Insights into the anticancer photodynamic activity of Ir(III) and Ru(II) polypyridyl complexes bearing β-carboline ligands.

{"full_text": " European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\n Contents lists available at ScienceDirect\n\n\n European Journal of Medicinal Chemistry\n journal homepage: www.elsevier.com/locate/ejmech\n\n\nResearch paper\n\nInsights into the anticancer photodynamic activity of Ir(III) and Ru(II)\npolypyridyl complexes bearing \u03b2-carboline ligands\nJuan Sanz-Villafruela a, 1 , Cristina Bermejo-Casadesus b, 1 , Elisenda Zafon b, 1 ,\nMarta Mart\u00ednez-Alonso a , Gema Dura\u0301 c , Aranzazu Heras a , Iva\u0301n Soriano-D\u00edaz d ,\nAngelo Giussani d , Enrique Ort\u00ed d, ** , Francesc Tebar e, *** , Gustavo Espino a, **** ,\nAnna Massaguer b, *\na\n Universidad de Burgos, Departamento de Qu\u00edmica, Facultad de Ciencias, Plaza Misael Ban\u0303uelos S/n, 09001, Burgos, Spain\nb\n Universitat de Girona, Departament de Biologia, Facultat de Cie\u0300ncies, Maria Aurelia Capmany 40, 17003, Girona, Spain\nc\n Universidad de Castilla-La Mancha, Departamento de Qu\u00edmica Inorga\u0301nica, Orga\u0301nica y Bioqu\u00edmica. Facultad de Qu\u00edmicas, Avda. Camilo J. Cela 10, 13071, Ciudad\nReal, Spain\nd\n Instituto de Ciencia Molecular, Universidad de Valencia, Catedra\u0301tico Jose\u0301 Beltra\u0301n 2, 46980, Paterna, Spain\ne\n Departament de Biomedicina, Unitat de Biologia Cel\u22c5lular, Facultat de Medicina i Cie\u0300ncies de la Salut, Centre de Recerca Biome\u0300dica CELLEX, Institut d\u2019Investigacions\nBiome\u0300diques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, 08036, Barcelona, Spain\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: Ir(III) and Ru(II) polypyridyl complexes are promising photosensitizers (PSs) for photodynamic therapy (PDT)\nCancer due to their outstanding photophysical properties. Herein, one series of cyclometallated Ir(III) complexes and\nPhotodynamic therapy two series of Ru(II) polypyridyl derivatives bearing three different thiazolyl-\u03b2-carboline N^N\u2032 ligands have been\nCyclometalated complexes\n synthesized, aiming to evaluate the impact of the different metal fragments ([Ir(C^N)2]+ or [Ru(N^N)2]2+) and\nMitochondria\n N^N\u2019 ligands on the photophysical and biological properties. All the compounds exhibit remarkable photo\u00ad\n stability under blue-light irradiation and are emissive (605 < \u03bbem < 720 nm), with the Ru(II) derivatives dis\u00ad\n playing higher photoluminescence quantum yields and longer excited state lifetimes. The Ir PSs display pKa\n values between 5.9 and 7.9, whereas their Ru counterparts are less acidic (pKa > 9.3). The presence of the\n deprotonated form in the Ir-PSs favours the generation of reactive oxygen species (ROS) since, according to\n theoretical calculations, it features a low-lying ligand-centered triplet excited state (T1 = 3LC) with a long\n lifetime. All compounds have demonstrated anticancer activity. Ir(III) complexes 1\u20133 exhibit the highest cyto\u00ad\n toxicity in dark conditions, comparable to cisplatin. Their activity is notably enhanced by blue-light irradiation,\n resulting in nanomolar IC50 values and phototoxicity indexes (PIs) between 70 and 201 in different cancer cell\n lines. The Ir(III) PSs are also activated by green (with PI between 16 and 19.2) and red light in the case of\n complex 3 (PI = 8.5). Their antitumor efficacy is confirmed by clonogenic assays and using spheroid models. The\n Ir(III) complexes rapidly enter cells, accumulating in mitochondria and lysosomes. Upon photoactivation, they\n generate ROS, leading to mitochondrial dysfunction and lysosomal damage and ultimately cell apoptosis.\n Additionally, they inhibit cancer cell migration, a crucial step in metastasis. In contrast, Ru(II) complex 6 exhibits\n moderate mitochondrial activity. Overall, Ir(III) complexes 1\u20133 show potential for selective light-controlled\n cancer treatment, providing an alternative mechanism to chemotherapy and the ability to inhibit lethal can\u00ad\n cer cell dissemination.\n\n\n\n\n * Corresponding author.\n ** Corresponding author.\n *** Corresponding author.\n **** Corresponding author.\n E-mail addresses: enrique.orti@uv.es (E. Ort\u00ed), tebar@ub.edu (F. Tebar), gespino@ubu.es (G. Espino), anna.massaguer@udg.edu (A. Massaguer).\n 1\n Equally contributed.\n\nhttps://doi.org/10.1016/j.ejmech.2024.116618\nReceived 24 January 2024; Received in revised form 31 May 2024; Accepted 22 June 2024\nAvailable online 28 June 2024\n0223-5234/\u00a9 2024 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC license\n(http://creativecommons.org/licenses/by-nc/4.0/).\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\n\n\n Abbreviations IC50 Half-maximal inhibitory concentration\n ICP:MS Inductively coupled plasma mass spectrometry\n 2D Two-dimensional LC Ligand centered\n AO Acridine orange LBPA Lysobisphosphatidic acid\n ATP Adenosine triphosphate LEs/Lys Late endosomes/lysosomes\n ATCC American type culture collection LLCT Ligand-to-ligand charge transfer\n BSA Bovine serum albumin MMP Mitochondrial membrane potential\n CCCP Carbonyl cyanide 3-chlorophenylhydrazone MLCT Metal-to-ligand charge transfer\n CPCM Conductor-like polarizable continuum model MTT 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium\n CT Charge transfer bromide\n DCF: 2\u2032,7\u2032 dichlorofluorescein NTOs Natural transition orbitals\n DCFDA 2\u2032,7\u2032 dichlorofluorescein diacetate PBS Phosphate buffered saline;\n DFT Density functional theory PEI Polyethylenimine;\n DCM Dichloromethane PES Potential energy surface\n DMEM Dulbecco\u2019s modified Eagle\u2019s medium PI Phototoxicity index\n DMSO Dimethyl sulfoxide PDT Photodynamic therapy\n EEA1 Early endosomal antigen 1 PrI Propidium Iodide;\n ECACC European collection of authenticated cell cultures PS Photosensitizer\n EEs Early endosomes RBC Red blood cells\n FBS Fetal bovine serum ROS Reactive oxygen species\n GFP Green fluorescent protein SD Standard deviation\n NMR Nuclear magnetic resonance TAP 1,4,5,8-tetraazaphenanthrene\n HR-MS High resolution mass spectrometry Tom20 Translocase of the outer membrane subunit 20\n\n\n\n1. Introduction one/two-photon excitation properties, and finally they also present\n excellent abilities to generate ROS in a photocatalytic manner [8]. These\n Chemotherapy is a fundamental component in the treatment of two families of metal complexes also show some differential properties.\ncancer, either used as a monotherapy or as an adjunct to surgery and Thus, the dipositive Ru(II) derivatives tend to exhibit higher water sol\u00ad\nradiotherapy. The discovery of cisplatin as an anticancer drug revealed ubility and wider absorption profiles in the visible region [9,10]. In\nthe potential of metal-based complexes as chemotherapeutic agents. contrast, the monopositive Ir(III) counterparts commonly display lower\nToday, platinum-based metal complexes such as cisplatin, oxaliplatin, water solubility and inferior absorption in the visible region, but they\nand carboplatin are extensively used in clinical oncology [1]. However, offer excellent photoluminescent quantum yields, large Stokes shifts,\nsome cancer types exhibit resistance to platinum-based treatments, and and longer excited-state lifetimes due to the heavy-atom effect (large\ntheir efficacy in sensitive cancers is often restricted by side effects and spin-orbit coupling constant of Ir) [11]. Indeed, polypyridine Ir(III) and\nthe emergence of drug resistance [2]. Moreover, platinum compounds Ru(II) metal complexes have demonstrated biological activity against\nhave limited effectiveness in treating metastasis, which accounts for various cellular organelles depending on their molecular structure. In\nover 90 % of cancer-related deaths [3]. Consequently, there is great particular, complexes fitted with a combination of cationic charge and\ninterest in developing new chemotherapeutic agents with novel mech\u00ad lipophilicity exhibit preferential accumulation within mitochondria,\nanisms of action aiming to reduce adverse effects, overcome multidrug due to the high membrane electric potential across the mitochondrial\nresistance, and exhibit antimetastatic activity. In this regard, photody\u00ad inner membrane, which is generated by the proton gradient during the\nnamic therapy (PDT) with metal-based photosensitizers has emerged as electronic chain transport [12\u201315].\na promising alternative. PDT is a clinically approved two-stage pro\u00ad Mitochondria have emerged as appealing targets for cancer treat\u00ad\ncedure that involves the administration of a photosensitizer (PS) fol\u00ad ment due to their central role in cellular metabolism and bioenergetics,\nlowed by its controlled activation in the tumors by light irradiation. programmed cell death regulation, and redox balance [16]. Cancer cells\nWhen photoactivated, the PS becomes electronically excited and dis\u00ad depend to a high extend on mitochondrial metabolism to proliferate and\nplays photocatalytic activity in different types of reactions with cellular survive, as it provides the major source of energy in form of ATP for\noxygen (O2). This process leads to the generation of reactive oxygen tumour progression and supplies the metabolic intermediates for mac\u00ad\nspecies (ROS) that damage essential biomolecules and ultimately cause romolecules biosynthesis. Furthermore, mitochondria are implicated in\ncancer cells death [4,5]. Moreover, PDT can activate an anti-cancer different steps of metastasis, including motility, invasion, plasticity, and\nimmune response [6]. The localized activation of PSs within tumors colonization [17,18]. Different mitochondrial-targeted agents have\ntherefore enables selective targeting of cancer cells while minimizing shown promising results for cancer treatment when applied alone or in\ndamage to healthy tissues. Furthermore, since ROS act within a limited combination with other chemotherapeutic agents [19,20]. In cancer\nrange of a few nanometers, it is possible to direct their activity towards cells, the mitochondria exhibit varying degrees of dysfunction, such as\nspecific subcellular organelles, such as mitochondria or lysosomes, increased mitochondrial membrane potential (MMP) and elevated pro\u00ad\nthrough careful molecular design [7]. duction of ROS [21], which significantly increase their susceptibility to\n Ru(II) complexes of formula [Ru(bpy)2(N^N)]A2 (bpy = 2,2\u2032-bipyr\u00ad the photodynamic effects of cationic polypyridine metal complexes.\nidine, N^N = diimine ligands, A = counteranion) and Ir(III) biscyclo\u00ad Lysosomes are also validated targets for anticancer therapy [22,23].\nmetalated complexes of formula [Ir(ppy)2(N^N)]A (ppy = 2- They are membrane-bound organelles characterized by a lumen with a\nphenylpyridinate) offer interesting possibilities for anticancer PDT. pH ranging from 4.5 to 5, with hydrolytic enzymes functioning opti\u00ad\nThese compounds have common advantages such as easy preparation mally at this acidic pH [24,25]. Lysosomes play a crucial role in intra\u00ad\nand modification due to the modular character of their synthesis, good cellular digestion and recycling processes and are involved in the\ncellular uptake and subcellular targeting capacity, photostability, regulation of cellular homeostasis, apoptosis, and autophagy. There are\nphosphorescent properties for bioimaging with long emission lifetimes, increasing evidences that lysosomes also contribute to chemoresistance\n\n 2\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\nby facilitating membrane trafficking of efflux transporters such as assessed relative to that of the untreated cells. To calculate the internal\u00ad\nP-glycoprotein (P-gp), promoting drug sequestration, and modulating ization kinetics, data were fitted to one-phase exponential association\ncell signalling [25]. Certain biscyclometalated Ir(III) complexes bearing curves using GraphPad Prism software (GraphPad Software, Inc.). Three\n\u03b2-carboline ligands exhibit a pH-responsive behaviour, which amplifies independent experiments were performed for each compound.\ntheir activity in the acidic pH of lysosomes. In this environment, the In addition, the internalization of the complexes was analyzed by\naforementioned complexes have demonstrated increased photo\u00ad determining the amount of metal present in the cells. To this end, HeLa\nluminescence and singlet oxygen generation upon light irradiation, as cells were seeded in 6-well plates (Sarstedt) at a density of 2 million cells\ncompared to neutral compartments [26,27]. Furthermore, it has been per well and allowed to attach overnight. Cells were then treated for 4 h\nshown that a dual photocytotoxic effect at the level of both mitochon\u00ad with complexes 1, 2, 3, and 6, diluted from the 1 mM solutions in culture\ndria and lysosomes notably enhances the antitumor effectiveness of medium at 5 \u03bcM, or with medium alone as a control. After removing the\nagents active in PDT, since lysosomal dysfunction significantly com\u00ad treatments, cells were washed with PBS and harvested by trypsinization.