Photoinduced synergistic cytotoxicity towards cancer cells via Ru(II) complexes

Zhong Han a, Yuncong Chen *ab, Yanjun Wang a, Xiangchao Shi a, Hao Yuan ab, Yang Bai a, Zhongyan Chen a, Hongbao Fang a, Weijiang He *a and Zijian Guo *ab
aState Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China. E-mail: chenyc@nju.edu.cn; heweij69@nju.edu.cn; zguo@nju.edu.cn
bChemistry and Biomedicine Innovation Center, Nanjing University, Nanjing 210023, China

Received 25th July 2020 , Accepted 11th September 2020

First published on 14th September 2020


Ru(II) complexes have been recognized as excellent anticancer candidates. However, the poor cell uptake of these complexes has been the major obstacle in improving their anticancer efficacy. Along with the development of the knowledge on the anticancer nature of the Ru(II) complexes, several strategies have been designed to increase the cellular accumulation of these complexes. Among them, the light-triggered drug uptake approach is most promising. In this study, a bioactive Ru(II)–polypyridyl complex Ru-1 is constructed via incorporating a modified phenanthroline ligand into the enlarged conjugate structure. The modulation of the ligand makes Ru-1 powerful in generating singlet oxygen and effective in photodynamic therapeutic activity. Moreover, the cell uptake is improved during this photodynamic process and induces further chemotherapeutic effect, which act synergistically to enhance its anticancer activity. The photoinduced synergistic cytotoxicity of Ru-1 towards cancer cells offers an effective way to sensitize the antitumor activity of Ru(II) complexes.


Cancer remains the second leading cause of death all over the world, behind cardiovascular disease, with almost 18 million newly diagnosed cases every year.1 The use of platinum-based anticancer drugs such as cisplatin, carboplatin and oxaliplatin has made great contributions toward the treatment of cancer in the past several decades. However, these chemotherapeutic agents suffer from incident serious dose-limiting systemic toxicity, debilitating, long-term side effects2 and intrinsic or acquired tumor resistance phenomena,3 which have boosted the research into metallopharmaceuticals in order to find good alternatives to platinum-based drugs. Among them, ruthenium(II) complexes have been proved to be efficacious at inhibiting the growth of certain cancer cell lines and are regarded as promising alternatives to cisplatin and its derivatives.4 Nevertheless, long cell permeation times and relatively high concentration requirements caused by the poor cell membrane penetration of the Ru(II) complexes hinder them from being used widely.

The poor uptake of anticancer agents is one of the possible reasons for drug resistance.5 As the first protective barrier of cancer cells, the cytomembrane is the only channel for exchanging substances and information between cancer cells and their environment.6 Recently, the promotion of the cell uptake of certain complexes via damaging the cell membranes by photoinduced reactive oxygen species (ROS) of photosensitizers has been reported and proved feasible.7 Hence, it may be an effective way to enhance the cell accumulation of Ru(II) complexes through structural modification to generate ROS. Taking this into account, ruthenium(II) complexes coordinated with polypyridyl ligands are among the most promising molecular photosensitizers owing to their strong absorbance in the visible spectrum and long-lived triplet metal-to-ligand charge transfer (3MLCT) excited states caused by the heavy atom effect.8 Furthermore, they exhibit high 1O2 quantum yields and their solubility can be optimized by adjusting the counterions.9 Thus, the promoted cell uptake and the synergistic cytotoxicity of photodynamic therapy and chemotherapy can be achieved.

Herein, we report the design, synthesis and anticancer performance of the Ru(II)–polypyridyl complex Ru-1 constructed with a modified phenanthroline compound L1 as the ligand. In addition, Ru(bpy)3, Ru(phen) and Ru(phen-NO2) are also prepared as control complexes with different 1O2 generation abilities. Due to the expanded conjugate structure of L1, Ru-1 showed the highest singlet oxygen generating ability and distinct photodynamic therapeutic (PDT) activity, which improved its cellular accumulation after short irradiation and it further exhibited a chemotherapeutic effect towards cancer cells. Therefore, the PDT and chemotherapeutic effects are combined together in one complex to kill the cancer cells synergistically (Fig. 1).


image file: d0dt02627a-f1.tif
Fig. 1 Molecular structures of Ru-1 and the control complexes.

