Structurally Simple Osmium(II) Polypyridyl Complexes as Photosensitizers for Photodynamic Therapy in the Near Infrared.

{"full_text": " Angewandte\n Forschungsartikel Chemie\n www.angewandte.org\n\n Zitierweise: Angew. Chem. Int. Ed. 2023, 62, e202218347\n Medicinal Inorganic Chemistry Internationale Ausgabe: doi.org/10.1002/anie.202218347\n Deutsche Ausgabe: doi.org/10.1002/ange.202218347\n\nStructurally Simple Osmium(II) Polypyridyl Complexes as\nPhotosensitizers for Photodynamic Therapy in the Near Infrared**\nAsma Mani+, Tao Feng+, Albert Gandioso+, Robin Vinck+, Anna Notaro, Lisa Gourdon,\nPierre Burckel, Bruno Saubam\u00e9a, Olivier Blacque, Kevin Cariou, Jamel-Eddine Belgaied,\nHui Chao,* and Gilles Gasser*\n\nIn memory of Dr. Franz Heinemann (1989\u20132022)\n\n\n Abstract: Five osmium(II) polypyridyl complexes of the general formula [Os(4,7-diphenyl-1,10-phenanthroline)2L]2 +\n were synthesized as photosensitizers for photodynamic therapy by varying the nature of the ligand L. Thanks to the\n pronounced \u03c0-extended structure of the ligands and the heavy atom effect provided by the osmium center, these\n complexes exhibit a high absorption in the near-infrared (NIR) region (up to 740 nm), unlike related ruthenium\n complexes. This led to a promising phototoxicity in vitro against cancer cells cultured as 2D cell layers but also in\n multicellular tumor spheroids upon irradiation at 740 nm. The complex [Os(4,7-diphenyl-1,10-phenanthroline)2(2,2\u2019-\n bipyridine)]2 + was found to be the most efficient against various cancer cell lines, with high phototoxicity indexes.\n Experiments on CT26 tumor-bearing BALB/c mice also indicate that the OsII complexes could significantly reduce\n tumor growth following 740 nm laser irradiation. The high phototoxicity in the biological window of this structurally\n simple complex makes it a promising photosensitizer for cancer treatment.\n\n\n\n\nIntroduction to their lack of selectivity for diseased tissues. For this\n reason, many studies were conducted to develop more\nCancer is a multifactorial global issue, assumed to be the efficient and selective alternative treatments, as evidenced\nsecond leading cause of death in the world. According to the by the fact that over 60 % of all current experimental trials\nWorld Health Organization, it was responsible for 9.6 mil- worldwide are focusing on cancer treatment.[3]\nlion deaths in 2018.[1] Different treatments, including radio- Photodynamic therapy (PDT) was discovered as a\ntherapy and chemotherapy, can be used to treat this disease, promising alternative/complementary technique to treat\nquite often in combination with surgery.[2, 3] However, these several types of cancer as well as infectious and skin\nadjuvant treatments usually lead to severe side effects due diseases.[4] It relies on the simultaneous action of a photo-\n\n\n[*] A. Mani,+ A. Gandioso,+ R. Vinck,+ A. Notaro, L. Gourdon, K. Cariou, P. Burckel\n G. Gasser Universit\u00e9 de Paris, Institut de physique du globe de Paris, CNRS\n Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for 75005 Paris (France)\n Life and Health Sciences, Laboratory for Inorganic Chemical B. Saubam\u00e9a\n Biology Cellular and Molecular Imaging Platform, US 25 Inserm, UMS 3612\n 75005 Paris (France) CNRS, Facult\u00e9 de Pharmacie de Paris, Universit\u00e9 Paris Cit\u00e9\n E-mail: gilles.gasser@chimeparistech.psl.eu 75006 Paris (France)\n Homepage: www.gassergroup.com\n O. Blacque\n A. Mani,+ J.-E. Belgaied Department of Chemistry, University of Zurich\n National Institute of Applied Sciences and Technology, Winterthurerstrasse 190, 8057 Zurich (Switzerland)\n Carthage University, EcoChimie Laboratory\n [+] These authors contributed equally to this work.\n Tunis (Tunisia)\n [**]A previous version of this manuscript has been deposited on a\n T. Feng,+ H. Chao\n preprint server (https://doi.org/10.26434/chemrxiv-2022-8gxvj).\n MOE Key Laboratory of Bioinorganic and Synthetic Chemistry,\n School of Chemistry, Sun Yat-Sen University \u00a9 2023 The Authors. Angewandte Chemie published by Wiley-VCH\n Guangzhou, 510006 (P. R. China) GmbH. This is an open access article under the terms of the\n E-mail: ceschh@mail.sysu.edu.cn Creative Commons Attribution Non-Commercial NoDerivs License,\n which permits use and distribution in any medium, provided the\n T. Feng,+ H. Chao\n original work is properly cited, the use is non-commercial and no\n MOE Key Laboratory of Theoretical Organic Chemistry and Func-\n modifications or adaptations are made.\n tional Molecule, School of Chemistry and Chemical Engineering,\n Hunan University of Science and Technology\n Xiangtan, 400201 (P. R. China)\n\nAngew. Chem. 2023, 135, e202218347 (1 of 11) \u00a9 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH\n\f 15213757, 2023, 20, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ange.202218347 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Angewandte\n Forschungsartikel Chemie\n\n\nsensitizer (PS), light and oxygen. Once administrated to the The promising results obtained with ruthenium led us to\npatient, the PS accumulated in the target tissues is exposed focus on other transition metals to further improve their\nto light at a specific wavelength. The PS then reaches a PDT potential. In this endeavor, osmium was shown to be\nrelatively unstable excited singlet state, followed by an the metal of choice to design new polypyridyl complexes\nintersystem crossing from the PS singlet state to its longer- with activation wavelengths in the NIR region.