Towards Ruthenium(II)‐Rhenium(I) Binuclear Complexes as Photosensitizers for Photodynamic Therapy

The search for new metal‐based photosensitizers (PSs) for anticancer photodynamic therapy (PDT) is a fast‐developing field of research. Knowing that polymetallic complexes bear a high potential as PDT PSs, in this study, we aimed at combining the known photophysical properties of a rhenium(I) tricarbonyl complex and a ruthenium(II) polypyridyl complex to prepare a ruthenium‐rhenium binuclear complex that could act as a PS for anticancer PDT. Herein, we present the synthesis and characterization of such a system and discuss its stability in aqueous solution. In addition, one of our complexes prepared, which localized in mitochondria, was found to have some degree of selectivity towards two types of cancerous cells: human lung carcinoma A549 and human colon colorectal adenocarcinoma HT29, with interesting photo‐index (PI) values of 135.1 and 256.4, respectively, compared to noncancerous retinal pigment epithelium RPE1 cells (22.4).


Introduction
Photodynamic therapy (PDT) is a minimally invasive tumor treatment approach based on the use of a photosensitizer (PS) that is irradiated at a precise wavelength corresponding to the absorption band of the PS.After activation, the PS transfers its energy to molecular oxygen ( 3 O 2 ) to form toxic singlet oxygen ( 1 O 2 ), leading to tumor cell death. [1] TLD 1433 is the most successful representative of this class and has entered Phase II clinical trials against bladder cancer. [6]A significant advantage of metal complexes over classical organic-based PS such as Photofrin or Hematoporphyrin (HpD) is their singlet oxygen production efficiency. [7]Transition metal complexes often benefit from relativistic heavy atom effects.The transition from an excited singlet state (S 1 ) to a triplet state (T 1 ), the so-called inter-system crossing (ISC), is spin-forbidden and therefore unlikely and slow.Spin-orbit coupling (SOC), a phenomenon that occurs in heavy elements, significantly increases the probability of ISC. [8] For these reasons, the combination of a Ru(II) polypyridyl complex and a Re(I) tricarbonyl complex in a single molecule would be beneficial for the design of a PDT PS as it would give a high probability for ISC.[22] For example, Re(CO) 3 Cl-based Ru complex (I) showed that the energy transfer from Re to Ru takes place with nearly 100 % efficiency (Figure 1). [23]Re(bpy)(CO) 3 -based Ru compound (II) exhibited long-lived photoinduced charge-separated states. [24]nother Re(bpy)(CO) 3 -based Ru compound (III) demonstrated that the combination of Ru and Re could change photophysical characteristics of the original Ru compound.Moreover, The excited state energy transfer from the Re(I) chromophore to the Ru(II) was observed. [25]To date, these compounds normally used bisimine ligands such as bipyridine along with halogenbound element (Cl) or a single ligand (pyridine derivatives), which are rarely used in biological fields.Recently, Re-(dppz)(CO) 3 based Ru compound (IV) displayed a significant phototoxicity towards A2740 ovarian cancer cells and selectively accumulated in mitochondria. [26]ith this in mind, in this work, we report the preparation and characterization of the bimetallic complex 5 (see Scheme 1).Importantly, both metal entities would be electronically coupled in order to allow for a potential shift of the absorption spectrum of the resulting complex.To the best of our knowledge, this is the first compound of this class, whose Re atom is coordinated in a tripodal fashion.

