Ruthenium(II) Polypyridyl Complexes Containing COUBPY Ligands as Potent Photosensitizers for the Efficient Phototherapy of Hypoxic Tumors

Abstract

Hypoxia, a hallmark of many solid tumors, is linked to increased cancer aggressiveness, metastasis, and resistance to conventional therapies, leading to poor patient outcomes. This challenges the efficiency of photodynamic therapy (PDT), which relies on the generation of cytotoxic reactive oxygen species (ROS) through the irradiation of a photosensitizer (PS), a process partially dependent on oxygen levels. In this work, we introduce a novel family of potent PSs based on ruthenium(II) polypyridyl complexes with 2,2'-bipyridyl ligands derived from COUPY coumarins, termed COUBPYs. Ru-COUBPY complexes exhibit outstanding in vitro cytotoxicity against CT-26 cancer cells when irradiated with light within the phototherapeutic window, achieving nanomolar potency in both normoxic and hypoxic conditions while remaining nontoxic in the dark, leading to impressive phototoxic indices (>30,000). Their ability to generate both Type I and Type II ROS underpins their exceptional PDT efficiency. The lead compound of this study, SCV49, shows a favorable in vivo pharmacokinetic profile, excellent toxicological tolerability, and potent tumor growth inhibition in mice bearing subcutaneous CT-26 tumors at doses as low as 3 mg/kg upon irradiation with deep-red light (660 nm). These results allow us to propose SCV49 as a strong candidate for further preclinical development, particularly for treating large hypoxic solid tumors.

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Rational design, synthesis, and characterization of Ru-COUBPY complexes. (A) Design of the COUBPY ligands and of the corresponding Ru­(II) polypyridyl complexes. (B) Synthetic route for the preparation of COUBPY ligands 1–3 and Ru-COUBPY complexes SCV42, SCV45, and SCV49. Reagents and conditions: (a) (1) LDA, THF, – 78 °C, 1 h, (2) TMSCl, – 78 °C, 10 s, (3) EtOH, – 78 °C to rt, 1 h, 76%; (b) (CCl₃)₂, CsF, ACN, 60 °C, 3.5 h, 57%; (c) KCN, 18-crown-6, ACN, rt to 50 °C, overnight, 64%; (d) (1) NaH, 6, ACN, rt, 3 h, (2) AgNO₃, rt, 2 h, 20–75%; (e) [Ru­(bpy)₂Cl₂], EtOH-H₂O (3:1), 80 °C, overnight, 62–93%. (C) Ground-state geometries of Ru-COUBPY complexes in ACN optimized by the PBE0/6-31+G­(d,p)/SDD method in ACN.

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Photophysical characterization of Ru-COUBPY complexes. (A) Absorption (left panel) and emission (λ_exc = 460 nm) (right panel) spectra of the Ru-COUBPY complexes in ACN. (B) Photostability of the complexes in supplemented cell culture medium at 37 °C after irradiation with green (λ₁ = 505 ± 35 nm, 100 mW cm^–2) or red (λ₂ = 620 ± 15 nm; 130 mW cm^–2) light. C ₀ and C_t represent the concentration of the compound at the beginning of the experiment (t = 0) and at various time points throughout the experiment, respectively. (C) Photographic images of Ru-COUBPY complex solutions (50 μM) in DCM under daylight (left panel) and in the dark (right panel) upon irradiation with a blue laser (405 nm).

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Photogeneration of ROS by Ru-COUBPY complexes studied using specific fluorogenic probes (A–C) and EPR spectroscopy (D–E). Left panels: Increase in fluorescence emission of probes SOSG (5 μM) (A), HPF (5 μM) (B), and DHR123 (10 μM) (C) occurred upon irradiation of Ru-COUBPY complexes (10 μM) in PBS (2% DMSO). Right panels: EPR spectra of Ru-COUBPY complexes trapped by 4-amino-TEMP (D) or DMPO (E) in MeOH, measured in the dark and after green light irradiation.

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Cellular uptake studies of Ru-COUBPY complexes in living HeLa cells by confocal microscopy. Single confocal planes of HeLa cells incubated with the compounds SCV42 (top panel), SCV45 (center panel), and SCV49 (bottom panel) for 30 min (10 μM) at 37 °C, imaged at t = 0 and after 2 min of first observation. Excitation was performed with a 514 nm laser line. White arrows point out mitochondria and white arrowheads point out vesicle staining. Black arrowheads on the right column indicate cell blebbings. Scale bar: 20 μm. LUT for fluorescence images: Fire. Intensity calibration bars are shown in the left central column. Left and right columns: merge of compound and brightfield images.

