[Ru(phen)2podppz]2+ significantly inhibits glioblastoma growth in vitro and vivo with fewer side-effects than cisplatin

Ruihao Li a, Yabin Ma b, Xiaochun Hu a, Wenjing Wu a, Xuewen Wu a, Chunyan Dong a, Shuo Shi *a and Yun Lin *a
aShanghai East Hospital, Shanghai Key Lab of Chemical Assessment and Sustainability, School of Chemical Science and Engineering, Tongji University, 1239 Siping Road, 200092 Shanghai, P. R. China. E-mail: ylin@tongji.edu.cn; shishuo@tongji.edu.cn
bDepartment of Pharmacy, Shanghai East Hospital, Tongji University, Shanghai 200120, PR China

Received 26th May 2020 , Accepted 12th June 2020

First published on 16th June 2020


To overcome the acquired resistance and the significant side-effects of the reported drugs, four new ruthenium(II) complexes with alkynyl (Ru1, Ru2, Ru3, Ru4) were designed and synthesized. Ru1, Ru2, Ru3 and Ru4 were characterized by ESI-MS, 1H NMR, 1H–1H COSY NMR and elemental analysis. Compared with Ru2, Ru3, Ru4 and cisplatin, the anti-tumor experiments in vitro and vivo confirmed that Ru1 could most effectively inhibit tumor growth. In the experiments of safety evaluation in vivo, Ru1 could avoid any detectable side-effects compared with cisplatin. DNA binding experiments and cell cycle experiments showed that Ru1 exhibited the strongest DNA binding ability and interfered with the cell cycle by inserting DNA to inhibit tumor growth. The study demonstrated that Ru1 had the potential to be an exciting new drug candidate for glioblastoma treatment.


Introduction

Glioblastoma multiforme (GBM), which emanates from the glial cell, is the most prevailing malignant tumor of the central nervous system and the most aggressive form of glioma.1 The median survival is less than 2 years with the current standard treatment of maximal safe resection, a combination of chemotherapy given with radiation therapy.2 Temozolomide (TMZ) was used to treat GBM for over a decade, but its therapeutic effects are limited by acquired resistance.3

In 1969, Bamett Rosenberg, American biophysicist, accidentally discovered the anti-tumor ability of cisplatin (DDP), and DDP gradually became the first-line drug in clinical practice.4,5 The major issues of DDP chemotherapy are resistance and side effects,6–9 such as renal toxicity, myelosuppression, nausea, vomiting, and toxicity, which limit its use in tumor therapy. Ruthenium and platinum belong to the 8 subgroup of transition metal elements and are of similar nature,10 but the richer valences(II,III,IV) of ruthenium result in more diverse physical and chemical properties.11–13 Recently, the good biocompatibility and powerful anti-tumor effect of ruthenium(II) complexes have shown that they are a good alternative to platinum drugs.14–21

Ru(II) complexes can intercalate into the structure of double-stranded DNA to interfere with the self-replication of DNA,22,23 thus affecting the progress of the cell cycle and inhibiting the division and proliferation of tumor cells, and finally inducing the apoptosis of tumor cells. They can also interfere with cellular transcription processes by inserting DNA, which eventually leads to preventing RNA polymerases from binding with DNA and inhibiting the growth of tumor cells.24,25

Although numerous previous studies have shown the anti-tumor effects and the ability of fluorescence imaging of Ru(II) complexes in in vitro and in vivo experiments,26,27 Ru(II) complexes have not been approved for clinical use. A recent study indicated that alkynyl groups could not only improve the ability of substances to enter cells but also increase the ability of Ru(II) complexes to bind to DNA.28–31

In this study, we have synthesized and characterized four new ruthenium(II) complexes with alkynyl, [Ru(phen)2podppz]2+, [Ru(bpy)2podppz]2+, [Ru(phen)2ppip]2+, and [Ru(bpy)2ppip]2+. In vitro studies confirmed their long-term stability in phosphate-buffered saline (PBS) and water. The anti-tumor mechanism of Ru1 was investigated via DNA binding experiments and a cell cycle assay. An in vivo safety evaluation demonstrated that Ru1 could avoid any detectable side-effects compared with cisplatin. In in vivo anti-tumor GBM models, Ru1 could effectively inhibit tumor growth, suggesting that Ru1 could be utilized as a potential drug for GBM treatment (Scheme 1).


image file: d0dt01877e-s1.tif
Scheme 1 (A) structures of Ru(II) complexes. (B) The schematic description of the anti-tumor mechanism of Ru1.