\npromises autophagy in response to cellular photodamage [28]. This The samples were then centrifuged and washed three more times with\ncytoprotective mechanism enables cells to eliminate damaged proteins PBS. The number of cells in each sample was determined with the\nand organelles by their engulfment in double-membraned vesicles or Novocyte flow cytometer. Subsequently, the amount of iridium or\nautophagosomes, which subsequently fuse with lysosomes for degra\u00ad ruthenium in each sample was determined by inductively coupled\ndation. In particular, clearance of oxidized or depolarized mitochondria plasma mass spectrometry (ICP-MS) analysis. Previously, cell pellets\n(mitophagy) plays a crucial role in cell survival preventing the release of were dissolved in 400 \u03bcL of 69 % v:v nitric acid (PanReac Applichem)\nmitochondrial cytochrome c, activation of caspase 3, and induction of and heated at 100 \u25e6 C for 18 h. After cooling, the samples were diluted\napoptosis [29,30]. with Milli-Q water to a final volume of 10 mL. The iridium or ruthenium\n With these ideas in mind and following our interest in developing content was quantified on an ICP-MS Agilent 7500c instrument at the\nnew metal-based photosensitizers for anticancer PDT [12,31,32], we Serveis Te\u0300cnics de Recerca, Universitat de Girona. The standards were\nhave synthesized three series of Ir(III) and Ru(II) cationic trischelate freshly prepared in Milli-Q water with 2 % HNO3 before each experi\u00ad\ncomplexes bearing pH-sensitive \u03b2-carboline ligands ([Ir(C^N)2(N^N\u2032)]+ ment. The concentrations used for the calibration curve were 0, 1, 2, 5,\nor [Ru(N^N)2(N^N\u2032)]2+) with the aim of studying their intracellular 10 and 20 ppb. The isotopes detected were 193 Ir and 101 Ru. Readings\ntargeting properties and their potential as PDT photosensitizers. More were conducted in triplicate. Rhodium was added as an internal stan\u00ad\nspecifically, we were interested in revealing the modulation ability of dard at a concentration of 10 ppb to all samples. Three independent\nboth the N^N\u2019 ligands (L1-L3) and the metal fragment, [Ir(C^N)2]+ or replicates were performed for each complex. The amount of metal was\n[Ru(N^N)2]2+, on the pKa of our complexes and consequently on their expressed in relation to the number of cells (1 x 106) in each sample.\nphotophysical properties and also on their intracellular distribution and\nphotocytotoxicity, to establish the respective structure-activity 2.3. Cell viability assays\nrelationships.\n Cells were seeded on 96-well plates 24 h prior to the experiment at\n2. Material and methods different concentrations depending on the cell line: 3000 PC-3 cells/\n well, 2500 A549 cells/well, 3500 MCF7 cells/well, 1500 HeLa cells/well\n The synthetic procedures and the characterization data are shown in or 5000 1BR.3.G cells/well. The compounds were diluted in sterile\nthe supporting information. dimethyl sulfoxide (DMSO) and Milli-Q water to obtain 1 mM stock\n solutions (20 % DMSO v/v). To treat the cells, solutions ranging from\n2.1. Cell lines 0 to 50 \u03bcM were prepared by diluting aliquots of the stock solutions in\n cell culture medium. This concentration range was expanded to 100 \u03bcM\n The biological activity of the complexes was evaluated in five human in cases where the IC50 values exceeded 50 \u03bcM. Cisplatin (1 mg/mL;\ncell lines. PC-3 prostate cancer, A549 basal lung adenocarcinoma, HeLa Accord Healthcare) was used as a positive control. Cells were incubated\ncervical carcinoma, and MCF-7 breast cancer cell lines were obtained with each solution for 6 h at 37 \u25e6 C to allow the internalization of the\nfrom the American Type Culture Collection (ATCC). 1BR.3.G human skin compounds. Subsequently, the plates were kept in the dark or irradiated\nfibroblasts were obtained from the European Collection of Authenticated with a light-emitting diode (LED) system (LuxLight) for 1 h at different\nCell Cultures (ECACC). Cells were cultured in Dulbecco\u2019s modified Ea\u00ad wavelengths (460 nm (blue), 515 nm (green), or 635 nm(red)),\ngle\u2019s medium (DMEM) (Corning), supplemented with 10 % fetal bovine providing a total light dose of 24.1 J cm\u25002. Each treatment was per\u00ad\nserum (FBS) (Gibco-BRL), 1 % L-glutamine (Corning), and 1 % penicillin- formed in triplicate. After 41 h, the treatments were removed, the cells\nstreptomycin (Corning) at 37 \u25e6 C in a humidified atmosphere containing 5 were washed with PBS, and the cell viability was determined using the\n% CO2. Cells were maintained by successive trypsinization and seeding. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)\nThe VenorH GeM Mycoplasma Detection Kit (Minerva Biolabs) was assay. Cells were incubated for 2 h with 100 \u03bcL of fresh culture medium\nregularly used to check for potential mycoplasma contamination. containing 10 \u03bcL of MTT solution (0.5 mg/mL) (Sigma-Aldrich). Then,\n the medium was discarded, and DMSO (Sigma-Aldrich) was added to\n2.2. Cellular internalization each well to dissolve the purple formazan crystals. The absorbance of\n each well at a wavelength of 570 nm was determined using a Multiscan\n The cellular uptake of the compounds was assessed by flow cytometry Plate Reader (Synergy 4, Biotek, Winooski, USA). For each compound,\nusing HeLa cells. Cells were seeded at a concentration of 100,000 cells/ the concentration that inhibits cell viability by 50 % (IC50) was deter\u00ad\nwell in 12-well plates and incubated for 24 h. Subsequently, cells were mined using the Gen5 Data Analysis Software (BioTeck). Compounds\ntreated with solutions of compounds 1, 2, and 3, at 5 \u03bcM, which were with IC50 values greater than 100 \u03bcM were considered inactive. The\nfreshly prepared by diluting aliquots of the corresponding 1 mM stock phototoxicity index (PI\u2013 \u2013IC50,dark/IC50,light) was assessed for each com\u00ad\nsolution (see section 2.3) in cell culture medium, or medium alone as a pound. All experiments were performed at least in triplicate.\ncontrol. Following incubation for different time intervals (1 min, 10 min, The cytotoxic activity of complex 1 was further evaluated against\n30 min, 1 h, 2 h, 4 h, and 6 h), cells were washed with phosphate buffered A549 cell spheroids. To generate the spheroids, cells were seeded at a\nsaline (PBS) (Corning) and harvested by trypsinization. After washing density of 1500 cells per well into 96-well plates coated with a thin\nwith PBS, the fluorescence emission of 10,000 cells was measured at 675 solidified layer of Geltrex\u2122 reduced growth factor basement membrane\nnm using a Novocyte flow cytometer (Agilent Technologies) equipped matrix (Gibco) and left to grow in culture medium supplemented with 2\nwith NovoExpress software. The median fluorescence of each sample was % Geltrex for 6 days. Then, spheroids were treated in the dark or under\n\n 3\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\nblue light irradiation with dilutions of the complex ranging from 50 to by flow cytometry using a Novocyte flow cytometer (Agilent Technol\u00ad\n0.05 \u03bcM in cell culture medium containing 2 % Geltrex, as described ogies) equipped with the NovoExpress software. The median fluores\u00ad\nabove. Non-treated cells were used as control. After 41 h of incubation, cence intensity of 10,000 cells was established, and the fold increase\nthe treatments were removed, cells were washed with PBS, and the % of versus untreated control cells was determined. Three independent ex\u00ad\nviability was determined with CellTiter-Glo 3D reagent (Promega), periments were carried out for each compound.\nfollowing manufacturer\u2019s instructions. Briefly, 100 \u03bcL of complete me\u00ad\ndium and 100 \u03bcL of CellTiter-Glo 3D reagent were added to each well 2.7. Plasmids and transfection\nand then cells were kept in agitation for 5 min and incubated for 25 min\nat room temperature. The luminescence of each well was determined HeLa cells were transfected with DNA using PEI (polyethylenimine)\nusing a Multiscan Plate Reader (Synergy 4, Biotek, Winooski, USA) to protocol. Cells were used for experiments 24 h after transfection. The\nassess the cell viability. Two independent experiments with duplicate Tom20(1\u201333)-GFP plasmid was obtained by subcloning DNA encoding\nsamples were performed. The IC50 values were calculated with the Gen5 Tom20 fragment corresponding to residues 1\u201333 into pEGFP-N1 (Clon\u00ad\nData Analysis Software (BioTeck). tech). The pEGFP-Rab5 and pEGFP-Rab7 vectors were kindly supplied by\n A. Sorkin (University of Pittsburgh, Pittsburgh, PA, USA). The GFP-CD63\n2.4. Clonogenic assays construct was kindly supplied by J. Kluperman (University Medical\n Center Utrecht, Utrecht, Netherlands). Lamp2A, from Lamp2A-3xFlag,\n PC-3 cells were seeded on 12-wells plates at a concentration of kindly provided by Mirka Allerding (Christian-Albrechts-Universita\u0308t zu\n100,000 cells/well. 24 h later, cells were incubated for 4h with solutions Kiel, Kiel, Germany), was subcloned into pEGFP plasmid (GFP-Lamp2A).\nof complexes 1, 2, 3, and 6 at the corresponding IC50,light, which were MitoRed plasmid was acquired from Clontech.\nfreshly prepared by diluting the corresponding 1 mM stock solution in\nculture medium. Cisplatin (5 \u03bcM) was used as a positive control. The cells 2.8. Immunofluorescence staining\nwere subsequently kept in the dark or irradiated with blue light (460 nm)\nfor 1 h. The treatments were then removed, and the cells were washed, HeLa cells grown on coverslips were treated with complex 1 at 5 \u03bcM,\ntrypsinized and counted. Three thousand cells were immediately plated prepared by diluting a 1 mM stock solution in cell culture medium, for\nin 5 cm diameter culture dishes and incubated for 10 days to allow the time periods ranging from 30 to 45 min. After the treatment, cells were\nformation of colonies of at least 50 cells. The colonies were then fixed and fixed with freshly prepared 4 % paraformaldehyde (Electron Microscopy\nstained with 1 % methylene blue in 70 % ethanol. Images of the plates Sciences) at room temperature for 15 min and mildly permeabilized\nwere obtained using the Alpha Innotech Imaging System (Alpha Inno\u00ad with PBS containing 0.1 % Triton X-100 or 0.1 % saponin and 0.1 %\ntech). The number of colonies in each plate was determined using the Fiji bovine serum albumin (BSA) at room temperature for 5 min. After a 5\nImageJ software [33]. Each compound was tested in triplicate. min incubation with blocking solution (PBS and 1 % BSA), the coverslips\n were incubated with the primary mouse anti-EEA1 (BD Biosciences) or\n anti-LBPA (clone6C4; Sigma-Aldrich) in PBS and 0.1 % BSA for 50 min\n2.5. Hemolysis assay\n at room temperature, washed intensively, and incubated with the\n appropriate secondary anti-bodies labeled with AlexaFluor-555 from\n The hemolytic activity of the compounds was determined by\n Molecular Probes (Invitrogen-Life Technologies). After staining, the\nmeasuring the hemoglobin release from red blood cells (RBD). Com\u00ad\n coverslips were mounted in Mowiol (Calbiochem). Complex 1 and\nmercial porcine blood with sodium polyphosphate as an anticoagulant\n TRITC images were acquired sequentially with 405 and 561 nm laser\n(Norfrisa, Spain), was diluted with PBS to a final concentration of 5 %.\n lines, an acoustic optical beam splitter, and emission detection ranges of\nAliquots of 150 \u03bcL were exposed to solutions of the complexes at the\n 630\u2013670 and 571\u2013625 nm, respectively (TCS SP5 laser scanning\ncorresponding IC50,light, which were freshly prepared by diluting the\n confocal microscope; Leica Microsystems). Image processing was per\u00ad\ncorresponding 1 mM stock solution in culture medium. Samples were\n formed with ImageJ software (U.S. National Institutes of Health) [34].