The synthesis details of Ru-1 are illustrated in the ESI.Ru-1 is characterized by 1H and 13C NMR spectroscopy, high-resolution mass spectroscopy, and further confirmed by single crystal X-ray analysis12 (Fig. S1, S2, Tables S2 and S3, see the ESI). The photophysical data of Ru-1 and other control complexes are summarized in the ESI (Table S1). In HEPES buffer solutions, the absorption bands at approximately 400–500 nm are assigned to metal-to-ligand charge-transfer (1MLCT) absorption for the four complexes and the enlarged conjugate structure of Ru-1 led to the increase of its molar extinction coefficient (log[thin space (1/6-em)]ε = 4.02). The phosphorescent emission spectra reveal that all the complexes except Ru(Phen-NO2) comprise maximum emission wavelength at 602 nm. The emission of Ru(Phen-NO2) was very weak due to the photoinduced electron transfer (PET) effect of the strong electron-withdrawing nitro group (Fig. S3). Moreover, Ru-1 exhibits a specific ratiometric response to pH (Fig. S4), showing the decreased emission at 510 nm and the concomitant enhanced emission at 602 nm when pH is decreased from 12.0 to 3.0. Furthermore, given the importance of modulating the pKa of phenol derivatives, we observed that the pKa value of Ru-1 is 5.36, spanning the intracellular pH range. It exhibits a reversible pH sensing ability and a linear response range from pH 3.92 to 6.77.

To determine singlet oxygen generation efficiency, the 1O2 quantum yields (ΦΔ) of the four complexes were quantified through an indirect method. By measuring the absorbance of the singlet oxygen scavenger 1,3-diphenyl isobenzofuran (DPBF) with Ru(bpy)3 as the reference, the singlet oxygen quantum yields were determined as 0.57, 0.06 and 0.81 for Ru(phen), Ru(Phen-NO2) and Ru-1, respectively (Fig. 2, Fig. S5 and S6). Ru-1 showed the strongest 1O2 generation ability and this could be ascribed to the enlarged conjugate structure of its ligand compared with those of the other control complexes. For Ru(Phen-NO2), the lowest 1O2 quantum yield is obtained and this is caused by the PET effect of the nitro group, which effectively quenched both singlet and triplet excited states. Moreover, the addition of ascorbic acid (VC) into Ru-1 solution effectively quenched the 1O2 generated by Ru-1.


image file: d0dt02627a-f2.tif
Fig. 2 1O2 production from a plot of changes in absorbance by DPBF at 411 nm against irradiation time (λex = 450 nm) in the presence of Ru(phen), Ru(Phen-NO2), Ru-1, and Ru-1 (with 2 mM ascorbic acid) in acetonitrile vs.Ru(bpy)3 as a standard.

The cell uptake process of Ru-1 with or without irradiation was monitored in MCF-7 cells via confocal fluorescence imaging. As is illustrated in Fig. 3, after incubation with Ru-1 in the dark for 4 hours, no evident phosphorescence was observed in the cells. Further incubation without photoirradiation for another 4 hours caused subtle changes in phosphorescence intensity, indicating the poor membrane permeability of Ru-1 under dark conditions. For comparison, upon treating MCF-7 cells with light irradiation (5 min at 450 nm, 30 J cm−2) and further incubating with Ru-1 for 4 hours, bright intracellular phosphorescence was observed even in the nucleus. All the above results indicated that the quick enhancement of intracellular phosphorescence could be attributed to photoirradiation, implying the accelerated cell uptake process of Ru-1 under light conditions.


image file: d0dt02627a-f3.tif
Fig. 3 Temporal tracking of intracellular phosphorescence in MCF-7 cells after incubating with 10 μM Ru-1via confocal fluorescence imaging. (a) Dark: The cells were kept under dark conditions for more than 4 hours after incubation; light: The cells were irradiated for 5 min (450 nm, 30 J cm−2) after incubation. (b) Average phosphorescence intensities of the cells under different conditions at diverse time points. The time points (0 h, 2 h, and 4 h) refer to the time after photoirradiation.

