Instigating Visible Light Inspired DNA Impairment by ROS Harvesting Ir(III)‐Cyclometallated Imidazophenanthroline Complexes Against MDA‐MB‐231 Cells

{"full_text": " Research Article\n doi.org/10.1002/ejic.202400769\n\n www.eurjic.org\n\n\nInstigating Visible Light Inspired DNA Impairment by ROS\nHarvesting Ir(III)-Cyclometallated Imidazophenanthroline\nComplexes Against MDA-MB-231 Cells\nSreejani Ghosh[a] and Priyankar Paira*[b]\n\nDifficulties to abate the vehemence of deleterious triple- negative breast cancer cells. The significant phototoxicity of the\nnegative breast cancer (TNBC) is an ardent issue in the current complexes has arisen due to the production of reactive singlet\ncontext of anticancer research due to the lack of a selective oxygen (1O2) following the type-II pathway (\u03a6s = 0.26). Also, the\ntreatment modality. To address this issue, herein we have complexes have shown the proficiency in oxidation of reduced\nendeavored to establish the imidazophenanthroline-based Ir- nicotinamide adenine dinucleotide (NADH) (TOF = 37.82 h 1)\n(III)-cyclometallated heteroleptic complexes, suited for the one- indicating the possibility of reactive oxygen species (O2 , OH)\n * *\n\n\n\n\nphoton photodynamic therapy (OP-PDT) under irradiation of generation through type-I pathway upon visible light irradia-\nvisible light (400\u2013700 nm) possessing long-lived excited triplet tion. Along with this, intracellular glutathione (GSH) depletion\nstate and significant photostability. Among three synthesized capabilities have endued the complexes to unarm the TNBC\ncomplexes, [L1Ir], [L2Ir], [L3Ir], the complex [L2Ir] has been cells in front of the profuse amount of ROS instigating the\nrecognized as the most competent complex to exhibit selective programmed cell death (PCD) through substantial DNA dam-\nphototoxicity (IC50 = 3.8 \u03bcM; PIc = 78.94) in MDA-MB-231 triple- age.\n\n\n\n1. Introduction breast cancer (TNBC) is deprived of the hormone receptors as\n well as the human epidermal growth factor receptors 2 (HER2)\nCancer renders as one of the most severe and perilous ailments and has been identified as the most hostile and heterogeneous\naffecting people throughout the world.[1] The avoidance of subtypes with poor prognosis among all types of breast\napoptosis, uncontrolled proliferation, hindrance to anti-growth cancer.[5] The gene expression profile of TNBC characterizes this\nsignals and provocation of own growth signals, amplified cancer as basal-like breast cancer (BLBC), which occurs due to\nangiogenesis as well as unbridled metastasis of tumour tissues mutation in BRCA1/2 gene patterns.[6] Chemotherapy is gen-\ncan be considered as the primary causes of mortality due to erally employed to cure the basal type of breast cancer, but\ncancer.[2] In 2024, the National Cancer Institute (NCI) unveiled a different gene patterns of the TNBC subtypes necessitate a\nstatistical report of 2,001,140 new cases of cancer in the United particular therapeutic approach instead of a generalized one.[7]\nStates and the number of deaths is 611,720. As a second Therefore in the current phase of anticancer research, there are\nleading cause of death, it is expected that the death toll for different therapeutic approaches for treating normal breast\ncancer will be more than 13.1 million by 2030.[3] Among various cancer, but there is no proper treatment regimen to subdue the\ntypes of cancer, breast cancer (BC) has been diagnosed as the unbridled triple-negative breast cancer.[8] Although the stupen-\nmost prevailing malignancy of women worldwide and has dous discovery of cisplatin by Rosenberg in the 1960s paved\nsecured the second place in the list of cancer-related mortality the way for metal complexes in cancer therapy, the usage of\nas per the satistical report of the World Health Organization platinum complexes in chemotherapy was restricted owing to\n(WHO) and Global Cancer Statistics of GLOBOCAN.[4] BC is their unavoidable side effects including drug resistance, neuro-\nbroadly categorized as human epidermal growth factor receptor toxicity, nephrotoxicity, hepatotoxicity, and treatment\n2 (HER-2) enriched, hormone receptor (HR) positive, and triple- selectivity.[9] Unfortunately, shortcomings associated with che-\nnegative based on the expression of biomarkers, viz Ki67, motherapy, radiation therapy, and surgery along with drug\nhuman epidermal growth factor receptor 2 (HER2), estrogen resistance, have made the treatment of cancers very challeng-\nreceptors (ER), and progesterone receptors (PR). Triple-negative ing. In recent times, photodynamic therapy (PDT) has garnered\n profound interest as a potentially superior cancer healing\n[a] S. Ghosh strategy.[10] Importantly, PDT unveils numerous benefits over\n Department of Chemistry, School of Advanced Sciences (SAS), Vellore conventional treatment modalities such as surgery or chemo-\n Institute of Technology (VIT), Vellore \u2013 632014, Tamil Nadu, India therapy. The lower invasiveness, great selectivity, and incredible\n[b] P. Paira efficiency, along with less detrimental side effects, have\n Department of Chemistry, School of Advanced Sciences (SAS), Vellore\n established this therapy as the most significant current strategy\n Institute of Technology (VIT), Vellore \u2013 632014, Tamil Nadu, India\n E-mail: priyankar.paira@vit.ac.in for treating cancer.[11] This method leverages light and photo-\n Supporting information for this article is available on the WWW under sensitizers (PSs) to transform molecular oxygen into reactive\n https://doi.org/10.1002/ejic.202400769 oxygen species (ROS), which can adeptly assassinate the\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (1 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f 10990682c, 2025, 10, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejic.202400769 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n doi.org/10.1002/ejic.202400769\n\n\naffected tissues in the body.[12] On the other hand, the luminescence lifetimes (100 ns) for time-resolved detection; and\ngeneration of ROS is closely linked to the non-radiative (iv) enhanced photostabilities (less photobleaching).[14] Addi-\nintersystem crossing (ISC) from excited singlet states (Sn) to tionally, several kinds of Ir(III) complexes have been investigated\nexcited triplet states (T1). PSs are activated and then change recently as anticancer theranostic drugs; and Ir(III) cyclometa-\ntheir states when they are exposed to light: they first move lated complexes have come to lime-light as powerful photo-\nfrom the ground state (S0) to the different excited singlet states dynamic therapeutic agents.[15] These complexes demonstrated\n(Sn) and then they change the spin state undergoing non- the ability to identify the cancer cells in the human body and\nradiative intersystem crossing (ISC) to move to the excited T1 then their destruction either by disrupting DNA or by triggering\nstate depending on the energy gaps (\u0394EST) between the singlet the mitochondrial malfunction via the release of reactive\n(S1) and triplet (T1) excited states. Therefore, this movement oxygen species (ROS).[16] Furthermore, given their high-lying\nmay either result in fluorescence induced by a radiation metal-centered states and flexibly modifiable highest occupied\npathway or a radiative phosphorescence pathway from the T1 molecular orbital (HOMO) and lowest unoccupied molecular\nto S0 state. Cancer cells are eliminated by the photosensitizer\u2019s orbital (LUMO) energy levels, heteroleptic Ir(III) complexes with\nexcited triplet state, which changes the ground state 3O2 into bidentate (N N) ligands and cyclometallation with (C N) ligands\nactive singlet oxygen (1O2). This step has been designated as a have demonstrated excellent photostability.[17] Cyclometalated\ntype II reaction. Electron transfer mechanisms, also known as Ir(III) complexes, exhibiting a high photostability, render them\ntype I reactions, are accountable for the generation of radical useful for continuous irradiation and real-time intracellular\nspecies, namely superoxide ion (O2 ) and hydroxyl radical\n *\n transport and accumulation monitoring.[18] The aforementioned\n( OH).