DNA-Targeting Ru^II -Polypyridyl Complex with a Long-Lived Intraligand Excited State as a Potential Photodynamic Therapy Agent.

Accepted Article Accepted Article Title: DNA Targeting Ru(II)-Polypyridyl Complex with Long-Lived 3IL Excited State as potential photodynamic therapy agent Authors: Wuyang Hua, Gang Xu, Jian Zhao, Jiapeng Lu, Wenfang Sun, and Shaohua Gou This manuscript has been accepted after peer review and appears as an Accepted Article online prior to editing, proofing, and formal publication of the final Version of Record (VoR). This work is currently citable by using the Digital Object Identifier (DOI) given below. The VoR will be published online in Early View as soon as possible and may be different to this Accepted Article as a result of editing. Readers should obtain the VoR from the journal website shown below when it is published to ensure accuracy of information. The authors are responsible for the content of this Accepted Article. To be cited as: Chem. Eur. J. 10.1002/chem.202003031 Link to VoR: https://doi.org/10.1002/chem.202003031 01/2020 10.1002/chem.202003031 Chemistry - A European Journal FULL PAPER DNA Targeting Ru(II)-Polypyridyl Complex with Long-Lived 3IL Excited State as potential photodynamic therapy agent Abstract: Subtle ligand modifications on Ru(II)-polypyridyl complexes may result in different excited-state characteristics, which provides the opportunity to tune their photo-physicochemical properties and subsequently change their biological functions. Here, a DNA targeting Ru(II)-polypyridyl complex (named Ru1) with highly photosensitizing 3IL (intraligand) excited state was designed based on a classical DNA-intercalator [Ru(bpy)2(dppz)]·2PF6 by incorporation of dppz ligand tethered with pyrenyl group, which has 4 orders of magnitude higher potency than the model complex [Ru(bpy)2(dppz)]·2PF6 upon light irradiation. This study provides a facile strategy for the design of organelle-targeting Ru(II)-polypyridyl complex with dramatically improved photobiological activity. Introduction Photodynamic therapy (PDT) has been emerging as a promising cancer treatment modality, due to its excellent spatiotemporal selectivity, negligible side effects and noninvasiveness. [1] Upon light activation, photosensitizers (PSs) can exert anticancer effects by generating cytotoxic reactive oxygen species (ROS) via energy or electron transfer from the triplet excited state of the PSs to ground-state oxygen to kill cancer cells. [1c,1d,2] Therefore, PSs with long-lived triplet excited states are desired for PDT. Profiting from the rich photophysics and salient biological properties,[3] Ru(II)-polypyridyl complexes have been widely studied for their potential applications in organelle imaging, tumor diagnosis, chemotherapy and PDT.[4] The notable example is polypyridyl Ru(II)-dppz (dppz = dipyrido[3,2-a:2′,3′c]phenazine) complexes, which have been widely known as DNA light switches and structure-specific DNA probes due to their two 3MLCT (metal-to-ligand charge transfer) states (a luminescent 3MLCTprox state and a dark 3MLCTdis state) and strong DNA intercalation abilities.[5,6] Besides, a Ru(II)polypyridyl complex, TLD1433, has successfully finished phase Ib clinical trial in 2018 and is currently in Phase 2 human clinical trial (ClinicalTrials.gov Identifier: NCT03945162) as a photosensitizer for bladder cancer PDT treatment, highlighting [a] [b] [‡] W. Hua, Dr. G. Xu, Dr. J. Zhao, Dr. Z. Wang and Prof. S. Gou Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research and Pharmaceutical Research Center, School of Chemistry and Chemical Engineering Southeast University, Nanjing 211189, China E-mail: 101010898@seu.edu.cn (Prof. S. Gou) and zhaojianzhaokuan@163.com (Dr. J. Zhao) J. Lu and Prof. W. Sun Department of Chemistry and Biochemistry North Dakota State University, Fargo, North Dakota 58108-6050, USA E-mail: wenfang.sun@ndsu.edu (Prof. W. Sun) These authors contributed equally to this work. the great potential utility of Ru(II)-polypyridyl complexes in PDT.[6] Cancer cells are more affected by DNA damage relative to normal cells due to their much faster division rates. [7] Therefore, DNA can serve as an effective target in PDT.[2a,8] Notably, it is generally accepted that binding of a PS to DNA is a vital step for efficient DNA photocleavage.[9] Consequently, Ru(II)-based PSs with DNA intercalation abilities could be favorable for PDT. [8,9] However, [Ru(bpy)2(dppz)]2+, a typical DNA light-switch complex, exhibited negligible photocytotoxicity due to its low singlet oxygen (1O2) generation efficiency.