Furthermore, the promoted cell accumulation of Ru-1via photoirradiation was confirmed by the inductively coupled plasma mass spectrometry (ICP-MS) determination of Ru content in MCF-7 cells (Fig. 4). The Ru content of cells incubated with Ru-1 under dark conditions was determined to be 6.65 ppb per 1 × 105 cells. However, upon treating the cells with 5 min photoirradiation and another 4 h incubation, the Ru content in the cells drastically increased to 89.56 ppb per 1 × 105 cells, showing an approximately 13.5 times enhancement. At the same time, the Ru content in cells without Ru-1 incubation was negligible (about 1.60 ppb per 1 × 105 cells). The remarkable enhancement of Ru content in cells with or without photoirradiation demonstrated that light was the key factor that promoted the cell accumulation of Ru-1. Moreover, adding ascorbic acid (2 mM) as a ROS scavenger before photoirradiation resulted in an intracellular Ru content of 9.59 ppb per 1 × 105 cells, which was only slightly higher than that without photoirradiation. The drop in the photoinduced enhancement of cell uptake suggested that the photoinduced cell uptake enhancement was dependent on ROS. Similar experiments were conducted for the analogues of Ru-1. As is shown in Fig. 4a, no obvious increments were observed for the control complexes, implying that a higher singlet oxygen generation efficiency is significant in the photo-promoted cell uptake of the Ru–polypyridyl complexes. At the same time, relevant experiments on the photoenhanced cell uptake of Ru-1 has been conducted in the presence of cisplatin. As is shown in Fig. 4b, the Ru content of the cells treated with light irradiation increased greatly while the Pt content remained the same. Thus, the photoenhanced cell uptake caused by ROS generation only works for Ru-1.


image file: d0dt02627a-f4.tif
Fig. 4 (a) Ru content in 1 × 105 cells determined by ICP-MS measurement of the digestion solutions of MCF-7 cells incubated, respectively, with Ru-1 (10 μM), Ru(bpy)3 (10 μM), Ru(phen) (10 μM), and Ru(Phen-NO2) (10 μM) with or without photoirradiation (5 min, 450 nm, 30 J·cm−2) at the fourth hour of incubation under dark conditions; (b) Pt and Ru contents in 1 × 105 cells determined by ICP-MS measurement of the digestion solutions of MCF-7 cells incubated, respectively, with cisplatin (5 μM) or Ru-1 (10 μM) co-dosed with cisplatin (5 μM) with or without photoirradiation (5 min, 450 nm, 30 J·cm−2) at the fourth hour of incubation under dark conditions. The incubated cells were digested for ICP-MS determination using a standard procedure after 24 h of incubation. Blank: Ru contents for the cells without any Ru complex incubation.

Practically, in vitro cell culture study is a valuable tool for screening chemotherapy agents and it provides preliminary data for further relative studies.10 The antitumor activity of Ru-1 towards different tumor cells including human breast cancer cell line (MCF-7) and murine melanoma cell line (B16F10) under both light (λ = 450 nm) and dark conditions was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As is shown in Fig. 5, for MCF-7 cells, the IC50 value of Ru-1 obtained in the dark is 21.33 μM. However, the cytotoxicity increased significantly after irradiation with 450 nm light for 5 min (30 J cm−2) and the IC50 value was then determined to be ∼1.75 μM, which is lower than that of cisplatin (∼6.5 μM). The high light toxicity of Ru-1 combined with its low dark toxicity gave a phototherapeutic index [PI = IC50 (dark)/IC50 (light)] of more than 11.5. The photoactivated cytotoxicity of Ru-1 has also been observed towards B16F10 cells, with the PI being ∼11.4. The antitumor cytotoxicity of cisplatin and other control Ru(II) complexes showed no obvious difference between incubation under dark conditions and incubation with photoirradiation (Fig. S8). This result confirmed that the higher ROS generation efficiency of Ru-1 is crucial in promoting its cytotoxicity.


image file: d0dt02627a-f5.tif
Fig. 5 Cytotoxicities of Ru-1 against MCF-7 (a) and B16F10 (b) cell lines determined after 24 h of incubation under dark conditions with (red) or without (black) photoirradiation (450 nm, 5 min, 30 J cm−2) at the 4th hour of incubation.