[2]\nlived triplet state.[4\u20137] From the excited triplet state, the Osmium-based complexes have been proposed as DNA-\nchromophore can then decay to the ground state by a binding agents in diagnostic probes for oncology. Some\nvibronic radiation-less relaxation, producing phosphores- osmium polypyridyl complexes containing a dppz (dipyrido-\ncence, or interact with the biological environment by two phenazine) ligand have shown a high luminescence response\ndifferent mechanisms referred to as type I or type II. In a at 750 nm when bound to DNA-quadruplexes.[22] Due to\ntype I mechanism, the exchange of a proton or electron their spectacular luminescent properties, osmium-based\noccurs between the triplet excited state of the PS and the complexes were applied to cancer treatment. Wang et al.\nsurrounding molecular oxygen to generate reactive oxygen have reported new dinuclear polypyridyl osmium complexes\nspecies (ROS), such as the superoxide anion radical (O2 ), *\n for photothermal therapy using a bridging ligand pppp\nthe hydroxyl radical and other free radicals. These ROS are ([1,10]phenanthrolino[5\u2019\u2019,6\u2019\u2019 : 5\u2019,6\u2019]pyrazino[2\u2019,3\u2019 : 5,6]-\npowerful oxidant agents capable of causing oxidative pyrazino [2,3-f][1,10]phenanthroline). The complex [(Os-\ndamage and hence cellular death. Alternatively, the type II (DIP))2pppp]4 + revealed a high photothermal activity upon\nmechanism is favored in oxygenated environments where irradiation at 808 nm toward human melanoma cells.[23]\nthe PS as a triplet state transfers its energy to ground-state McFarland et al. have also reported osmium-based PSs with\nmolecular oxygen (3O2) to produce singlet oxygen (1O2), exceptional photophysical properties. These complexes re-\nknown for its high reactivity leading to the formation of vealed high phototoxicity toward cancer cells upon irradi-\nadducts with organic substrates.[6] With its low lifetime ation at 730 nm.[24] However, this remarkable PDT activity is\n(\u03c4 < 3 \u03bcs), singlet oxygen exhibits a low intracellular diffusion limited by the sophisticated structure of the oligothiophene-\ndistance estimated to be in the range of 2\u20134 \u00d7 10 6 cm2 s 1 derivatized phenanthroline ligand used to prepare these\nleading to a low distance of action ( \ufffd 0.01\u20130.155 \u03bcm).[7],[8] complexes. In addition, no proof of in vivo efficacy was\nThe characteristics of 1O2 impart PDT with a localized established for these complexes.[24] Lazic et al. have also\neffect, making it a highly selective cancer treatment. developed an osmium-based PS called TLD 1829 containing\n Porphyrin-based compounds were the first developed the auxiliary \u03c0-extended 2,2\u2019-biquinoline (biq) ligand. This\nand the most investigated PSs. However, these first-gener- PS demonstrated high in vivo activity against murine colon\nation PSs are often insoluble in water, have poor light cancer after irradiation at 808 nm.[2]\nabsorption and induce prolonged skin photosensitivity The selection of complexes bearing ligands with a\nfollowing treatment.[7] To tackle these problems, in addition simpler structure, such as the DIP, is therefore of high\nto a second generation of organic PSs, metal-based PSs were interest. We have thus decided to develop new osmium-\ndeveloped, using different metals such as RuII, PtII, PtIV, OsII, based PSs, with the potential to be irradiated in the NIR,\nReI or IrIII. Among these, RuII remains undoubtedly the enabling potentially a higher penetration depth during PDT\nmost studied metal in PDT, especially RuII polypyridyl treatments. In this work, we present the synthesis and\ncomplexes, thanks to their tunable photophysical and bio- characterization of five new osmium (II) polypyridyl com-\nlogical properties.[9\u201313] RuII polypyridyl complexes have also plexes 1\u20135 (Figure 1). These complexes were generated from\nemerged as promising agents in Photo-Activated Chemo- a common osmium precursor complex bearing two DIP\nTherapy (PACT) thanks to their photolabile properties.[12, 14] ligands, coordinated to different bipyridine or phenanthro-\nMany Ru-based PSs were evaluated in our group and others, line derivatives: 2,2\u2019-bipyridine (bpy), 4,4\u2019-dimethyl 2,2\u2019-\nespecially coordinated with substituted 1,10-phenanthroline bipyridine (dmbpy), 4,4\u2019-diamine-2,2\u2019-bipyridine (dnbpy),\nand 2,2\u2019-bipyridine ligands, unveiling compounds with prom- 1,10-phenanthroline (phen). The homoleptic complex 5 was\nising photodynamic properties.[4, 9, 15\u201318] RuII based complexes obtained by coordination of the precursor with a third DIP\nusing 4,7-diphenyl-1,10-phenanthroline (DIP) ligand were ligand. The PDT potential of the newly synthesized com-\nefficient as PDT agents upon irradiation at 595 nm.[19] To plexes was then evaluated in vitro against various cancer cell\nensure optimal light penetration in the tissues to treat deep- lines: cervical cancer cells A2780, human and mouse colon\nseated or large tumors, the excitation wavelength of the PS cancer cells (HT29 and CT26). The cellular uptake and\nshould ideally lie in the range of 630\u2013850 nm. RuII com- phototoxicity on 3D multicellular tumor spheroids (MCTS)\nplexes involving a tris-heteroleptic scaffold were used as were also investigated in CT-26 cells. Encouraged by\nimmunoprotective photosensitizers upon irradiation at promising in vitro results, we investigated the efficacy of the\n733 nm.