\nincubated for 1 h at 37 \u25e6 C in the dark or with blue light irradiation under\nconstant agitation at 220 rpm in an orbital shaker. Treatment with\n 2.9. Time-lapse microscopy\nTween 0.2 % in PBS was used as the positive control to induce complete\nred blood cells (RBC) lysis. Subsequently, the samples were centrifuged\n HeLa cells expressing the different fluorescent fusion proteins or\nto pellet the cells and 80 \u03bcL of supernatant was transferred to a 96-well\n transferrin conjugated with tetramethylrhodamine, or stained with the\nplate and diluted with H2O (80 \u03bcL). The absorbance of each well was\n LysoTracker Red DND-99 or MitoTracker Green (M7514) dyes (Molec\u00ad\nmeasured with a Synergy 4 plate reader (Biotek) at 540 nm. The per\u00ad\n ular Probes; Invitrogen-Life Technologies), were grown on 25 mm\ncentage of hemolysis H (%) was calculated with the formula: H(%) =\n An) diameter glass coverslips (Warner Instruments) and mounted in an\n100 \u00d7 (Ax\u2212\n (Ap\u2212 An). Attofluor chamber (Invitrogen-Life Technologies). Subsequently, cells\n where Ax represents absorption of the sample, An represents ab\u00ad were treated with complex 1 freshly prepared at 5 \u03bcM by diluting the 1\nsorption of the untreated negative control and Ap represents absorption mM stock solution in cell culture medium. Time-lapse images were ac\u00ad\nof the positive control. quired every 1\u20132 min, up to a maximum of 30 min, using a Leica TCS SP5\n laser-scanning confocal spectral microscope (Leica Microsystems)\n2.6. ROS measurement equipped with a DMI6000 inverted microscope and an incubation con\u00ad\n trol system (37 \u25e6 C, 5 % CO2). Complex 1, GFP, and DsRed images were\n Cellular ROS content was determined using the 5(6)-carboxy-2\u2032,7\u2032- sequentially acquired with the 405, 488 and 561 nm laser lines and the\ndichlorofluorescein diacetate (Carboxy-DCFDA) (Sigma-Aldrich). HeLa emission detection ranges 630\u2013670, 500\u2013555 and 571\u2013625 nm,\ncells were seeded on 12-well plates at a density of 100,000 cells/well. respectively. Image processing was performed with ImageJ software. In\n24 h later, the cells were treated for 4 h at 37 \u25e6 C with solutions of vivo co-localization between complex 1 and mitochondria or endoly\u00ad\ncompounds 1, 2, 3, and 6 at a dose corresponding to the IC50,light, ob\u00ad sosomes compartments was analyzed from confocal images acquired\ntained by diluting the corresponding 1 mM stock solution in culture after 30\u201360 min of the complex 1 incubation in cells expressing MitoRed\nmedium, or with medium alone as a control. After washing with PBS, or GFP-Rab7, respectively. Briefly, images were background substracted\ncells were stained with 10 \u03bcM Carboxy-DCFDA for 30 min, washed using FIJI-Image J software (Wayne Rasband, NIH) and the co-\nagain, and then photoactivated with blue light or incubated in the dark localization analysis performed with the plugin JACoP to determine\nfor 1 h. Cells were immediately collected by trypsinization and analyzed the Pearson\u2019s Correlation Coefficient (PCC) in 20\u201325 cells per condition.\n\n 4\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\n2.10. Determination of mitochondrial membrane potential Photographs of each side of the cross wound were obtained at 0, 16, and\n 24 h using an Olympus CKX41 Microscope equipped with the LCmicro\n HeLa cells were seeded on 12-well plates at a concentration of software (Olympus). Analyses of cell migration were performed using the\n100,000 cells per well. After 24 h, the cells were incubated for 4 h with MRI Wound healing tool macro of Image J. Three independent experi\u00ad\nsolutions of compounds 1, 2, 3, and 6 at the corresponding IC50,light, ments were performed for each compound.\nwhich were freshly prepared by diluting the corresponding 1 mM stock\nsolution in culture medium. As a positive control, cells were co- 2.14. Statistics\nincubated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) at\n50 \u03bcM. Cells were then photoactivated with blue light or incubated in Statistical analysis was performed using GraphPad Prism (GraphPad\nthe dark for 1 h. Cells were immediately harvested by trypsinization and Software). Quantitative variables were expressed as mean or median\nincubated with the JC-1 dye (Biotium) according to the manufacturer\u2019s and standard deviation (SD). Statistical differences were analyzed by the\ninstructions. The fluorescence of the cells was analyzed using a Novocyte Mann-Whitney non-parametric test. A value of p < 0.05 was considered\nflow cytometer. JC-1 was detected at 590 nm (FL2), to identify healthy statistically significant.\nmitochondria, and at 529 nm (FL1), to identify depolarized mitochon\u00ad\ndria. Each compound was tested in three independent experiments. 2.15. Computational details\n\n2.11. Evaluation of lysosomal damage All the triplet and singlet state minima of complexes 1\u25009 were\n optimized, without imposing any symmetry restriction, at the density\n HeLa cells were seeded on glass-bottom chambered coverslips functional theory (DFT) level of theory. Calculations were performed\n(\u03bc-slide 8 well, Ibidi) at a concentration of 50,000 cells per well and using the B3LYP exchange\u2013correlation functional [35,36], which was\nallowed to attach for 24 h. Cells were then treated for 4 h with solutions previously employed to describe similar metal complexes [37,38]. The\nof compounds 1, 2, 3, and 6 at the corresponding IC50,light, obtained by DEF2-SVP basis set [39\u201341] was selected for all the atoms in the com\u00ad\ndiluting the corresponding 1 mM stock solution in culture medium, or plexes. The inner electrons for the Ir and Ru atoms were exchanged by\nwith medium alone as a negative control. Cells were subsequently the Stuttgart\u2013Dresden effective core potential while explicitly treating\nmaintained in the dark or irradiated with blue light for 1 h. After the outer core electrons. Calculations involving triplet states were all\nremoving the treatments, the cells were incubated with 5 \u03bcM of Acridine carried out using the unrestricted approximation, checking that the spin\nOrange (AO) (Sigma-Aldrich) at 37 \u25e6 C for 15 min. Lysosomal damage contamination was between 1.95 and 2.05. Frequency calculations were\nwas evaluated by confocal microscopy using a Nikon A1R confocal mi\u00ad executed to ensure the absence of imaginary frequencies within the\ncroscope. The cell cytoplasm and nucleoli were visualized in green (\u03bbem optimized minima. This critical condition confirms the authenticity of\n= 510 nm), while acidic cellular compartments such as lysosomes were the minima. To account for the influence of the surrounding solvent\nvisualized in red (\u03bbem = 625 nm). The images were analyzed using the (H2O), the conductor-like polarizable continuum model (CPCM) was\nNIS-Elements AR (Nikon, Japan) and Fiji/ImageJ software. employed [42]. All the calculations were performed with ORCA 5 soft\u00ad\n ware [43] and analyzed with the help of TheoDORE software [44]. To\n2.12. Apoptosis assay assign the nature of the different excited triplet states, time-dependent\n DFT (TD-DFT) calculations, at the same level of theory, were carried\n The cell death pathway was analyzed with the Vybrant\u00ae Apoptosis out as implemented in ORCA 5. To facilitate this assignment, natural\nAssay Kit (Molecular Probes). HeLa cells were seeded on 12-well plates transition orbitals (NTOs) [45] were obtained with TheoDORE software\nat a concentration of 100,000 cells per well and treated under both dark together to its fragment-based analysis.\nand blue light conditions with solutions of compounds 1, 2, 3, and 6 at Additional TD-DFT calculations were carried out to calculate the\nthe corresponding IC50,light, prepared by diluting the corresponding 1 phosphorescence lifetimes (\u03c4PL) for the emitting triplet states at\nmM stock solution in culture medium, as previously described. Cisplatin minimum-energy geometries obtained as described above. These cal\u00ad\nwas used as the positive control at a concentration of 50 \u03bcM. After 24 h culations were performed using a SARC-ZORA-TZVP basis set [41,46]\nof treatment, the cells were collected by trypsinization and stained with for Ir and Ru atoms and a ZORA-DEF2-SVP basis set [39] for all the other\nAnnexin-V-FITC and propidium iodide according to the manufacturer\u2019s atoms, together with the ZORA Hamiltonian [47] to consider relativistic\ninstructions. Samples were immediately analyzed using a Novocyte flow effects. In order to calculate \u03c4PL we followed the methodology employed\ncytometer. Annexin-FITC staining was detected at a wavelength of 520 in a previous work [38] for a similar family of iridium complexes.\nnm (FL1), and propidium iodide was detected at 617 nm (FL2). The\nfluorescence emission of 10,000 cells per sample was measured, and the 3. Results and discussion\npercentage of live, early apoptotic, late apoptotic, and necrotic cell\npopulations was determined. Three independent experiments were 3.1. Synthesis of ligands and complexes\nconducted.\n A total of nine complexes (1\u20139) were synthesized aiming to evaluate\n2.13. Wound healing assay their potential use as photosensitizers in PDT. The synthesis of the\n ancillary N^N\u2032 ligands L1-L3 and the Ir(III) complexes 1\u20133 was already\n A549 cells were selected for migration assays since they showed lower described in a previous study about their photocatalytic activity\nproliferation rate than HeLa cells. Cells were seeded at a density of (Scheme 1) [48]. Herein, the synthesis of six new Ru(II) polypyridyl\n750,000 cells/well on 6 wells plates and allowed to attach for 24 h. Cells complexes of general formula rac-[Ru(bpy)2(N^N\u2032)](PF6)2 (4\u20136) and\nwere incubated for 4 h with solutions of the compounds at the corre\u00ad rac-[Ru(TAP)2(N^N\u2032)](PF6)2 (7\u20139), where bpy = 2,2\u2032-bipyridine, TAP =\nsponding IC50,light, which were prepared by diluting the corresponding 1 1,4,5,8-tetraazaphenanthrene and N^N\u2019 = L1-L3, is presented. Thus, we\nmM stock solution in culture medium. Samples were photoactivated or intend to assess the role of both the N^N\u2032 ligands and the metal fragments\nkept in the dark for 1 h. Cells were then washed twice with PBS, and a ([Ir(C^N)2]+ and [Ru(N^N)2]2+) on the biological properties of the new\ncross-shaped wound was made by scratching the confluent monolayer photosensitizers. Indeed, the electron-deficient TAP ligand was chosen\nwith a 200 \u03bcL pipette tip. Wells were washed again twice to remove the due to its potential to increase cellular photodamage [49].\ndetached cells and incubated in incomplete media (DMEM supplemented The Ru(II) derivatives 4\u20139 were synthesized by heating rac-cis-Ru\nwith 0,5 % FBS, 1 % L-glutamine, and 1 % penicillin-streptomycin) for 24 (bpy)2Cl2 or rac-cis-Ru(TAP)2Cl2 with the corresponding N^N\u2019 ligand,\nh to avoid cell division. Untreated cells were used as control samples. L1-L3, in a water:ethanol mixture (1:1; v/v) as shown in Scheme 2 [50].\n\n 5\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\n\n\n Scheme 1. Synthesis and molecular structure of the thiazolyl-\u03b2-carboline ligands L1-L3.\n\n\nThese reactions were carried out using a high pressure round bottom angles are given in Table 1. The unit cells of both crystal structures exhibit\nflask under a nitrogen atmosphere. The desired products were isolated two pairs of the optical isomers (\u039b and \u0394), but Fig. 1 only shows the\nas hexafluorophosphate salts in the form of racemic mixtures (\u0394 and \u039b molecular structures of the respective \u039b enantiomers. Two PF\u22126 counter\u00ad\nisomers) and are dark-red solids. All the complexes were isolated in good ions and one or two water molecules per metal complex are also present in\nyields and purities according to analytical and spectroscopic data. TAP, the unit cell. The molecular structure of these complexes shows the\nrac-cis-Ru(bpy)2Cl2, and rac-cis-Ru(TAP)2Cl2 were prepared according classical pseudo octahedral geometry with three N,N-donor ligands\nto published procedures [51\u201353]. Analytical HPLC experiments adopting bidentate chelate coordination modes. The Ru\u2013N bond dis\u00ad\nconfirmed the high purity of all PSs (>98 %, see Fig. S59). tances (2.051\u20132.101 \u00c5) and coordination angles (78.1\u201379.53\u25e6 ) have\n standard values [50],54\u201356. Nonetheless, the Ru\u2013N distances for the\n3.2. Characterization of complexes \u03b2-carboline and thiazolyl entities are longer than those for the pyridine\n rings. The five-membered chelate rings of L2 and L3 in the coordination\n The identity and purity of the ligands and complexes were estab\u00ad polyhedrons are essentially planar as shown by the respective torsion\nlished by multinuclear nuclear magnetic resonance (NMR), high reso\u00ad angles N\u2013C\u2013C\u2013N (2.21 and 5.47\u25e6 , respectively). Moreover, the crystal\nlution mass spectrometry (HR-MS), and elemental analysis. The structure is held together mainly through hydrogen bonding interactions,\nmolecular structure of 2 and 3 was previously confirmed by X-ray involving the dicationic complexes as donors, the PF\u22126 counterions as\ndiffraction [48]. In this work, the molecular structure of 5 and 6 was also acceptors and the water molecules as both donors and acceptors\nconfirmed by X-ray diffraction. The 1H and 13C{1H} NMR spectra of Ir\n(III) and Ru(II) complexes were recorded in DMSO\u2011d6. The 1H and 13C\n Table 1\n{1H} NMR spectra of Ru(II) complexes 4\u20139 confirmed the coordination Selected bond distances (\u00c5) and angles (\u25e6 ) for compounds rac-[Ru(bpy)2(L2)]\nof the ligand to the metal center and showed two sets of resonances for (PF6)2\u22c5(H2O) (rac-5\u22c5(H2O)) and rac-[Ru(bpy)2(L3)](PF6)2\u22c5(H2O)2 (rac-6\u22c5\nthe bpy or TAP attributed to the asymmetry of these complexes. The (H2O)2).