[13] The remarkable attributes of Ir(III) organometallic\n*\n complexes can also disturb the cell cycle by oxidizing NADH\ncompounds enabled researchers to formulate various kinds of and demolishing the energy currency in cancer cells. Also,\ncationic cyclometalated Ir(III)-based metal complexes that are glutathione (GSH), an important antioxidant elevated in cancer\nexcellent cellular imaging agents. The following are the reasons cells, can be depleted by cyclometalated iridium (III) complexes,\nfor their use in imaging: (i) large Stokes shifts (> 100 nm) for propelling the cells more towards oxidative stress, which\nminimizing inner filter effects; (ii) rapid transmembrane activity promotes substantial programmed cell death (Figure 1).[19]\n(short incubation time and less potential toxicity); (iii) long\n\n\n\n\nFigure 1. Design of the cyclometallated complexes.\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (2 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f 10990682c, 2025, 10, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejic.202400769 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n doi.org/10.1002/ejic.202400769\n\n\n2. Results and Discussion magnetic resonance (NMR) spectroscopy, Fourier transform\n infrared (FT-IR) spectroscopy, high-resolution mass spectrome-\n2.1. Synthesis and Characterization try (HRMS), and purity of the ligands were analyzed by C, H, N\n analytical technique, which have been already reported from\nThe process described in Scheme 1 was followed to synthesize our group. Bright yellow-coloured iridium precursor ([IrCP]) was\nthe ligands L1\u2013L3 as well as their corresponding complexes, taken in a pear-shaped flask and it was dissolved properly in\n[L1Ir], [L2Ir], [L3Ir]. To synthesize the ligands (L1\u2013L3), we 10 ml of 4: 1 toluene/methanol solvent mixture to get a clear\nfollowed our previously established protocol.[20] After 30 h of yellowish solution. Then 2.1 equivalents of the previously\nreflux reaction involving [1,10]-phenanthroline-5, 6-dione and prepared pure ligand (L1/L2/L3) were added to the solution of\nthree respective aryl aldehydes (naphthaldehyde, anthranalde- iridium precursor ([IrCP]) and sonicated for 5 minutes to get a\nhyde, and chromone aldehyde) in 1 : 1 molar ratio by dissolving clear brownish-coloured solution upon thoroughly mixing the\nthe reacting components in minimum volume of glacial acetic reactants. Thereafter, the reaction mixture was refluxed for 6 h\nacid with excess ammonium acetate, we were able to detect in N2-atmosphere at 120 \u00b0C. The progression of the reaction was\nthe formation of desired ligands (L1\u2013L3) by monitoring the periodically monitored by thin-layer chromatography (TLC)\nprogression of the reaction through thin layer chromatography using 100 % methanol as a solvent system. At around 6 h of\n(TLC). Then the product was precipitated out upon neutraliza- reflux, we observed a significant change in the colour of the\ntion by ice-cold ammonium hydroxide solution and we reaction mixture from pale brown to dark brown, and the\nobtained the crude product after filtration. The crude product completion of the reaction was confirmed by re-performing the\nwas washed thoroughly with hexane and diethyl ether to TLC. After cooling the reaction mixture at room temperature,\nremove the impurities and a recrystallization technique was the product was seen to be precipitated out. Then, we obtained\nused to obtain the pure crystalline products. The structural the crude product after filtration. The crude product was\nconfirmation of the ligands was obtained by 1H and 13C nuclear repeatedly washed with hexane as well as hexane-ethyl acetate\n\n\n\n\nScheme 1. Synthetic route for the formation of [L1Ir], [L2Ir] and [L3Ir] complexes.\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (3 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f 10990682c, 2025, 10, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejic.202400769 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n doi.org/10.1002/ejic.202400769\n\n\nmixture (3 : 1) 5\u20136 times to remove impurities and the purity of charge transfer transition (1MLCT), observed at \u03bbabs: 358\u2013425 nm\nthe product was checked by performing the TLC. The cleaned (\u03bbmax~390 nm) indicating the charge transfer transition from\nproduct was then dried and subjected to recrystallization from filled d\u03c0 (t2g) MOs of metal to high energy vacant \u03c0*-MOs of\nmethanol/diethyl ether to obtain more purified product. The imidazophenanthroline N^N ligand [1MLCT: d\u03c0(Ir)!\u03c0* (N N)]. The\ncrystalline product of each complex was weighed in a weighing insignificant low-intensity weakest absorption tails at higher\nbalance and the % yield was calculated. In due course, the wavelength region \u03bbabs: 425\u2013515 nm (Figure 2a inset) might be\ncomplexes [L1Ir], [L2Ir], and [L3Ir] were obtained as deep indicated as the admixture of spin-forbidden 3MLCT and 3ILCT/\n 3\nyellow to reddish brown coloured crystals with high yields (94\u2013 LLCT transitions as a result of spin-orbit coupling (SOC)\n96 %). The structures of all the complexes were assured by 1H, allowing intersystem crossing (ISC) from singlet excited to\n13\n C NMR, FT-IR spectroscopy, and HRMS. The purity of the triplet excited states, which was essential for enabling PDT\ncomplexes was scrutinized by ultraperformance liquid chroma- (Figure 2a). The theoretical investigation of electronic transi-\ntography (UPLC) as well as with C, H, N analysis. tions for complex, [L2Ir] from highest occupied molecular\n orbital (HOMO) to lowest unoccupied molecular orbital (LUMO)\n also supported the significant metal-to-ligand charge transfer\n2.1.1. Photophysical Characterization transition as an exclusive phenomenon for these complexes\n (Figure 2b). All the plausible electronic transitions associated\nSusceptibility towards the light is an important asset for with these complexes have been picturized schematically in\ndesigning a phototherapeutic anticancer drug. To ratify the Figure 2c.\nphotosensitizing ability of the synthesized complexes, namely\n[L1Ir], [L2Ir], and [L3Ir], we precisely evaluated their absorption\nand emission characteristics in a de-aerated acetonitrile 2.1.1.2. Emission Spectra\nmedium at a constant pH of 7.4 and a temperature of 25 \u00b0C.\n Emission spectral analysis of these cyclometallated complexes\n was very crucial to get a clear vision of the probable pathways\n2.1.1.1. UV-Visible Spectra of ROS formation during PDT and hence we investigated the\n emission properties of each complex in de-aerated acetonitrile\nIn the absorption spectra, a similar type of absorption band by exciting the molecules at respective 1MLCT regions\nwith five different regions has been identified for each of the ([L1Ir]\u03bbex = 390 nm, [L2Ir]\u03bbex = 382 nm, [L3Ir]\u03bbex = 395 nm). It was\nthree complexes with different intensities (Figure 2a). These observed that complexes, [L1Ir] and [L3Ir] displayed a single\nabsorption bands can be broadly categorized as \u03c0!\u03c0*, LMCT, intense emission band at \u03bbem = 572 nm with very large Stokes\u2019\nand MLCT. The more intense higher energy absorption bands at shift of 180 nm and 177 nm, respectively indicating the\nlower wavelength UV region (240\u2013312 nm) had arisen due to possibility of phosphorescence as an emission phenomenon\nthe spin-allowed ligand-centered (1LC) \u03c0!\u03c0* transition from the lowest-lying triplet excited state to singlet ground\n(1LLCT/1ILCT), then lower energy comparatively less intense state (Figure 2e). On the other hand, complex, [L2Ir] unveiled\nabsorption bands at next higher wavelength region \u03bbabs: 312\u2013 dual emission representing two emission bands: one is at a\n358 nm, was observed because of spin-allowed ligand-centered higher energy region (\u03bbem = 438 nm) with Stokes\u2019 shift of 56 nm\n(1LC) ligand to metal charge transfer (1LMCT) transitions. The and another is at a lower energy region (\u03bbem = 572) with Stokes\u2019\nleast intense lower energy absorption bands at higher wave- shift of 190 nm, which attributed the complex, [L2Ir] a special\nlength visible region (358\u2013515 nm) were envisioned for spin- characteristic for exhibiting both fluorescence and phosphor-\nallowed metal-centered (1MC) metal-to-ligand charge transfer escence phenomena simultaneously (Figure 2e). The emission\n(1MLCT) transitions. In the ligand-centered (1LC) electronic at 438 nm with a lower Stokes\u2019 shift can be considered\ntransition, the first absorption bands at shorter wavelength fluorescence due to the radiative decay from the singlet excited\nregion, \u03bbabs: 240\u2013282 nm ([L2Ir]\u03bbmax = 253 nm; [L1Ir]\u03bbmax = 260 nm state to the singlet ground state and the emission at 572 nm\nand [L1Ir]\u03bbmax = 273 nm) was designated as spin-allowed charge with a higher Stokes\u2019 shift value was the indication of\ntransfer transition from filled \u03c0-MOs of 2-phenyl pyridine C N phosphorescence as an emission phenomenon due to radiative\nligand to vacant \u03c0*-MOs of imidazophenanthroline N^N ligand decay from triplet excited state to singlet ground state. This\n(1LLCT: \u03c0ppy!