[10] To improve the photocytotoxicity of [Ru(bpy)2(dppz)]2+, Gasser and co-workers designed a number of [Ru(bpy)2(dppz)]2+ derivatives as PSs by incorporation of functional moieties on the dppz ligand. [11] This approach not only retained the DNA-binding affinity of the complexes, but also improved their photocytotoxicity and promoted the potential application of these complexes as PDT agents. However, the anticancer mechanism is still based on the similar 3MLCT excited state as that in [Ru(bpy)2(dppz)]2+. The lowest-energy triplet excited state (T1) for [Ru(bpy)2(dppz)]2+ is 3MLCT state,[6] which is less sensitive to O2 due to its relatively short excited-state lifetime. One of the effective strategies to achieve the long-lived excited state of the Ru(II)-polypyridyl complex is to access the triplet intraligand (3IL) state, which is extremely sensitive to O2 or other excited-state quenchers.[3] Based on the reported work, we speculate that subtle modifications on the structures of Ru(II)-polypyridyl complexes may lead to different excited-state behaviors. This would provide an opportunity to tune the photo-physicochemical properties of the Ru(II)-polypyridyl complexes and improve their biological functions based on a rational design of the ligand. Furthermore, many Ru(II) complexes attached with pyrenyl groups were reported and showed greatly increased photocytotoxicity.[12] To examine that hypothesis, the pyrenyl group was selected as the functional moiety to lower the energy of the 3IL state of [Ru(bpy)2(dppz)]2+ through substitution on the dppz ligand (Scheme 1). We hypothesize that introduction of the pyrenyl group would enable the access of the low-lying, longlived pyrene-based 3IL state of the resultant complex, and thus significantly improve the ROS production and photocytoxicity of [Ru(bpy)2(dppz)]2+ without affecting its DNA-binding ability. Herein we report the synthesis, photophysics and photobiological activities of the new complex Ru1 that has strong DNA intercalating ability and high photocytotoxicity. The possible mode of actions for this complex was also investigated. Besides the N^N Ru(II) complexes, plenty of cyclometalated Ru(II) complexes have been studied[13] and explored as photocytotoxic agents.[13a,b] For comparison with Ru1, complex Ru2 (structure shown in Scheme 2) bearing the cyclometalating (C^N) analogue of dppz was synthesized and studied. Supporting information for this article is given via a link at the end of the document. This article is protected by copyright. All rights reserved. Accepted Manuscript Wuyang Hua‡,[a] Gang Xu‡,[a] Jian Zhao,*[a] Jiapeng Lu,[b] Wenfang Sun,*[b] and Shaohua Gou*[a] 10.1002/chem.202003031 Chemistry - A European Journal FULL PAPER nm and 450 - 600 nm, which can be assigned to the 1 MLCT/1LLCT (ligand-to-ligand charge transfer) transitions involving the N^N and C^N ligands, respectively.[15,16] Results and Discussion Synthesis and photophysical properties Ru1 was synthesized by following the procedures shown in Schemes 2. The intermediate 1 was obtained by Suzuki coupling reaction. Then [Ru(bpy)2(1,10-phenanthroline-5,6dione)]·2PF6 was refluxed with intermediate 1 in methanol to afford Ru1. The cyclometalated organometallic Ru(II) analogue (Ru2) was prepared for comparison purpose, which existed as two stereoisomers (Scheme 2) and the mixture of isomers were used in this study. The synthesized complexes were characterized by 1H and 13C NMR spectroscopy along with elemental analysis and ESI-MS spectrometry. Scheme 2. Synthetic procedure of Ru1 and Ru2. i). N2, Pd(PPh3)4, K2CO3, toluene, ethanol, H2O, reflux, yield 83 %; ii). N2, methanol, reflux, yield 90 %; iii. N2, methanol, reflux, yield 88 %. The UV-Vis absorption and emission spectra of Ru1 and Ru2 were studied in CH3CN and CH2Cl2, respectively. As shown in Figure 1a, Ru1 exhibited a strong absorption in the region of 400 - 500 nm, with an absorption maximum at 450 nm (26900 M-1 cm-1). The molar extinction coefficient of this band in Ru1 is much stronger than those in [Ru(bpy) 3]2+ (13000 M-1 cm-1)[10] and [Ru(bpy)2(dppz)]2+ (16300 M-1 cm-1)[10], indicating the enhanced light-harvesting ability of Ru1 due to the extended -conjugation via incorporation of pyrenyl group on dppz. Different from Ru1, two broad absorption bands were observed for Ru2 at 370 - 450 Figure 1. (A) UV-Vis absorption spectra of Ru1 and Ru2 in CH3CN and CH2Cl2, respectively. (B) Emission spectra of Ru1 (ex = 534 nm) and Ru2 (ex = 590 nm) in CH3CN and CH2Cl2, respectively. (C) Emission spectra of Ru1 in aerated and deaerated acetonitrile. (D) Time-resolved triplet transient absorption spectra of Ru1 in deaerated CH3CN, ex = 355 nm, A355 = 0.4 in a 1-cm cuvette. The emission characteristics were investigated in acetonitrile for Ru1 and in dichloromethane for Ru2 due to its poor solubility in acetonitrile. As shown in Figure 1b, Ru1 and Ru2 emitted at 632 nm and 759 nm, respectively. Compared with [Ru(bpy)2(dppz)]·2PF6 and other pyrenyl-containing Ru(II) complexes,[12c,e] the maximum emission wavelength of Ru1 showed a bathochromic shift, possibly attributing to the increased delocalization of the π-system of the ligand. However, Ru2 exhibited a blue shift of the emission peak as compared with its analogue [Ru(bpy)2(ppy)]+ (825 nm in acetonitrile, ppy = 2-phenylpyridine).