Results and discussion

Synthesis and characterization

Initially, the compounds pip, hdppz, cis-[Ru(phen)2Cl2]·2H2O and cis-[Ru(bpy)2Cl2]·2H2O were synthesized according to the literature references.32–35 Subsequently, the reported complexes RA, RB, RC and RD were similarly obtained according to the literature procedures (Fig. S1).34–38

The synthetic route to the alkynyl complexes is summarized in Scheme 2. RA, RB, RC and RD were individually reacted with propargyl bromide in the presence of anhydrous potassium carbonate in DMF, resulting in the formation of alkynyl [Ru(phen)2podppz]2+, [Ru(bpy)2podppz]2+, [Ru(phen)2ppip]2+, and [Ru(bpy)2ppip]2+ (Ru1, Ru2, Ru3, and Ru4) respectively, in very good yield. All new compounds were fully characterized by 1H NMR, 1H–1H COSY, TOF-MS, and elemental analysis (Fig. S2–S5).


image file: d0dt01877e-s2.tif
Scheme 2 Synthetic route for Ru1, Ru2, Ru3 and Ru4.

These complexes exhibit weak solubility in water, but show high solubility in DMSO. All Ru(II) complexes displayed remarkable stability in PBS or water (containing 0.5% DMSO) at 298 K for at least 5 days, as confirmed by UV-Vis spectroscopy (Fig. S6).

UV-Vis Absorption Titration

To clarify the nature of the different cytotoxicities induced by the complexes, UV absorption spectroscopy studies were carried out to investigate the DNA binding ability of the Ru(II) complexes. When calf-thymus DNA (CT-DNA) was added into Ru1 solution, an obvious hypochromism (H) was observed for both intra-ligand charge transfer (ILCT) (λ = 250–260 nm) band and metal-to-ligand charge transfer (MLCT) (λ = 400–420 nm). However, a slight hypochromism was observed for the other complexes. The extent of the hypochromism commonly parallels the intercalative binding affinity. These results demonstrated that Ru1 exhibits the strongest DNA binding ability (Fig. S8).

Since the octahedral complex binds to DNA in three dimensions, both its ancillary ligand and intercalative ligand can tune the DNA binding affinity.39–41 Increasing the surface area of the ancillary ligand and intercalative ligand leads to a substantially increased intercalative binding affinity. Proceeding from the ancillary ligand phen to bpy, the hydrophobicity of the ancillary ligand phen of Ru1 is greater than that of the bpy ligand of Ru2. Meanwhile, the surface area of the intercalative ligand podppz is bigger than that of ppip, which is advantageous to the DNA binding of Ru1, too. Therefore, by synthetically considering these factors, the fact of Ru1 exhibiting the strongest DNA binding ability and the best anti-proliferative effect could be well understood. Meanwhile, the results showed a significant difference in the ability of DDP and Ru1 to reduce cell viability. The antitumor ability of Ru1 is the most prominent candidate among the four new synthesized Ru(II) complexes. Therefore, further experiments were performed to assess the antitumor capacity of Ru1.

