (100 mW, 80 MHz, 100 fs) for 3 min per section in advance. The inset scale bar represents 200 mm.

Fig. 7. (a) Volume change curves of 3D MCSs after treatment with DMEM (control), RuL1e4 (10 mM), H2TPP (10 mM) and platinum (Pt-10: 10 mM, Pt-30: 30 mM), respectively, over 6 days. The MCSs were subjected to two-photon laser irradiation (100 mW, 80 MHz, 100 fs) at 800 nm (H2TPP, Control, Pt-10, Pt-30)/810 nm (RuL2)/820 nm (RuL1, RuL3)/830 nm (RuL4) for 3 min/section on Day 3. The error bars denote standard deviation of three replicate trials. (b) Representative images of Calcein AM staining on various pre-treated HeLa MCSs. aec: before laser irradiation (i.e., on Day 3); def: 48 h after TPA-PDT treatment (i.e., on Day 5); a, d: Bright field; b, e: Calcein AM channel; c, f: Overlay. Inset scale bars: 200 mm.

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To further confirm the TPA-PDT therapeutic efficacy, the MCSs with the various treatments above were stained by calcein AM (Viability/Cytotoxicity Kit for mammalian cells, Life Technologies) before (i.e., on Day 3) and 48 h (i.e., on Day 5) after receiving TPAPDT treatment. The virtually non-fluorescent membrane permeant calcein AM can initiate fluorescence when it interacts with intracellular esterases within living cells. Therefore, the survival of HeLa cells in MCSs can be reflected by fluorescence. Fig. 7b gives a visual assessment of the cellular viability in MCSs. Neither of the MCSs treated with DMEM and H2TPP showed any obvious fluorescence changes after irradiation, suggesting that the majority of cells survived. Cisplatin exhibited a chronic cytotoxity towards MCSs. In particular, better therapeutic effects were achieved with

151

higher concentrations of cisplatin. It is noteworthy that even at a concentration of 30 mM and an exposure time of 5 days, HeLa cells in MCSs were not totally eliminated by cisplatin. As for RuL1eRuL4, the majority of the cells in MCSs survived after being exposed to the complexes for three days, indicating that the complexes were essentially nontoxic to cells in the absence of light. On the contrary, no fluorescence can be observed in the MCSs after irradiation, implying that the HeLa cells in 3D MCSs were mostly eradicated. Obviously, RuL1eRuL4 achieved much better therapeutic efficacy than H2TPP and cisplatin, primarily because of their superior penetration ability and sufficient TPA photosensitization activity in mitochondria, which triggers cell death more efficiently.

Fig. 8. One-photon and two-photon PDT doseeresponse curves for RuL4 and H2TPP in 3D MCSs. The conditions for the dark (black), OPA-PDT (blue) and TPA-PDT (red) experiments were identical except that the PDT-treated samples were irradiated with a laser beam (100 mW, 80 MHz, 100 fs) at 800 nm (H2TPP)/830 nm (RuL4) for 3 min per section of MCSs in TPA-PDT and an LED area light source (20 mW cm2) at 450 nm for 10 min in OPA-PDT. The error bars denote standard deviation of three replicate trials. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3.8. Photodynamic activity in 3D MCSs