\nHR-MS showed mass/charge ratios and isotopic distributions that\n Distances/angles rac-5\u22c5(H2O) rac-6\u22c5(H2O)2\ncorroborated the molecular structures proposed.\n Ru(1)\u2013N(1) 2.095(5) 2.092(4)\n Ru(1)\u2013N(2) 2.084(5) 2.101(4)\n3.3. X-ray crystal structures Ru(1)\u2013N(4) 2.055(6) 2.056(4)\n Ru(1)\u2013N(5) 2.056(6) 2.051(4)\n Ru(1)\u2013N(6) 2.068(5) 2.071(4)\n The crystal structures of rac-[Ru(bpy)2(L2)](PF6)2\u22c5(H2O) and rac-[Ru\n Ru(1)\u2013N(7) 2.064(5) 2.067(4)\n(bpy)2(L3)](PF6)2\u22c5(H2O)2 were resolved by X-ray diffraction analysis. In N(1)\u2013Ru(1)\u2013N(2) 78.1(2) 78.32(15)\nboth cases, high quality single crystals were obtained by slow evaporation N(4)\u2013Ru(1)\u2013N(5) 78.9(2) 79.53(17)\nfrom methanol solutions of 5 and 6. Crystallographic refinement pa\u00ad N(6)\u2013Ru(1)\u2013N(7) 78.8(2) 78.64(17)\nrameters are collected in Table S1. Selected bond distances and bond\n\n\n\n\n Scheme 2. Synthesis of complexes with L1 as illustrative examples and molecular structure of complexes 1\u20139.\n\n 6\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\n\n\nFig. 1. ORTEP diagrams for the molecular structures of \u039b-[Ru(bpy)2(L2)]2+ and \u039b-[Ru(bpy)2(L3)]2+ in the crystal network of rac-5\u22c5(H2O) and rac-6\u22c5(H2O)2.\nThermal ellipsoids are shown at the 30 % probability level. Hydrogen atoms (except N\u2013H), PF\u22126 counterions, and water molecules are not shown for the sake\nof clarity.\n\n\n(Table S2a and Fig. S31). However, double \u03c0,\u03c0-stacking contacts\ninvolving the \u03b2-carboline fragments are also observed (Table S2b and\nFig. S31).\n\n3.4. Photostability experiments\n\n The photostability of aerated solutions (1.510\u25002, or 5 \u00d7 10\u25003 M,\nDMSO\u2011d6:D2O (3:2)) of our complexes was studied by 1H NMR. The\nsolutions were exposed to blue-light irradiation (\u03bbirr = 460 nm, 24 W)\nover a period of 24 h at room temperature. To our delight, no symptoms\nof photodegradation were observed, revealing that all the complexes\nexhibit an outstanding photostability, meaning that no structural alter\u00ad\nations occur under these conditions (Figs. S32\u2013S40).\n\n3.5. Absorbance\n\n The UV\u2013Vis absorption spectra of the new complexes (10\u25005 M) were\nrecorded at room temperature in both acetonitrile and in the mixture\nH2O:DMSO (99:1). In both solvent systems, the absorption profiles are Fig. 2. Overlaid absorbance spectra of complexes 1, 4, and 7 in H2O:DMSO\nsimilar (Figs. S41\u2013S42). The spectra of complexes 1, 4, and 7 in H2O: (99:1, v:v) (10\u2212 5 M) at 25 \u25e6 C.\nDMSO are shown in Fig. 2 as representative examples. All the complexes\nexhibited strong bands with maxima between 240 and 290 nm that are phosphorescent emission. Fig. 3 shows the emission spectra of 1, 4, and 7\nattributed to spin-permitted ligand-centered transitions (1LC, \u03c0 \u2192 \u03c0*). In in H2O:DMSO and Table 2 collects the photophysical data for all com\u00ad\nthe 350\u2013600 nm region, the Ir(III) complexes present an absorption band plexes in acetonitrile. These general features suggest a dominant charge-\nwith maxima about 380 nm followed by a weak band that extends up to transfer (CT) character and a triplet nature for the emitting excited state\n550 nm. These bands are attributed to mixed spin-allowed and spin- [59]. The \u03bbem for some complexes is slightly red-shifted in H2O:DMSO\nforbidden metal-to-ligand charge transfer (1MLCT and 3MLCT) and (99:1) compared to the respective \u03bbem in acetonitrile. Besides, the emis\u00ad\nligand-to-ligand charge transfer (LLCT) transitions [57,58]. The ab\u00ad sion band of the Ru PSs is, in general, red-shifted with respect to the Ir\nsorption profile of complex 3 in water differs from that recorded in analogues, except in the case of 9. Regarding the effect of the N^N\u2019 ligand\nacetonitrile due to the presence of an absorption band centered at on the maximum emission wavelength, the experimental data indicate\naround 550 nm which spreads up to 650 nm (Figs. S41\u201342). This feature that L2 causes a blue shift in \u03bbem for complexes 2, 5, and 8 in comparison\nis attributed to the partial deprotonation of the N\u2500H of the \u03b2-carboline to derivatives with L1 (1, 4, and 7). In the case of L3, no clear trend can be\nligand (vide infra) as predicted by theoretical calculations (Fig. S56). established since a red shift is observed within the Ir series (\u03bbem = 648 nm\nComplexes 4, 5, and 6 present two absorption bands centered at around for 1 vs \u03bbem = 678 nm for 3 in H2O:DMSO, 99:1) and the Ru-bpy series\n400 and 475 nm, respectively, and complexes 7, 8, and 9 display a main (\u03bbem = 682 nm for 4 vs \u03bbem = 723 nm for 6 in H2O:DMSO, 99:1), while an\nabsorption band centered at around 420 nm. These bands are also almost negligible blue shift is noticed for the Ru-TAP series (\u03bbem = 680 nm\nattributed to 1MLCT, 3MLCT, and LLCT transitions. for 7 vs \u03bbem = 678 nm for 3 in H2O:DMSO, 99:1).\n The excited state lifetimes (\u03c4) of 1\u20139 were determined in both\n3.6. Emission and photophysical properties deoxygenated CH3CN and aerated H2O:DMSO (99:1) solutions. In\n deoxygenated CH3CN, all complexes showed moderate or long \u03c4 values\n The emission spectra of 1\u20139 were recorded for both deoxygenated (125\u20131525 ns) compatible with phosphorescent emission. In particular,\nacetonitrile solutions (10\u25005 M) and aerated solutions in H2O:DMSO complexes bearing ligand L2 (2, 5, and 8) exhibit the longest \u03c4 values in\nmixtures (99:1) (10\u25005 M) at 25 \u25e6 C. All the complexes exhibit a single each series, and complexes of the Ru-TAP series showed the highest \u03c4\nbroad emission band (Figs. S43\u2013S44) and Stokes shifts larger than 192 nm values from 1090 to 1525 ns (Table 2). As expected, in aerated H2O:\n(Table S3) in both solvent systems, which is consistent with\n\n 7\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\n monocationic form is predominant, but it should coexists with the neutral\n form in a 3:1 ratio, approximately. In contrast, complex 3, with a lower\n pKa of 5.9, should adopt its neutral deprotonated form within most\n cellular organelles and its cationic protonated form in acidic organelles,\n such as lysosomes (Fig. S52). Indeed, 3 could be used to label lysosomes\n profiting from the expected pH-responsive emission enhancement at the\n acidic pH of these organelles. We interpret that the enhanced acidity of\n the N\u2013H group in complex 3 is due to the higher conjugation provided by\n the extra benzene ring in L3, which stabilizes the negative formal charge\n of the ligand in the neutral form of 3 [63,64]. However, this is not the only\n factor ruling the acidity of this group (vide infra).\n The pKa values for selected Ru(II) complexes were determined by\n UV-VIS spectroscopy (Figs. 4 and 5 and Figs. S48\u2013S51), since the vari\u00ad\n ations of the emission intensity as a function of pH were not obvious for\n these compounds. The pKa inferred for 4 and 6 (10.3 and 10.0, respec\u00ad\n tively) and for 7 and 9 (9.3 in both cases) were remarkably higher than\n those obtained for the corresponding Ir(III) counterparts, meaning that\n the Ru(II) complexes persist protonated in the cells. These results reveal\nFig. 3. Overlaid normalized emission spectra of complexes 1, 4, and 7 in H2O: that the [Ir(ppy)2]+ fragment increases the acidity of the N\u2013H group\nDMSO (99:1, v:v) (10\u2212 5 M) at 25 \u25e6 C. more than the [Ru(bpy)2]2+ or [Ru(TAP)2]2+ fragments. Overall, the\n acidity of the N\u2013H group depends on electronic effects stemming mainly\n from the metallic fragment, but also from the thiazolyl scaffold at some\nTable 2 degree within the iridium derivatives.\nPhotophysical properties of complexes in acetonitrile (10\u25005 M) at 25 \u25e6 C under a\nnitrogen atmosphere.\n 3.8. Singlet oxygen quantum yields\n Complex \u03bbex (nm) \u03bbem (nm) \u03c4 (ns) \u03d5PL(%)\n\n 1 405 641 125 2.08 Singlet oxygen quantum yields (\u03a6\u0394 ) were experimentally deter\u00ad\n 2 405 605 668 1.83 mined for representative complexes 1, 4 and 7 using ABDA as a specific\n 3 405 652 183 0.54\n probe and Rose Bengal as the standard reference (Figs. S57\u2013S58). The\n 4 450 679 679 16.40\n 5 450 677 758 32.01 results obtained revealed that 1 is by far the most efficient photosensi\u00ad\n 6 450 720 284 7.46 tizer (0.95) in good agreement with its high photo-induced cytotoxicity\n 7 450 650 1482 17.66 against cancer cells under light irradiation, while 4 and 7 provided\n 8 445 647 1525 20.10 lower \u03c6\u0394 values (0.25 and 0.61, see Table S8). A control experiment in\n 9 450 642 1090 14.10\n the absence of PS was also performed to ruled out ABDA\n photobleaching.\nDMSO (99:1), the \u03c4 values are shorter for all the complexes due to the\nquenching effect attributed to O2 (Table S3). 3.9. Theoretical characterization\n Regarding the photoluminescence quantum yields (\u03d5PL), the values\nrecorded for the Ir(III) complexes in acetonitrile are very low (<2.1%). DFT-based electronic structure calculations on the Ir(III) and Ru(II)\nBy contrast, the Ru(II) complexes exhibit higher \u03d5PL values (from 7.46 to complexes 1\u20139 were performed in order to describe the photophysical\n32.01%) and complexes 5 and 8 stand out with \u03d5PL values of 32.01 and properties of their lowest-energy triplet state (T1 state), T1 being strictly\n20.10%, respectively. The \u03d5PL in aerated H2O:DMSO are low for all the related to the capacity of a complex to act as photosensitizer. Since the\ncomplexes (Table S3). key process of PDT is the transfer of energy from the T1 state of the\n photosensitizer to molecular oxygen (which is the process resulting in\n\n3.7. Determination of pKa\n\n The complexes reported herein are expected to behave as weak acids\ndue to the presence of a polar N\u2013H bond in the \u03b2-carboline fragment. In\nparticular, for the Ir(III) complexes under physiological conditions, the\ncoordinated \u03b2-carboline ligands could undergo deprotonation of the N\u2013H\ngroup to give either the neutral forms of the complexes or the equilibria\nbetween the cationic and neutral forms of the complexes depending on\nthe pH. Indeed, the protonation state of the complexes could affect not\nonly its global charge but also their solubility, aggregation state, and\ncellular uptake, as well as their absorption and emission profiles [60]. The\npKa values of Ir(III) complexes 1, 2, and 3 were experimentally deter\u00ad\nmined by analyzing the variation of their emission intensity as a function\nof pH. As a matter of fact, the emission intensity of 1, 2, and 3 gradually\ndecreases with increasing pH (Figs. S45\u2013S47), as previously reported for\nsimilar derivatives [60\u201362]. The pKa values determined experimentally\nwere 7.9, 7.5, and 5.9 for 1, 2, and 3, respectively. Therefore, we\nconcluded that 2, with a pKa value very close to physiological pH\n(7.35\u20137.45), should be present in an equilibrium between its mono\u00ad\ncationic and neutral form, with a proportion close to 1:1. In the case of Fig. 4. Overlay of the absorbance spectra of 4 in H2O:DMSO (99:1, v:v) (10\u2212 5\ncomplex 1, with a pKa value higher than physiological pH, the M) at 25 \u25e6 C recorded at different pH values from 3 to 12.\n\n 8\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\n\n\nFig. 5. Profile of the absorbance of 4 at 475 nm in H2O:DMSO (99:1, v:v) (10\u2212 5\nM) at 25 \u25e6 C recorded as a function of pH between 6 and 12.