[20] In contrast, using two-photon instead of one- most promising complex (1) in a CT26 tumor-bearing mouse\nphoton irradiation enables the excitation of the complexes model. Our findings suggest that, with their simple structure,\nwith less energetic radiations, and thus at higher wave- these new complexes have the potential to become clinically\nlengths. As such, different polypyridyl complexes based on efficient one-photon NIR PDT PSs.\nRuII or IrII as well as bimetallic RuII-PtII complexes were\nshown to be photoactive in the NIR region, upon two-\nphoton irradiation at up to 830 nm.[21]\n\n\nAngew. Chem. 2023, 135, e202218347 (2 of 11) \u00a9 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH\n\f 15213757, 2023, 20, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ange.202218347 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Angewandte\n Forschungsartikel Chemie\n\n\n\n\nFigure 1. General synthesis route for the osmium complexes 1\u20135.\n\n\n\nResults and Discussion (CH3CN), respectively. Single crystal X-ray diffraction\n studies were carried out.[29] Crystal data, structure refine-\nSynthesis and characterization ment parameters and molecular structures are provided in\n the Supporting Information as well as in Figure 2 and\nThe synthesis of the dichloride osmium complex Os- Figure 3. For complex 4, the Os atom is coordinated to two\n(DIP)2Cl2 has already been reported in previous works, bathophenanthroline ligands and one substituted phenan-\nwithout detailed characterization.[25, 26] However, preventing throline ligand through the N atoms in a distorted octahe-\nthe formation of the byproduct [Os(DIP)3]2 + [27] while dral geometry. For complex 5, the Os is coordinated to three\nrepeating the described procedure appeared challenging. bathophenanthroline ligands through the N atoms in a\nAfter several purification attempts by silica gel chromatog- distorted octahedral geometry.\nraphy, the precursor complex was successfully isolated from\nbyproducts by precipitation from acetone in an ethanol/\nacetone mixture (50 : 1) (v/v). All the ligands were commer- Photophysical properties\ncially available except dnbpy, which was synthesized as\npreviously reported.[28] The final compounds were obtained The UV/Visible spectrum of the different polypyridyl\nby heating the precursor complex at 90 \u00b0C with the complexes was recorded to investigate their electronic\ncorresponding ligand in degassed ethylene glycol. Detailed behavior in the desired phototherapeutic window (i.e. 600\u2013\nprocedures and characterizations are provided in the 850 nm).[30] We note that the baseline for the precursor is\nSupporting Information. The structure of all complexes was not ideal potentially due to the formation of nanoparticles.\nconfirmed by 1H and 13C NMR spectroscopy (Figure S1\u2013 The absorption spectrum of complexes 1\u20135 shows a\nS12) and high-resolution mass spectrometry and their purity panchromatic absorption from 240 to 840 nm, Figure 4. All\nwas evaluated by elemental analysis and HPLC (Fig- five complexes show a similar profile, suggesting that the\nure S13). Complexes 4 and 5 were successfully crystallized accessible electronic transitions, and the ground and excited\nby slow diffusion of diethyl ether in acetone or acetonitrile states between the compounds are qualitatively similar.\n\nAngew. Chem. 2023, 135, e202218347 (3 of 11) \u00a9 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH\n\f 15213757, 2023, 20, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ange.202218347 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Angewandte\n Forschungsartikel Chemie\n\n\n\n\nFigure 2. X-ray molecular structure of complex 4, with hydrogen atoms omitted.\n\n\n\n\nFigure 3. X-ray molecular structure of complex 5, with hydrogen atoms omitted.\n\n\nTheir spectrum is however different from that of their confirms our assumptions.[24, 27] Unlike their ruthenium\nprecursor Os(DIP)2Cl2. Complexes 1\u20135 exhibit three major counterparts, these complexes have therefore the potential\nabsorption bands, as is usually observed for related com- to be phototoxic upon irradiation at wavelengths above\nplexes. They show a sharp and intense peak at 280 nm, 595 nm.[19]\nwhich can be assigned to the IL \u03c0\u03c0* transitions of DIP, two Singlet oxygen (1O2) is the main toxic species for a PS\nbroad peaks (with maxima at ca. 450 and 500 nm, respec- working through the type II mechanism.[33, 34] Therefore, the\ntively) attributed to the Metal to Ligand Charge Transfer production of singlet oxygen upon irradiation of the PSs was\n(MLCT) Os(d\u03c0)- ligand(\u03c0*), and finally a weaker broadband quantitatively evaluated in CH3CN and Phosphate Buffer\ncovering the region 650\u2013750 nm.[25\u201328] This latter can be Saline (PBS) using two methods.[35, 36] As an indirect method,\nexplained by the spin-forbidden MLCT transitions due to the decrease in absorbance of p-nitrosodimethylaniline in\nthe direct singlet-triplet transition of the PS.[2, 31] These the presence of the photosensitizers and imidazole as 1O2\ntransitions are explained by the strong spin-orbit coupling of scavenger was monitored as a function of the irradiation\nosmium, often encountered in heavy atoms.