[13c] The emission of Ru1 can be quenched by O2 (Figrue 1c), indicating that the luminescence is phosphorescence. However, no obvious change in emission intensity of Ru2 was observed between aerated and deaerated solutions (Figure S1a), probably owing to the short-lived emitting state. Based on the much red-shifted emission bands with respect to the corresponding excitation wavelengths, the sensitivity of the emission to air quenching, and referring to the similar Ru(II) complexes reported in the literature,[15] the observed emission should be phosphorescence from the triplet excited states for both Ru1 and Ru2. However, the emission lifetimes of the two complexes were too short to be measured on our instrument. Considering the microsecond lifetime for the lowest triplet excited state (T1) of Ru1 discussed below, the emitting excited state in Ru1 should not be its T1 state. Ru(II) dyads tethered with -expansive organic chromophores such as pyrene that possessed a long-lived nonemissive T1 state and a high-lying emitting state have been reported previously. [10,16] This article is protected by copyright. All rights reserved. Accepted Manuscript Scheme 1. Design Rationale for the DNA Targeting Ru(II)-polypyridyl PS (Ru1). 10.1002/chem.202003031 Chemistry - A European Journal FULL PAPER Complexes λabs/nm (ε/103 M-1cm-1) λem/nm Φema Φb τT/ μsc [Ru(bpy)3]·2PF6 450(13.0), 423(9.8), 286(92.1) 621 0.10 d 0.81 0.2 e [Ru(bpy)2(dppz)]·2PF6 445(16.3), 421(13.9), 366(17.9), 283(140.4) 622 0.08 f 0.16 0.8 g Ru1 450(26.9), 343(4.6), 328(39.4), 284(115.3) 632 0.11 0.93 7.0 h Ru2 533(27.0), 479(29.6), 297(160.3), 286(164.0) 759 - - - 404(65.1), 341(91.1), a. Luminescence quantum yield; b. 1O2 quantum yield in methanol; c. Triplet excited-state lifetime; d. Cited from Ref. 18; e. Cited from Ref. 19; f. Cited from Ref. 10; g. Cited from Ref. 17; h. In deaerated CH3CN. The triplet excited-state characteristics of Ru1 and Ru2 were further studied by the nanosecond transient absorption (TA) spectroscopy (Figure 1d). The triplet lifetime of Ru1 deduced from the decay of TA signals was about 7 μs, almost 1 order of magnitude longer than that of [Ru(bpy) 2(dppz)]2+ (0.8 μs).[17] This manifests that introduction of the pyrenyl group to [Ru(bpy)2(dppz)]2+ greatly prolongs its triplet excited-state lifetime. However, the triplet excited-state lifetime of Ru2 was unable to be reliably detected due to its instability upon 355-nm laser excitation (Figure S1b). Density functional theory calculation (DFT) DFT calculations were performed to understand the parentage of the T1 state[15] of Ru1 by calculating the spin density surface. As shown in Figure 2, the triplet spin density of T1 is exclusively distributed on the pyrene and dppz motifs, which makes the T1 state 3,* in nature and thus longer lived. In contrast, the spin density surface of [Ru(bpy)2(dppz)]2+ is delocated on the Ru metal center and the dppz and bipyridine ligands, indicating a 3 MLCT/3LLCT configuration in its T1 state. Ru1 [Ru(bpy)2(dppz)]2+ Figure 2. Isosurfaces for the spin density of Ru1 and [Ru(bpy)2(dppz)]2+ at their optimized triplet-state geometries. Reactive oxygen species (ROS) generation The ROS generation abilities of the Ru(II) complexes were determined using a chemical trapping method by monitoring the decrease of absorption of 1,3-diphenylisobenzofuran (DPBF) at 410 nm in methanol.[10] As we expected, Ru1 greatly reduced the absorption of DPBF at 410 nm upon 460-nm light irradiation (Figure S2d), whereas a faint decrease of DPBF absorbance was observed for [Ru(bpy)2(dppz)]·2PF6, suggesting that Ru1 is Figure 3. (A) Live/dead cell co-staining assays using calcein AM and propidium iodide as fluorescence probes. Cells were incubated with 0.05 μM of Ru1 for 4 h and then with the fluorescence probes for 30 min without light irradiation (-hv). The cells were incubated with 0.05 μM of Ru1 for 4 h followed by 460-nm light (6.0 mW/cm2) irradiation for 2 min and then incubated with fluorescence probes for 30 min (+hv). Calcein AM and propidium iodide (5 μM, respectively) were used as fluorescence probes. Green channel: λex = 488 nm, λem = 500-540 nm. Red channel: λex = 561 nm, λem = 600-640 nm. Scale bar = 80 μm. (B, C) Molecular docking study of (B) Ru1 and (C) Overlapped Ru1 and [Ru(bpy)2dppz]2+ with DNA. (D, E) Dihedral angles between the pyrenyl substituent and the phen motif of Ru1 (D) before and (E) after docking with DNA. This article is protected by copyright. All rights reserved. Accepted Manuscript Table 1. Photophysical parameters of the related compounds. 