\n\n\nsinglet-oxygen formation which in turn is responsible of cellular dam\u00ad\nage) it is essential to have a T1 state that allows and promotes such an\nenergy transfer. A prerequisite is that the T1 state must have an energy Fig. 6. A) Possible decay paths from the T1 minimum. B) Representation of the\ngreater than 1 eV (i.e., the T1\u2500S0 energy difference computed at the T1 radiative and non-radiative decay paths from the T1 minimum along the PES,\nminimum must be larger than 1 eV), because that is the energy required and the computed magnitudes (phosphorescence lifetime (\u03c4) and adiabatic\nfor promoting molecular oxygen from its triplet ground state to its energy difference between T1 and 3MC (\u0394ET1 \u2212 3 MC )) to estimate their rela\u00ad\nreactive singlet excited state. Once such a condition is fulfilled, the tive importance.\nlonger the T1 state lives without having the possibility to decay through\nradiative and/or non-radiative processes, the higher is the probability to shown in Table 3, Ru(II) complexes have very low \u0394ET1 \u2212 3 MC energy\ninteract with molecular oxygen and then produce singlet oxygen. The\n differences, much lower than those calculated for Ir(III) complexes, thus\nideal case would then be a low emissive T1 state, well separated from all\n suggesting that the population of the non-emitting 3MC states is more\nother electronic states and in particular from the ground state, so that\n likely for the former. We then obtained that Ru(II) complexes have\nthe excitation remains \"trapped\" in the T1 minimum, \"waiting for\u201d the\ninteraction with molecular oxygen. This reflects a situation in which the\nsystem takes a long time to emit (large phosphorescence lifetime) and at Table 3\nthe same time has no accessible non-radiative decay path (high emission Vertical emission energies (Ever, eV) from the optimized T1 minima, adiabatic\nquantum yield). One of the most important non-radiative decay oper\u00ad energy differences between each T1 minimum and the ground state S0 at its\nating in Ir(III) and Ru(II) cationic complexes is related to the thermal minimum (Eadi, eV), adiabatic energy differences between each T1 minimum and\npopulation of triplet metal-centered (3MC) states, which can easily cross the lowest MC minimum (\u0394ET1 \u2212 3 MC , eV), and phosphorescence lifetimes (\u03c4, \u03bcs)\nwith the ground state, then decaying along [37]. calculated for complexes 1\u20139. The electronic nature of each T1 minimum is also\n We then proceeded to the evaluation of radiative and non-radiative specified (see Table S7). \u201cd\u201d for 1\u20133 denotes the deprotonated structure of the\ndecays for the here studied complexes (see Fig. 6). Through the explo\u00ad complex.\nration of the T1 potential energy surface (PES), a single T1 minimum of Complex State Ever (eV) Eadi (eV) \u0394ET1 \u2212 3 MC (eV) \u03c4 (\u03bcs)\nligand-to-ligand charge-transfer (3LLCT) character was located for Ir(III) 3\n 1 LLCT 1.92 2.16 0.69 1.33\ncomplexes except for complex 2, which presents a ligand-centered (3LC) 1d 3\n LC 1.57 1.74 1.16 155.27\nnature. In contrast, three T1 minima of metal-to-ligand CT (3MLCT) and 2 3\n LC 1.84 2.18 0.73 9.15\n3\n LC nature were characterized for Ru(II) complexes (see Table S7 and 2d 3\n LC 1.59 1.76 1.14 174.04\n 3\nSection 8 in the SI). LC states are in general considered to be slower 3\n 3\n LLCT 1.85 2.08 0.61 1.65\n 3d LC 1.52 1.69 1.22 247.47\nemitters than CTs states, although such a vision has been recently revis\u00ad 3\n 4 MLCT1 1.61 1.84 0.31 17.19\nited [38]. In order to evaluate the emission propensities, phosphores\u00ad 4 3\n MLCT2 1.80 2.04 0.11 153.31\ncence emission lifetimes were computed for all the T1 minima. As shown 4 3\n MLCT3 1.81 2.05 0.10 280.87\nin Table 3, Ru(II) complexes are predicted to have lifetime values 5 3\n MLCT1 1.64 1.87 0.20 16.43\n 3\none-to-three orders of magnitude larger than Ir(III) complexes. When 5 MLCT2 1.79 2.01 0.05 776.99\n 3\n 5 MLCT3 1.81 2.04 0.02 1929.43\nconsidering only the lowest-energy T1 minimum, the difference is 6 3\n MLCT1 1.50 1.73 0.31 24.52\nreduced in most cases to one order of magnitude, however still reflecting a 6 3\n MLCT2 1.81 2.03 0.01 122.81\nsignificantly slower emission process in Ru(II) complexes than in the Ir 6 3\n MLCT3 1.83 2.06 \u2212 0.01 195.93\n 3\n(III) analogues. 7 LC 1.81 2.10 0.02 78.69\n 3\n 7 MLCT1 1.79 2.00 0.13 803.48\n MC states were also characterized for the nine complexes. The 3\n 7 MLCT2 1.79 2.00 0.12 747.92\nadiabatic energy difference between each T1 minimum and the lowest 8 3\n LC 1.80 2.10 0.00 143.55\n3\n MC minimum (hereafter \u0394ET1 \u2212 3 MC ) are also reported in Table 3. Such a 8 3\n MLCT1 1.77 1.97 0.13 479.71\n 3\nvalue provides an estimation of how probable is to access from a T1 8 MLCT2 1.80 2.00 0.10 797.14\n 3\n 9 LC 1.70 2.01 0.07 80.71\nminimum to the lowest non-emitting 3MC state, and consequently de\u00ad 3\n 9 MLCT1 1.79 1.99 0.09 1253.97\ntermines the importance of non-radiative decays for each complex. As 9 3\n MLCT2 1.80 2.00 0.07 1361.32\n\n\n 9\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\n Table 4\n Phototoxicity of the complexes against PC-3 cancer cells.\n Compound IC50 (\u03bcM) PI\n\n Dark Light\n\n 1 2.08 \u00b1 0.48 0.021 \u00b1 0.007 99.0\n 2 1.49 \u00b1 0.38 0.017 \u00b1 0.004 87.6\n 3 2.30 \u00b1 0.86 0.017 \u00b1 0.012 135.3\n 4 37.27 \u00b1 18.89 2.65 \u00b1 0.03 14.1\n 5 35.55 \u00b1 3.40 2.20 \u00b1 0.63 16.1\n 6 39.46 \u00b1 6.60 3.32 \u00b1 1.70 11.9\n 7 31.60 \u00b1 2.13 6.77 \u00b1 1.05 4.7\n 8 59.99 \u00b1 13.63 8.48 \u00b1 1.39 7.1\n 9 49.25 \u00b1 20.90 2.97 \u00b1 1.93 16.6\n Cisplatin 2.53 \u00b1 0.73 n.d. \u2013\n\n PC-3 cells were treated with the compounds for 6 h at 37 \u25e6 C to ensure their\n maximum internalization, and then kept in the dark or exposed to blue light for\n 1 h (460 nm, 24.1 J cm\u25002). Cell viability was assessed 43 h later by MTT assays.\n Data represent the mean \u00b1 SD of at least three independent experiments, each\n performed in triplicate. n.d.: not determined. PI: phototoxicity index = IC50,dark/\n IC50,light.\n\n\n systems (vide infra, Table 4), and in particular the highest PI value ob\u00ad\n tained for complex 3, for which the pKa has the lowest value and a more\n significant presence of the deprotonated form is therefore expected.\n\n\n 3.10. Cellular uptake of the complexes\n\n The efficient cellular uptake of the complexes is essential for their\n biological activity as photosensitizers. To assess cell internalization, the\n Ir(III) compounds 1, 2, and 3 were selected because of their demon\u00ad\n strated photoluminescence properties under cell culture conditions.\n Cells were incubated with the complexes at a concentration of 5 \u03bcM, and\n intracellular fluorescence was measured at various time points using\n flow cytometry. As shown in Fig. 7A, fluorescence emission from the\nFig. 7. A) Representative flow cytometry histograms showing the intracellular complexes was detected in over 90 % of the cells after just 1 min of\nfluorescence of HeLa cells after 1 min of incubation with compounds 1, 2, and exposure. This rapid internalization suggests that the compounds enter\n3 at 5 \u03bcM. Untreated control cells were used as a reference. B) Cells were the cells through diffusion across the cell membrane [65]. Moreover,\nincubated for the indicated times with the compounds at 5 \u03bcM, and the median since the fluorescence emission of complex 3 was dramatically sup\u00ad\nfluorescence intensity at each time point was determined by flow cytometry to\n pressed at pH higher than 5.9, this finding suggests its rapid accumu\u00ad\nassess the internalization kinetics. Data represents the mean \u00b1 SD of three in\u00ad\n lation within acidic cellular compartments, such as lysosomes.\ndependent experiments.\n Intracellular fluorescence was monitored for 6 h, and similar internali\u00ad\n zation kinetics were observed for the three compounds (Fig. 7B). Within\nslower radiative processes but faster non-radiative decays, then pre\u00ad\n the first 2 h, a linear increase in cellular fluorescence was observed,\ncluding a direct explanation of the higher PI values characterizing Ir(III)\n indicating a high uptake rate of the complexes during this period. After\ncomplexes (vide infra).\n 2 h, the intracellular fluorescence levels began to stabilize, approaching\n An additional factor that needs consideration is the significantly lower\n saturation at 4 h. Based on these results, incubation times of at least 4 h\npKa values that characterize Ir(III) complexes compared to Ru(II) com\u00ad\n were established to ensure maximum intracellular accumulation of the\nplexes (see Table S4). The pKa values estimated for the three Ir(III)\n compounds before photoactivation, with the aim of achieving the most\ncomplexes suggest that both the protonated and deprotonated forms\n effective biological response.\nshould be present at the physiological pH value of 7, in particular for\ncomplex 3 having a pKa of 5.9. We then studied the photophysical\nproperties of the three deprotonated Ir(III) complexes (hereafter named 3.11. Effect on cell viability\nas 1d, 2d, and 3d), and found a two-order of magnitude increase in the\ntriplet state lifetimes, and an increase of \u0394ET1 \u2212 3 MC of around 0.5 eV The anticancer activity of the complexes was evaluated against PC-3\n cells in both dark and light-irradiation conditions. Cells were treated at\n(Table 3). The deprotonation actually causes a large stabilization of the\n concentrations ranging from 0.001 to 50 \u03bcM, and the concentration at\nLC state located on the ancillary ligand (i.e., where the deprotonation\n which cell viability was inhibited by 50 % (IC50) was determined for\ntakes place), which becomes the T1 state, while leaving unaffected the MC\n each compound. Compounds with IC50 values above 50 \u03bcM were further\nstate. So, the deprotonation globally determines an increase in the energy\n evaluated at a wider concentration range (up to 100 \u03bcM). The selection\nseparation with the non-emitting MC states. In addition, the LC state of\n of the most promising candidates for PDT was based on the calculation\nthe deprotonated form resulted significantly lower emissive than the\n of the phototoxicity index (PI\u2013\u2013IC50,dark/IC50,light). As shown in Table 4,\nLLCT state of the protonated form, as reflected by the much higher triplet\n the three Ir(III) complexes exhibited notable antiproliferative effects in\nstate lifetime calculated for the former (Table 3). Globally, the three\n the dark, with IC50,dark values ranging from 1.49 to 2.30 \u03bcM, which are\ndeprotonated Ir(III) complexes display T1 states that live longer, since\n similar to the IC50 of the chemotherapeutic drug cisplatin under the\nboth the radiative and non-radiative decays are slower in comparison\n same experimental conditions. In contrast, the Ru(II) complexes showed\nwith the protonated iridium complexes. This result can then rationalize\n more moderate anticancer activity, with IC50,dark values between 35.55\nthe higher PI values (PI\u2013 \u2013IC50,dark/IC50,light) reported for the Ir(III) and 39.46 \u03bcM for the complexes with bpy ligands and between 31.60\n\n 10\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\nand 59.99 \u03bcM for complexes with TAP ligands. Importantly, upon irra\u00ad effects on non-irradiated healthy tissues. In addition, it should be taken\ndiation with blue light, the activities of complexes 1, 2, and 3 were in consideration that PDT is being successfully applied to treat derma\u00ad\nsignificantly increased, resulting in IC50,light values in the low nanomolar tological malignancies, which involve the local irradiation of the skin.\nrange and PI values of 99.0, 87.6, and 135.3, respectively. In contrast, Consequently, the light selectivity index (LSI = IC50,light skin fibroblasts/\ndespite their higher light absorption at 460 nm, the Ru(II) complexes IC50,light cancer cells) was also calculated for all the complexes. Complex\nexhibited IC50,light values ranging from 8.48 to 2.20 \u03bcM and markedly 3 was the only one that exhibited significant light selectivity in all tested\nlower PI values (between 4.7 and 16.6). cancer cell lines, with LSI values of 5.7, 6.9, 4.9, and 8.1, in PC-3, HeLa,\n The significant increase in the antiproliferative activity of the Ir(III) A549, and MCF7 cells, respectively.