Acknowledgments

To quantify the photodynamic activity of the complexes RuL1eRuL4 towards 3D HeLa MCSs, we used the fast-response and highly sensitive CellTiter-Glo® 3D Cell Viability kit (Promega) to measure the IC50 value of these compounds in MCSs with cisplatin as the positive control and H2TPP as a comparison. This kit can efficiently lyse MCSs and fully release intracellular ATP and convert the amount of ATP in living cells into a chemiluminescence intensity. Fig. 8 and Figs. S20e22 depict the OPA- and TPA-PDT dosedependent MCS survival rates, and MCSs treated with RuL1eRuL4 and H2TPP, respectively, using the same conditions as were used for the monolayer cells. The resulting IC50 values are summarized in Table 3. Relative to OPA-PDT in the two biomodels, we observe an obvious gap of IC50 values between the monolayer cells and 3D MCSs. It appears that all compounds showed less cytotoxicity to MCSs than the monolayer cells. The IC50 values in the MCSs were approximately double those recorded for the monolayer cells. Despite an ECM that hinders anticancer drug penetration, MCSs possess a hypoxic microenvironment that cultivates a more reducing environment and, more importantly, tissue depth that can fade visible light, leading to a decline of the formation of singlet oxygen; both of these factors are ignored in the monolayer model. These criteria impact the final PDT therapeutic outcome in 3D MCSs, resulting in a rise of the IC50 values. Although H2TPP exhibits a rather high singlet oxygen yield (0.70) [26], it showed very low photocytotoxicity in MCSs because of its weak absorption at 450 nm. In contrast with one-photon PDT, two-photon PDT manifested more impressive inhibition towards 3D MCSs because, as noted previously, the two-photon excitation of the Ru(II) complexes can obtain deeper penetration depth and thus prevent laser decline with depth. The IC50 values in these cases were only one-third the values in one-photon PDT. Strikingly, RuL4 exhibited an IC50 value of 1.9 mM in two-photon PDT in MCSs with a photocytotoxicity index value over 52. As expected, H2TPP showed only a small difference between the presence or absence of irradiation as a result of poor penetration in MCSs and less TPA photosensitization activity.

This work was supported by the 973 Program (Nos. 2014CB845604 and 2015CB856301), the National Science Foundation of China (Nos. 21172273, 21171177, and 21471164), the Program for Changjiang Scholars and Innovative Research Team at the University of China (No. IRT1298), and the Research Fund for the Doctoral Program of Higher Education (20110171110013).

4. Conclusion In conclusion, four ruthenium(II) polypyridyl complexes, Ru1eRu4, have been synthesized and utilized as mitochondriatargeted two-photon photodynamic anticancer agents. The cytotoxicity of all four complexes were examined with 3D HeLa MCSs. To the best of our knowledge, most PDT agents have been screened using monolayer cells and received similar therapeutic outcomes, which is likely to give different results in vivo. To date, examples of MCSs as metallic photosensitizer screening models remain very rare. In this work, 3D HeLa MCSs were utilized as the screening model and gave a better therapeutic outcome than one-photon PDT when combined with two-photon PDT. Possessing remarkably high singlet oxygen quantum yields (0.74e0.81), significant TPA cross sections (124e198 GM), substantial cellular uptake, rapid MCSs penetration and remarkable mitochondria-targeting, the complexes Ru1eRu4 are well-suited to be used as efficient two-photon PDT agents. In particular, RuL4 demonstrated the most promising PDT potential and may be considered to be an efficient two-photon PDT candidate. Conflict of interest The authors of this paper reported no financial conflict of interest.

Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2015.04.002.