In vitro toxicity

To screen the most powerful anti-tumor capability of the Ru(II) complexes, we assess their cytotoxicity against GBM in vitro and in vivo. The in vitro cytotoxicity of Ru1, Ru2, Ru3, Ru4 and DDP against U87 MG cells was investigated by using the CCK-8 assay. Compared with Ru2, Ru3, Ru4 and DDP, Ru1 exhibited the strongest anti-proliferative effect due to the strongest DNA binding ability inducing cell apoptosis and inhibiting the cell cycle (Fig. 1A). Because of the poor solubility of Ru complexes in water, these complexes were first dissolved in DMSO and then diluted into the test solution appropriately. Meanwhile, the in vitro cytotoxicities of 0.25% and 0.5% DMSO in controlled buffers against the U87 MG cells were also investigated. The results confirmed that the addition of small amounts of DMSO was not toxic to U87, and that the anti-tumor effect was caused by the ruthenium complexes themselves (Fig. S7).
image file: d0dt01877e-f1.tif
Fig. 1 (A) In vitro cytotoxicity of Ru1, Ru2, Ru3, Ru4 and DDP against U87 MG cells with different drug dosages for 24 h via the CCK-8 assay. (B) Fluorescent inverted microscope images of the U87 MG cells treated with PBS, DDP and Ru1 for 24 h. Live and dead cells were stained by Calcein AM (AM)/Propidium Iodide (PI) for 30 min and presented in green and red colors in those images, respectively. The scale bar is 100 μm.

Live & dead viability assay

Moreover, to observe the anti-proliferative ability of DDP and Ru1, the U87 MG cells were stained with AM/PI after being administered with the same concentration of DDP and Ru1. It was found that most U87 MG cells remained alive (represented by green color) after treatment with DDP, suggesting that DDP has no significant efficacy for damaging U87 (Fig. 1B). In contrast, the treatment with Ru1 resulted in a large quantity of dead cells (represented by red color). This result sufficiently demonstrates that Ru1 has a stronger ability of killing U87 MG cells than DDP.

Cellular apoptosis

Flow cytometry was used to continue our exploration on the mechanism of cytotoxicity of Ru1 against tumor cells. Apoptotic and necrotic cells were detected in the U87 MG cells via dual fluorescence staining of Annexin V/PI. As shown in Fig. 2A, only 23.7% of the U87 MG cells were apoptotic after being administered with DDP (75 μmol L−1), while almost 48.6% of the U87 MG cells were apoptotic after being treated with an equal concentration of Ru1. This result demonstrated that Ru1 possessed a stronger ability of inducing the cellular apoptosis of U87 than DDP, which was consistent with the results of the cytotoxicity tests.
image file: d0dt01877e-f2.tif
Fig. 2 Cellular apoptosis and cell cycle analysis in U87 MG cells in response to PBS, DDP and Ru1. (A) Flow cytometry of 5 μL Annexin V and 5 μL PI labelled U87 MG cells exposed to PBS, DDP or Ru1 for 24 h. (B) Flow cytometry of PI labelled U87 MG cells incubated with PBS, DDP, or Ru1 for 24 h. Quantitative flow cytometry analysis of cell cycle ratios. (Single asterisks indicate p < 0.05, double asterisks indicate p < 0.01, and triple asterisks indicate p < 0.001.)

Cell cycle analysis

The completion of the cell cycle is crucial for DNA replication. Also, the dynamic balance of cell cycle activities is an important factor to maintain the stable transmission of genetic information. To further investigate the mechanisms of inhibiting tumor cell proliferation of Ru1, the effect of Ru1 and DDP on the cell cycle were assessed in the U87 MG cells by flow cytometry. 33.9% of cells were blocked in the S phase by DDP, while 48.6% of cells treated with Ru1 in the S phase were observed, which confirmed that Ru1 and DDP could play an anti-tumor role through the inhibition of the cell cycle stranded in the S phase (Fig. 2B). Moreover, Ru1 showed excellent inhibition ability of the cell cycle compared to DDP. Taken together, these results demonstrated that DNA damage caused by Ru1 can efficiently inhibit cell cycle arrest and induce cellular apoptosis.