\ncomplexes after photoactivation highlighted their potential for PDT and\nprompted their selection for further investigation of their activity and\ntheir mechanism of action. The Ru (II) complex 6 was included in the 3.12. Hemolytic activity\nstudy to examine the impact of the metallic fragment on the biological\nactivity of these complexes. To further assess the potential toxicity of complexes 1, 2, 3, and 6,\n The photocytotoxic behavior of 1, 2, 3, and 6 was next investigated their activity against red blood cells (RBCs) was assessed. Complexes\nin response to irradiation with green (530 nm) and red (655 nm) light, as were incubated with RBCs freshly obtained from porcine blood at a\nlonger wavelengths have deeper tissue penetration capacities (Table 5) concentration corresponding to their IC50,light in PC-3 cells (Table 4) and\n[66,67]. After irradiation with green light, PI values between 16 and hemoglobin release was measured as an indicator of RBCs damage. None\n19.2 were obtained for the Ir(III) complexes, revealing a certain pho\u00ad of the complexes displayed significant hemolytic activity, both in dark\ntoactivation capacity at 530 nm. Conversely, following irradiation with conditions and when exposed to blue light, as evidenced by hemolysis\nred light, the PI values of complexes 1 and 2 decreased significantly, percentages below 2 % in all cases (Table S9). These results indicate the\nwhich is consistent with the low light absorption observed for complex excellent blood compatibility of the complexes at the photocytotoxic\n1 at wavelengths above 550 nm (Fig. 2). In contrast, complex 3 exhibited concentrations. Furthermore, the absence of hemoglobin release from\na PI of 8.5. The deprotonated form of complex 3 presents one absorption the RBCs indicates that the complexes do not cause direct damage to the\nband centered at 550 nm (Fig. S42) which spreads widely up to 650 nm cell membrane. This suggests that they interact with intracellular targets\nand could explain its higher photoactivation by red light. In the case of that are not present in RBCs, such as mitochondria or DNA.\nthe Ru(II) complex, an IC50,light value of 5.51 \u00b1 1.76 \u03bcM was obtained\nupon irradiation with green light, resulting in a PI of 7.2. This value is\nslightly lower than the PI value obtained with blue light (11.9), indi\u00ad 3.13. Colony formation assays and effect on cell spheroids\ncating that complex 6 better maintains its photoactivation capacity\nunder green light, which is consistent with its higher absorption at 530 The impact of the treatments on the long-term viability and growth\nnm (Fig. S42, Table 5). potential of cancer cells was assessed using clonogenic assays. PC-3 cells\n The anticancer efficacy of complexes 1, 2, 3, and 6 was further were exposed to complexes 1, 2, 3, and 6 at their respective IC50,light for\nevaluated in human cancer cell lines of different origins: cervical (HeLa), 4 h, followed by either irradiation with blue light or incubation in the\nlung (A549), and breast (MCF-7), as well as in non-malignant fibroblasts dark for 1 h. Cisplatin was used as a positive control. The treatments\n(1BR.3.G). As shown in Table 6, upon photoactivation with blue light, were removed, and the cells were seeded at low density and incubated to\nthe IC50,light values for the three Ir(III) complexes remained in the low- allow colony formation. As shown in Fig. 8A, the number of colonies was\nnanomolar range in all cancer cell lines and PI values were between notably reduced by cisplatin and the photoactivated complexes. Spe\u00ad\n70 and 201. The Ru(II) complex 6 also exhibited good phototoxicity in cifically, in comparison to the untreated cells, the number of colonies\nthe different cell lines, with notable effectiveness against HeLa cells, was reduced to 14 \u00b1 3 % after cisplatin treatment, and to 53 \u00b1 2 %, 28\nalthough its activity was consistently lower than that of the structurally \u00b1 3 %, 35 \u00b1 2 %, and 56 \u00b1 7 % after exposure to photoactivated\nrelated Ir(III) complex 3. It is worth noting that the IC50 values obtained complexes 1, 2, 3, and 6, respectively. However, none of the treatments\nagainst the 1BR.3.G fibroblast were, in general, slightly higher than in the dark induced any inhibition of colony formation (Fig. 8A and B).\nthose obtained in the cancer cell lines, especially for compounds 3 and 6. These findings confirm the photocytotoxicity of the complexes at their\nMoreover, when comparing the cytotoxicity of the complexes towards respective IC50,light and suggest that cell damage occurs during the\nfibroblast not exposed to the light and their activity against cancer cells photoactivation of the complexes. Hence, prolonged exposure to the\nupon irradiation, high photoselectivity indexes (PSI: IC50,dark in non- compounds following photoactivation would not be necessary to inhibit\ncancer cells/IC50,light in cancer cells) were observed. Specifically, a PSI cell proliferation.\nof 543, 659, 461, and 769 were obtained for complex 3 in PC-3, HeLa, Finally, the antitumor effect of complex 1 was tested using spheroids,\nA549, and MCF7 cells, respectively. Therefore, the dose applied for which better mimic tumor biology than the two-dimensional (2D) cell\nphotodynamic therapy (PDT) of these cancers would not have harmful cultures. Spheroids consist of microaggregates of cancer cells that\n recapitulate some important features of solid tumors, such as nutrient,\n growth factor, and oxygen gradients, as well as cell-cell and cell-\nTable 5 extracellular matrix interactions [68,69]. Spheroids were generated\nPhotocytotoxicity of complexes 1, 2, 3, and 6 against PC-3 cancer cells upon from A549 cells, which were allowed to grow to form spherical aggre\u00ad\nphotoactivation with green and red light. gates ranging in size from 50 to 100 \u03bcm. The spheroids were subse\u00ad\n Compound Green Light Red Light quently treated with varying concentrations of complex 1 to determine\n IC50 (\u03bcM) PI IC50 (\u03bcM) PI its IC50 in this 3D model. Upon treatment under both dark and light\n conditions, a visible decrease in spheroid size was observed, albeit at\n 1 0.13 \u00b1 0.08 16 0.83 \u00b1 0.20 2.5\n 2 0.085 \u00b1 0.04 17.5 0.46 \u00b1 0.05 3.2\n different concentrations (Fig. 8C). Under dark conditions, an IC50,dark\n 3 0.12 \u00b1 0.02 19.2 0.27 \u00b1 0.08 8.5 value of 11.95 \u00b1 0.80 \u03bcM was obtained, which is approximately two\n 6 5.51 \u00b1 1.76 7.2 13.69 \u00b1 0.34 2.9 times higher than the value obtained in the 2D cultures (Table 6). After\nPC-3 cells were incubated with the compounds for 6 h at 37 \u25e6 C, then kept in the irradiation, the IC50,light value was 0.36 \u00b1 0.08 \u03bcM, resulting in a PI of\ndark or exposed to green (530 nm) or red (655 nm) light for 1 h (24.1 J cm-2). 33.2. This value was 5.8 times higher than the IC50,light value obtained in\nCell viability was assessed 41 h later by MTT assays. Data represent the mean \u00b1 2D models, but was still within the nanomolar range, demonstrating the\nSD of at least three independent experiments, each performed in triplicate. PI: strong tumor growth inhibition capacity of the complex upon\nphototoxicity index = IC50,dark/IC50,light. photoactivation.\n\n 11\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\nTable 6\nPhotocytotoxicity of 1, 2, 3, and 6 against HeLa, A549, and MCF7 cancer cells and 1BR.3.G fibroblasts.\n Complex HeLa A549 MCF7 1BR.3.G\n\n IC50 (\u03bcM) PI IC50 (\u03bcM) PI IC50 (\u03bcM) PI IC50 (\u03bcM) PI\n\n Dark Light Dark Light Dark Light Dark Light\n\n 1 2.84 \u00b1 0.90 0.018 \u00b1 158 4.64 \u00b1 3.25 0.062 \u00b1 75 2.23 \u00b1 0.47 0.025 \u00b1 89 5.07 \u00b1 0.83 0.056 \u00b1 90\n 0.007 0.029 0.022 0.006\n 2 1.42 \u00b1 0.72 0.016 \u00b1 89 2.40 \u00b1 1.56 0.035 \u00b1 70 4.22 \u00b1 0.28 0.021 \u00b1 201 4.12 \u00b1 0.78 0.032 \u00b1 129\n 0.014 0.022 0.017 0.007\n 3 1.29 \u00b1 0.34 0.014 \u00b1 92 1.53 \u00b1 0.24 0.02 \u00b1 0.005 76 1.32 \u00b1 0.40 0.012 \u00b1 110 9.23 \u00b1 0.46 0.097 \u00b1 95\n 0.004 0.002 0.010\n 6 56.62 \u00b1 1.32 \u00b1 0.24 43 35.12 \u00b1 1.83 \u00b1 1.32 19 28.27 \u00b1 1.37 \u00b1 0.52 21 92.81 \u00b1 3.68 \u00b1 0.27 25\n 5.68 11.47 6.51 5.82\n\nHeLa, A549, MCF7 and 1BR.3.G cells were incubated with the compounds for 6 h at 37 \u25e6 C, then kept in the dark or exposed to blue light for 1 h (460 nm, 24.1 J cm\u25002).\nCell viability was assessed 41 h later by MTT assays. Data represent the mean \u00b1 SD of at least three independent experiments, each performed in triplicate. PI:\nphototoxicity index = IC50,dark/IC50,light.\n\n\n damaging different biomolecules, ultimately leading to cell death [70,\n 71]. The generation of intracellular ROS was analyzed using the\n carboxy-DCFDA probe, which is oxidized by various ROS within cells to\n the green fluorescent DCF product. Flow cytometry experiments showed\n that the fluorescence emission of the cells remained unaltered under\n dark conditions after the treatments with the complexes at their\n respective IC50,light (Fig. 9), indicating that ROS levels were not modi\u00ad\n fied. However, upon irradiation, the fluorescence of the cells was\n significantly increased by complexes 1, 2, and 3, with fold changes of\n 13.2 \u00b1 0.9, 13.5 \u00b1 2.6, and 9.6 \u00b1 0.4, respectively. In the case of the Ru\n (III) complex 6, fluorescence was increased by 6.2 \u00b1 2.1-fold.\n Finally, to ensure that the probe fluorescence accurately reflected the\n levels of ROS, the potential interference from the intrinsic fluorescence\n of the complexes was evaluated. To this end, HeLa cells were incubated\n with complexes 1, 2, 3 and 6 at their corresponding IC50,light and the\n cellular fluorescence was compared to that of untreated control cells by\n flow cytometry. The histograms corresponding to the different treat\u00ad\n ments showed no differences compared to control cells (Fig. S62), which\n confirmed that the fluorescence emission by the complexes is minimal at\n the IC50,light. Therefore, any disturbance of the complexes with these\n flow cytometry measurements could be excluded.\n These findings collectively demonstrate the efficient generation of\n intracellular ROS by the photoactivated complexes, suggesting that their\n photocytotoxic activity is primarily attributed to their prooxidant\n properties.\n\n 3.15. Cellular internalization and localization\n\n To ascertain whether the observed differences in the photocytotoxic\n\n\n\n\nFig. 8. Antitumoral activities A) Colony formation after exposure of PC-3 cells\nto complexes 1, 2, 3, and 6 at the corresponding IC50,light in the dark or with\nblue-light irradiation (1 h, 460 nm, 24.1 J cm\u2212 2). Control cells were incubated\nwith the medium alone. Cisplatin at 5 \u03bcM was used as the positive control. B)\nBar charts represent the percentage of colonies after each treatment relative to\ncontrol cells (mean \u00b1 SD of 3 experiments. ***p < 0.001). C) Representative\nmicroscopy images of A549 spheroids treated with complex 1 at 20 \u03bcM in the\ndark or at 0.5 \u03bcM with blue-light irradiation. Untreated control A549 cells\nseeded on Geltrex\u2122 formed rounded spheroids. Images show the growth-\nsuppressing effect of the complex on spheroids in dark and light conditions\n Fig. 9. Cellular ROS generation. HeLa cells were incubated with complexes 1,\nafter 48h of treatment. The scale bar represents 100 \u03bcm.\n 2, 3, and 6 at the corresponding IC50,light for 4 h and then maintained in the\n3.14. Intracellular ROS generation dark or exposed to blue-light irradiation for 1 h (460 nm, 24.1 J cm\u2212 2). ROS\n levels were determined by flow cytometry using the carboxy-DCFDA probe. The\n The efficient generation of ROS within the cells is crucial for mean ROS elevation \u00b1SD relative to untreated control cells determined in three\n independent experiments are presented. *p < 0.05, **p < 0.01 and ***p <\nachieving successful outcomes in PDT. ROS are responsible for\n 0.001 versus control cells.\n\n 12\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\nactivity of the complexes, particularly between the Ir(III) complexes (1, mitochondria. To mitigate the impact of fixation on the fluorescence\n2, and 3) and the Ru(II) complex (6), were due to variations in their emission of the complex, further characterization of its subcellular\ncellular internalization, HeLa cells were incubated with the complexes at localization was analyzed in vivo. Co-localization studies were per\u00ad\n5 \u03bcM for 4 h and the intracellular metal content was quantified by ICP- formed in HeLa cells expressing recombinant proteins specific to cell\nMS. The results demonstrated a significantly lower level of internaliza\u00ad compartments (EEs, LEs/Lys, and mitochondria) fused with green (GFP),\ntion for the Ru(II) complex (6) compared to its Ir(III) counterpart cherry, or dsRed fluorescent living colors. After a 30-min incubation\n(complex 3) (0.7 \u00b1 0.2 ng metal/106 cells vs 137.2 \u00b1 31.7 ng metal/106 with complex 1, in vivo confocal images revealed a high degree of\ncells) (Fig. 10), which highlights the key influence of the metal on the co-localization with dsRed-Mito (MitoRed) labeled mitochondria\ncapacity of the complexes to enter the cells. Similar high values were (Pearson\u2019s Correlation Coefficient, PCC = 0.723 \u00b1 0.049). (Fig. 11B).