References [1] Dolmans DEJGJ, Fukumura D, Jain RK. Photodynamic therapy for cancer. Nat Rev Cancer 2003;3:380e7. [2] Bonnett R. Photosensitizers of the porphyrin and phthalocyanine series for photodynamic therapy. Chem Soc Rev 1995;24:19e33. [3] Henze K, Martin W. Evolutionary biology: essence of mitochondria. Nature 2003;426:127e8. [4] McBride HM, Neuspiel M, Wasiak S. Mitochondria: more than just a powerhouse. Curr Biol 2006;16:R551e60. [5] Green DR. Apoptotic pathways: the roads to ruin. Cell 1998;94:695e8. [6] Pervaiz S, Olivo M. Art and science of photodynamic therapy. Clin Exp Pharm Physiol 2006;33:551e6. [7] Fulda S, Galluzzi L, Kroemer G. Targeting mitochondria for cancer therapy. Nat Rev Drug Discov 2010;9:447e64. [8] Robin AJ, Smith RCH, Murphy MP. Mitochondria-targeted small molecule therapeutics and probes. Antioxid Redox Signal 2011;15:18. [9] Hoye AT, Davoren JE, Wipf P, Fink MP, Kagan VE. Targeting mitochondria. Acc Chem Res 2008;41:87e97. [10] Dickinson BC, Chang CJ. A targetable fluorescent probe for imaging hydrogen peroxide in the mitochondria of living cells. J Am Chem Soc 2008;130: 9638e9. [11] Leung CWT, Hong Y, Chen S, Zhao E, Lam JWY, Tang BZ. A photostable AIE luminogen for specific mitochondrial imaging and tracking. J Am Chem Soc 2012;135:62e5. [12] Armstrong JS. Mitochondrial medicine: pharmacological targeting of mitochondria in disease. Br J Pharmacol 2007;151:10. [13] Hamilton G. Multicellular spheroids as an in vitro tumor model. Cancer Lett 1998;131:29e34. [14] Friedrich J, Seidel C, Ebner R, Kunz-Schughart LA. Spheroid-based drug screen: considerations and practical approach. Nat Protoc 2009;4:309e24. [15] Wang X, Zhen X, Wang J, Zhang J, Wu W, Jiang X. Doxorubicin delivery to 3D multicellular spheroids and tumors based on boronic acid-rich chitosan nanoparticles. Biomaterials 2013;34:4667e79. [16] Gary-Bobo M, Mir Y, Rouxel C, Brevet D, Basile I, Maynadier M, et al. Mannosefunctionalized mesoporous silica nanoparticles for efficient two-photon photodynamic therapy of solid tumors. Angew Chem Int Ed 2011;123: 11627e31. [17] Zhao J, Wu W, Sun J, Guo S. Triplet photosensitizers: from molecular design to applications. Chem Soc Rev 2013;42:5323e51. [18] Li Y, Tan C-P, Zhang W, He L, Ji L-N, Mao Z-W. Phosphorescent iridium(III)-bisN-heterocyclic carbene complexes as mitochondria-targeted theranostic and photodynamic anticancer agents. Biomaterials 2015;39:95e104. [19] Ye RR, Tan CP, He L, Chen MH, Ji LN, Mao ZW. Cyclometalated Ir(III) complexes as targeted theranostic anticancer therapeutics: combining HDAC inhibition with photodynamic therapy. Chem Commun 2014;50:10945e8. [20] Lincoln R, Kohler L, Monro S, Yin H, Stephenson M, Zong R, et al. Exploitation of long-lived 3IL excited states for metaleorganic photodynamic therapy: verification in a metastatic melanoma model. J Am Chem Soc 2013;135: 17161e75. [21] Zou Q, Fang Y, Zhao Y, Zhao H, Wang Y, Gu Y, et al. Synthesis and in vitro photocytotoxicity of coumarin derivatives for one- and two-photon excited photodynamic therapy. J Med Chem 2013;56:5288e94. [22] Andrasik SJ, Belfield KD, Bondar MV, Hernandez FE, Morales AR, Przhonska OV, et al. One-and two-photon singlet oxygen generation with new fluorene-based photosensitizers. ChemPhysChem 2007;8:399e404.
nez-Banzo A, Paterson MJ, Nonell S, Borrell JI, Christiansen O, [23] Arnbjerg J, Jime et al. Two-photon absorption in tetraphenylporphycenes: are porphycenes better candidates than porphyrins for providing optimal optical properties for two-photon photodynamic therapy? J Am Chem Soc 2007;129:5188e99. [24] Ke H, Wang H, Wong W-K, Mak N-K, Kwong DWJ, Wong K-L, et al. Responsive and mitochondria-specific ruthenium(II) complex for dual in vitro applications: two-photon (near-infrared) induced imaging and regioselective cell killing. Chem Commun 2010;46:6678e80.