\u03c0imidazophen*); the second absorption band in 1LC exceptional dual emission property of [L2Ir] may be ascribed to\ntransition at next shorter wavelength region, \u03bbabs: 282\u2013312 nm increased \u03c0-conjugation of anthracene moiety in ancillary (N N)\n(\u03bbmax~295 nm) was due to spin-allowed charge transfer tran- ligand. The fluorescence lifetime of the complexes, [L1Ir], [L2Ir],\nsition from filled \u03c0-MOs to vacant \u03c0*-MOs of imidazophenan- and [L3Ir] were 0.056 \u03bcs, 0.049 \u03bcs, and 0.055 \u03bcs, respectively.\nthroline N^N ligand (1ILCT: \u03c0imidazophen!\u03c0imidazophen*). The absorp- The measurement of decay constant for the respective com-\ntion band at \u03bbabs: 312\u2013358 nm (\u03bbmax~332 nm) appeared due to plexes showed high non-radiative decay constants ((Knr)[L1Ir] =\nspin-allowed electronic transition from filled \u03c0-MOs of \u03c0-donor 17.45, (Knr)[L2Ir] = 13.88, and (Knr)[L3Ir] = 16.76, respectively) as well\n2-phenyl pyridine C N ligand to metal vacant d\u03c0 (t2g/eg) MOs as low radiative decay constants ((Kr)[L1Ir] = 0.54, (Kr)[L2Ir] = 6.53,\n[1LMCT: \u03c0(C N)!d\u03c0(Ir)]. In the MLCT region (\u03bbabs: 358\u2013515 nm), and (Kr)[L3Ir] = 1.46, respectively). The significant fluorescence\nthe first low intensity most important weak absorption bands lifetime in the microsecond domain and high Knr as compared\nfor all the complexes were due to spin-allowed metal to ligand to that of Kr also authenticated the feasibility of the phosphor-\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (4 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f 10990682c, 2025, 10, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejic.202400769 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n doi.org/10.1002/ejic.202400769\n\n\n\n\nFigure 2. (a) Absorption spectra of [L1Ir], [L2Ir], and [L3Ir]; (b) HOMO to LUMO electronic transition considering the complex, [L2Ir]; (c) Probable electronic\ncharge transfer transitions between different MOs associated with the respective complexes; (d) Jablonski diagram depicting the pathways of ROS generation;\n(e) Emission spectra of the complexes, [L1Ir], [L2Ir], and [L3Ir].\n\n\n\n\nescence phenomenon upon excitation at the MLCT wavelength phosphorescence promoting the generation of reactive oxygen\nregion. Interestingly, the high Kr of 6.53 for [L2Ir] in comparison species (ROS) in either of two ways: (1) energy transfer upon\nto the other two complexes suggested the possibility of emission from low-lying excited 3MLCT to ligand centered\nfluorescence as an emission process concomitant with the singlet ground state (1LC) (conversion of 3O2!1O2) or electron\nphosphorescence. The emission quantum yield for MLCT transfer upon emission from lowest-lying excited 3ILCT to\nexcitation, (\u03a6f)MLCT of each complex was determined considering ground 1LC state (generation of O2 , OH, H2O2), described in\n * *\n\n\n\n\nrhodamine B as a standard. In reference to the quantum yield Figure 2d during implementation of photodynamic therapy\nvalue of 0.31 of rhodamine B in aqueous solution, complexes, (PDT) under irradiation of visible light. The phosphorescence as\n[L1Ir], [L2Ir], and [L3Ir] showed the quantum yields of 0.03, 0.32, an emission mechanism for all complexes can be rationalized as\nand 0.08, respectively (Figure S13). The highest quantum yield a consequence of spin-orbit coupling (SOC) and the presence of\nvalue, large Stokes\u2019 shift, and significantly high non-radiative Ir(III) heavy metal triggering the rapid non-radiative inter-system\ndecay constant of [L2Ir] suggested that this complex was highly crossing (ISC) to reach an excited triplet state from excited\nefficient among the other two complexes for taking part in singlet state.[21,22]\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (5 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f 10990682c, 2025, 10, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejic.202400769 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n doi.org/10.1002/ejic.202400769\n\n\n2.1.2. Electrochemical Characterisation by Cyclic Voltammetry and they had good solubility in methanol, ethanol, and\n acetonitrile whereas they were mostly insoluble in water\nThe measurement of redox efficiencies of complexes [L1Ir], medium. However, prior to the accomplishment of biological\n[L2Ir], and [L3Ir] is very essential to rationalize their competency and photobiological studies, the determination of stability in\ntowards the formation of reactive oxygen species (ROS) under- dark as well as in light irradiation was a crucial factor in\ngoing various intracellular redox reactions. To comprehend the understanding the stability in different biological environments\nelectron-transfer ability of these complexes, an electrochemical of cells and the photo durability of any photodynamic\nexperiment was conducted using a highly conductive carbon therapeutic agent in exposure to light. Therefore, we checked\nblack-modified glassy carbon electrode (GCE/CB) as the working the stability of the complexes in 10 % DMSO-PBS buffer\nelectrode. Figure S14 depicts the cyclic voltammetry (CV) solution, 1 mM GSH medium, DMEM medium, and 10 % FBS\nresponses of [L1Ir], [L2Ir], and [L3Ir] adsorbed onto GCE/CB solution at 25 \u00b0C and pH 7.4. This study demonstrated that all\nmodified electrodes in phosphate buffer solution at pH 7.4. The the complexes were highly stable in all tested mediums up to\nredox potentials for the complexes [L1Ir], [L2Ir], and [L3Ir], were 72 hours of study (Figure S18\u2013S21). Then we conducted the\nnoted at 0.227 V, 0.293 V, and 0.159 V vs Ag/AgCl, photostability study for all the complexes with the help of UV-\nrespectively corresponding to the redox transitions of the IrIII/IV visible spectroscopy.[24] Interestingly, all complexes exhibited\nas an electron-transfer reaction. The electrode potential energy good stability under visible light irradiation (400\u2013700 nm,\ndiagram also displays the reactive oxygen (ROS) generation 10 Jcm 2) for up to 45 minutes (Figure S22). We further\ncapability of the complexes [L1Ir], [L2Ir], and [L3Ir] (Fig- investigated the photostability in time-dependent 1H NMR\nure S14b). spectroscopy and time-dependent HRMS using complex [L2Ir]\n as representative of other complexes. The photostability results\n in NMR spectroscopy and HRMS unveiled excellent photo\n2.2. DFT Study endurance up to 45 minutes of study (Figure S23).[25]\n\nDensity Functional Theory (DFT) computations were conducted\nwith the complex [L2Ir] as a representative of other complexes 2.4. Hemolysis Study\nin a gas phase environment using the B3LYP exchange-\ncorrelation function as implemented in the Gaussian 09 W After ensuring the stability, the blood toxicity of the complexes\nsoftware suite. The LANL2DZ basis set was selected for these was assessed by evaluating their hemocompatibility in terms of\nanalyses.[23] The optimized structures of these complexes hemolysis study. Erythrocytes function as ideal osmometers,\nemphasizing key bond lengths, electron localization function and their lysis can be triggered by variations in the blood\u2019s\n(ELF), and covalent interactions between donor and acceptor osmotic pressure and physical state. Hemoglobin is released\natoms have been illustrated in Figure S15\u2013S17. For the complex, when erythrocytes are destroyed, and when this hemoglobin is\n[L2Ir], the bond angles C Ir N and N Ir N were found at 78.9\u00b0 excreted in the urine, it is diagnosed as hemoglobinuria, or\nand 78.59\u00b0, respectively. The distances between the iridium and \u201cblood poisoning.\u201d In this case, erythrocyte lysis serves as a\nthe nitrogen atoms of the donor ligands were as measured at reliable marker of the toxicity of any foreign substance to the\n2.07 \u00c5, 2.08 \u00c5, and 2.16 \u00c5, where the distances between iridium blood cells. Moreover, erythrocyte bridging by metal complexes\nand co-ordinated carbon atoms of this cyclometallated complex may be the cause of hemagglutination. Humans are severely\nwere 2.02 \u00c5, and 2. 