10.1002/chem.202003031 Chemistry - A European Journal FULL PAPER Table 2. IC50 values of the Ru(II) complexes against A549 and MCF-7 cancer cell lines. A549 MCF-7 Dark (μM) Light (μM) PIa Dark (μM) Light (μM) PI Ru1 10.3 ± 1.2 0.010 ± 0.001 1030 13.4 ± 1.5 0.004 ± 0.001 3350 Ru2 10.9 ± 0.2 8.30 ± 0.71 1.3 15.2 ± 1.7 12.2 ± 1.3 1.2 [Ru(bpy)3]·2PF6 57.6 ± 3.5 8.30 ± 0.4 6.9 53.9 ± 4.3 7.20 ± 0.5 7.5 [Ru(bpy)2(dppz)]·2PF6 168 ± 9.8 147 ± 2.3 1.1 200 ± 4.5 152 ± 3.0 1.3 Cisplatin 5.40 ± 0.3 4.92 ± 0.2 1.1 6.20 ± 0.4 6.60 ± 0.6 0.9 a PI = dark IC50 value/Light IC50 value. capable of producing ROS more efficiently than [Ru(bpy)2(dppz)]·2PF6. The ROS quantum yields of Ru1 and [Ru(bpy)2(dppz)]·2PF6 were measured to be 0.93 and 0.16 (Table 1), respectively, using [Ru(bpy)3]·2PF6 (Φs=0.81)[20] as the standard. The ROS quantum yields of [Ru(bpy)2(dppz)]·2PF6 was in good agreement with those reported in the literature. [10] In contrast, no obvious changes of DPBF absorbance were observed for Ru2, implying the negligible ROS generation under the test condition. The drastic difference in ROS generation efficiency of Ru1 and Ru2 likely reflects the difference in the T1 lifetimes of these two complexes. The longer the T1 lifetime, the higher the ROS generation efficiency. In vitro photocytotoxicity Live/dead cell co-staining assay Photocytotoxicity of Ru1 was further studied by live/dead cell costaining assay.[21] Calcein AM and propidium iodide (PI) were used to label the living and dead cells, respectively. Because of the stronger photocytotoxicity of Ru1 toward the MCF-7 cell line, this assay and the following photobiological studies were carried out only on the MCF-7 cell line. As shown in Figure 3a, very few cell death was observed for MCF-7 cells without light irradiation, whereas cells were almost killed upon light irradiation as revealed by the intense red fluorescence. This experiment further demonstrates the potent PDT capability of Ru1. DNA interactions The cytotoxicity and photocytotoxicity of Ru1 and Ru2 were evaluated against A549 (human pulmonary adenocarcinoma cell) and MCF-7 (human breast cancer cell) cancer cell lines by MTT assay,[2b] together with [Ru(bpy)3]·2PF6, [Ru(bpy)2 (dppz)]·2PF6 and cisplatin as controls. Initially, the concentrations ranging from 0.25 to 200 μM were chosen for the study. Unexpectedly, all the cancer cells were killed by Ru1 even at the lowest concentration (0.25 μM) upon 460-nm irradiation (6.0 mW/cm2, 3.6 J/ cm2), demonstrating the dramatic photocytotoxicity of Ru1. Therefore, Ru1 was further tested with concentrations in the range of 0.001 - 0.125 μM. According to the IC50 values (Table 2), Ru1 exhibited markedly high phototoxic activities against A549 and MCF-7 cancer cells with exceptionally large phototherapeutic indices (PIs) of 1030 and 3004, respectively. Notably, Ru1 possesses the same core structure as that of [Ru(bpy) 2(dppz)]·2PF6, except for a pyrenyl group pendant on the skeleton of dppz. This structural modification enables Ru1 to be 4 orders of magnitude more potent than [Ru(bpy)2(dppz)]·2PF6 under 460-nm light irradiation. In contrast, Ru2 produced no discernible phototoxic activity due to its negligible ROS generation efficiency. Collectively, incorporation of a pyrene moiety on the dppz ligand of [Ru(bpy)2(dppz)]·2PF6 allows for the access of the long-lived 3 IL state of the resultant complex and greatly increases its triplet excited-state lifetime, which in turn significantly improved the phototoxicity of the new complex. DNA is an effective cancer therapeutic target for metal-based complexes. In many cases photoinduced DNA damage is responsible for the photobiological activity of Ru(II)-polypyridyl PSs.[8] Since [Ru(bpy)2dppz]·2PF6 was reported to be able to interact with DNA,[10,11] the DNA binding and photodamaging properties of the new complexes Ru1 and Ru2 were explored. DNA binding abilities of the complexes were studied by the fluorescence competitive binding experiments. [22,23] As shown in Figure S3, the emission intensities of DNA-bound ethidium bromide (EtBr) were greatly decreased upon addition of Ru1, Ru2 and [Ru(bpy)2(dppz)] · 2PF6, possibly attributing to the replacement of EtBr by these Ru(II) complexes. However, It is worth noting that the Ru(II) complexes can absorb the excitation light in this assay. With the increased amount of the Ru(II) complexes, the excitation light could be absorbed, resulting in fluorescence quenching. To further confirm the interactions of the Ru(II) complexes with DNA, electronic absorption titration was conducted with results shown in Figure S4. Addition of ctDNA caused hypochromicity in the absorption spectra of all Ru(II) complexes, probably due to the intercalations of the Ru(II) complexes with ctDNA.