[32] A similar time (450 nm). The results obtained by this indirect method\nresult recently reported on [Os(phen)3]2 + and [Os(DIP)3]2 + were confirmed by a direct method based on the quantifica-\n\nAngew. Chem. 2023, 135, e202218347 (4 of 11) \u00a9 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH\n\f 15213757, 2023, 20, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ange.202218347 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Angewandte\n Forschungsartikel Chemie\n\n\n\n\nFigure 4. UV/Vis/NIR spectra of complexes 1\u20135 and their precursor complex in CH3CN.\n\n\n\ntion of the characteristic luminescence produced by the Despite low 1O2 production yields in polar aqueous\nrelaxation of 1O2.[35, 36] The singlet oxygen quantum yields media, our compounds could still prove to be efficient PSs\nwere then calculated using [Ru(bpy)3]Cl2 and phenalenone since cells include apolar environments. Indeed, it was\nas references in PBS and CH3CN, respectively. previously demonstrated that, upon interaction with hydro-\n Both methods showed that all complexes but 3 efficiently phobic components of the cell such as DNA, PSs that\nsensitize oxygen with quantum yields of 35\u201350 % in aerated otherwise produce a low level of 1O2 in water can efficiently\nCH3CN and 2.6\u20136.2 % in aerated PBS, Table 1. The lower sensitize molecular oxygen.[40]\nyields obtained with complex 3 may be explained by the\npresence of a diamine group, which is able to quench the\nsinglet oxygen, as reported previously.[37, 38] The quenching (Photo-)toxicity\nmechanism is due to a charge transfer between the ground\nstate of a nitrogen-containing compound and singlet To evaluate the (photo-)cytotoxic effect of the synthesized\noxygen.[38] The quantum yield in aqueous media appeared complexes, a screening was performed on cervical cancer\nlower than in CH3CN. The low singlet oxygen production in cells (A2780) and non-cancerous retinal pigment epithelial\naqueous media can be related to the strong quenching (RPE-1) cells. For this purpose, cells were incubated with\nproperties of water.[39] Unfortunately, the singlet oxygen 0.1, 1, or 10 \u03bcM of compounds 1\u20135 in the dark for 4 hours.\nproduction yield could not be determined in deuterated After washing, they were then either kept in the dark or\nwater D2O using the direct method as the luminescence irradiated for 1 h at 620 nm (spectral half-width: 32 nm,\nsignal was under the detection limit. 60 min, 1.88 mW cm 2, 6.7 J cm 2), 645 nm (spectral half-\n width: 32 nm, 60 min, 2.50 mW cm 2, 9.0 J cm 2), 670 nm\n\n\nTable 1: 1\u20135 singlet oxygen quantum yields (\u03a6(1O2)) in CH3CN and aqueous solutions determined by direct and indirect methods upon excitation\nat 450 nm.\n\nCompound CH3CN CH3CN D2O PBS\n\n Direct method [%] Indirect method [%] Direct method [%] Indirect method [%]\n\n1 35 39.7 n.d 6.2\n2 37 41.5 n.d 4.3\n3 9 20.7 n.d 1.7\n4 40 39.9 n.d 5.9\n5 50 43.4 n.d 2.6\n\nn.d. = not determinable.\n\nAngew. Chem. 2023, 135, e202218347 (5 of 11) \u00a9 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH\n\f 15213757, 2023, 20, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ange.202218347 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Angewandte\n Forschungsartikel Chemie\n\n\n(spectral half-width: 32 nm, 60 min, 3.75 mW cm 2, Since complex 1, as a PF6 salt, was revealed to be the\n13.5 J cm 2), 740 nm (spectral half-width: 32 nm, 60 min, most efficient PS at 740 nm, and in view of future in vivo\n3.50 mW cm 2, 12.6 J cm 2). The cell viability was determined experiments in a colon tumor mouse model, an additional\n2 days later using a fluorometric assay. For comparative phototoxicity evaluation was performed on human and\npurposes, the clinically approved PDT photosensitizer mouse colon adenocarcinoma (HT29 and CT26) cells. For\nProtoporphyrin IX (PpIX) was included in this study. this purpose, the PF6 counter ion was exchanged by\n This first screening showed that compounds 1\u20135 have no chloride ions Cl , using an Amberlite IRA-410, to ensure\ncytotoxic effect at up to > 10 \u03bcM in the dark in A2780 and better solubility in biological fluids, mainly later for in vivo\nRPE-1 cells (Figure S20). In contrast, all compounds tests. The results presented in Table 2 show that complex 1\ninduced high phototoxicity upon light exposure at all tested displays similar and high phototoxicity in the two cell lines\nwavelengths in the two cell lines (Figure S21). While (IC50 = 0.33 \ufffd 0.03 \u03bcM and 0.34 \ufffd 0.06 \u03bcM for HT29 and\ncomplexes 1, 3 and 4 led to a potent reduction of cell CT26, respectively). Of note, the same phototoxicity was\nviability at concentrations as low as 1 \u03bcM, complexes 2 and 5 observed with the structurally similar ruthenium complex\nonly revealed their phototoxic effect at 10 \u03bcM. Of note, no [Ru(DIP)2dmbpy]2 + in CT26 cells upon irradiation at\nsignificant selectivity towards cancer cells was observed in 540 nm using the same low light dose (14.2 J cm 2) (IC50 =\nthis preliminary assay. Importantly, we could demonstrate 0.34 \ufffd 0.005 \u03bcM).[19] In contrast, the phototoxicity of the\nthat complexes 1\u20134 were able to have a phototoxic effect at osmium complex [Os(DIP)2(dpp)]2 +, where dpp = 2,3-bis(2-\nup to 740 nm. In light of these promising results, the pyridyl)pyrazine, was previously tested in the rat glioma\nphototoxicity of the complexes was evaluated in more detail. cells F98, following irradiation at 625 nm and showed a\nBecause of their structural similarity with 1 (for 2) and their significantly lower anticancer activity (IC50 = 86.1 \ufffd 8.5 \u03bcM)\nrelatively poor phototoxicity (for 5), both complexes 2 and 5 than that our complex 1.