In vivo anti-tumor study

To investigate the in vivo anti-tumor efficacy of Ru1, mice bearing subcutaneous U87 MG tumor were treated with PBS, DDP and Ru1 at a single dosage of 5 mg kg−1via intraperitoneal injection. The in vivo antitumor experiment further confirmed excellent tumor growth inhibition achieved by Ru1 (Fig. 3A). Tumor volumes of mice were measured every 2 days for 10 days after administration. Ru1 could inhibit tumor growth more effectively, compared with the PBS group and the DDP group (Fig. 3B).
image file: d0dt01877e-f3.tif
Fig. 3 (A) The image of tumors isolated from the nude mice. (3 groups, (1) PBS, (2) DDP, (3) Ru1). (B) Tumor growth curves and (C) histogram of average tumor weight measured on day 10 after various treatments. (D) Apoptotic tumor cells in the tumor sections were stained with TUNEL (green) and observed by fluorescence microscopy. Cell nuclei were stained with DAPI (blue). (E) H&E stains of the tumors of mice from each group. (Single asterisks indicate p < 0.05, double asterisks indicate p < 0.01, and triple asterisks indicate p < 0.001.) Scale bars: 100 μm.

The tumor weight was measured to assess the therapeutic effect, too. The results were consistent with the tumor volume as expected: tumor weights for the Ru1 group and DDP group on day 10 were 300 ± 17 mg and 770 ± 18 mg, respectively (Fig. 3C). The sections from tumors in each group were stained with TUNEL42 for the evaluation of apoptosis at the histological level (Fig. 3D). Compared with the DDP groups, tumors treated with Ru1 showed a higher apoptosis due to the suppression of DNA replication and transcription. H&E staining of tumors in mice from each group showed that the necrosis degeneration of tumor cells of the Ru1 group was more obvious than that of the other groups (Fig. 3E).

In vivo safety evaluation of DDP and Ru1

To investigate the in vivo safety and biocompatibility of Ru1 and DDP, the blood of all mice was collected after treatment on day 10 for biochemistry tests and blood routine examination. Blood biochemistry analysis showed that blood urea nitrogen (BUN), creatinine (CREA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels of mice treated with DDP were significantly higher than that of the other two (Fig. 4A–D). There was no significant difference in alkaline phosphatase (ALP) between the DDP group and other groups (Fig. S9). According to the results, the function of the liver and kidneys was obviously damaged in the DDP group. No significant hematological toxicity was caused by Ru1. The mice treated with DDP presented a significant decrease in white blood cell (WBC) count (Fig. 4E), suggesting the occurrence of myelosuppression, which is one of the main side-effects of DDP. The decrease in WBC count was significantly attenuated with Ru1 treatment. Other major indicators of blood routine were not statistically different among these three groups (Fig. S10). We also measured the changes of body weight to evaluate the systemic toxicity of the chemotherapeutic agents. Mice treated with Ru1 presented no significant weight loss compared with the PBS group, whereas the therapy of DDP reduced the weight of mice noticeably (Fig. 4F).
image file: d0dt01877e-f4.tif
Fig. 4 In vivo toxicity and safety studies of DDP and Ru1 in nude mice. Mice were injected with PBS, DDP and Ru1 at a single dosage of 5 mg per kilogram of animal body weight. (A) ALT and (B) AST levels represent liver function. (C) BUN and (D) CREA represent kidney function. (E) WBC counts. Data were expressed as mean ± SD (n = 3). (F) The relative body weight of mice. Data were expressed as mean ± SD (n = 4). (Single asterisks indicate p < 0.05, double asterisks indicate p < 0.01, and triple asterisks indicate p < 0.001.)

Histopathological examination

H&E staining of the major organs showed liver damage caused by DDP, including cell edema, small vacuolar degeneration in the cytoplasm, and pyknosis in the cell nucleus, via our histological observations. There was no abnormality of the renal pathology observed in the DDP group, and there was only renal dysfunction instead of obvious tissue damage. The Ru1 group showed negligible pathological injury in line with the PBS group (Fig. 5A–E). Biochemistry tests, blood routine examination and histological results further confirmed the lower systemic toxicity and better in vivo safety of Ru1.
image file: d0dt01877e-f5.tif
Fig. 5 (A–E) H&E stained histological sections. (A) The heart, (B) liver, (C) spleen, (D) lungs and (E) kidneys of mice from each group were observed. Scale bars: 100 μm.