\nobtained for Ir(III) complex 1 (141.8 \u00b1 16.5 ng metal/106 cells), while On the other hand, while co-localization of complex 1 with LEs/Lys\nthe Iridium content was lower in the case of complex 2 (56.2 \u00b1 10.8 ng markers CD63, Rab7, or Lamp2A was detected after 20\u250030 min of in\u00ad\nmetal/106 cells). In general, the higher cellular accumulation of Ir(III) cubation by time-lapse video microscopy (Fig. 12A), no co-localization\ncomplexes correlated well with their enhanced photocytotoxic activity with Rab5 (EEs marker) was observed at any time (Fig. 12B). In line\ncompared to the Ru(II) complex. Nevertheless, a direct correlation be\u00ad\ntween Iridium content and the IC50 values of the complexes could not be\nestablished. Overall, these findings suggest that a minimum level of\ncellular uptake is essential for the photocytotoxic efficacy of these\ncomplexes.\n Subsequently, the specific subcellular distribution of the compounds\nwas examined, as it has a strong influence on their biological activity\ndue to the short action radius of ROS once generated [72,73]. The\nstudies were carried out by confocal microscopy with complex 1, since it\ndisplays the highest quantum yield and fluorescence emission at phys\u00ad\niological pHs. HeLa cells were chosen for the experiments, since they are\nmorphologically spread, have a large cytoplasm, and are easy to trans\u00ad\nfect and express proteins, which makes them suitable for studying the\nentry and subcellular localization of the complex. Cells were incubated\nwith complex 1 at 5 \u03bcM for 30\u201345 min and, after fixation with 4 %\nparaformaldehyde, permeabilized with 0.1 % Triton (TX-100) or 0.1 %\nsaponin and stained with antibodies against early endosomal antigen 1\n(EEA1) and lysobisphosphatidic acid (LBPA), which are early endo\u00ad\nsomes (EEs) and late endosomes/lysosomes (LEs/Lys) markers, respec\u00ad\ntively. Images acquired with a confocal microscope (Leica TCS SP5)\nindicated that the fluorescence of complex 1 was highly sensitive to\nfixation and Triton permeabilization and only faint staining was\nobserved after saponin permeabilization. Notably, co-localization of\ncomplex 1 with few LBPA-positive endosomes (LEs/Lys) was detected,\nprincipally after 45 min of incubation (Fig. 11A). Interestingly, the\ndiffuse pattern exhibited by complex 1 resembled that of the\n\n\n\n\n Fig. 11. Complex 1 localizes in lysosomes and mitochondria. A) HeLa cells\n were incubated with complex 1 (5 \u03bcM) at the indicated times and after fixation,\n LBPA was detected with a specific antibody and the secondary Alexa-555 anti-\n mouse. Images acquired with a confocal microscope (Leica TCS SP5) show some\n colocalization between LBPA (red channel) and the fluorescent complex 1\n (green channel) (white arrows point selected colocalization staining). B) HeLa\n cells transiently expressing DsRed-Mito (MitoRed) were incubated with com\u00ad\n plex 1 (5 \u03bcM) during 30 min. In vivo images of complex 1 and MitoRed were\n sequentially acquired, using the inverted SP5-confocal microscope equipped\nFig. 10. Cellular internalization of complexes. HeLa cells were incubated with with an incubation control system (37 \u25e6 C, 5 % CO2), with the 405 and 561 nm\ncomplexes 1, 2, 3, and 6 at 5 \u03bcM. The amount of iridium or ruthenium per laser lines and the emission detection ranges 630\u2013670 and 571\u2013625 nm,\nmillion cells after 4 h of treatment was determined by ICP-MS. Each bar in the respectively. Inset shows high magnification images of complex 1 localization\ngraph represents the mean \u00b1 SD of three independent experiments. in MitoRed-positive mitochondria.\n\n 13\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\nwith the previous cellular uptake data obtained by flow cytometry a result of the photoactivation of complex 1 by the irradiation at 405 nm.\n(Fig. 7), altogether suggest that complex 1 predominantly enters into Given the cationic and lipophilic nature of the complex, its photody\u00ad\ncells by fast diffusion across the cell membrane and is then incorporated namic activity could importantly affect mitochondria function and ul\u00ad\nand retained in the acidic LEs/Lys without the need to follow the timately induce cell death. Therefore, the localization and delivery of\nestablished endocytic internalization pathway. complex 1 into mitochondria was analyzed in more detail using confocal\n In these microscopy experiments, rounded cells with blebbing (un\u00ad video microscopy. GFP-Tom20 (Translocator Outer Membrane subunit)\nhealthy cells) were detected at the end of the time-course experiment, as or MitoRed were ectopically transiently expressed in HeLa cells and\n\n\n\n\nFig. 12. HeLa cells expressing indicated recombinant GFP fusion proteins of the endolysosomal compartment (LEs/Lys) A) or Rab5 as a marker of early endosomes\n(EEs) B) were incubated at 37 \u25e6 C with complex 1 (5 \u03bcM) and images acquired at the indicated times with in vivo SP5-confocal microscopy. After 20-30 min incubation\ntime, colocalization among complex 1 (red channel) and the different markers of LEs/Lys (Rab7, CD63, and Lamp2A) but not Rab5 (green channel) was evident\n(white arrows).\n\n 14\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\ncomplex 1 loading into mitochondria was visualized by confocal mi\u00ad compound decorated the edges, but not the MitoRed positive matrix of\ncroscopy. Fig. 13A and B shows a fast incorporation of complex 1 in GFP- fragmented and rounded mitochondria. Complex 1 localization on\nTom20 and MitoRed labeled mitochondria, respectively (1 min or less). mitochondria was further analyzed with MitoTracker Green, a selective\nImportantly, after repeated irradiation to visualize the compound, the fluorescent labelling dye of this organelle, which passively diffuses\nmitochondria began to lose their original morphology by progressively across the plasma membrane and then accumulates in active mito\u00ad\nfragmenting and rounding. chondria in a potential-dependent manner. MitoTracker Green labels the\n It is to note that the acquired images revealed that the fluorescence of mitochondrial matrix by reacting with free thiol groups of mitochondrial\nGFP-Tom20 was more sensitive to the phototoxicity generated by proteins [71,74]. HeLa cells pre-stained with MitoTracker Green were\ncomplex 1 (as it progressively loses fluorescence emission) than that of incubated with complex 1 and then irradiated (Fig. 13D). Complex 1\nMitoRed (Fig. 13A and B). This could be explained by the different promptly labeled mitochondria and, after acquiring images, Mito\u00ad\nsensitivity of the fluorescent proteins to ROS generated by complex 1 or Tracker Green fluorescence rapidly disappeared. This result suggests\nby the fact that GFP-Tom20 is closer to complex 1 than MitoRed. In fact, that the phototoxicity generated by complex 1 affects mitochondrial\nGFP-Tom20 is bound to the mitochondrial outer membrane and membrane potential and functionality, which is in accordance with the\nMitoRed is localized into the mitochondrial inner membrane and matrix. proximity generation of ROS triggered by complex 1, as described above\nIn addition, the fluorescence of complex 1 was also affected by the ROS (Fig. 9), in this organelle. In this line, fluorescence intensity of preloaded\ngenerated by the compound (auto-bleaching). Fig. 13C shows in more MitoTracker Green in HeLa cells was reduced after mitochondrial de\u00ad\ndetail that after 30 min incubation and irradiation of complex 1, the polarization induced by carbonyl cyanide 3-chlorophenylhydrazone\n\n\n\n\nFig. 13. Time-lapse video microscopy was performed in HeLa cells expressing GFP-Tom20 A) or MitoRed B, C) incubated with complex 1 (5 \u03bcM) during 30 min at 37\n\u25e6\n C. Images were acquired at the indicated times with SP5-confocal microscopy and insets show high magnification areas to visualize complex 1 localization in labeled\nmitochondria. C) Images show the effect of complex 1 on mitochondrial morphology after 30 min of photoactivation. D) HeLa cells pre-stained with MitoTracker\nGreen (30 min, 1 \u03bcM) were incubated with complex 1 (5 \u03bcM) and images acquired in a time-lapse confocal microscopy (TCS SP5-Leica). Images show that complex 1\nrapidly incorporates into MitoTracker Green-labeled mitochondria and that after complex 1 irradiation MitoTracker Green quickly dissociates.\n\n 15\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\n(CCCP) treatment [75]. In agreement with the cellular entry of the dark conditions (Fig. 16 and Fig. S60). Notably, blue-light irradiation\ncompound by diffusion and its rapid incorporation into mitochondria, significantly reduced the population of red fluorescent cells by 32.7 %,\nindependently of the endocytic pathway, the inhibition of dynamin 29.2 %, and 26.0 % for complexes 1, 2, and 3, respectively, compared to\nusing dynasore, which impaired transferrin internalization mediated by the dark treatments. A similar effect was observed in cells exposed to the\nclathrin- and dynamin-dependent endocytosis, had no impact on the electronic chain uncoupler CCCP [77], demonstrating that the photo\u00ad\nlocalization of complex 1 within GFP-Tom20 or MitoRed-labeled mito\u00ad dynamic activity of these complexes induces mitochondrial membrane\nchondria (Fig. 14A). Consequently, dynasore also failed to alter the depolarization. These results are consistent with the degeneration of the\ndisruption of mitochondrial membrane potential induced by complex 1 mitochondria observed by confocal microscopy (Fig. 14B). In contrast,\n(Fig. 14B). Ru(II) complex 6 only reduced the red fluorescent population by 17.2 %,\n Finally, double staining with markers of mitochondria, MitoRed or indicating a lower impact on mitochondrial functionality.\nGFP-Tom20, and LEs/Lys compartments, GFP-Rab7 or Lysotracker-Red,\nwas performed after 1 h of complex 1 incubation (Fig. 15A and B, 3.17. Lysosomal damage\nrespectively). Confocal microscopy images confirmed the accumulation\nof complex 1 in both mitochondria and LEs/Lys (PCC = 0.707 \u00b1 0.058 Microscopy experiments also revealed the accumulation of complex\nand 0.762 \u00b1 0.073, respectively). 1 in LEs/Lys compartments (Fig. 12). Thus, the impact of the treatments\n on lysosomal integrity was evaluated using Acridine Orange (AO),\n3.16. Mitochondrial damage which readily diffuses through cell and organelle membranes, binding\n with high affinity to nucleic acids. Due to its weak basic property, AO is\n After observing the rapid accumulation and effect of complex 1 in protonated and sequestered in acidic environments such as lysosomes,\nmitochondria, the impact of complexes 1, 2, 3, and 6 on mitochondrial shifting its green fluorescence emission towards red [78]. Microscopic\nfunction was investigated. Mitochondrial membrane depolarization was images in Fig. 17 show that cells exposed to complexes 1, 2, 3, and 6 at\nanalyzed as an indicator of mitochondrial damage, given its essential the IC50,light in the absence of light irradiation exhibited green fluores\u00ad\ninvolvement in ATP synthesis and regulation of apoptosis [76]. Changes cent staining throughout the nucleus and cytoplasm along with cytosolic\nin membrane polarization were assessed by flow cytometry using the granular red fluorescence indicating intact lysosomes. After exposure to\nJC-1 fluorescent dye, which accumulates in healthy mitochondria in an blue light, a similar pattern was observed in untreated control cells.\nMMP-dependent manner, undergoing a shift in its fluorescence emission However, cells exposed to the irradiated complexes showed a significant\nfrom green to red. Red fluorescence emission was detected in 44.8 % of decrease in AO red fluorescence, indicating lysosomal damage. These\nthe control cells and similar fluorescence patterns were observed in cells findings are consistent with the lysosome-targeting activity described\ntreated with complexes 1, 2, 3, and 6 at their respective IC50,light under for other Ir(III) complexes bearing \u03b2-carboline ligands [60].\n\n\n\n\nFig. 14. A) HeLa cells transiently transfected with GFP-Tom20 (upper panels) or MitoRed (lower panels) were preincubated 20 min at 37 \u25e6 C with dynasore (5 \u03bcM)\nand then further incubated 10 min with labeled transferrin (Tf-A555, 50 \u03bcg/mL; upper panels) and complex 1 (5 \u03bcM). In vivo SP5-confocal images, acquired with the\ncorresponding settings, show effectiveness of the dynamin inhibitor dynasore avoiding transferrin endocytosis but not complex 1 association with GFP-Tom20 (upper\npanels) or MitoRed (lower panels) positives mitochondria. B) HeLa cells preincubated 30 min with dynasore (5 \u03bcM) and MitoTracker Green (1 \u03bcM) were further\nincubated with complex 1 (5 \u03bcM) at 37 \u25e6 C for 10 min. Acquired images show MitoTracker Green fast dissociation from mitochondria after complex 1 photoactivation\nindependently of dynamin activity.