[4I] Red shifts were also observed in the spectra of [Ru(bpy)2(dppz)]·2PF6 and Ru1 treated group. The DNA interaction was further confirmed by circular dichroism (CD) spectrum. As prescented in Figure S5A, addition of Ru1 and Ru2 dramatically changed the CD spectrum of ctDNA, demonstrating that these two complexes effectively binded with DNA. This article is protected by copyright. All rights reserved. Accepted Manuscript Complex 10.1002/chem.202003031 Chemistry - A European Journal FULL PAPER 0.05 μM of Ru1 for 4 h and irradiated by 460-nm light (6.0 mW/cm2) for 2 min. The cells were further incubated for 4 h before evaluation. Relative gray intensity = (gray intensity of indicated protein)/(gray intensity of β-actin). The data are representative of three independent experiments and the mean ± S.D. is shown. ***p < 0.001, **p < 0.01. Two-sided Student’s t-test. The DNA photocleavage abilities of Ru1 and Ru2 were investigated by agarose gel electrophoresis, with pBR322 plasmid DNA as the target and [Ru(bpy)3]·2PF6 and [Ru(bpy)2(dppz)]·2PF6 as the controls. As revealed in Figure 4a, without light irradiation, no changes were observed for the [Ru(bpy)3]·2PF6 group. However, the intensity of plasmid DNA bands gradually disappeared with the increased concentrations of Ru1 and Ru2, indicating that these complexes effectively inhibited the intercalation of EtBr into plasmid DNA. This result is in accordance with the result from the EtBr competitive binding experiment. It should be noted that although [Ru(bpy)3]·2PF6 possesses weak DNA binding ability and [Ru(bpy) 2(dppz)]·2PF6 has low ROS generation efficiency (as discussed in previous sections), they are still able to induce DNA damage at high concentrations upon 460-nm irradiation. Ru1 showed efficient DNA photocleavage upon irradiation, which induced a complete single-strand break in pBR322 DNA at the concentration of 6.25 μM (drug-to-nucleotide ratio: 0.041) and thus led to the formation of nicked circular form (Form II) of the DNA. In contrast, Ru2 did not show any observable DNA cleavage under 460-nm light irradiation because of its inability to form ROS. These results demonstrate that a strong DNA binding ability and a high 1O2 generation efficiency are the two essential factors for efficient DNA photocleavage. Cellular ROS production The in vitro ROS generation of Ru1 was evaluated in MCF-7 cells with DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) as the ROS probe. As shown in Figure 4b, no ROS signal was observed for Ru1 treated MCF-7 cells without irradiation. On the contrary, an obvious green fluorescence was observed upon 460-nm irradiation, demonstrating the effective ROS generation ability of Ru1 in MCF-7 cells. Endoplasmic reticulum stress activation Figure 4. (A) Agarose gel mobility pattern of pBR322 plasmid DNA incubated with the Ru(II) complexes at the concentrations of 0 (0 μM), 1 (0.39 μM), 2 (0.78 μM), 3 (1.56 μM), 4 (3.12 μM), 5 (6.25 μM), 6 (12.5 μM), 7 (25.0 μM), 8 (50.0 μM), 9 (100 μM), respectively. The pBR322 DNA incubated with the complexes was then irradiated with a 460-nm LED light (6.0 mW/cm2) for 5 min and incubated overnight. (B) Cell images of MCF-7 cells treated with Ru1 and DCFH-DA. (-hv) The cells were loaded with Ru1 (0.05 μM) and DCFH-DA (5 μM) successively without light irradiation. (+hv) The cells were incubated with Ru1 (0.05 μM) for 4 h, loaded with DCFH-DA (5 μM) and irradiated with 460-nm light for 2 min. λex = 488 nm. λem=520-550 nm. Scale bar: 80 μm. (C, D) Results of ER stress evaluation of MCF-7 cells after treatment with Ru1. (C) Blots; (D) Relative gray intensity analysis. MCF-7 cells were incubated with Endoplasmic reticulum (ER) is vulnerable to ROS damage.[25] To evaluate whether Ru1 induced the ER stress upon light activation, expression levels of the phosphorylated RNAdependent protein kinase-like endoplasmic reticulum kinase (PERK), phosphorylated eukaryotic initiation factor 2α (P-eif2α)[25c] and C/EBP homologous protein (CHOP) in MCF-7 cells were measured by western blot assay. As shown in Figures 4c and 4d, Ru1 up-regulated the expression of all the tested proteins upon light irradiation, indicating that Ru1 can induce ER stress via PERK/eif2α pathway in cancer cells. This article is protected by copyright. All rights reserved. Accepted Manuscript DNA intercalation was proven by the DNA melting temperature assay.