[41]\nwere excluded from further investigations. The concentra- The dinuclear osmium complexes [(Os(DIP))2pppp]4 +,\ntion of complexes 1, 3, and 4 needed to kill 50 % of the cells described previously, demonstrated a high photothermal\n(IC50) was determined following 4 hours of incubation and activity toward human melanoma cells, using higher com-\n1 hour of irradiation at 740 nm or 4 hours of incubation in plex concentrations (\ufffd 10 \u03bcM) upon irradiation at 808 nm.[23]\nthe dark in A2780 and RPE-1 cells, Table 2. All complexes Furthermore, complex 1 with a Cl counterion was found to\nappeared less toxic than PpIX in the dark (IC50 = 3 \ufffd 2 \u03bcM), be cytotoxic after irradiation with near-infrared light in the\nwith 1 and 3 exhibiting the lowest cytotoxicity (IC50 = 58 \ufffd micromolar range in hypoxic conditions (IC50, 740 nm =\n9 \u03bcM and 62 \ufffd 10 \u03bcM, respectively). With the exception of 4.75 \ufffd 0.30 \u03bcM, IC50, dark = 46.44 \ufffd 1.0 \u03bcM, PI = 10) (Fig-\nPpIX, the IC50 of every compound significantly decreased ure S23\u2013S25). Importantly, we could demonstrate that\nupon light irradiation at 740 nm, confirming their potential complex 1 exhibits a phototoxic effect at up to 740 nm, even\nas PDT PSs. Of note, 1 displayed the highest phototoxicity under hypoxic conditions, an important requirement for a\nindex (PI = 118), defined as the ratio between dark toxicity PDT agent.\nand phototoxicity, against A2780 cells. Similar results were The impressive results obtained with complex 1 against\nobtained on healthy RPE-1 cells, confirming the absence of the three tested cell lines A2780, CT-26, and HT29 at\nselectivity towards cancer cells. 740 nm, a NIR wavelength, prompted us to submit it to\n additional experiments before initiating in vivo studies.\n\n\nTable 2: IC50 values in normoxic and hypoxic conditions, in the dark and upon irradiation at 740 nm (spectral half-width: 32 nm, 60 min,\n3.50 mW cm 2, 12.6 J cm 2) for 1, 3, and 4 compared to PpIX on non-cancerous retinal pigment epithelium (RPE-1), cervical cancer (A2780) cells,\nhuman colon cancer (HT29) cells and mouse colon adenocarcinoma (CT-26) cells. Average of three independent measurements.\n\nNormoxic conditions Dark 740 nm (1 h, 12.6 J cm 2) PI\n\nPpIX A2780 3 \ufffd 2 \u03bcM 2.2 \ufffd 0.2 \u03bcM 2\n RPE-1 4 \ufffd 2 \u03bcM 6 \ufffd 2 \u03bcM n.d\n1 A2780 58 \ufffd 9 \u03bcM 0.49 \ufffd 0.09 \u03bcM 118\n RPE-1 47 \ufffd 2 \u03bcM 0.54 \ufffd 0.03 \u03bcM 87\n3 A2780 62 \ufffd 10 \u03bcM 1.3 \ufffd 0.3 \u03bcM 48\n RPE-1 76 \ufffd 9 \u03bcM 2.9 \ufffd 0.6 \u03bcM 27\n4 A2780 19 \ufffd 2 \u03bcM 0.51 \ufffd 0.07 \u03bcM 37\n RPE-1 26.7 \ufffd 0.5 \u03bcM 0.77 \ufffd 0.06 \u03bcM 35\n1* HT29 14 \ufffd 1 \u03bcM 0.33 \ufffd 0.03 \u03bcM 42\n CT26 13.1 \ufffd 0.3 \u03bcM 0.34 \ufffd 0.06 \u03bcM 39\n\n\nHypoxic conditions Dark 740 nm (1 h, 12.6 J cm 2) PI\n\nPpIX CT-26 > 100 \u03bcM 35.5 \ufffd 0.6 \u03bcM > 2.8\n1[a] CT-26 46 \ufffd 1.0 \u03bcM 4.8 \ufffd 0.3 \u03bcM 10\n\nn.d. = not determinable. [a] The counter ion was exchanged from PF6 to chloride Cl .\n\nAngew. Chem. 2023, 135, e202218347 (6 of 11) \u00a9 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH\n\f 15213757, 2023, 20, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ange.202218347 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Angewandte\n Forschungsartikel Chemie\n\n\nPhotostability incorporation of NH2 into the bpy (complex 3) decreases the\n internalization of the complex. Unfortunately, we could not\nAn important parameter to assess is the (photo-)stability of establish a relationship between the accumulation and their\na PS. For this reason, the stability of 1\u20135 was evaluated. The photo-toxicity values because all the complexes described\nstability was first determined in the dark by observing herein present a similar photo-toxicity.\nchanges in the UV/Vis spectrum of the complexes over To get further insight into the internalization mechanism\n48 hours of incubation in different media. This experiment of complex 1, its subcellular localization was determined by\nsuggests that all complexes are stable in CH3CN, PBS, and confocal microscopy using the intrinsic luminescence of our\nDMSO (Figure S15\u2013S17). Of note, complexes were found to OsII complex (\u03bbex = 448 nm, \u03bbem = 645\u2013730 nm). In CT26 cells\nbe poorly soluble in PBS, requiring supplementation with incubated with 5 \u03bcM complex 1 for 4 hours, the lumines-\n1 % DMSO. Since PDT requires light application, the cence appeared as both a diffuse and a punctate signal\nphotocytotoxicity activity of photosensitizer candidates is suggesting the accumulation of the complex in both the\nstrongly influenced by their photostability upon irradiation. cytosol and some vesicular compartments (Figure 5). How-\nFor this reason, we performed photo-stability studies of ever, the complex did not accumulate in the nucleus and\ncomplexes 1\u20135 at 37 \u00b0C in biologically relevant media (i.e. mitochondria as shown by the absence of colocalization with\nPBS and DMEM medium (Gibco) supplemented with 10 % Hoechst 33342 and MitoTracker Green respectively.\nFBS) (Figure S18 and S19) upon irradiation at 740 nm for\n1 h. No significant changes in the absorption spectra of\ncomplexes 1\u20135 were observed in PBS and DMEM after 1 h Phototoxicity on 3D multicellular tumor spheroids (MCTS)\nof irradiation.