04 \u00c5. The molecular electrostatic potential affected by this impact, which causes blood cell blockage,\nmap of this complex unveiled the different colour zones blood osmolarity imbalance, and erythrocyte depletion that\nindicating the electron density and internal charge transfer that prevents proper blood function.[26] In order to inspect the\noccurred in the complexes, where the red region indicated the hemolytic qualities of complexes [L1Ir], [L2Ir], and [L3Ir], a\nhigh electron density, the blue zone indicated the low electron hemolysis study was performed at several concentrations (10,\ndensity, while the greenish blue colour pointed the positive 25, 50 \u03bcg/ml). The positive and negative controls were saline\nregion (Figure S16). In Figure S16a, a 3D map of the complex solution, and Milli-Q water, respectively. After one hour of\n[L2Ir] displays non-covalent interactions, where a red-blue circle incubation with the complexes, it was observed that all three\nindicates the coordination bond, red spots stand for resonance, complexes were almost non-hemolytic up to 10 \u03bcg/ml (eqn. iii)\nand green with golden tint stands for hydrogen bonds. More- according to the ISO/TR 7406.\nover, the RGD scatter plot and 2D map of the electron\nlocalization function (ELF) of the complex [L2Ir] reveal various\nbonding and non-bonding interactions that prevailed in this 2.5. Binding Propensity towards Human Serum Albumin (HSA)\nmolecule (Figure S17a, b).\n Binding propensity between drugs and plasma proteins is an\n important fact in pharmacology and chemical biology. The\n2.3. Solubility, Dark- and Photo-Stability binding of a drug with serum albumin, enhance the solubility\n as well as the transportation of drug in blood plasma reducing\nEnvisioning the solubility of the complexes [L1Ir], [L2Ir], and the toxicity of the bound drug. Human serum albumins (HSAs)\n[L3Ir] revealed that all complexes were highly soluble in DMSO are produced in liver and are the key globular proteins present\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (6 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f 10990682c, 2025, 10, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejic.202400769 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n doi.org/10.1002/ejic.202400769\n\n\nin blood plasma of human. It has been studied that human further validated the binding propensity as well as the binding\nserum albumin (HSA) is widely distributed in the cell\u2019s modes of [L2Ir] with HSA by dint of molecular docking (MD)\nsurroundings by creating a more active coordinate complex study. The Autodock 4.2 computational program with the\nwith the drug. This makes the precise admission of the drug Lamarckian genetic algorithm (LGA) was used to study the blind\neasier into the cell after transportation through blood stream. docking experiment. The heart-shaped crystal structure of HSA\nTherefore, it is very crucial to rationalize the binding efficiencies (PDB ID: 1AO6) was taken from the Protein Data Bank, and then\nof these complexes with HSA for confirming their facile trans- the structure was refined by using the online tool Swiss model.\nportation to the target cells.[27] HSA molecule inherits the HSA is comprised of a single polypeptide chain with 585 amino\nfluorescence property due to the presence of tryptophan acid residues, possessing three homologous \u03b1-helical domains:\nmoiety or fluorescence resonance energy transfer (FRET) from domain I (amino acid residues 1\u2013195), domain II (amino acid\ntyrosine to tryptophan and it exhibits fluorescence emission at residues 196\u2013383), domain III (amino acid residues 384\u2013585),\n330 nm upon excitation at 280 nm. Therefore, to comprehend and each domain contains two sub-domains, A and B (Figure 3).\nthe complex-HSA interaction phenomenon, we accomplished Different subdomains of human serum albumin are responsible\nthe emission quenching experiment upon gradual addition of for binding with innumerable endogenous and exogenous\nthe complexes, [L1Ir], [L2Ir] and [L3Ir] to the HSA solution at substances. The main binding sites of metallodrugs are located\n25 \u00b0C and at constant pH of 7.4 (Figure S25). It was vividly in between the subdomains IIA and IIIA of HSA, where metal-\nobserved that initial fluorescence intensity of the HSA was lodrugs interact with HSA in non-covalent ways, especially in\nstarted to decline significantly upon gradual addition of the the hydrophobic binding pockets lined by amino acid residues\nrespective complexes with increased concentration (5\u201355 \u03bcM). in subdomain IB. It was visualized that complex [L2Ir] was\nThe progressive lowering in fluorescence intensity firmly bound to HSA at the site below the subdomain IB, flanked by\nindicated the substantial interactions of the complexes with the subdomains IIA and IIIA. The significantly high binding\nHSA, which was nicely corroborated by estimating the Stern\u2013 energy of 11.11 kcal/mol pleasantly certified the experimental\nVolmer quenching constant (KHSA), quenching rate constant (Kq), observation. The docked structure of [L2Ir] was stabilized by\nand binding constant (K) by applying the Equation (iv) and (v). the various non-covalent interactions arising from alkyl and \u03c0-\nThe KHSA values for the complexes [L1Ir], [L2Ir] and [L3Ir] were alkyl interactions with arginine residues (ARG-114, ARG-145), \u03c0-\nfound to be 0.45\u00d7106 M 1, 0.83\u00d7106 M 1 and 0.27\u00d7106 M 1, carbon interaction with arginine residue (ARG 186), amide-\u03c0\nrespectively. The Kq values for these complexes were observed stack interaction with histidine residue (HIS-146) of domain I\nas 4.5\u00d71013 M 1 s 1, 8.3\u00d71013 M 1 s 1 and 2.7\u00d71013 M 1 s 1, respec- along with \u03c0-\u03c3 interaction with isoleucine residue (ILE-142) and\ntively. Static quenching, efficient bimolecular quenching, and glycine residue (GLY 189). Moreover, amide-\u03c0 stacking and \u03c0\u2013\u03c0\nbimolecular binding with these molecules were all validated by interactions of leucine residue (LEU-185) with the anthracenyl\nfinding Kq values greater than the highest value of dynamic moiety stabilized the docked structure of [L2Ir] with HSA\nquenching (2.0\u00d71010 mol 1 s 1). The Scatchard Equation (vi) (Figure 3).[28]\nallowed us to calculate the total number of binding sites (n)\nand binding constants (K) for these complexes. In this study, we\nfound that the complex [L2Ir] attained very strong binding\nefficacy with human serum albumin and facilitated the trans-\nportation of that complex towards the cell membrane. We\n\n\n\n\nFigure 3. (a)\n 3D view of the best pose of the complex [L2Ir] within the hydrophobic pocket of HSA; (b) 2D representation indicating the multiple interactions of different\namino acid residues of HSA with the complex [L2Ir].\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (7 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f 10990682c, 2025, 10, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejic.202400769 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n doi.org/10.1002/ejic.202400769\n\n\n2.6. Lipophilicity and Cellular Accumulation Table 1. Cytotoxicity of the Complexes [L1Ir], [L2Ir], and [L3Ir] against\n MDA-MB-231 under light and dark conditions.\nPermeability is an essential characteristic of any drug candidate Complexes IC50 (\u03bcM)[a] MDA-MB-231 PIc\nto comprehend the potential distribution of drug to the Dark Light\npharmacological target after considerable cellular accumulation\n [L1Ir] 130.91 \ufffd 2.72 \u03bcM 2.68 \ufffd 0.25 \u03bcM 48.84\nby penetrating the cell membrane as well as to identify the\nability of drug to traverse through the gastrointestinal (GI) tract. [L2Ir] > 300 \u03bcM 3.8 \ufffd 0.34 \u03bcM 78.94\nAs the cell membrane consists of protein-lipid-protein bilayer, a [L3Ir] > 300 \u03bcM 14.15 \ufffd 0.05 \u03bcM 21.20\nsuitable drug candidate should possess at least a threshold [a] IC50 is the concentration of the synthesized complexes at which 50 %\namount of lipophilicity (hydrophobicity) to be absorbed to the of cells undergo cytotoxic cell death under the treatment of a drug. PIc\nphospholipid membrane. Therefore, lipophilicity is an important (phototoxicity index) = ratio of IC50 in the dark to IC50 in light.