[12c] As shown in Figure S5B, the DNA melting temperatures of [Ru(bpy)2(dppz)]·2PF6, Ru1, and Ru2 treated groups were 69 ℃, 66 ℃, and 65 ℃, respectively, which were higher than that of free ctDNA (Tm = 61 ℃), demonstrating the intercalation effect of Ru1, and Ru2. It was reported that intercalation of the ligand into DNA could cause significant viscosity increase of the DNA solution.[12c] Therefore, viscosity of the DNA solutions treated with the Ru(II) complexes was carried out. As shown in Figure S5C, with the addition of Ru(II) complexes, the viscosity of ctDNA increased apparently, further confirming that Ru1 and Ru2 binded to DNA via intercalation. The DNA binding mode of Ru1 was further studied by molecular docking using AutoDock 4.2.[24] As shown in Figures 3b and 3c, the most stable binding conformation of Ru1 and DNA manifested that Ru1 can intercalate with DNA base pairs. Moreover, Ru1 overlapped well with the co-crystallized [Ru(bpy)2(dppz)]2+ in the DNA crystal structure, indicating the similar binding mode of Ru1 to that of [Ru(bpy)2(dppz)]2+ (Figure 3c).[24b] Importantly, the intercalation induced conformational constrain of Ru1, which reduced the dihedral angle between the pyrenyl substituent and the dppz ligand from 54o in the optimized ground-state geometry for the unbound Ru1 to 12o upon binding with DNA. This result indicates that intercalation increased the coplanarity of the pyrenyl-substituted dppz ligand in Ru1 (Figure 3d and 3e), which would increase the 3π,π* configuration in the T1 state and prolong the excited-state lifetime of Ru1. This change would facilitate its application for PDT. 10.1002/chem.202003031 Chemistry - A European Journal FULL PAPER Cell death pathways Figure 5. Cell images of MCF-7 cells treated with 0.05 μM of Ru1 for 4 h in dark (-hv) or irradiated with 460-nm light (6.0 mW/cm2) for 2 min (+hv) and loaded with JC-10. Red channel: λex = 561 nm, λem = 580-680 nm. Green channel: λex = 488 nm, λem = 500-540 nm. Scale bar: 20 μm. Mitochondrial membrane potential change Mitochondria is another common target for PDT. To assess whether PDT using Ru1 caused mitochondrial damage, the mitochondrial membrane potential (MMP) change of MCF-7 cells was evaluated with or without light irradiation. [26] When the MMP is at a high level, JC-10 aggregates in the matrix of mitochondria, resulting in a red fluorescence. Once the MMP decreases, JC10 exists as the monomer and emits green fluorescence. As shown in Figure 5, the Ru1 group showed red emission without light irradiation. However, an intense green fluorescence from the MCF-7 cells was observed after light irradiation, indicating the decrease of MMP in MCF-7 cells. The loss of MMP should be attributed to the significant photocytotoxicity of Ru1. Conclusions A Ru(II)-polypyridyl complex Ru1 was designed by tethering the pyrenyl substituent to the dppz ligand on [Ru(bpy) 2(dppz)]·2PF6. Both spectroscopic study and DFT calculation revealed that Ru1 possessed a long-lived 3IL T1 state. In vitro PDT studies indicated that Ru1 displayed a remarkable photocytotoxicity against A549 and MCF-7 cells, with IC50 values of 10 and 4 nM, respectively, which led to exceptionally large phototherapeutic indices (PIs) of 1030 and 3004, respectively. Notably, Ru1 was 4 orders of magnitude more potent than [Ru(bpy) 2(dppz)]·2PF6 upon light irradiation, demonstrating the dramatic improvement of the photobiological activity via incorporation of the pyrenyl substituent to [Ru(bpy)2(dppz)]·2PF6. Biological studies indicated that Ru1 induced cell death through photoinduced DNA damage, ER stress and MMP change. This study manifests that the photophysical and biological activities of the organelle-targeting Ru(II)-polypyridyl complexes can be significantly altered via subtle ligand structural modifications. This design strategy could stimulate the development of more efficient organelle-targeting Ru(II)-polypyridyl complexes with improved excited-state properties for potential clinical PDT applications. Experimental Section The details for complex synthesis, photophysical studies are provided in the Supporting Information. Photophysical measurements Figure 6. Apoptosis inducing property of (A) Control in dark. (B) Control with irradiation. (C) Ru1 in dark. (D) Ru1 with irradiation. The cells were incubated with 0.05 μM of Ru1 for 4 h and irradiated by 460-nm (6.0 mW/cm2) light for 2 min. MCF-7 cells were stained by Annexin V-FITC/PI. The Y-axis shows the PI-labeled population and the X-axis shows FITC-labeled Annexin V-positive cells. Ru1 and Ru2 were dissolved in CH3CN and CH2Cl2, respectively. The UV-Vis absorption measurements were conducted on a Varian Cary 50 spectrophotometer. The emission spectra of Ru1 and Ru2 were recorded on a Horiba Jobin-Yvon FluoroMax-4 fluorometer/phosphorometer. The solutions were degassed with N2 for 30 min prior to measurement. The excitation wavelength was 534 nm for Ru1 