J. Liu et al. / Biomaterials 56 (2015) 140e153 [25] Collins HA, Khurana M, Moriyama EH, Mariampillai A, Dahlstedt E, Balaz M, et al. Blood-vessel closure using photosensitizers engineered for two-photon excitation. Nat Photon 2008;2:420e4. [26] Allison RR, Downie GH, Cuenca R, Hu X-H, Childs CJH, Sibata CH. Photosensitizers in clinical PDT. Photodiagnosis Photodyn Ther 2004;1:27e42. [27] Ishi-i T, Taguri Y, S-i Kato, Shigeiwa M, Gorohmaru H, Maeda S, et al. Singlet oxygen generation by two-photon excitation of porphyrin derivatives having two-photon-absorbing benzothiadiazole chromophores. J Mater Chem 2007;17:3341e6.
V, Granet R, Guilloton M, Spiro M, et al. Synthesis, [28] Sol V, Blais JC, Carre spectroscopy, and photocytotoxicity of glycosylated amino acid porphyrin derivatives as promising molecules for cancer phototherapy. J Org Chem 1999;64:4431e44. [29] Koo CK, So LKY, Wong KL, Ho YM, Lam YW, Lam MHW, et al. A triphenylphosphonium-functionalised cyclometalated platinum(II) complex as a nucleolus-specific two-photon molecular dye. Chem Eur J 2010;16: 3942e50. [30] Zhou W, Wang X, Hu M, Zhu C, Guo Z. A mitochondrion-targeting copper complex exhibits potent cytotoxicity against cisplatin-resistant tumor cells through a multiple mechanism of action. Chem Sci 2014;5:2761e70. [31] Bergamo A, Sava G. Ruthenium complexes can target determinants of tumour malignancy. Dalton Trans 2007:1267e72. ~ a B, Dunbar KR, Turro C. New cyclometallated Ru (II) complex [32] Albani BA, Pen for potential application in photochemotherapy? Photochem Photobiol Sci 2014;13:272e80. ~ a B, Leed NA, de Paula NABG, Pavani C, Baptista MS, et al. [33] Albani BA, Pen Marked improvement in photoinduced cell death by a new tris-heteroleptic complex with dual action: singlet oxygen sensitization and ligand dissociation. J Am Chem Soc 2014;136:17095e101. [34] Sgambellone MA, David A, Garner RN, Dunbar KR, Turro C. Cellular toxicity induced by the photorelease of a caged bioactive molecule: design of a potential dual-action Ru (II) Complex. J Am Chem Soc 2013;135:11274e82. [35] Higgins SLH, Brewer KJ. Designing red-light-activated multifunctional agents for the photodynamic therapy. Angew Chem Int Ed 2012;51:11420e2. [36] Pierroz V, Joshi T, Leonidova A, Mari C, Schur J, Ott I, et al. Molecular and cellular characterization of the biological effects of Ruthenium(II) complexes incorporating 2-pyridyl-2-pyrimidine-4-carboxylic