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Colocalization studies of SCV42 in living HeLa cells by confocal microscopy. Single confocal planes of HeLa cells incubated with the SCV42 compound (10 μM, green) and Mitoview 650 (0.1 μM, red), or Lysoview 633 (1×, red). Left panel: Merge of the two staining. Center panel: SCV42 signal. Right panel: Mitoview (top) or Lysoview (bottom) signal. White arrows and arrowheads indicate positive and negative colocalization, respectively. Scale bar: 20 μm.

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In vitro photobiological characterization of Ru-COUBPY complexes in CT-26 2D monolayer cell cultures and 3D multicellular tumor spheroid (MCTS) models. (A) Dose–response curves for SCV42 (green), SCV45 (orange), SCV49 (purple), and PpIX (blue) in CT-26 cells, after 4 h of incubation, upon deep-red light (645 nm, 9.0 J cm^–2) irradiation (filled symbols) or in the dark (unfilled symbols) under normoxic conditions. (B) Activity plots illustrating the chromatic (photo)­cytotoxicity screening of compounds SCV42 and SCV49 in CT-26 cells under green (540 nm, 9.0 J cm^–2), deep-red (645 nm, 9.0 J cm^–2), far-red (670 nm, 13.5 J cm^–2), and NIR (740 nm, 12.6 J cm^–2) light irradiation, as well as in the dark, under normoxic (21% O₂) and hypoxic (2% O₂) conditions. The plots highlight IC₅₀ values (left panel) and phototherapeutic indexes (PIs) (right panel). Detailed IC₅₀ values with standard deviations and corresponding PI values are provided in Table . (C) Evolution of the CT-26 MCTS diameter over a 9-day period. On day 3, MCTSs were treated with varying concentrations of SCV49 (0.1 to 100 μM) or drug-free cell culture medium (n.t.) for 36 h in the dark, followed by 1 h of deep-red light (645 nm, 9.0 J cm^–2) irradiation. Data are presented as mean ± SD from three replicates. Statistical significance on day 9 was determined using one-way ANOVA followed by Bonferroni’s multiple comparison test (Asterisks: **p < 0.02, ***p < 0.002). (D) Brightfield micrographs of CT-26 MCTS treated with SCV49 (100 μM) or drug-free cell culture medium (nt) for 36 h, followed by 1 h of deep-red light (645 nm, 9.0 J cm^–2) irradiation. Scale bar: 1 mm.

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In vivo pharmacokinetic (PK) and toxicological evaluation of SCV49 in healthy CD1 mice. PK includes: (A) Plasma concentration–time curve, and (B) biodistribution profile of ruthenium (Ru) in major organs quantified by ICP-MS at various time points following IP administration of SCV49 at 5 mg/kg. Data are presented as mean ± SD (n = 3 males). Toxicological evaluation includes: (C) Body weight (g) and (D) food intake (g/animal) of mice treated IP with vehicle or SCV49 (10 or 30 mg/kg) on day 1, with sacrifice on day 5. Data are presented as mean ± SD (n = 3 males, n = 3 females).

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In vivo evaluation of SCV49 PDT antitumor efficacy in a subcutaneous CT-26 tumor model in BALB/c mice. (A) Experimental design: Eight-week-old female BALB/c mice were injected subcutaneously with 1.15 × 10⁶ CT-26 cells on day −10. By day 0, when tumors reached 50–100 mm³, mice were randomly divided into 7 groups (n = 5/group, Table ). On days 1 and 3, each group received the assigned treatment and was either exposed to light irradiation or not (660 nm, 15 or 20 min, Table , 100 mW/cm²). On day 9, animals were sacrificed, and organs and blood samples were collected. (B) Body weight (g) and (C) relative tumor volume (RTV) curves of mice over the 9-day study period. (D) Average tumor weights of mice on the day of sacrifice. Data are presented as mean ± SEM (n = 5 females). RTV values on day 9, and average tumor weight data were analyzed using a one-way ANOVA followed by Bonferroni’s multiple comparison test (Asterisks: * p < 0.05, ** p < 0.001). (E) Representative images of tumors from mice in group G2 (vehicle control, light 2x) and group G7 (SCV49, 6 mg/kg, light 2×) at the study endpoint.