Results and Discussion
The synthesis of the Ru(II) complex 3 and the Ru(II)-Re(I) complex 5 investigated in this work is detailed in Scheme 1. Regarding the design of complex 5, we decided to use 4,7diphenyl-1,10-phenanthroline (Bphen) as an auxiliary ligand for the ruthenium in order to allow accessing a better light absorption (i.29][30][31][32] Interestingly, the total charge of the binuclear complex could be easily modulated depending on the Re(I) chelator nature, which allows tuning the solubility of the whole complex.For the Re(I)CO 3 chelating part, we turned our attention towards dipicolylamine (DPA).[Re(I)(CO) 3 DPA] complexes were found to be stable.A strong influence on the ISC within 5 can be expected due to the close proximity of the two metal centers (SOC scales approximately with Z 4 r À 3 , where Z = atomic number and r = distance). [8]Overall, the Bphen ligands together with the overall charge of + 3 for complex 5 should provide a wellbalanced hydro-and lipo-philicity.
Compound 3 was obtained by refluxing ruthenium precursor Ru(Bphen) 2 Cl 2 (2) [33] with the known ligand 1 [34] in MeOH/ H 2 O (1 : 1) for 15 h and was precipitated as its dihexafluorophosphate salt.This novel Ru(II) complex was then reacted with the tricarbonyl Re(I) salt 4 [35] in MeOH at 80 °C to yield complex 5, Figure 1.25][26] which was also precipitated as its hexafluorophosphate salt.Both complexes were characterized by 1 H, 13 C NMR and FT-IR as well as ESI-HRMS (Figures S1-S12).For 5, the expected carbonyl bands in the IR were observed at 2040 cm À 1 and 1920 cm À 1 .Worthy of note, the structure of Ru(II) complex 3 was confirmed by single crystal X-ray crystallography (Figure 2 and Table S1), which grew from acetonitrile/diethyl ether containing a ruthenium(II) dicationic complex, two PF 6 À counterions and two solvents of acetonitrile.One pyridine group is disordered over two sets of positions with site-occupancy factors of 0.143 (10)  and 0.857 (10).One pyridin-2-ylmethyl ligand is also disordered over two sets of positions with site-occupancy factors of 0.273(9) and 0.727 (9).The Ru(II) ion is coordinated by two bathophenanthroline ligands and one substitute phenanthroline ligand through the N atoms in a distorted octahedral geometry.
In order to better understand the photophysical properties of complex 3 and complex 5, the UV-Vis absorption spectrum was recorded (Figure 3a).Compared to Ru(bpy) 3 Cl 2 , complex 3 demonstrated similar distinct photophysical characteristics, which exhibited absorption peaks centered around 290 nm in the UV region, attributed to bipyridine centered transitions.In  the visible region, it displayed an absorption band characteristic of metal to ligand charge transfer (MLCT) centered around 450 nm.The emission spectrum of complex 3 ranges from approximately 540 to 800 nm, indicating its emission properties in the red to near-infrared region (NIR).Complexation with the rhenium did not seem to strongly affect the absorption of the complex as the UV spectrum for complex 5 did not showcase any major differences (see Figure S14).However, an important decrease in the fluorescence intensity was observed (Figure 3b).In order to gain a deeper understanding of the photophysical properties, a series of photostability experiments were conducted using PBS and acetonitrile as solvents.The results (detailed in the Figure S13), revealed that compound 3 demonstrated a relatively stable behavior when exposed to 540 nm irradiation.To assess the effect of the overall charge on the hydrophilicity of complexes, their partition coefficient between PBS and octanol (LogP) was determined (see Figure 3c).Compound 5 demonstrated an improved partition in PBS in comparison to compound 3, thanks to the presence of the Re moiety, which suggested that the overall charge of + 3 of compound 5 could provide an interesting hydro-and lipophilicity balance.