acid. J Am Chem Soc 2012;134:20376e87. [37] Mari C, Pierroz V, Ferrari S, Gasser G. Combination of Ru(II) complexes and light: new frontiers in cancer therapy. Chem Sci 2015. http://dx.doi.org/ 10.1039/C4SC03759F. [38] Joshi T, Pierroz V, Mari C, Gemperle L, Ferrari S, Gasser G. A bis(dipyridophenazine)(2-(2-pyridyl)pyrimidine-4-carboxylic acid)ruthenium(II) complex with anticancer action upon photodeprotection. Angew Chem 2014;126:3004e7. [39] Stephenson M, Reichardt C, Pinto M, W€ achtler M, Sainuddin T, Shi G, et al. Ru(II) dyads derived from 2-(1-pyrenyl)-1H-imidazo[4,5-f][1,10]phenanthroline: versatile photosensitizers for photodynamic applications. J Phys Chem A 2014;118:10507e21. [40] Boca SC, Four M, Bonne A, van der Sanden B, Astilean S, Baldeck PL, et al. An ethylene-glycol decorated ruthenium(ii) complex for two-photon photodynamic therapy. Chem Commun 2009:4590e2. [41] Truillet C, Lux F, Moreau J, Four M, Sancey L, Chevreux S, et al. Bifunctional polypyridyl-Ru(ii) complex grafted onto gadolinium-based nanoparticles for MR-imaging and photodynamic therapy. Dalton Trans 2013;42:12410e20. [42] Yu H, Huang S, Kou J, Li L, Jia H, Chao H, et al. Synthesis, DNA-binding and photocleavage studies of ruthenium(II) complexes [Ru(btz)3]2þ and [Ru(btz)(dppz)2]2þ. Sci China Ser B Chem 2009;52:1504e11. [43] Xu W, Zuo J, Wang L, Ji L, Chao H. Dinuclear ruthenium(II) polypyridyl complexes as single and two-photon luminescence cellular imaging probes. Chem Commun 2014;50:2123e5. [44] Sun D, Liu Y, Yu Q, Zhou Y, Zhang R, Chen X, et al. The effects of luminescent ruthenium(II) polypyridyl functionalized selenium nanoparticles on bFGFinduced angiogenesis and AKT/ERK signaling. Biomaterials 2013;34:171e80. [45] Puckett CA, Barton JK. Methods to explore cellular uptake of ruthenium complexes. J Am Chem Soc 2006;129:46e7. [46] Cardinaels T, Ramaekers J, Nockemann P, Driesen K, Van Hecke K, Van Meervelt L, et al. Imidazo[4,5-f]-1,10-phenanthrolines: Versatile ligands for the design of metallomesogens. Chem Mater 2008;20:1278e91. [47] Jayabharathi J, ThanClam V, Venkatesh Perumal M, Srinivasan N. Synthesis, crystal structure, kamlet-taft and catalan solvatochromic analysis of novel imidazole derivatives. J Fluoresc 2012;22:409e17. [48] Caspar JV, Meyer TJ. Photochemistry of tris(2,20 -bipyridine) ruthenium(II) ion (Ru(bpy)2þ 3 ). Solvent effects. J Am Chem Soc 1983;105:5583e90.