\n Complex 1 was found to be the most promising candidate\n among the series of osmium complexes investigated in a 2D\nCellular uptake and localization studies cell model. Due to its remarkably high and promising\n photocytotoxicity, we explored its activity in a multicellular\nThe cellular uptake of our osmium complexes 1\u20135 was then tumor spheroid (MCTS) model. In 3D spheroids, MCTS\ninvestigated in CT-26 cells by determining the amount of Os simulates the conditions in clinically treated tumors, includ-\ninside the cells using inductively coupled plasma mass ing an hypoxic environment, and the extracellular matrix\nspectrometry (ICP-MS) after 4 h of incubation at 10 \u03bcM, deposition. Additionally, the growth pattern, metabolism,\n(Figure 5). The Os complex [Os(DIP)3] (PF6)2 (5) was found and gene expression mimic the complexity of the initial\nto have the highest uptake, almost three times higher than stages of solid tumors. These features allow for a reasonable\nthe rest of the complexes. This is probably due to the higher estimation of in vivo antitumor activity, qualifying MCTS as\nlipophilicity of the DIP ligand compared to bpy and phen. a more reliable model than monolayer cell cultures for\nThe uptake of 1 and 2 is also 2-fold higher in comparison to advanced cancer research.[42, 43]0.\ncomplexes 3 and 4. The lower uptake of complexes 3 and 4 An experiment was therefore performed to evaluate the\nmight be due to the lower solubility of these complexes. The time-dependent effect on the growth of MCTSs treated with\n\n\n\n\nFigure 5. (Left) Cellular uptake of compounds 1\u20135 by ICP-MS (Right) Cellular uptake of complex 1 by confocal microscopy. A) Mitochondria-specific\ndye MitoTracker Green (green, exc: 488 nm, em: 513\u2013550 nm) B) Complex 1 (red, exc: 448 nm, em: 645\u2013730 nm) C) Overlay plus nucleus-specific\ndye Hoechst 33342 (blue, exc: 405 nm, em: 409\u2013448 nm) D) Higher magnification of the boxed area in (C).\n\nAngew. Chem. 2023, 135, e202218347 (7 of 11) \u00a9 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH\n\f 15213757, 2023, 20, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ange.202218347 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Angewandte\n Forschungsartikel Chemie\n\n\ncomplex 1. CT26 MCTSs (ca. 550 \u03bcm in diameter) were presence of salt decreases the repulsive electrostatic inter-\ntreated with increasing concentrations of 1 (0,1, 1, 10, 30, action between the RuII complexes, resulting in their\nand 100 \u03bcM). After 36 h of incubation, a luminescence signal aggregation.[45] This aggregation was prevented in culture\nis observed in the spheroids (Figure S22). The medium was medium by the coating effect provided by plasmatic\nthen exchanged with fresh medium, and cells were kept in proteins, leading to the formation of sub-micron\nthe dark or irradiated for 1 h at 740 nm. Following this nanoparticles.[46] As this aggregation behavior could affect\ntreatment, half of the medium in the wells was exchanged the biodistribution of the complexes in vivo, we performed a\nevery two days, and pictures of the spheroids were taken Dynamic Light Scattering (DLS) analysis to investigate the\n(Figure 6). Importantly, CT26 MCTSs treated with the aggregation behaviour of complex 1 with a Cl counterion in\nhighest concentrations of 30 \u03bcM and 100 \u03bcM of complex 1 10 % FBS in PBS. The DLS analysis of 10 % FBS in PBS\nhad a reduced diameter. In contrast, no effect was seen in without the complex revealed the existence of nanometric\nMCTSs treated with the complex and kept in the dark objects with a mean diameter of 8.87 nm, which could\n(100 \u03bcM) in comparison to untreated MCTS. In addition, correspond to serum albumin.[47] In the presence of complex\ncomplex 1 was tested via a luminescent cell viability assay in 1 with a Cl counterion, a subtle shift in the mean diameter\nCT-26 MCTS (single graphs are available in Figure S23). of the objects from 8.87 to 12.68 could be observed\nComplex 1 displayed high cytotoxicity toward CT-26 MCTSs (Figure S14). This small shift could be explained by the\nwith IC50 \ufffd 31 \ufffd 6 \u03bcM. This result is comparable to the IC50 binding of the complex to albumin, as observed previously\nobtained with [Ru(DIP)2dmbpy]2 + in HeLa MCTS.[19] with other drugs.[48] In 100 % PBS, complex 1 with the Cl\n Overall, the outstanding activities shown by complex 1 in counterion tends to form larger particles that sediment,\nthe monolayer cell model were confirmed in an MCTS which can be observed with the naked eye and is confirmed\nmodel. This is of high interest since the center of spheroids by the high polydispersity index obtained by DLS. There-\nis considered hypoxic (i.e., with a low concentration of fore, in contrast to our previously described RuII complexes,\noxygen) and it could be anticipated that our complex would for which nanoparticles of up to 350 nm were observed in\nbe efficient in further experiments in vivo These findings are the presence of plasmatic proteins, complex 1 appears to be\na powerful motivation for further investigation of complex 1 soluble in this medium, probably thanks to its binding to\nas a novel potential photosensitizer agent in photodynamic plasmatic proteins.\ntherapy.\n\n In Vivo Studies\nDynamic Light Scattering (DLS)\n Encouraged by the promising results obtained in vitro with\nWe previously observed that RuII complexes incorporating complex 1 in 2D and 3D models, its PDT efficacy was\ntwo DIP ligands could form aggregates in isotonic aqueous further investigated in a CT26 tumor-bearing BALB/c\nsolutions.