Given that the high photosensitizing activity of Ru-1 could promote its cell uptake and improve its cytotoxicity toward cancer cells, further experiments were employed to analyze its mode of action during the light-promoted antitumor progress. Different incubation conditions were tested for the Ru complexes as well as cisplatin and a reported photoactivatable reagent, methylene blue (MB, ΦΔ = 0.52 in H2O).11 As is shown in Fig. 6, for Ru-1, the removal of the complex after irradiation led to an IC50 value of ∼7.5 μM for MCF-7 cells, which is much larger than that of the cells treated with Ru-1 in the whole incubation progress. Similar tests were conducted using the B16F10 cell line and analogical results were obtained, that is, the IC50 value rose to ∼6.3 μM when the complex was removed after irradiation. However, for MB and other control Ru–polypyridyl complexes (Fig. S9 and S10), the same tests did not affect their cytotoxicity towards MCF-7 cells (IC50 values changed from 28.2 μM to 25.6 μM for MB and other Ru complexes showed no obvious cytotoxicity). Considering these data, we can conclude that a further chemotherapeutic effect exists during the antitumor activity of Ru-1 upon entering the cells.


image file: d0dt02627a-f6.tif
Fig. 6 Cytotoxicities of Ru-1 (a, 1–4), cisplatin (a, 5–6), and MB (b) against MCF-7 cell lines under different treatment conditions. (1) MCF-7 cells were incubated with Ru-1 or MB for 24 h under dark conditions; (2) light irradiation (450 nm for Ru-1 and 635 nm for MB, 5 min, 30 J cm−2) was employed at the fourth hour of incubation of MCF-7 cells with Ru-1 or MB and the cells were further incubated for 20 h; (3) light irradiation (450 nm for Ru-1 and 635 nm for MB, 5 min, 30 J cm−2) was employed at the fourth hour of incubation with Ru-1 or MB. After that, the cells were washed three times with PBS and then the culture medium was replaced with new medium without Ru-1 or MB for further incubation for 20 h; (4) the cells were washed three times with PBS and the culture medium was replaced with new medium without Ru-1 or MB at the fourth hour. After that, light irradiation (450 nm for Ru-1 and 635 nm for MB, 5 min, 30 J cm−2) was employed and the cells were further incubated for 20 h; (5) MCF-7 cells were incubated with cisplatin for 24 h under dark conditions; (6) light irradiation (450 nm, 5 min, 30 J cm−2) was employed at the fourth hour of incubation of MCF-7 cells with cisplatin and the cells were further incubated for 20 h.

In summary, a Ru(II)–polypyridyl complex Ru-1 was designed and synthesized for sensitizing the antitumor activity of Ru(II)-based antitumor agents through promoting their cell accumulation. The singlet oxygen generation ability of Ru-1 was significantly enhanced via enlarging the conjugation of ligand, which leads to higher cellular uptake in cancer cells than the control complexes. Through light irradiation, Ru-1 showed not only obvious photodynamic antitumor activity but also a further chemotherapeutic effect. Overall, our results demonstrated a strategy for the design of Ru(II)-based antitumor agents, that is, synergistically combining their chemotherapeutic activities with photodynamic ability using one Ru(II) complex with high photosensitizing ability to be an efficient method for antitumor therapy.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This received financial support from the National Natural Science Foundation of China (Grants 21907050, 21977044, 21731004, and 91953201), the Natural Science Foundation of Jiangsu Province (BK20190282) and the Excellent Research Program of Nanjing University (ZYJH004).

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Footnote

Electronic supplementary information (ESI) available. CCDC 1502447. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/D0DT02627A

This journal is © The Royal Society of Chemistry 2020