153

[49] Crosby GA, Demas JN. Measurement of photoluminescence quantum yields. Review. J Phys Chem 1971;75:991e1024. [50] Xu C, Webb WW. Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 to 1050 nm. JOSA B 1996;13: 481e91. [51] Ding HY, Wang XS, Song LQ, Chen JR, Yu JH, Chao L, et al. Aryl-modified ruthenium bis(terpyridine) complexes: quantum yield of 1O2 generation and photocleavage on DNA. J Photochem Photobiol A Chem 2006;177:286e94. [52] Li GY, Du KJ, Wang JQ, Liang JW, Kou JF, Hou XJ, et al. Synthesis, crystal structure, DNA interaction and anticancer activity of tridentate copper(II) complexes. J Inorg Biochem 2013;119:43e53. [53] Abdel-Shafi AA, Beer PD, Mortimer RJ, Wilkinson F. Photosensitized generation of singlet oxygen from vinyl linked benzo-crown-ether-bipyridyl ruthenium(II) complexes. J Phys Chem A 1999;104:192e202. [54] Fowley C, Nomikou N, McHale AP, McCarron PA, McCaughan B, Callan JF. Water soluble quantum dots as hydrophilic carriers and two-photon excited energy donors in photodynamic therapy. J Mater Chem 2012;22:6456e62. [55] Huang H, Zhang P, Yu B, Chen Y, Wang J, Ji L, et al. Targeting nucleus DNA with a cyclometalated dipyridophenazineruthenium(II) complex. J Med Chem 2014;57:8971e83. [56] Steck EA, Day AR. Reactions of phenanthraquinone and retenequinone with aldehydes and ammonium acetate in acetic acid solution. J Am Chem Soc 1943;65:452e6. [57] Makarov NS, Drobizhev M, Rebane A. Two-photon absorption standards in the 550-1600 nm excitation wavelength range. Opt Express 2008;16:4029e47. [58] Velusamy M, Shen JY, Lin JT, Lin YC, Hsieh CC, Lai CH, et al. A new series of quadrupolar type two-photon absorption chromophores bearing 11,12dibutoxydibenzo[a,c]-phenazine bridged amines; their applications in twophoton fluorescence imaging and two-photon photodynamic therapy. Adv Funct Mater 2009;19:2388e97. [59] Botchway SW, Charnley M, Haycock JW, Parker AW, Rochester DL, Weinstein JA, et al. Time-resolved and two-photon emission imaging microscopy of live cells with inert platinum complexes. Proc Natl Acad Sci U S A 2008;105:16071e6. [60] Koo CK, Wong KL, Man CWY, Lam YW, So LKY, Tam HL, et al. A bioaccumulative cyclometalated platinum(II) complex with two-photoninduced emission for live cell imaging. Inorg Chem 2009;48:872e8. [61] Lo KKW, Louie MW, Zhang KY. Design of luminescent iridium(III) and rhenium(I) polypyridine complexes as in vitro and in vivo ion, molecular and biological probes. Coord Chem Rev 2010;254:2603e22. [62] Young RH, Wehrly K, Martin RL. Solvent effects in dye-sensitized photooxidation reactions. J Am Chem Soc 1971;93:5774e9. [63] Celli JP, Spring BQ, Rizvi I, Evans CL, Samkoe KS, Verma S, et al. Imaging and photodynamic therapy: mechanisms, monitoring, and optimization. Chem Rev 2010;110:2795e838. [64] Cho S, You Y, Nam W. Lysosome-specific one-photon fluorescence staining and two-photon singlet oxygen generation by molecular dyad. RSC Adv 2014;4:16913e6. [65] Yang Y, Guo Q, Chen H, Zhou Z, Guo Z, Shen Z. Thienopyrrole-expanded BODIPY as a potential NIR photosensitizer for photodynamic therapy. Chem Commun 2013;49:3940e2. [66] Adam W, Kazakov DV, Kazakov VP. Singlet-oxygen chemiluminescence in peroxide reactions. Chem Rev 2005;105:3371e87. [67] Frederiksen PK, Jørgensen M, Ogilby PR. Two-photon photosensitized production of singlet oxygen. J Am Chem Soc 2001;123:1215e21. [68] Gomes A, Fernandes E, Lima JL. Fluorescence probes used for detection of reactive oxygen species. J Biochem Biophys Methods 2005;65:45e80. [69] Majumdar P, Yuan X, Li S, Le Guennic B, Ma J, Zhang C, et al. Cyclometalated Ir(III) complexes with styryl-BODIPY ligands showing near IR absorption/ emission: preparation, study of photophysical properties and application as photodynamic/luminescence imaging materials. J Mater Chem B 2014;2: 2838e54. [70] Li SPY, Lau CTS, Louie MW, Lam YW, Cheng SH, Lo KKW. Mitochondria-targeting cyclometalated iridium(III)ePEG complexes with tunable photodynamic activity. Biomaterials 2013;34:7519e32. [71] Pluen A, Boucher Y, Ramanujan S, McKee TD, Gohongi T, di Tomaso E, et al. Role of tumorehost interactions in interstitial diffusion of macromolecules: cranial vs. subcutaneous tumors. Proc Natl Acad Sci U S A 2001;98:4628e33. [72] Netti PA, Berk DA, Swartz MA, Grodzinsky AJ, Jain RK. Role of extracellular matrix assembly in interstitial transport in solid tumors. Cancer Res 2000;60: 8. [73] Goodman TT, Ng CP, Pun SH. 3-D tissue culture systems for the evaluation and optimization of nanoparticle-based drug carriers. Bioconjug Chem 2008;19: 1951e9.