\n\nparameter to measure the membrane permeability. It is\nmention-worthy; a balance between hydrophobicity and hydro-\nphilicity is also required for intra-cellular interaction after\nsuccessful membrane penetration. Consequently, we precisely 2.7.2. Photoinduced Singlet Oxygen(1O2) Generation\nstudied the lipophilic competency of [L1Ir], [L2Ir], and [L3Ir]\ncomplexes to understand their membrane penetrating ability The singlet oxygen (1O2) generation efficiency of a photo-\nand considerable cellular accumulation. A lipophilicity study sensitizer (PS) upon light irradiation is an essential factor to\nwas performed in an n-octanol-water (PBS buffer) medium with rationalize its capability for undergoing type II photodynamic\nthe varied ratio of n-octanol and water to calculate the n- therapy (PDT) at the target-site of cancer cells. Metal complexes\noctanol/water partition coefficient (Po/w) following the shake- as photosensitizer (PS), transfer energy to triplet molecular\nflask method at 25 \u00b0C and pH 7.4 (Figure S26 a, b, c). Upon oxygen (3O2) during returning back to singlet ground state (1GS)\nestimation of lipophilicity (Log Po/w) for each of the three from excited triplet state (3MLCT) and 3O2 utilizes this energy for\ncomplexes, it was observed that complexes [L1Ir], [L2Ir], and spin paring, which results in the formation of highly reactive\n[L3Ir] possessed the lipophilicity values of 1.12 \ufffd 0.01, 1.22 \ufffd singlet oxygen (1O2) as depicted in Figure 2d. Therefore, we\n0.014, and 1.03 \ufffd 0.169, respectively (Figure S26d). This conse- used a standard protocol to quantify the singlet oxygen (1O2)\nquence was also reflected in the cellular accumulation, which generation efficiency of the complexes by UV-Visible spectro-\nwas also quantified by inductively coupled plasma mass scopy upon photosensitization of the complexes [L1Ir], [L2Ir],\nspectrometry (ICP-MS) (Figure S26e).[26] [L3Ir] with respect to Rose Bengal (RB), an established photo-\n sensitized singlet oxygen generator using 1,3-diphenylisoben-\n zofuran (DPBF) as 1O2 sensitive photodegradable dye. As soon\n2.7. Photobiology as 1O2 starts to liberate in the solution at room temperature\n upon photoexcitation of the photosensitiser, the fluorescent\n2.7.1. Cytotoxicity Under Dark and Light probe (DPBF) reacts with 1O2 and then immediately degrades to\n non-fluorescent 1, 2-dibenzoylbenzene via endoperoxide for-\nThe complexes [L1Ir], [L2Ir], and [L3Ir] were examined for mation, which can be detected by the gradual decrease in\nin vitro cytotoxicity utilizing the 3-(4,5-dimethylthiazol-2-yl)-2,5- optical density of DPBF in UV-Visible spectrum. In this study, we\ndiphenyl-tetrazolium bromide (MTT) assay in TNBC cell line observed that absorbance of DPBF at 417 nm was gradually\n(MDA-MB-231) under dark and light exposure. The complex- declined over a period of time in the precence of respective\ntreated cells were exposed to 4 mW cm 2 of yellow light at a complexes under visible light irradiation (400\u2013700 nm,\ndosage of 0\u20132 J cm 2 from a 400 W tungsten lamp equipped 10 Jcm 2) (Figure 4). This same analysis was also conducted for\nwith a 500 nm long pass filter and a heat isolation filter for Rose Bengal to compare the 1O2 generation competency of the\napproximately 4 hours. Complexes [L1Ir], [L2Ir], and [L3Ir] complexes. The relative changes in absorbance of DPBF (A/A0)\nexhibited a very nominal degree of dark cytotoxicity with IC50 was plotted against exposer times to visible light in second,\nvalues of 130.91 \ufffd 2.72 \u03bcM, > 300 \u03bcM, and > 300 \u03bcM respec- where A0 stands for absorbance of DPBF at zeroth time (t =\ntively (Table 1). In contrast, the complexes exhibit noteworthy 0 sec) and A is the absorbance of DPBF at a particular exposer\nIC50, 2.68 \ufffd 0.25 \u03bcM for [L1Ir] (through Type I and Type II PDT), time. The linearly declined plot (A/A0 vs time) indicated the\n3.8 \ufffd 0.34 \u03bcM for [L2Ir] (through Type I and Type II PDT), and significant degradation of DPBF through conversion of 3O2 to\n 1\n14.15 \ufffd 0.05 \u03bcM for [L3Ir] (through only Type II PDT) when O2 in favour of type II photodynamic process. Noteworthily,\nexposed to light. Even though the toxicity of [L1Ir] is the there was no such decrease in DPBF absorbance at 417 nm in\nhighest, the phototoxicity index (PI) of [L2Ir] (PIc[L2Ir] = 78.94) is the presence of the complexes in the dark condition under\nmuch more prominent than [L1Ir], and [L3Ir] (PIc[L1Ir] = 48.84 similar experimental conditions, which suggested the singlet\nand PIc[L3Ir ] = 21.20), consequently making it the most potent in oxygen generation was specifically under light condition but\nthe series (Figure S27). was reluctant to occur under dark condition. The calculated\n singlet oxygen quantum yields of complexes [L1Ir], [L2Ir], and\n [L3Ir] with reference to rose bengal [(\u03a6\u0394) = 0.76], were found to\n be 0.23, 0.26, and 0.29, respectively (eqn. (vii) and (viii)).\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (8 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f 10990682c, 2025, 10, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejic.202400769 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n doi.org/10.1002/ejic.202400769\n\n\n\n\nFigure 4. Singlet oxygen generation study by UV-visible spectroscopy in the presence of DPBF under visible light: For (a) Rose Bengal (RB); (b) Complex [L1Ir];\n(c) Complex [L2Ir]; (d) Complex [L3Ir] at 298 K. (e) A competitive plot depicting singlet oxygen generation between Rose Bengal (RB) and complexes [L1Ir],\n[L2Ir], [L3Ir] as (A0\u2013A)/A0 vs time in second; (f) Plots exhibiting the relative change in absorbance by DPBF for Rose Bengal and complexes [L1Ir], [L2Ir], [L3Ir] as\n(A/A0) vs time in second at 417 nm.\n\n\n\n\nTherefore, the substantial singlet oxygen quantum yield values (25 \u03bcM) and NADH (150 \u03bcM) (Figure 5). This observation clearly\nof concerned complexes affirmed their probable abilities to indicated that visible light irradiation triggered the oxidation of\ntake part in photodynamic therapy following type II pathway.[29] NADH to NAD + catalysed by the activated complex. As a\n possible mechanism of photocatalytic NADH oxidation by the\n complexes [L1Ir], [L2Ir], and [L3Ir], it can be depicted that at\n2.7.3. Photocatalytic NADH Oxidation first iridium in + 3 state [Ir(III)] in each complex is excited to\n activated state [*Ir(III)] upon visible light irradiation. The\nNicotinamide adenine dinucleotide (NAD) is the main coenzyme activated *Ir(III) state in each complex releases electron and\nfor regulating the redox reactions in cellular metabolic process. oxidised to Ir(IV) state. This unstable Ir(IV) state compel NADH\nAs the NADH (reduced form of NAD), plays an important role in to release an electron from N-centre of pyridine ring to form\nthe mitochondrial electron transport chain (ETC) system, the unstable NADH + intermediate. Then Ir(IV) takes a single\n *\n\n\n\n\noxidation of NADH to NAD + interrupt the electron transport electron to return to stable Ir(III) state and continue the catalytic\nchain and thereby hinder the energy production during cycle. The unstable NADH + releases one proton to form NAD ,\n * *\n\n\n\n\nmetabolic process, which results in the significant cellular which in turn releases electron to form ultimately NAD +. In this\ndamage. To certify the NADH oxidation capability of complexes way, the successive catalytic cycles triggered the conversion of\n[L1Ir], [L2Ir], and [L3Ir], we performed the UV-Visible absorption NADH to NAD + upon visible light irradiation and we estimated\nstudy following titration method. Interestingly, there was no the turn over frequencies (TOFs) for the conversion of NADH to\nsignificant change in absorption bands of the complex-NADH NAD +, where complex [L2Ir] showed the highest TOF of\nsolution with time under dark condition. However, a significant 37.82 h 1 and complexes [L1Ir], [L3Ir] displayed the TOF values\n *\n\n\n\n\nincrease in absorption band at \u03bbmax = 256 nm was visualised for of 11.13 h 1, 27.46 h 1, respectively (Equation (viii)). Therefore,\n * *\n\n\n\n\nthe successive production of NAD + due to gradual destruction this study agreeably corroborates the photocatalytic capabilities\nof NADH upon irradiation of visible light (400\u2013700 nm, of all the complexes attributing the complex [L2Ir] as the best\n10 Jcm 2) at a particular time interval in the solution of complex photocatalyst for conversion of NADH to NAD +, which in turn\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (9 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f 10990682c, 2025, 10, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejic.202400769 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n doi.org/10.1002/ejic.202400769\n\n\n\n\nFigure 5. Photocatalytic oxidation of NADH (150 \u03bcM) exhibiting progressive changes in absorption band under visible light irradiation (400\u2013700 nm, 10 Jcm 2):\nBy (a) Complex [L1Ir]; (c) Complex [L2Ir]; (d) Complex [L3Ir] at 298 K; (e) Plausible mechanism of catalytic photooxidation of NADH by complexes [L1Ir], [L2Ir],\n[L3Ir].