[44] While they form large aggregates in PBS, we mouse model. One hour following intratumoral injection,\nrecently showed that they can form smaller nanoparticles in the mice were either kept in the dark or exposed to one-\nthe presence of plasmatic proteins. As it is the case with photon irradiation using a 740 nm laser (50 mW, 12.6 J cm 2,\npositively charged gold nanoparticles, we postulated that the 300 s). The tumor volume and body weight of each mouse\n\n\n\n\nFigure 6. (Left) Changes in the growth kinetics of MCTSs treated with complex 1 at different concentrations (0,1, 1, 10, 30, and 100 \u03bcM). Images\nwere collected on days 0, 2, 4, 7, 9, and 12. (Right) MCTS diameter was measured at different time points. Average of three independent\nmeasurements.\n\nAngew. Chem. 2023, 135, e202218347 (8 of 11) \u00a9 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH\n\f 15213757, 2023, 20, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ange.202218347 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Angewandte\n Forschungsartikel Chemie\n\n\nwas measured and recorded every two days for two weeks. compared to TLD1829 (808 nm, 600 J cm 2, 4 h). Overall,\nAccording to the tumor growth inhibition curve (Figure 7A), this study demonstrates the important potential of complex\nthe tumors treated with complex 1 and light were found to 1 for photodynamic therapy in the biological window.\nbe nearly eradicated within a single procedure. On day 14,\nthe normalized tumor volume in the complex 1 + Light\ngroup was 4.67, 4.56 and 4.36-fold smaller than the control Conclusion\ngroup, the group treated with light only and the group\ntreated with complex 1 without irradiation, respectively In summary, we were able to prepare and characterize\n(Figure 7A, C and D). Importantly, the animals treated with structurally simple OsII polypyridyl complexes bearing two\ncomplex 1 behaved normally, without signs of pain, stress or bathophenanthroline ligands. These complexes showed ex-\ndiscomfort and did not lose weight, suggestive of the high cellent photophysical properties, including high 1O2 produc-\nbiocompatibility of the compound (Figure 7B). After the tion quantum yields. Importantly, they displayed a panchro-\ntreatment, all major organs (i.e., heart, liver, spleen, lung, matic absorption which enables the irradiation of the PS at\nkidney, brain, intestine) as well as the tumor tissues were wavelengths up to 740 nm. This wavelength is much higher\nhistologically examined by the hematoxylin-eosin stain. in comparison to the maximum wavelength at which the\nWhile no pathological alterations or injuries were observed ruthenium analog [Ru(DIP)2dmbpy]2 + can be excited. Cell\nfor all organs (Figure S24), significant damages including experiments on all complexes in non-cancerous retinal\nkaryopyknosis and widespread areas of apoptotic nuclei pigment epithelium (RPE-1) and cervical cancer cells A2780\nwere noticed in the tumor tissue (Figure 7E). Terminal showed no cytotoxicity in the dark and intense toxicity\ndeoxynucleotidyl transferase dUTP nick end labeling (TU- following light irradiation. Importantly, complex 1, with its\nNEL) stain was employed to analyze tumor tissues. The simple structure, was found to have a promising PI value at\ngreen fluorescence signals, indicative of DNA strand breaks 740 nm with low dark toxicity and an IC50 in the nanomolar\nduring apoptosis, for the treatment with complex 1 + Light range following irradiation. It also proved to be extremely\nwere observed, indicative of the strong therapeutic effect. stable and highly phototoxic against human and mouse\nAs discussed in the introduction, the Os-based complex colon cancer cells (HT29 and CT26). The high 1O2\nTLD 1829 showed high survival in murine population production quantum yield and absorption properties of\nbearing colon cancer as well, after irradiation at 808 nm complex 1 endow it with an excellent PDT efficacy in vivo.\n(600 J cm 2, 4 h).[2] TLD 1829 and complex 1 showed Such simple OsII polypyridyl complexes may indeed become\ncomparable in vivo activity with the advantage of a lower promising antitumor therapeutic agents for future clinical\nlight exposure of complex 1 (740 nm, 12.6 J cm 2, 1 h) applications.\n\n\n\n\nFigure 7. A) Average tumor growth curves after the respective treatment (n = 5) Dose: 5.0 mg kg 1; Irradiation (740 nm; 50 mW, 12.6 J cm 2, 300 s).\nB) Time-dependent change in body weight after various treatments on mice. C) Representative pictures of the tumor after the respective\ntreatments. D) Individual tumor growth curves after the respective treatments. E) Haematoxylin-eosin (H&E) stain (scale bar: 200 \u03bcm) and TUNEL\nstain (scale bar: 100 \u03bcm) of the tumor tissues after the respective treatments. *** P < 0.001. Average of five independent measurements.\n\nAngew. Chem. 2023, 135, e202218347 (9 of 11) \u00a9 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH\n\f 15213757, 2023, 20, Downloaded from https://onlinelibrary.wiley.com/doi/10.1002/ange.202218347 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Angewandte\n Forschungsartikel Chemie\n\n\nSupporting Information [3] Z. Abbas, S. Rehman, Neoplasma 2018, 139\u2013157.\n [4] F. Heinemann, J. Karges, G. Gasser, Acc. Chem. Res. 2017, 50,\n1\n H and 13C NMR spectra (Figure S1\u2013S12). HPLC traces 2727\u20132736.\n [5] C. Marie, V. Pierroz, S. Ferrari, G. Gasser, Chem. Sci. 2015, 6,\n(Figure S13). Size distribution by volume (Figure S14).