Experimental section

Materials

Cisplatin was obtained from Dalian Meilun Biotechnology Co. Ltd (Dalian, China). Phosphate Buffered Saline (PBS), Dulbecco's Modified Eagle Medium (DMEM/high glucose) and trypsin-EDTA were purchased from HyClone (USA). Fetal bovine serum (FBS) was obtained from Clarkbio FB25015 (Shanghai, China). Cell counting Kit-8 (CCK-8), Annexin V-FITC Apoptosis Detection Kit, Cell Cycle Detection Kit and Live & Dead Viability Assay Kit were purchased from Jiangsu KeyGEN BioTECH Corp. Ltd (Jiangsu, China). Ethanol and dimethyl sulfoxide (DMSO) were obtained from Sinopharm Chemical Reagent Co. Ltd. BALB/c nude mice were purchased from the Silaike Experimental Animal Centre (Shanghai, China). This study was approved by the Ethics Committee of Tongji University.

Physical measurements

1H NMR spectra were collected at 300 K on a Bruker spectrometer. 1H–1H COSY NMR spectra were recorded on a Bruker AV-400 M spectrometer. Mass spectra were measured on an Agilent TOF-G6230B mass spectrometer. The microanalysis of the elements (C, H, and N) was carried out using a Thermo Flash 2000 analyzer.