\n\n\n\nconveys the possibility of the formation of super oxide radical type I ROS produced under light irradiation (Figure S28b).\n(O2 ) as the reactive oxygen species (ROS) via reaction of\n *\n Therefore, this experiment supports the ability of the complexes\nmolecular oxygen and free electron liberated in NADH to NAD + for type I ROS production, which may also involve the type I\nconversion [O2-2e!O2 ] (Figure 5).[30]\n *\n pathway of photodynamic therapy to exterminate triple neg-\n ative breast cancer.[31]\n\n2.7.4. Reactive Oxygen Species Detection by TMB Assay\n 2.7.5. GSH Depletion Study\n3,3\u2019,5,5\u2019-Tetramethylbenzidine (TMB) is a non-toxic and non-\nmutagenic colourless dye and its solution acquires a distinctive Cancer cells are sheltered by the shielding effect of the high\nblue colouration upon oxidation. When TMB comes in contact antioxidant content and hence the suppression of antioxidant\nwith ROS, it produces TMB +, which can be detected by a system opens an easy and effective way to attack cancer cells\nsignificant characteristic absorption band of TMB + at \u03bbmax = via a ROS-mediated mechanism. Glutathione (GSH) is regarded\n652 nm. Hence, the oxidation of TMB dye can be used as a as the most prevalent endogenous antioxidant, which possess\ndetection tool to demonstrate the complexes\u2019 ability in the the main source of nonprotein thiol groups in cells and\nproduction of type I ROS species, viz hydroxyl radical ( OH) and *\n maintain the redox homeostasis in cells. In this regard, breaking\nsuperoxide radical (O2 ) when subjected to light irradiation. At\n *\n of redox homeostasis via intracellular GSH depletion finds an\nfirst, we obtained the absorption spectrum of TMB solution inevitable aspect to make the cancer cells more vulnerable to\nunder light irradiation and then solutions of the complexes ROS harvesting metallodrugs. Therefore, we tested the capa-\n[L1Ir], [L2Ir], and [L3Ir] (25 \u03bcM) were exposed to visible light bility of three complexes [L1Ir], [L2Ir], and [L3Ir] for depletion of\n(400\u2013700 nm, 10 Jcm 2) for 60 minutes followed by incubation intracellular glutathione level as this is very crucial analysis to\nwith 100 \u03bcM of TMB solution in PBS buffer medium at 25 \u00b0C support the ROS-mediated DNA damage as one of the\n(pH = 7.4). After that, we immediately checked the absorption important pathways of cancer cell death. We accomplished this\nspectrum of the complex-TMB mixture (Figure S28). The study in UV-Visible absorption spectroscopy using the Ellman\u2019s\nemergence of a new absorption band at 652 nm was the reagent, 5, 5-dithiobis-(2-nitrobenzoic acid) (DTNB) as a specific\nindication for TMB + formation upon oxidation of TMB by the detector of thiol group in a sample. DTNB possesses the\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (10 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f 10990682c, 2025, 10, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejic.202400769 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n doi.org/10.1002/ejic.202400769\n\n\ncharacteristic absorption band at around \u03bbmax = 325 nm. When bathochromic shift was only observed for complex [L1Ir] at\nDTNB comes in contact with GSH, GSH readily reacts with DTNB \u03bbmax = 258 nm and a decent bathochromic shift for complex\nand cleaves the S S linkage forming the oxidized GS-TNB [L3Ir] at \u03bbmax = 272 nm, whereas bathochromic was not visual-\nadducts. Consequently, TNB is set free in the solution, and it ized in the absorption spectrum of complex [L2Ir]. The bath-\nexhibits the absorption band at \u03bbmax = 412 nm. Therefore, ochromic shift in the absorption spectrum of [L1Ir] at \u03c0\u2013\u03c0*\ngradual shifting of \u03bbmax from 325 nm to 412 nm is an indication region can be attributed to the fact of overlapping the vacant\nof reaction of free GSH with DTNB. The steady increase in \u03c0S orbitals of this complex with the filled \u03c0 orbitals of DNA\nabsorption intensity at 412 nm measures the higher rate of TNB bases diminishing the overall energy gap of \u03c0!\u03c0S transition,\nformation and hence the concentration of GSH in solution. which leads to the shift of absorbance at longer wavelength\nInitially, we followed the titration method to quantify the total (bathochromic shift). The significant hypochromic shift man-\nGSH content in the sample keeping the DTNB concentration ifested sturdy intercalative interactions for all complexes, while\nfixed at 55 \u03bcM and made a standard curve by plotting the complex [L1Ir] exhibited electrostatic as well as intercalative\ndifferent absorption values at 412 nm versus known concen- interactions with ct-DNA. The calculation of intrinsic binding\ntration of GSH (0\u201360 \u03bcM) and we obtained a linear curve for constants (Kb) (eqn. ix) displayed the values as 1.59\u00d7105 M 1,\nfurther analysis of GSH concentration in unknown sample 1.45\u00d7105 M 1, and 0.73\u00d7105 M 1, respectively for complexes\n(Figure S29a, b). In the next step, we studied the GSH depletion [L1Ir], [L2Ir], and [L3Ir] (Figure 6a). The intrinsic binding\ncapability of the respective complexes by incubating each constant values in the 103 to106 order strongly support favour\ncomplex (50 \u03bcM) with 55 \u03bcM DTNB and 55 \u03bcM GSH for of the intercalative mode of binding interaction between\n2 minutes in DMSO-PBS buffer medium at 25 \u00b0C (pH 7.4). From complexes and DNA,[33] which was also validated by the\nthe respective absorbance values at 412 nm for the complexes negative values of Gibbs free energy [(\u0394Go)[L1Ir] = 29. 68 KJ/\n[L1Ir], [L2Ir], and [L3Ir], we quantified the corresponding free mol; (\u0394Go)[L2Ir] = 29.44 KJ/mol and (\u0394Go)[L1Ir] = 27.74 KJ/mol]\nGSH concentration from the standard curve and then calculated indicating the spontaneous drug-DNA intercalation process.\nthe amount of depleted GSH for each complex. The calculated An EtBr displacement assay was also performed using a\nresult showed that [L1Ir], [L2Ir], and [L3Ir] depleted the GSH by spectrofluorometer to rationalize the intercalative mode of\n37.5 \u03bcM, 40.5 \u03bcM and 29 \u03bcM, respectively (Figure S30). These interaction of the synthesized complexes with ct-DNA. After\nfindings demonstrated the high competency of the metal addition to ct-DNA, EtBr exhibited significant fluorescence at\ncomplexes to destroy the cancer cells by reactive oxygen \u03bbems = 604 nm upon excitation at \u03bbabs 485 nm, as a result of its\nspecies hindering the activity of intracellular GSH.[26] intercalative manner of interacting with a DNA base pair. With\n each subsequent rise in the concentration of the metal\n complexes, the fluorescence intensity of the EtBr-DNA adduct\n2.7.6. Study of DNA Impairment Under Dark and Light was steadily reduced (hypochromic nature). It implies that EtBr\n is successively removed from the EtBr-DNA adduct, upon\nDeoxyribonucleic acid (DNA) plays a pivotal role in regulating intercalative interaction between DNA and the metal com-\ncellular growth, survival, and reproduction as well as in carrying plexes. We obtained the Kapp values as 2.67\u00d7106 M 1,\ngenetic material from generation to generation. Therefore, DNA 3.2\u00d7106 M 1, and 1.45\u00d7106 M 1for complexes [L1Ir], [L2Ir] and\ndamaging strategy has become a topic of interest in this [L3Ir], respectively using Equation (x) under the condition of\npresent work to develop a new approach to treating triple- 50 % reduction of emission intensity. The Stern-Volmer quench-\nnegative breast cancer. Here, we have tried to develop a TNBC ing constant (KSV) values of complexes [L1Ir], [L2Ir] and [L3Ir],\npreventive therapeutic pathway through measuring the DNA were determined as 3.8\u00d7104 M 1, 3.9\u00d7104 M 1 and 1.5\u00d7104 M 1,\ndamaging potential of imidazophenenthroline-based simple respectively (Equation (xi)). The highest Kapp value of complex\nand small cyclometallated Ir(III)-complexes upon generation of [L2Ir] revealed the most powerful intercalative nature of this\nsinglet oxygen or type I reactive oxygen species (ROS). To scale complex compared to [L1Ir], and [L3Ir]. We also calculated the\nthe propensity of complex-DNA interaction and ROS-mediated C50 values for the complexes by plotting the % fluorescence vs\nDNA damage, we performed the UV-visible absorption study in concentration of complexes to measure their relative intercala-\nthe presence and absence of visible light irradiation as UV\u2013Vis tive competency in interaction with DNA. In Figure 6b, we\nspectroscopy offers the simplest and highly insightful technique observe the C50 values as 28.84 \u03bcM for [L1Ir], 24.06 \u03bcM for [L2Ir],\nfor comprehending the complex-DNA interaction. In this study, and 50.71 \u03bcM for [L3Ir]. The lowest C50 value of [L2Ir] strongly\nwe periodically added the calf thymus DNA (ct-DNA) solution corroborates the previous findings in support of the most\nwith increasing concentration (0 \u03bcM!20 \u03bcM for [L1Ir]; 0 \u03bcM! intercalative nature of the complex [L2Ir]. Therefore, the\n30 \u03bcM for [L2Ir]; 0 \u03bcM !35 \u03bcM for [L3Ir]) to the fixed intercalative mode of complex-DNA interaction can be consid-\nconcentration of the complexes (25 \u03bcM) at 25 \u00b0C. Consequently, ered the predominant mechanism for DNA damage in dark\nwe observed significant hypochromism at \u03c0\u2013\u03c0*, LMCT and conditions.