\n\n 16\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\n\n\nFig. 15. HeLa cells expressing MitoRed and GFPRab7 (A) or GFP-Tom20 with Lysotracker-Red labeled-lysosomes (30 min, 100 nM) (B) were incubated 60 min with\ncomplex 1 and in vivo images acquired with the SP5-confocal microscope. Images show colocalization of complex 1 with GFPRab7 (A) or Lysotracker-Red (B) positive\nendolysosomes (red arrows) and MitoRed (A) or GFP-Tom20 (B) labeled mitochondria.\n\n\n\n\nFig. 16. Effect of the complexes on the mitochondrial membrane potential. HeLa cells were treated with complexes 1, 2, 3, and 6 at the corresponding IC50,light in the\ndark or under light irradiation (1 h, 460 nm, 24.1 J cm\u2212 2). The mitochondrial membrane uncoupler CCCP (5 \u03bcM) was used as a positive control. Cells incubated with\nmedium alone were the negative control (CTRL). The percentage of cells showing JC-1 green and red fluorescence are represented (mean \u00b1 SD of three idependent\nexperiments). Loss of MMP can be detected by the reduction of the percentage of cells showing red fluorescence. *p < 0.05 and **p < 0.01 vs control cells.\n\n\n3.18. Cell death mechanism 8.2 %, and 60.9 \u00b1 2.9 % of the total cell population in cells treated with\n complexes 1, 2, and 3, respectively, compared to 4.4 \u00b1 0.1 % in control\n The mechanism of action of the complexes was further elucidated by cells. However, the percentage of necrotic cells (annexin -/PrI +) was\ninvestigating the type of cell death induced upon their photoactivation. between 5 % and 10 % for all treatments. In the case of complex 6, very\nDisruption of both mitochondrial and lysosomal functions can initiate a few apoptotic or necrotic cells were detected at 24 h. However, at a\nprogrammed cell death through apoptosis [31,79]. Thus, cells were longer incubation time (48 h) and higher concentration (IC50, light x 5),\nstained with annexin V-FITC, which enables the differentiation of 12.3 \u00b1 6.3 % of cells were found in early apoptosis and 16.9 \u00b1 10.1 % in\napoptotic from necrotic cells, based on the presence of phosphati\u00ad late apoptosis, while the percentage of necrotic cells remained un\u00ad\ndylserine on the outer cell membrane of apoptotic cells (annexin +). changed (Fig. S61). These results are consistent with a reduced effect of\nAdditionally, by measuring the permeability of the cell membrane to complex 6 on MMP (Fig. 16), which would result in a reduced release of\npropidium iodide (PrI), it was possible to distinguish between cytochrome c and other proteins involved in the activation of apoptosis\nearly-stage apoptosis (PrI-) and late-stage apoptosis or necrosis (PrI+). A [80]. Taken together, these results suggest that the photocytotoxic ac\u00ad\npositive control for apoptosis was established using cisplatin (5 \u03bcM). tivity of the compounds primarily induces programmed cell death.\nFlow cytometry analysis of the cells after 24 h of treatment showed an Subsequently, ROS accumulation could cause massive oxidative damage\nincrease in the percentage of cells in early apoptosis (annexin V+/PrI-) to cellular structures, ultimately compromising cell membrane integrity\nfrom 2.9 \u00b1 0.3 % in control cells to 7.9 \u00b1 6.9 %, 10.6 \u00b1 9.0 %, and 12.2 and leading to late apoptosis or secondary necrosis [81]. Another\n\u00b1 8.8 % in cells treated with photoactivated complexes 1, 2, and 3, interesting issue is about the excretion of complexes following photo\u00ad\nrespectively (Fig. 18). A higher percentage of cells in late apoptosis therapy. Since the complexes have demonstrated the ability to induce\n(annexin +/PrI +) was detected, accounting for 23.5 \u00b1 1.2 %, 65.1 \u00b1 apoptosis in target cells, excretion of the complexes is expected to be\n\n\n 17\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\n\n\nFig. 17. Lysosomal damage. HeLa cells were treated with the complexes 1, 2, 3, and 6 at the corresponding IC50,light in the dark or with light irradiation (1 h, 460 nm,\n24.1 J cm\u2212 2). Control cells were treated with medium alone under light conditions. Lysosomal damage was evaluated by confocal microscopy using AO staining (\u03bbex\n= 488 nm). Cell cytoplasm and nucleoli were visualized in green (\u03bbem = 510 nm) while acidic cellular compartments, such as lysosomes, were visualized in red (\u03bbem\n= 625 nm). The scale bar represents 20 \u03bcm.\n\n\n\n\nFig. 18. Cell death mechanism. A) Representative flow cytometry histograms of HeLa cells treated with photoactivated complexes 1, 2, 3, and 6 for 24 h at the\ncorresponding IC50,light and double stained with propidium iodide and Annexin V-FITC. Cisplatin (5 \u03bcM) was used as positive control. B) Percentages of healthy, early\napoptotic, late apoptotic, and necrotic cells (mean \u00b1 SD) after each treatment determined in two independent experiments.\n\n\nfacilitated by cellular processes mainly involving apoptotic pathways findings suggested that the activity of the complexes could effectively\nand subsequent clearance mechanisms in the organism. inhibit the tumor cell migration.\n\n3.19. Cell migration 4. Conclusions\n\n Cell migration is an initial step in cancer metastasis. Mitochondria We have prepared three series of Ir(III) (1\u20133) and Ru(II) (4\u20139) tris-\nplay an essential role in this process by providing the necessary energy chelate polypyridyl complexes and studied their photophysical and\nsupply for the modification of focal adhesions and remodeling of the biological properties as potential PDT PSs. All the compounds are pho\u00ad\ncytoskeleton [17,18]. Considering the mitochondrial-targeted effects of tostable and emissive in both acetonitrile and H2O/DMSO (99:1) solu\u00ad\nthe Ir(III) complexes, the impact of the treatments on the cell migration tions. The N\u2013H groups of these complexes are slightly acidic, which\ncapacity was investigated using wound healing assays. In these assays, endows them with pH-responsive properties. The Ir(III) derivatives are\nartificial wounds or \"scratches\" were created on confluent A549 cell more acidic than their Ru(II) analogues, (5.9 < pKa < 7.9 for 1\u20133 vs 9.3\nmonolayers, and cell movement was tracked using microscopy. As < pKa < 10.3 for 4\u20139). Singularly, complex 3 exhibits a pKa value of 5.9\nshown in Fig. 19, after the treatment in dark conditions, cell migration and, as a result, it is deprotonated in most cell organelles at the\neffectively allowed wound closure at 24 h (Fig. 19A), demonstrating a respective physiological pH values. Indeed, the deprotonation at pH >\nmigration rate similar to that of the control cells (Fig. 19B). However, 5.9 increases the electron density on the iridium center, which in turn\nupon photoactivation, the wound healing percentage was significantly causes a red-shift in its absorption profile and a dramatic emission\nreduced, with the migration rate decreasing by 70\u250080 % in all cases quenching. Consistently, theoretical calculations performed for the\ncompared to the control cells and cells treated in the dark. These deprotonated forms of the three Ir(III) complexes predict a large\n\n 18\n\fJ. Sanz-Villafruela et al. European Journal of Medicinal Chemistry 276 (2024) 116618\n\n\n\n\nFig. 19. Effect on cell migration. A) In vitro wound assay images showing the antimigratory effect of 1, 2, and 3 at the corresponding IC50,light on A549 cells 24 h\nafter treatment. B) Cell migration rate (\u03bcm2/h) of cells exposed to the Ir(III) complexes under dark and light conditions. The bars represent the mean \u00b1 SD of three\nindependent experiments. **p < 0.01 and ***p < 0.001 versus cells treated in the dark.\n\n\nstabilization of ligand-centered states, resulting in T1 (3LC) states with Declaration of generative AI and AI-assisted technologies in the\nlonger lifetimes and higher \u0394ET1 \u2212 3 MC energy gaps compared to their writing process\nprotonated forms. This adjustment leads to less favorable radiative and\nnon-radiative decays for the deprotonated forms of the Ir(III) derivatives During the preparation of this work the authors used the DeepL and\nand enhances their ability to generate ROS. Grammarly tools in order to improve language and readability. The\n The Ir(III) complexes show high intrinsic cytotoxicity against authors subsequently reviewed and edited the content as needed and\ndifferent cancer cells, which increases significantly upon blue-light take full responsibility for the content of the publication.\nirradiation, reaching phototoxicity indices between 70 and 201 and\nIC50,light values in the low nanomolar range. Despite being less effective,\n Declaration of competing interest\ngreen light irradiation could enhance the cytotoxic activity by 15\u201319\ntimes for the Ir(III) complexes and 7.2 times for Ru(II) complexes\n The authors declare that they have no known competing financial\ncompared to treatments in dark conditions. In the case of complex 3, its\n interests or personal relationships that could have appeared to influence\nactivity is also significantly enhanced by red-light irradiation. Regarding\n the work reported in this paper.\nthe mechanism of action, extensive microscopy experiments with com\u00ad\nplex 1 have described that Ir(III) complexes can rapidly enter cells,\n Data availability\nindependently of the endocytic pathway, and associate with the endo\u00ad\nlysosomal and mitochondrial cellular compartments. Photoactivation of\n Data will be made available on request.\nthe complexes triggers potent ROS generation and severely affects the\nmitochondrial and lysosomal functionality, ultimately leading to pro\u00ad\n Acknowledgements\ngrammed cell death by apoptosis. In addition, Ir(III) complexes have\nbeen shown to effectively inhibit important cancer processes, including\n This work was supported by the Ministerio de Ciencia e Innovacio\u0301n/\ncell migration and colony formation. These results demonstrate the\n Agencia Estatal de Investigacio\u0301n of Spain (MCIN/AEI/10.13039/\npotential of these compounds for PDT of various types of cancer and\n 501100011033) (projects PID2021-127187OB-C21, PID2021-\ntheir ability to inhibit processes associated with cancer malignancy,\n 128569NB-I00, PID2020-115910RB-I00, PID2021-127187OB-C22, and\nsuch as migration preceding metastasis or new tumor generation.\n CEX2019-000919-M). PhD students acknowledge their predoctoral\n grants to Universidad de Burgos (J.S.V., 2019/00002/008/001), Uni\u00ad\nCRediT authorship contribution statement\n versity of Girona (C.B., IFUdG 2021), Generalitat de Catalunya (E.Z.,\n AGAUR; 2021 FI_B 01036) and Generalitat Valenciana (I. S.D., CIACIF/\n Juan Sanz-Villafruela: Writing \u2013 original draft, Investigation.\n 2021/438), respectively. We thank M. Calvo, E. Coll and G. Mart\u00edn and\nCristina Bermejo-Casadesus: Methodology, Investigation, Formal\n acknowledge the use of the Advanced Optical Microscopy Facility of the\nanalysis. Elisenda Zafon: Methodology, Investigation. Marta Mart\u00ed\u00ad\n University of Barcelona (Spain).\nnez-Alonso: Investigation, Data curation. Gema Dura\u0301: Supervision.\nAranzazu Heras: Supervision, Funding acquisition. Iva\u0301n Soriano-D\u00edaz:\nData curation. Angelo Giussani: Methodology, Data curation. Enrique Appendix A. Supplementary data\nOrt\u00ed: Writing \u2013 original draft, Supervision, Funding acquisition,\nConceptualization. Francesc Tebar: Writing \u2013 original draft, Method\u00ad Supplementary data to this article can be found online at https://doi.\nology, Investigation. Gustavo Espino: Writing \u2013 original draft, Super\u00ad org/10.1016/j.ejmech.2024.116618.\nvision, Methodology, Funding acquisition, Conceptualization. Anna\nMassaguer: Writing \u2013 original draft, Validation, Supervision, Method\u00ad References\nology, Investigation, Funding acquisition.\n [1] A.M.P. Romani, Cisplatin in cancer treatment, Biochem. Pharmacol. 206 (2022)\n 115323, https://doi.org/10.1016/j.bcp.2022.115323.\n [2] R. Oun, Y.E. Moussa, N.J. Wheate, The side effects of platinum-based\n chemotherapy drugs: a review for chemists, Dalton Trans. 47 (2018) 6645\u20136653,\n https://doi.org/10.1039/C8DT00838H.\n\n\n 19\n\fJ. Sanz-Villafruela et al. 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Photobiol. 96 (2020) 280\u2013294, https://doi.org/10.1111/\n php.13219.\n\n\n\n\n 21\n\f", "pages_extracted": 21, "text_length": 160428}