Synthesis and characterization

[Ru(phen)2podppz]2+ (Ru1). [Ru(phen)2hdppz]2+ (RA) was prepared and characterized according to the literature procedure.36 Firstly, RA (0.4 mmol, 420 mg) and K2CO3 (0.4 mmol, 55 mg) were suspended in DMF (4 mL). Then, propargyl bromide (0.8 mmol, 66 μL) was added and the reaction mixture was stirred at room temperature for 36 h under argon. Upon the addition of 10 mL of water, the precipitated complex was washed with water (4 × 30 mL) and purified by chromatography over alumina by using MeCN as the eluent; yield: about 55% (236 mg). 1H NMR (600 MHz, CD3CN) δ 9.42 (dd, J = 8.2, 1.0 Hz, 1H), 9.26 (dd, J = 8.2, 0.9 Hz, 1H), 8.71 (dd, J = 8.3, 1.2 Hz, 1H), 8.69–8.65 (m, 3H), 8.40 (dd, J = 5.3, 1.2 Hz, 1H), 8.34–8.30 (m, 4H), 8.27 (dd, J = 5.3, 1.2 Hz, 1H), 8.17 (dd, J = 5.4, 1.2 Hz, 1H), 8.08 (dddd, J = 21.9, 16.4, 6.9, 4.5 Hz, 4H), 7.98 (dd, J = 8.7, 0.9 Hz, 1H), 7.80 (dd, J = 8.2, 5.3 Hz, 1H), 7.75–7.68 (m, 4H), 7.57 (dd, J = 7.8, 0.8 Hz, 1H), 7.51 (dd, J = 8.2, 5.4 Hz, 1H), 5.18 (d, J = 2.4 Hz, 2H), 3.02 (t, J = 2.4 Hz, 1H). TOF-MS for C45H28N8ORu2+: calcl. 798.14, found m/z 399.0804 (M2+/2). Anal. data for C45H28F12N8OP2Ru: calc (%): C, 49.69; H, 2.59; N, 10.30. Found (%): C, 49.55; H, 2.57; N, 10.27.
[Ru(bpy)2podppz]2+ (Ru2). [Ru(bpy)2hdppz]2+ (RB) was prepared and characterized according to the literature procedure.37Ru2 was synthesized similarly to Ru1. RB (0.4 mmol, 400 mg) and K2CO3 (0.4 mmol, 55 mg) were suspended in DMF (4 mL). Then, propargyl bromide (0.8 mmol, 66 μL) was added and the reaction mixture was stirred at room temperature for 36 h under argon. Upon the addition of 10 mL of water, the precipitated complex was washed with water and purified by chromatography over alumina by using MeCN as an eluent; yield: 52.5% (215 mg). 1H NMR (600 MHz, CD3CN) δ 9.42 (dd, J = 8.2, 1.1 Hz, 1H), 9.27 (dd, J = 8.2, 1.0 Hz, 1H), 8.69–8.57 (m, 4H), 8.24 (dd, J = 5.3, 1.2 Hz, 1H), 8.23–8.16 (m, 3H), 8.09 (dddd, J = 16.4, 14.3, 8.2, 4.6 Hz, 3H), 8.00–7.91 (m, 4H), 7.87 (dd, J = 8.2, 5.3 Hz, 1H), 7.78 (dd, J = 5.7, 0.7 Hz, 1H), 7.71 (dd, J = 8.2, 5.3 Hz, 1H), 7.61–7.49 (m, 3H), 7.42 (ddd, J = 7.3, 5.8, 1.2 Hz, 1H), 7.34 (ddd, J = 7.3, 5.8, 1.2 Hz, 1H), 5.17 (d, J = 2.4 Hz, 2H), 3.03 (t, J = 2.4 Hz, 1H). TOF-MS for C41H28N8ORu2+: calcl. 750.14 found m/z 375.07 (M2+/2). Anal. data for C41H28F12N8OP2Ru: calcd (%): C, 47.36; H, 2.71; N, 10.78. Found (%): C, 47.45; H, 2.69; N, 10.81.
[Ru(phen)2ppip]2+ (Ru3). [Ru(phen)2pip]2+ (RC) was similarly obtained according to the literature procedure.38 Firstly, RC (0.4 mmol, 419 mg) and K2CO3 (0.4 mmol, 55 mg) were suspended in DMF (4 mL). Propargyl bromide (0.8 mmol, 66 μL) was then added and the reaction mixture was stirred at room temperature for 48 h under argon. Upon the addition of 10 mL of water, the precipitated complex was washed with water and purified by chromatography over alumina by using MeCN as the eluent; yield: 50% (0.2I7 mg). 1H NMR (600 MHz, DMSO) δ 9.19 (d, J = 8.6 Hz, 1H), 9.10 (d, J = 7.3 Hz, 1H), 8.82–8.76 (m, 4H), 8.41 (d, J = 5.4 Hz, 4H), 8.19 (d, J = 5.3 Hz, 1H), 8.10 (t, J = 5.0 Hz, 4H), 8.08 (d, J = 5.4 Hz, 1H), 7.97–7.94 (m, 2H), 7.90 (dd, J = 8.6, 5.3 Hz, 1H), 7.83–7.71 (m, 8H), 5.62–5.51 (m, 2H), 3.89 (t, J = 2.3 Hz, 1H). TOF-MS for C46H30N8Ru2+: calcl. 796.16, found m/z 398.08 (M2+/2). Anal. data for C46H30F12N8P2Ru: calcd (%): C, 50.88; H, 2.79; N, 10.32. Found (%): C, 50.95, H, 2.80; N, 10.34.
[Ru(bpy)2ppip]2+ (Ru4). [Ru(bpy)2pip]2+ (RD) was prepared and characterized according to the procedure described in the literature.35Ru4 was synthesized similarly to Ru3. RD (0.4 mmol, 400 mg) and K2CO3 (0.4 mmol, 55 mg) were suspended in DMF (4 mL). Propargyl bromide (0.8 mmol, 66 μL) was added and the reaction mixture was stirred at room temperature for 48 h under argon. Upon the addition of 10 mL of water, the precipitated complex was washed with water and purified by chromatography over alumina by using MeCN as an eluent; yield: about 55.8% (223 mg). 1H NMR (600 MHz, DMSO) δ 9.22 (d, J = 8.6 Hz, 1H), 9.14 (d, J = 8.2 Hz, 1H), 8.90 (dd, J = 8.2, 3.9 Hz, 2H), 8.86 (dd, J = 11.0, 8.5 Hz, 2H), 8.24 (t, J = 7.9 Hz, 2H), 8.17–8.09 (m, 4H), 8.02 (dd, J = 8.5, 5.3 Hz, 1H), 7.97–7.93 (m, 3H), 7.87 (t, J = 4.8 Hz, 2H), 7.76–7.71 (m, 3H), 7.66 (d, J = 5.5 Hz, 1H), 7.63–7.58 (m, 3H), 7.36 (dt, J = 17.1, 6.7 Hz, 2H), 5.57 (ddd, J = 48.3, 19.3, 2.2 Hz, 2H), 3.90 (t, J = 2.3 Hz, 1H). TOF-MS for C42H30N8Ru2+: calcl. 748.16, found m/z 374.08 (M2+/2). Anal. data for C42H30F12N8P2Ru: calcd (%): C, 48.61; H, 2.91; N, 10.80. Found (%): C, 48.69; H, 2.89; N, 10.78.