\nMLCT regions in the absorption spectra of the complexes [L1Ir], After the comprehensive insight into the DNA damaging\n[L2Ir], and [L3Ir] (Figure S30), which can be considered as a capabilities of the complexes in the absence of light, we\nresult of the lowering of absorption intensity upon accumu- investigated the visible light-inspired DNA photocleavage in\nlation of \u03c0-electrons into the DNA base pair.[32] In association UV-visible spectroscopy. It was observed that complexes were\nwith the hypochromic effect for all complexes, a considerable capable of progressively damaging the DNA upon irradiation of\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (11 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f 10990682c, 2025, 10, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejic.202400769 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n doi.org/10.1002/ejic.202400769\n\n\n\n\nFigure 6. (a) Complex-DNA interaction study by UV-Vis spectroscopy and respective Kb values for complexes [L1Ir], [L2Ir], and [L3Ir]; (b) Ethidium bromide\n(EtBr) displacement by relative quenching of fluorescence intensities and respective C50 values for complexes [L1Ir], [L2Ir] and [L3Ir]. (c) Comparative study of\nDNA photo-cleavage by UV-Vis spectroscopy. (d) 3D view of [L2Ir]-DNA interaction; (e) 2D view of [L2Ir]-DNA interaction.\n\n\n\n\nvisible light (400\u2013700 nm, 10 Jcm 2) for 60 minutes. The relative light irradiation in comparison to the other two complexes\ncompetency of DNA photocleavage was also identified by through the generation of toxic reactive oxygens species (ROS).\nplotting A/A0 vs time concerning the DNA damage in dark To ratify the complex-DNA interaction theoretically, we also\nconditions (Figure 6c). The A/A0 vs time plot revealed that performed a molecular docking study with the best DNA-\ncomplex [L2Ir] was highly capable of damaging DNA under damaging complex [L2Ir]. In theoretical outlook, complex [L2Ir]\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (12 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f 10990682c, 2025, 10, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejic.202400769 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n doi.org/10.1002/ejic.202400769\n\n\ncomes out as the robust DNA binding moiety with a remarkable [L3Ir]. The meticulous investigations have revealed that com-\nbinding constant of 10.4 kcal/mol by undergoing different plexes are eligible to release reactive oxygen species upon\ninteractions (Figure 6e). visible light irradiation instigating the damage of DNA. The\n complexes have shown remarkable photostability, which can\n help them to withstand visible light. The phototoxicity index\n2.8. Proposed Mechanism of Cell Death by PDT Based on the revealed that complex [L2Ir] is more phototoxic compared to\nObtained Result the others. The human serum albumin interactions and DNA\n binding studies also justify the best efficiency of the complex\nBased on the obtained result we delineated a clear mechanism [L2Ir] towards biomolecular interactions.\nof DNA impairment in favor of triple-negative breast cancer cell\ndeath (Figure 7). The redox potential of the complexes showed\nthat they can fluctuate from + 3 to-4 oxidation state depending Experimental Section\nupon the conditions and the complexes can generate singlet\n Materials and Methods. In this study, high-quality reagents and\noxygen as well as superoxide or hydroxyl radicals upon solvents of commercial grade were employed. All chemicals and\nirradiation of visible light (400\u2013700 nm, 10 Jcm 2). Therefore, we biochemical substances were sourced from Sigma-Aldrich Chem-\nhave postulated that visible light irradiation may unveil two icals Limited, Spectrochem, TCI Chemicals, and E-Merck. Cell lines\npathways (type I and type II) of photodynamic therapy, where utilized in the experiments were obtained from NCCS, Pune. A\n Bruker DPX spectrometer with high power (400 MHz) was\nthe type II mechanism predominates more than type I. In the\n employed to record all of the NMR spectra, with tetramethyl silane\ntype II process, significant DNA impairment occurs due to the (TMS) serving as the standard. A TDS conductometer-307 was used\nformation of 8-oxo-deoxyguanosine (8-Oxo-dG) from the deox- to measure the complexes\u2019 experimental conductivity. A Shimadzu\nyguanosine moiety by the toxic impact of singlet oxygen, which Affinity FT-IR spectrometer was utilized to record the complexes\u2019\nindicates the damage of guanine base in DNA as depicted in infrared (IR) spectra across the range of 4000\u2013400 cm 1. JASCO\nFigure 7. On the other hand, complexes also show the capability 8440 fluorescence spectrophotometer with a xenon lamp was\n selected for the fluorescence experiment and a JASCO V-730\nof energy transfer to generate other reactive oxygen species via\n spectrophotometer with a 1 cm quartz cell was utilized for the UV\u2013\nthe type I pathway. As a consequence, the effect of both visible absorption experiment. An Elisa reader and 96 wallplates\nmechanisms triggers DNA impairment leading to apoptosis of were utilized for the MTT assay. MDA-MB-231 (TNBC) cells were\ntriple-negative breast cancer cells. purchased from the National Centre for Cell Sciences (NCCS, Pune,\n India). Cell culture supplies such as Dulbecco\u2019s Modified Eagle\u2019s\n Medium (DMEM) along with 1 % penicillin, streptomycin, and 1 %\n Glutamax, were procured from Gibco. Fetal Bovine Serum (FBS),\n3. Conclusions and Dimethyl Sulfoxide (DMSO), antibiotic mixture, Trypsin,\n Reduced Glutathione, DTNB were purchased from Hi-Media labo-\nIn conclusion, we can depict that we have successfully ratories in Mumbai.\nsynthesized and characterized the complexes [L1Ir], [L2Ir], and\n\n\n\n\nFigure 7. Proposed mechanism of DNA impairment by visible light-induced ROS generation.\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (13 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f 10990682c, 2025, 10, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejic.202400769 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n doi.org/10.1002/ejic.202400769\n\n\nGeneral Synthetic Procedure for the Preparation of (strong, Arm C=C stretching), 1472 (strong, C N stretching), 1163\nIridium(III)-Cyclometallated Imidazophenanthroline Complexes (medium, C=C bending), 1027 (medium, C=C bending), 755 (strong,\n([L1Ir], [Ir2Ir], [L3Ir]) C H bending); HRMS (MeOH) m/z: 897.2318 [M Cl] +.\n\n30 mg (0.02 mmol, 1 equiv.) of bright yellow coloured iridium [L3Ir]. 