During the analysis of compound 5, we discovered that its stability in solution was far from ideal.Therefore, its stability was first investigated by 1 H NMR spectroscopy by recording 1 H spectra in CD 3 CN at different time points.3 has a characteristic singlet peak at 4.80 ppm in CD 3 CN (Figure 4a), which can be ascribed to the four H atoms of the two CH 2 groups of the DPA chelator.Due to the linkage of N to Re, the two CH 2 groups of 5 displayed distinct shifts at 5.33 and 5.76 ppm, respectively (Figure 4b).However, when 5 was placed in CD 3 CN for 12 h, some changes occurred in the NMR spectrum, with three peaks appearing in the same region (5.34 and 5.76 ppm belong to 5, 4.72 ppm belong to 3, Figure 4c).Furthermore, the integral ratio of CH 2 in complex 3 and CH 2 of complex 5 is 1 : 5.2, it proves that 16 % of complex 5 was transformed into complex 3 after 12 h, which suggested that there are some issues with the stability of 5 with regards to the Re(I) complexation, which can be slowly converted back to 3 if kept in solution.
In order to have further insight into the stability of 5, HPLC was used to monitor the stability of complexes 3 and 5.The retention time of 3 is 11.369 min (Figure 5a) while the one of 5 is 10.456 min (Figure 5b).Some degradation of compound 5 in CH 3 CN was indeed observed after 16 h, as indicated by the appearance of a peak 11.269 min (Figure 5c).To ascertain that the measured peak was actually 3, pure 3 was added to the solution, which confirmed our hypothesis (Figure 5d).Furthermore, after analyzing the integrated areas of 10.395 and 11.268 in Figure 5c and Table S2, it was demonstrated that ca.20 % of compound 5 was converted into compound 3 after 16 h.Overall, this additional experiment suggests that 5 is not very stable and is slowly converted by to 3 in solution.This observation is consistent with that observed by NMR.Due to the stability issue of 5, we decided to only test the biological activity of 3. The phototoxicity of complex 3 was assessed by incubating the complex with human lung carcinoma A549 and human colon colorectal adenocarcinoma HT29 cells, as well as non-cancerous retinal pigment epithelium RPE1 cells.After incubation, the cells were irradiated with light at a wavelength of 540 nm for 40 minutes.Additionally, the toxicity of the complex in the absence of light (dark condition) was evaluated by incubating the cells with the complex for 4 hours without subsequent light exposure.The phototoxicity index (PI) is derived from the ratio of the IC 50 (concentration that causes 50 % cell death) in the dark to the IC 50 following light irradiation.It quantifies the relative increase in cell-killing effectiveness of the complex when exposed to light.Protoprophyrin IX (PPIX), a clinically-approved PDT PS, was used as the positive control for comparison purposes.As shown in Table 1 and Figure S15-17, complex 3 was found to be non-toxic in the dark (> 100 μM).However, complex 3 exhibited notable and comparable phototoxicity against cancer cell lines HT29 and A549, with submicromolar IC 50 values of 0.39 μM and 0.74 μM, respectively.These values were 6 to 12 times lower than the phototoxic IC 50 value observed on the normal cell line RPE1, indicating a favorable selectivity towards cancer cells.Very interestingly, complex 3 was significantly more phototoxic to cancer cells than PPIX, while less phototoxic to normal cells.The PI values of 135.2 for A549 and 256.4 for HT29 are interesting.These results highlight the promising photodynamic properties of complex 3 for targeting cancer cells.
These very exciting results prompted us to further investigate the internalization mechanism of complex 3. Its subcellular localization was determined by confocal microscopy using the intrinsic luminescence of Ru(II) compound.A549 cells were co-incubated with complex 3 for 4 h, followed by the  addition of a cell membrane targeting tracker (Green Mask) and a mitochondrial targeting tracker (MTG).This allowed for monitoring the localization of the compound within the cells.The results depicted in Figure 6 and Figure S18 clearly indicated internalization of complex 3 within the cells.The colocalization coefficients between complex 3 and the cell membrane/ mitochondria were measured as 0.14 and 0.59, respectively.These coefficients suggested that compound 3 primarily localized within the mitochondria of the cells.