and 590 nm for Ru2. The time-resolved triplet transient absorption spectra and triplet lifetimes were measured on an Edinburgh LP920 laser flash photolysis spectrometer. The excitation light was the third harmonic output (355 nm) of a Brilliant Nd:YAG laser with the repetition being set up to 1 Hz. The solutions were degassed with N2 for 30 min. The concentrations of sample solutions were adjusted to reach an absorbance of 0.4 in a 1-cm cuvette at 355 nm. DPBF assay to evaluate 1O2 production of Ru1 in solution This article is protected by copyright. All rights reserved. Accepted Manuscript To evaluate the cell death pathways induced by Ru1 PDT on MCF-7 cells, flow cytometry assay was conducted with or without light irradiation. As revealed in Figure 6, 460-nm light irradiation alone had no effect on the cell death rates (apoptosis and necrosis). Negligible cell death rates were observed for Ru1-treated MCF-7 cells without light irradiation. Once light irradiation was conducted, the cell death rates induced by Ru1 were greatly increased to 59.9%, further confirming the dramatic photocytotoxicity of Ru1. This result also indicates that apoptosis, especially late apoptosis is the dominant cell death pathway. 10.1002/chem.202003031 Chemistry - A European Journal FULL PAPER Live/dead cell co-staining assay The subscripts x and s refer to the sample and [Ru(bpy)3]2+, respectively. S stands for the slope of plot of the -ln(Abs 410 nm) vs irradiation time (in this study, we chose the initial 5 points). F stands for the absorption correction factor, F=1-10-OD. OD represents the optical density at 460 nm. MCF-7 cells were treated with 0.05 μM of Ru1 and incubated in a glass bottom dish for 4 h. For irradiated group, the cells were triggered by 460nm LED light (6.0 mW/cm2) for 2 min. The cells were further incubated for 1 h. Then the culture medium was removed, and the cells were washed with PBS (10 mM, pH=7.4). Next, the cells were incubated with live/dead cell fluorescence probes calcein AM and propidium iodide (5 μM respectively) for 30 min, followed by washing with PBS (10 mM, pH = 7.4). Finally, the cells were detected by LSCM. For the red channel, the sample was excited with a 561 nm laser and the emission was collected at 600-640 nm. For the green channel, the excitation wavelength was 488 nm and the emission was detected at 500-540 nm. MTT assay Agarose gel electrophoresis study In vitro anticancer activity of Ru1 and Ru2 against A549 and MCF-7 cancer cells were carried out by the MTT assay. Cancer cells were plated in 96-well plates at density of 105/mL per well. After an overnight culturing, cells were exposed to culture medium containing Ru1, Ru2, [Ru(bpy)3]·2PF6, [Ru(bpy)2dppz]·2PF6 and Cisplatin separately at a series of concentrations and incubated overnight. Then the cells were exposed to 460-nm light (6.0 mW/cm2) for 10 min or not. Then the cells were incubated for further 24 h. After that, 10 μL of MTT solution was added and the medium was removed. After a 4 h incubation, 130 μL of DMSO was added then the cells was taken to microplate reader to record the absorption at 490 nm. pBR322 plasmid DNA (0.1 μg/μL) was incubated with complexes at a concentration of 0, 0.39, 0.78, 1.56, 3.12, 6.25, 12.50 and 25.00, 50.00 and 100.00 μM, separately. For irradiated group, the samples were triggered by 460-nm LED light (6.0 mW/cm2) for 5 min. Then the samples were incubated at 37 ℃ in dark overnight. After that, the samples were loaded with loading buffer (10 %, v/v) and separated by agarose gel (100 V, 1h). Next, the gel was incubated by ethidium bromide (0.75 μg/mL) aqueous solution for 30 min. Finally, the gel was washed by water and taken to gel imaging system. Cellular ROS produce ability of Ru1 1.5 mL of ctDNA (50 μM) and 1.5mL of EB (50 μM) were mixed. After every 2 μL of complexes (1 mM) was added and incubated for 5 min, the fluorescence emission spectra were record by the λEX=540 nm. The concentration of ctDNA was determined by UV absorbance at 260 nm, [ctDNA]=(Abs260 nm)/(1 cm·6600 M-1cm-1). The concentrations of complexes were 0, 0.7, 1.3, 2.0, 2.7, 3.3, 4.0, 4.7, 5.3, 6.0, 6.7 μM, respectively. The apparent binding constants were calculated following literature reported before.