\n 2660\u20132686.\nAbsorption spectra for stability evaluation in the dark\n [6] I. Macdonald, T. Dougherty, J. Porphyrins Phthalocyanines\n(Figure S15\u2013S17). Absorption spectra for photobleaching 2001, 5, 105\u2013109.\nevaluation (Figure S18 and S19). Viability tests (Figure S20 [7] A. O\u2019Connor, W. Gallagher, A. Byrne, Photochem. Photobiol.\nand S21). Fluorescence microscopy of CT-26 spheroids 2009, 85, 1053\u20131074.\ntreated with complex 1 (Figure S22). Fluorometric cell [8] P. Ogilby, Chem. Soc. Rev. 2010, 39, 3181\u20133209.\nviability assay in normoxic conditions in CT26 MCTS after [9] S. Monro, K. Colon, H. Yin, J. Roque, P. Konda, S. Gujar, R.\nirradiation at 740 nm (Figure S23). Fluorometric cell viabil- Thummel, L. Lilge, C. Cameron, S. McFarland, Chem. Rev.\nity assay in hypoxia conditions in CT26 MCTS after 2019, 119, 797\u2013828.\n [10] R. Caspar, C. Cordier, J. Werner, C. Guyard-Duhayon, M.\nirradiation at 740 nm (Figure S24 and S25). Hypoxia cham-\n Gruselle, P. Le Floch, H. Amouri, Inorg. Chem. 2006, 45,\nber (Figure S26). Histological examination of all major 4071\u20134078.\norgans by a hematoxylin-eosin stain (Figure S27). [11] B. Howerton, D. Heidary, E. Glazer, J. Am. Chem. Soc. 2012,\n 134, 8324\u20138327.\n [12] L. Conti, E. Macedi, C. Giorgi, B. Valtancoli, V. Fusi, Coord.\n Chem. Rev. 2022, 469, 214656.\nAcknowledgements [13] L. He, Y. Li, C. Tan, R. Ye, M. Chen, J. Cao, L. Ji, Z. Mao,\nThis work was financially supported by an ERC Consolida- Chem. Sci. 2015, 6, 5409.\n [14] S. Bonnet, Dalton Trans. 2018, 47, 10330\u201310343.\ntor Grant Photo-MedMet to G.G. (GA 681679), has\n [15] S. McFarland, A. Mandel, R. Dumoulin-White, G. Gasser,\nreceived support under the program \u201cInvestissements Curr. Opin. Chem. Biol. 2020, 56, 23\u201327.\nd\u2019Avenir\u201d launched by the French Government and imple- [16] L. McKenzie, H. Bryant, J. Weinstein, Coord. Chem. Rev.\nmented by the ANR with the reference ANR-10-IDEX- 2019, 379, 2\u201329.\n0001-02 PSL (G.G.) and by a Qlife pr\u00e9-maturation funding [17] L. Zeng, P. Gupta, Y. Chen, E. Wang, L. Ji, H. Chao, Z. Chen,\n(G.G. and R.V.). A.G. thanks the ARC Foundation for Chem. Soc. Rev. 2017, 46, 5771\u20135804.\ncancer research for a postdoctoral Research Fellowship. [18] J. Karges, Angew. Chem. Int. Ed. 2022, 61, e202112236.\nPart of the ICP-MS measurements was supported by IPGP [19] J. Karges, F. Heinemann, M. Jakubaszek, F. Maschietto, C.\n Subecz, M. Dotou, R. Vinck, O. Blacque, M. Tharaud, B.\nmultidisciplinary program PARI, and by Paris-IdF region\n Goud, E. Vi\u00f1uelas Zah\u00ednos, B. Spingler, I. Ciofini, G. Gasser,\nSESAME Grant no. 12015908. The authors thank Mathilde\n J. Am. Chem. Soc. 2020, 142, 6578\u20136587.\nChaboud and Dr. Philippe Goldner for their help in the [20] L. Lifshits, J. Roque, P. Konda, S. Monro, H. Cole, D.\ndetermination of singlet oxygen quantum yield as well as von Dohlen, S. Kim, G. Deep, R. Thummel, C. Cameron, S.\nDr. Gregory Lefevre for lending the DLS apparatus. This Gujar, S. McFarland, Chem. Sci. 2020, 11, 11740\u201311762.\nwork was also supported by the National Natural Science [21] J. Karges, H. Chao, G. Gasser, J. Biol. Inorg. Chem. 2020, 25,\nFoundation of China (No. 22120102002), and the Science 1035\u20131050\nand Technology Innovation Program of Hunan Province of [22] K. Peterkov\u00e1, M. Stitch, R. Boota, P. Scattergood, P. Elliot, M.\nChina (No. 2021RC5028). L.G. acknowledges the ENS-PSL Towrie, P. Podbev\u0161ek, J. Plavec, S. Guinn, Chem. Eur. J. 2022,\n 28, e202203250.\nfor her PhD fellowship.\n [23] M. Wang, Y. Deng, Q. Li, S. Tang, R. Yang, R. Zhao, F. Liu,\n X. Ren, D. Zhang, F. Gao, Chem. Commun. 2022, 58, 12676\u2013\n 12679.\nConflict of Interest [24] J. Roque, P. Barrett, H. Cole, L. Lifshits, G. Shi, S. Monro, D.\n von Dohlen, S. Kim, N. Russo, G. Deep, C. Cameron, M.\nThe authors declare no competing financial interests. Alberto, S. McFarland, Chem. Sci. 2020, 11, 9784\u20139806.\n [25] B. Carlson, G. Phelan, W. Kaminsky, L. Dalton, X. Jiang, S.\n Liu, A. Jen, V. Uni, J. Am. Chem. Soc. 2002, 124, 14162\u201314172.\n [26] E. Kober, J. Caspar, P. Sullivan, T. Meyer, Inorg. Chem. 1988,\nData Availability Statement\n 27, 4587\u20134598.\n [27] Y. Wei, M. Zheng, L. Chen, X. Zhou, S. Liu, Dalton Trans.\nThe data that support the findings of this study are available 2019, 48, 11763\u201311771.\nfrom the corresponding author upon reasonable request. [28] O. Maury, J. Gu\u00e9gan, T. Renouard, A. Hilton, P. Dupau, N.\n Sandon, L. Toupet, H. Le Bozec, New J. 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Lux, O. Tillement, J. Renault, P. Serge, F. Wien, S.\n[39] I. Belousova, O. Danilov, V. Kiselev, I. Kislyakov, T. Kris\u2019ko, Lacombe, Int. J. Mol. Sci. 2020, 21, 4673.\n T. Murav\u2019eva, D. Videnichev, Laser Optics 2006: Wavefront [48] T. Hushcha, A. Luik, Y. Naboka, Talanta 2000, 53, 29\u201334.\n Transformation and Laser Beam Control 2007, 6613, 76\u201387.\n[40] C. Mari, V. Pierroz, R. Rubbiani, M. Patra, J. Hess, B. Manuscript received: January 19, 2023\n Spingler, L. Oehninger, J. Schur, I. Ott, L. Salassa, S. Ferrari, Accepted manuscript online: March 14, 2023\n G. Gasser, Chem. Eur. J. 2014, 20, 14421\u201314436. Version of record online: April 12, 2023\n\n\n\n\nAngew. Chem. 2023, 135, e202218347 (11 of 11) \u00a9 2023 The Authors. Angewandte Chemie published by Wiley-VCH GmbH\n\f", "pages_extracted": 11, "text_length": 62724}