Conclusions
In this work, we designed and characterized a Ru(II) complex 3 that bears a dipicolylamine moiety to allow chelation of a second metallic center.The Ru(II)-Re(I) complex 5 was thus prepared and characterized.During the course of this work, we found out that this dimetallic complex did not exhibit suitable stability in solution for use in biological systems as decoordination of the rhenium was observed.Yet, the parent Ru(II) complex (3) itself was further studied in terms of its biological activity.Complex 3 demonstrated selective phototoxicity towards A549 and HT29 cancer cells compared to normal cells, with excellent phototoxicity indexes that surpass those of the approved drug PPIX.This complex was also found to localize preferentially in the mitochondria, an organelle that plays a crucial role in various cellular processes.These findings provide valuable insights into the development of targeted photosensitizers for photodynamic therapy and in particular pave the way for further studies to investigate: 1. the role of the dipicolylamine moiety in the biological properties of 3 and 2. the possibility to construct more stable bimetallic scaffolds using this chelating moiety.

X-ray diffraction study
A single crystal of compound 3 was analysed by X-ray diffraction at 160(1) K on a Rigaku OD XtaLAB Synergy, Dualflex, Pilatus 200 K diffractometer using a single wavelength X-ray source (Cu K α radiation: λ = 1.54184Å) from a micro-focus sealed X-ray tube and an Oxford liquid-nitrogen Cryostream cooler.The selected suitable crystal was mounted using polybutene oil on a flexible loop fixed on a goniometer head and immediately transferred to the diffractometer.Pre-experiment, data collection, data reduction, and analytical absorption correction [37] were performed with the program suite CrysAlisPro. [38]Using Olex2, [39] the structure was solved with the SHELXT [40] small molecule structure solution program and refined with the SHELXL2018/3 program package [41] by full-matrix least-squares minimization on F. [38] PLATON [42] was used to check the result of the X-ray analysis.Deposition Number 2269280 contains the supplementary crystallographic data for this paper.These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.

Octanol/water partition coefficient (log P o/w ) measurements
Octanol (1 mL) and PBS (1 mL) were mixed by continuous shaking at room temperature for 24 h.The complexes (50 μM) were then dissolved in 1 mL octanol, and the same volume of PBS was added.The mixture was continually agitated for another 24 h before the two layers are separated after settling down.The concentrations of the complex in the two phases were measured by UV-Vis spectroscopy.

Cell culture
Human lung carcinoma (A549) cells were grown using F12 media, human colon colorectal adenocarcinoma (HT29) cells were used by McCoy's 5 A Medium, and retinal pigment epithelium (RPE1) cells using DMEM/F-12 medium supplemented with 10 % FBS and 1 % Penstrep.The cells were cultivated and maintained in a cell culture incubator at 37 °C with 5 % CO 2 and 20 % O 2 atmosphere.Before an experiment, the cells were passaged three times.

Phototoxicity
The phototoxicity of the complexes was assessed by measuring the cell viability using a fluorometric resazurin assay.The cultivated cells were seeded in sextuplicate in 96 well plates with a density of 4000 cells per well in 100μL of media.After 24h, the medium was removed and the cells were treated with increasing concentrations of the complex diluted in cell media achieving a total volume of 100 μL at 37 °C with 5 % CO2 and 20 % O2 atmosphere for 4 h.Then the media was replaced with 100 μL fresh medium, plates were irradiated at light wavelength (540 nm for 40 min (light dose 1.2 mW) at 37 °C using a LUMOS-BIO photoreactor (Atlas Photonics).As a control, a plate was kept in the dark for 40 min at the same condition.Then cells were cultivated for another 44 h, the media was changed with fresh media containing resazurin with a final concentration of 0.2 mg/mL.After 4 h of incubation at 37 °C, the fluorescence signal of resorufin product was measured (λ ex : 540 nm and λ em : 590 nm) in a BioTek®.IC 50 values were then calculated using GraphPad Prism software.Each experiment was performed in duplicate and an average IC 50 value (in μM) was reported with a standard deviation.

Cellular uptake
CT26 cells (1×10 5 cells) were seeded in 35 mm culture dishes for 24 h.Cell medium was then replaced by fresh medium containing 5 μM of Complex 3.After incubation for 4 h in the dark at 37 °C, cells were washed with PBS.Cells were then stained with Hoechst 33342 (1 μg/mL), and Green Mask (100 nM) or MTG (100 nM) at room temperature for 10 min.Live cells were imaged in a confocal laser scanning microscope (Leica SP8) equipped with an x63/1.40plan apochromat objective.The excitation/emission wavelengths were 405/420-450 nm (Hoechst), 488/670-800 nm (Complex 3), and 488/500-550 nm (Green Mask and MTG).Care was taken to use laser intensities as low as possible to avoid any phototoxicity.

Figure 2 .
Figure 2. Molecular structure of one of the two dicationic species present in the asymmetric unit of 3. The disorder and all hydrogen atoms are omitted for clarity.