[23] Φx=Φs·Sx·Fs/(Ss·Fx) To a glass bottom cell culture dish containing MCF-7 cells, 1 μL of Ru1 (50 μM) in PBS (10 mM, pH=7.4, containing 10% DMSO) was added into 999 μL of culture medium to make the final concentration of Ru1 as 0.05 μM and the cells was cultured for 4 h. Then the medium was removed, and the cells was washed by 1 mL PBS (10 mM, pH=7.4). After that, 1 mL of DCFH-DA solution (5 μM) was added and the cells were incubated for 10 min. Then the cells were irradiated by 460-nm light (6.0 mW/cm2) for 2 min and washed by PBS (10 mM, pH=7.4), followed by detection with LSCM. The excitation wavelength was 488 nm. The emission fluorescence was collected at 500-600 nm. Mitochondrial membrane potential decrease MCF-7 cells in a glass bottom cell culture dish were treated with 0.05 μM of Ru1 for 4 h. For irradiation experiment, the cells were triggered by 460-nm LED light (6.0 mW/cm2) for 2 min. The medium was removed, and the cells were washed with 1 mL of PBS (10 mM, pH=7.4). Then 500 μL of JC-10 working solution was added to each dish. After a 15-min incubation, the cells were washed by PBS (10 mM, pH=7.4) and moistened with 300 μL deionized water. The cells were detected by LSCM. The excitation and emission wavelength for monomer was 488 nm and 500-540 nm. For aggregation, the excitation and emission wavelength were 561 nm and 580-640 nm. DNA-EB fluorescence competing experiments Absorption titration experiments Ru complexes (20 μM) were incubated with 0, 4, 8, 12, 16, 20, 24, 28, 32, 36, 40 μM of ctDNA in Tris-HCl buffer (pH = 7.4) for 10 min at 25 °C. Then the absorption spectra were recorded. DNA melting experiments The DNA melting experiments were carried out on a Shimadzu UV-Vis spectrophotometer and the temperature was recorded with a platinum resistance thermometer. Equal concentration of ctDNA and Ru complexes were incubated and the absorption of the mixture was recorded at 260 nm at indicated temperature. The data was presented as (A-A0)/(Af-A0) vs. T. A = absorption at identified temperature; Af = absorption at 56 °C; A0 = absorption at 90 °C. Viscosity assay Cell apoptosis analysis MCF-7 cells were treated with 0.05 μM of Ru1 4 h. For irradiated group, the cells were triggered by 460-nm LED light (6.0 mW/cm2) for 2 min. After further incubated for 1 h, cells were collected by trypsinization and washed with PBS (10 mM, pH=7.4) twice, and then suspend cells in 1× Binding Buffer (0.1 M Hepes/NaOH (pH=7.4), 1.4 M NaCl, 25 mM CaCl2). Then the cells were stained with FITC Annexin V (100 ng/mL) and propidium iodide (PI, 2 μg/mL) using annexin-V FITC apoptosis kit for 30 min at room temperature (25 ℃ ) in dark. The apoptosis ratio was quantified by system software (Cell Quest; BD Biosciences). Viscosity assay was performed with an Ubbelohde viscometer at 30 °C. Ru complexes with a concentration of 0, 5, 10, 15, 20, 25, 30 μM were added into the degassed DNA solution (100 μM). Flow time (η) was recorded with a stopwatch. The data was presented as (η/η0)1/3 vs. [Complex]/[DNA]. Circular dichroism spectra The CD spectra were obtained on a Jasco-810 spectropolarimeter. Equal amount of ctDNA and Ru complexes were mixed and equilibrated for 5 min before the spectra were recorded. This article is protected by copyright. All rights reserved. Accepted Manuscript 60 μM DPBF in methanol and 10 μM Ru1 in methanol were mixed in an equal volume. Then the solution was taken to record the UV-Vis absorption spectrum after irradiated by 460-nm LED light (2.6 mW/cm2) for every 15 s. The 1O2 quantum yield was calculated by using [Ru(bpy)3]2+ (Φs=0.81) as the standard following the equation: 10.1002/chem.202003031 Chemistry - A European Journal Western Blots assay [3] MCF-7 cells were cultured to the cell density reached 80% and cultured with Ru1 (0.05 μM) for 4 h at 37 °C. After irradiated by 460-nm LED light (6.0 mW/cm2) for 2 min, the cells were further incubated for 24 h. Proteins were extracted by lysis buffer. The protein concentration was measured with the BCA (bicinchoninic acid) assay on a Varioskan multimode microplate spectrophotometer (Thermo, Waltham, MA). Then proteins (20 mg/lane) in equal concentration were separated by 8-12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferred onto polyvinylidene difluoride (PVDF) ImmobilonP membrane (Bio-Rad) in transblot apparatus (Bio-Rad). The blots were blocked with 5% defatted milk powder in PBST (Tris-buffered saline plus 0.1% Tween 20) for 1 h, and then incubated with a series of primary antibodies against CHOP, P-eif2α, P-ERK and β-actin overnight at 4 °C. The membrane was washed with PBST (1 ml×3) and incubated with IRDye 800 conjugated secondary antibody for 1 h at 37 °C. The blots were detected by an Odyssey scanning system (Li-COR, Lincoln, Nebraska). β-actin was used as loading control. [4] Acknowledgements J. Zhao and S. Gou are grateful to the National Natural Science Foundation of China (Grant Nos. 21601034 and 21571033) for financial aid for this work. This work was also supported by the Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX18-0129). J. Zhao also thanks for the support of “Zhi-Shan” project of Southeast University. 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All rights reserved. Accepted Manuscript Page No. – Page No.