UV-Vis absorption titration

2000 μL solutions of the blank buffer and Ru(II) complexes sample (8 μM) were placed in the reference and sample cuvettes (1.0 cm path length), respectively, and then the first spectrum was recorded in the range of 200–600 nm. During the titration, we added an equal amount of DNA buffer to each cuvette to eliminate the absorbance of the DNA itself, and mixed the solution by inverting it repeatedly. Before recording the absorption spectrum, complex-DNA solutions were incubated for 5 min. We repeated the titration processes until there was no change in the spectrum of at least 4 titrations, demonstrating that binding saturation had been reached. At the end of each titration, the change in the concentration of the Ru(II) complexes due to dilution was negligible.

Cell culture

The human glioblastoma cell line U87 MG cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were incubated in 25 mL of cell culture flask with high glucose DMEM with 10% FBS and 1% penicillin–streptomycin solution. The cells were cultured in an incubator at 37 °C under a humidified atmosphere containing 5% carbon dioxide.

Cytotoxicity assay

The in vitro cytotoxicities of Ru1, Ru2, Ru3, Ru4 and DDP were measured by using the CCK-8 assay. The U87 MG cells were seeded into 96-well plates at a density of 104 cells and 100 μL of DMEM in each well. After incubation for 24 h, tumor cells were treated with different concentrations of Ru1, Ru2, Ru3, Ru4 and DDP for 24 h. 100 μL of DMEM (without FBS) containing 10% CCK-8 was added into each well. After 2 h of culture, the cytotoxicity was determined by using the CCK-8 assay.

Cellular apoptosis

The U87 MG cells were seeded into 6-well plates at a density of 5 × 105 cells and 1 mL of DMEM in each well. After a 24 h incubation, tumor cells were treated with different concentrations of Ru1 and DDP for 24 h. The old DMEM was transferred to a 15 mL centrifuge tube. The cells were washed with PBS and digested with Trypsin Solution without EDTA. After digestion, the cells were transferred into the previous 15 mL centrifuge tube for centrifugation at 2000 rpm for 5 min. After centrifugation, the supernatant was discarded. The cells were washed by PBS twice (centrifuged at 2000 rpm for 5 min) and then collected. 500 μL binding buffer was added to suspend the cells. Subsequently, 5 μL Annexin V-FITC and 5 μL PI were added and mixed at room temperature and out of light for 15 minutes. Flow cytometry was performed within 1 h.

Live & dead viability assay

U87 MG cells were seeded at a density of 1 × 105 cells per well in 6-well plates overnight. The cells were administered with Ru1, PBS and DDP for 24 h. Next, the cells were stained with AM/PI solution (2 μL AM, 8 μL PI, 10 mL PBS) for 30 min and imaged by an inverted fluorescence microscope (AM Ex = 495 nm, Em = 520 nm; PI Ex = 530 nm, Em = 620 nm).

Cell cycle analysis

The U87 MG cells were seeded at a density of 1 × 105 cells per well in 6-well plates and left to adhere overnight. The cells were treated with PBS, DDP and Ru1 for 24 h, after which they were trypsinized, washed, and fixed with 70% alcohol at −4 °C for 12 h. Next, the fixed cells were stained with PI/RNase A staining solution (50μL RNase A 450μL PI) for 45 min at 37 °C in the dark. Flow cytometry was performed within 1 h.

In vivo safety evaluation of DDP and Ru1

To investigate the in vivo toxicity of DDP and Ru1, mice were euthanized and the blood from each group was collected after treatment on day 10 for a routine blood test and biochemistry tests. Meanwhile, the heart, liver, spleen, lungs and kidneys from each group were removed and stained by H&E for histological analysis.

In vivo anti-tumor study

To establish the tumor subcutaneous model, U87 MG cells (5 × 106) were subcutaneously injected in the right back of male nude mice (20–22 g). When the volume of these tumors reached approximately 150 mm3, the mice were randomly divided into three groups (4 in each) and respectively received the intravenous injection of DDP and Ru1 at a dosage of 5 mg kg−1 or PBS. The diameter of the tumors was measured with a vernier caliper every 2 days along with therapy administration. The volumes of the tumors were calculated via the formula: tumor volume = (length) × (width)2/2. Mice body weight was measured every 2 days after treatment. All mice were sacrificed on day 10 and the final tumor weight was monitored directly. The tumors and major organs (the heart, liver, spleen, lungs and kidneys) were excised and stored in 4% paraformaldehyde for histological examination: hematoxylin and eosin (H&E) staining and TUNEL staining.

Histopathological examination

The tumors and organs were embedded in paraffin and the tissue sections of each group were stained with hematoxylin–eosin. The sections of H&E staining were observed by using an optical microscope.

Immunofluorescence histochemical analysis

The tumors and major organs (the heart, liver, spleen, lungs and kidneys) were fixed with 4.0% paraformaldehyde, embedded in paraffin, sectioned and stained with H&E. TUNEL staining was used to detect apoptotic cells according to a previously reported method.

Statistical analysis

Analysis of variance (ANOVA) was performed for all data analysis and values were presented as means ± standard deviation. Differences were considered statistically significant at P < 0.05 (*) P < 0.01 (**) and P < 0.001 (***). All data were analyzed by using SPSS 21.0.

Conclusions

In summary, we have successfully synthesized four new Ru(II) complexes, [Ru(phen)2podppz]2+, [Ru(bpy)2podppz]2+, [Ru(phe-n)2ppip]2+, and [Ru(bpy)2ppip]2+. They were characterized by ESI-MS, 1H NMR, 1H–1H COSY NMR and elemental analysis. Firstly, the anti-tumor experiments in vitro and vivo confirmed that Ru1 exhibited excellent antitumor efficacy on GBM. Furthermore, we confirmed the strongest binding ability and the anti-tumor mechanism of Ru1via DNA binding experiments and cell cycle experiments. Moreover, the study in vivo safety and biocompatibility of Ru1 was investigated and the results demonstrated that Ru1 could avoid any detectable side-effects compared with cisplatin. The results indicate that Ru1 is a very promising drug candidate and provides a new idea for the follow-up treatment of GBM.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Nature Science Foundation of China (21671150, 81860547, 21877084, 81573008), Joint projects of Health and Family Planning Committee of Pudong New Area (PW2017D-10), the Fundamental Research Funds for the Central Universities (kx0150720173382), Science and Technology Commission of Shanghai Municipality Fund (16DZ1930509), Science and Technology Commission of Shanghai Municipality Fund (no. 16DZ1930509), and Shanghai Pudong New Area Science and Technology Development Fund, Innovation Fund (PKJ2017-Y18).

Notes and references

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Footnotes

Electronic supplementary information (ESI) available. See DOI: 10.1039/D0DT01877E
Contribute equally to this work.

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