46 mg (0.051 mmol, 91 %); Mr (C44H28N6O2IrCl) = 900.40 g/mol;\nprecursor ([IrCP]) was taken in a pear-shaped flask and it was Anal. calcd for C44H28N6O2IrCl: C 58.69, H 3.13, N 9.33; found: C\ndissolved properly in 10 ml of 4: 1 toluene/methanol solvent 58.68, H 3.12, N 9.40; Yield: 91 %; Colour: Reddish Brown; Mp: 215\u2013\nmixture to get a clear yellowish solution. Then 2.1 equivalents of 220 \u00b0C; Rf (100 % methanol): 0.26; UPLC purity (99.5 %, eluent: 50 %\nthe previously prepared pure ligand (L1/L2/L3) were added to the H2O/ACN, Rt: 2.741 min); 1H NMR (DMSO-d6, 400 MHz): \u03b4 8.33 (d, J =\nsolution of iridium precursor ([IrCP]) and sonicated for 5 minutes to 7.6 Hz, 1H, ArH), 8.28 (d, J = 8.0 Hz, 2H, ArH), 8.16 (brs, 2H, ArH),\nget a clear brownish-coloured solution upon thoroughly mixing the 8.07\u20138.10 (m, 2H, ArH), 7.97 (t, J = 8.0 Hz, 4H, ArH), 7.88 (t, J = 8.0 Hz,\nreactants. Thereafter, the reaction mixture was refluxed for 6 h in 4H, ArH), 7.68 (t, J = 7.6 Hz, 1H, ArH), 7.52 (d, J = 6.0 Hz, 2H, ArH),\nN2-atmosphere at 120 \u00b0C. The progression of the reaction was 7.07 (t, J = 7.6 Hz, 2H, ArH), 7.00\u20136.95 (m, 5H, ArH), 6.31 (d, J =\nperiodically monitored by thin-layer chromatography (TLC) using 7.6 Hz, 2H, ArH); 13C NMR (DMSO-d6, 100 MHz): \u03b4 168.1, 158.5, 148.4,\n100 % methanol as a solvent system. At around 6 h of reflux, we 148.2, 143.6, 138.0, 134.3, 131.9, 130.9, 126.6, 125.8, 124.9, 123.1,\nobserved a significant change in the colour of the reaction mixture 122.8, 119.7; IR (cm 1, KBr): \u03bd 3058 (weak, Arm sp2 C H stretching),\nfrom pale brown to dark brown, and the completion of the reaction 2922 (weak, sp3 C H stretching), 2030 (medium, C=O stretching),\nwas confirmed by re-performing the TLC. After cooling the reaction 1894 (), 1603 (medium, Arm C=C stretching), 1479 (medium, C N\nmixture at room temperature, the product was seen to be stretching), 1275 (strong, C=C bending), 1120 (C=C bending), 761\nprecipitated out. Then, we obtained the crude product after (strong, C H bending); HRMS (MeOH) m/z: 865.1904 [M Cl] +.\nfiltration. The crude product was repeatedly washed with hexane as\nwell as hexane-ethyl acetate mixture (3 : 1) 5\u20136 times to remove\nimpurities and the purity of the product was checked by perform-\ning the TLC. The cleaned product was then dried and subjected to 4. Supporting information Summary\nrecrystallization from methanol/diethyl ether to obtain more\n \u20201\npurified product. The crystalline product of each complex was H,13C NMR, FT-IR spectra, UPLC, HRMS of all compounds;\nweighed in a weighing balance and the %yield was calculated. In Quantum yield plot; DFT computed optimized structure; CV\ndue course, we obtained the deep yellow to reddish brown responses; Stability study plots; %Hemolysis plots, HSA binding\ncoloured crystals of complexes [L1Ir], [L2Ir], and [L3Ir] with high\n plots; Representative diagram of lipophilicity; ICP-MS Analysis;\nyields (91\u201397 %). The structures of all the complexes were assured\nby 1H, 13C NMR, FT-IR spectroscopy, and HRMS. The purity of the plots of NADH oxidation, TMB oxidation plots; plots for\ncomplexes was scrutinized by ultraperformance liquid chromatog- glutathione depletion, plots of DNA binding, EtBr quenching;\nraphy (UPLC) as well as with C, H, N analysis. Experimental Procedure.\n[L1Ir]. 48 mg (0.054 mmol, 97 %); Mr (C45H30N6IrCl) = 882.43 g/mol;\nAnal. calcd for C45H30N6IrCl: C 61.25, H 3.43, N 9.52; found: C 61.23,\nH 3.48, N 9.48; Yield: 97 %; Colour: Deep Yellow; Mp: 220\u2013225 \u00b0C; Rf 5. Abbreviations\n(100 % methanol): 0.22; UPLC purity (61.9 %, eluent: 50 % H2O/ACN,\nRt: 2.087 min); 1H NMR (DMSO-d6, 400 MHz): \u03b4 9.29 (s, 2H, ArH), 9.02 TLC thin-layer chromatography\n(d, J = 7.6 Hz, 1H, ArH), 8.28 (d, J = 8. 4 Hz, 3H, ArH), 8.21\u20138.12 (m,\n FT-IR Fourier transform-infra red\n4H, ArH), 8.11\u20138.08 (m, 3H, ArH), 7.97 (d, J = 7.6 Hz, 2H, ArH), 7.89 (t,\nJ = 7.2 Hz, 2H, ArH), 7.78 (t, J = 8.0 Hz,1H, ArH), 7.72\u20137.65 (m, 2H, HRMS high-resolution mass spectrometry\nArH), 7.54 (d, J = 5.6 Hz, 2H, ArH), 7.08 (t, J = 6.8 Hz, 2H, ArH), 7.02\u2013 ILCT intra-ligand charge transfer\n6.94 (m, 4H, ArH), 6.32 (d, J = 7.2 Hz, ArH); 13C NMR (DMSO-d6, LLCT ligand-ligand charge transfer\n100 MHz): \u03b4 168.5, 151.7, 143.7, 137.9, 136.2, 134.2, 131.9, 130.9, LMCT ligand-metal charge transfer\n130.7, 130.6, 130.0, 129.1, 128.5, 126.9, 126.4, 125.9, 124.9, 123.7, MLCT metal-ligand charge transfer\n123.0, 122.8, 122.1, 121.3, 118.4; IR (cm 1, KBr): \u03bd 3362 (medium,\nN H stretching), 3063 (weak, Arm sp2 C H stretching), 2910 (weak,\n CV cyclic voltammetry\nsp3 C H stretching), 1580 (strong, Arm C=C stretching), 1472 DFT density functional theory\n(strong, C N stretching), 1406 (strong, C=C bending), 1167 (C=C DMSO dimethyl sulphoxide\nbending), 762 (strong, C H bending); HRMS (MeOH) m/z: 847.2162 DMF dimethyl formamide\n[M Cl] +. PBS phosphate buffer saline\n[L2Ir]. 50 mg (0.054 mmol, 96 %); Mr (C49H32N6IrCl) = 932.49 g/mol; FBS fetal bovine serum\nAnal. calcd for C49H32N6IrCl: C 63.11, H 3.46, N 9.01; found: C 63.12, DMEM dulbecco\u2019s modified eagle\u2019s medium\nH 3.45, N 8.95; Yield: 96 %; Colour: Brown; Mp: 235\u2013240 \u00b0C; Rf (100 % HSA human serum albumin\nmethanol): 0.21; UPLC purity (96.9 %, eluent: 50 % H2O/ACN, Rt: MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium\n2.883 min); 1H NMR (DMSO-d6, 400 MHz): \u03b4 9.14 (d, J = 8.0 Hz, 2H,\n bromide\nArH), 8.87 (S, 1H, ArH), 8.29\u20138.23 (m, 4H, ArH), 8.14 (d, J = 4.8 Hz, 2H,\nArH), 8.04\u20138.00 (m, 2H, ArH), 7.98 (d, J = 7.6 Hz, 2H, ArH), 7.91 (t, J = NADH nicotinamide adenine dinucleotide (reduced)\n7.2 Hz, 2H, ArH), 7.86 (d, J = 9.2 Hz, 2H, ArH), 7.59 (t, J = 6.4 Hz, 4H, TMB 3,3\u2019,5,5\u2019- Tetramethylbenzidine\nArH), 7.49 (t, J = 8.0 Hz, 2H, ArH), 7.09\u20137.03 (m, 4H, ArH), 6.97 (t, J = GSH glutathione\n7.6 Hz, 2H, ArH), 6.35 (d, J = 7.6 Hz, 2H, ArH); 13C NMR (DMSO-d6, DTNB 5, 5-dithiobis-(2-nitrobenzoic acid)\n100 MHz): \u03b4 168.5, 154.8, 150.9, 149.2, 146.6, 142.9, 141.0, 137.8,\n ct-DNA calf-thymus DNA\n136.7, 134.2, 133.8, 131.9, 131.0, 128.4, 126.8, 124.9, 123.1,122.2,\n119.5; IR (cm 1, KBr): \u03bd 3360 (weak, N H stretching), 3044 (weak, EtBr ethidium bromide.\nArm sp2 C H stretching), 2915 (weak, sp3 C H stretching), 1602\n\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (14 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f 10990682c, 2025, 10, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/ejic.202400769 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\n doi.org/10.1002/ejic.202400769\n\n\n6. Funding Sources [4] a) N. M. Almansour, Front. Mol. Biosci. 2022, 9, 836417; b) R. L. Siegel,\n K. D. Miller, N. S. Wagle, A. Jemal, Cancer J Clin. 2023, 73, 17\u201348; c) H.\n Sung, J. Ferlay, R. Siegel, M. Laversanne, I. Soerjomataram, A. Jemal, F.\nDST CRG project grant (CRG/2021/002267), Government of Bray, Cancer J Clin. 2021, 71, 209\u2013249.\nIndia. VIT SEED funding. [5] S. Zhu, Y. Wu, B. Song, M. Yi, Y. Yan, Q. Mei, K. Wu, J. Hematol. Oncol.\n 2023, 16, 100.\n [6] L. Yin, J.-J. Duan, X.-W. Bian, S.-C. Yu, Breast Cancer Res. 2020, 22, 61.\n [7] O. Obidiro, G. Battogtokh, E. O. Akala, Pharmaceutica 2023, 15, 1796.\nAuthor Contributions [8] J. Wang, S.-G. Wu, Breast Cancer 2023, 15, 721\u2013730.\n [9] a) Q. Zhang, Q.-B. Lu, Sci. Rep. 2021, 11, 788; b) R. Oun, Y. E. Moussa,\n N. J. Wheate, Dalton Trans. 2018, 47, 6645\u20136653.\nThe project was developed and supervised by PP (Vellore [10] N. Alvarez, A. Sevilla, Int. J. Mol. Sci. 2024, 25, 1023.\nInstitute of Technology). Synthesis, characterization, analytical, [11] D. E. Dolmans, D Fukumura, R. K. Jain, Nat. Rev. Cancer 2003, 5, 380\u2013387.\n [12] P. Agostinis, K. Berg, K. A. Cengel, T. H. Foster, A. W. Girotti, S. O.\nand preliminary biological tests were executed by SG (Vellore\n Gollnick, S. M. Hahn, M. R. Hamblin, A. Juzeniene, D. Kessel, M. Korbelik,\nInstitute of Technology). CA: a Cancer J Clinicians 2011, 4, 250\u2013281.\n [13] N. Sobhani, A. A. Samadani, J Egypt. National Cancer Institute 2021, 33,\n 1\u201313.\n [14] S. Abbas, I. U. D. Din, A. Raheel, Appl. Organometallic Chem. 2020, 3,\nAcknowledgements 5413.\n [15] L. C. C. Lee, K. K. W. Lo, J. Am. Chem. Soc. 2022, 32, 14420\u201314440.\n [16] Z. T. Chu, N. Xu, Y. Su, H. Fang, Z. Su, Dalton Transactions 2024, 53,\nThe authors are grateful to the Department of Science and\n 18585\u201318591.\nTechnology (DST), Government of India, for supporting the [17] K. Choroba, J. Palion-Gazda, M. Penkala, P. Rawicka, B. Machura, Dalton\nwork through the DST-SERB CRG project grant. The authors Trans. 2024, 53, 17934\u201317947.\n [18] S. Jing, X. Wu, D. Niu, J. Wang, C. H. Leung, W. Wang, Molecules 2024,\nthank the Vellore Institute of Technology (VIT) for arranging VIT\n 29, 256.\nSEED funding. The authors are also grateful to the Department [19] P. Barretta, G. Mazzone, Inorg. Chem. Frontiers 2023, 10, 3686\u20133698.\nof Science and Technology, New Delhi, India, for providing [20] A. Mondal, S. Shanavas, U. Sen, U. Das, N. Roy, B. Bose, P. Paira, RSC Adv.\n 2022, 12, 11953\u201311966.\nfinancial support to acquire \u201cInductively Coupled Plasma Mass\n [21] A. R. Smith, P. L. Burn, B. J. Powell, ChemPhysChem 2011, 12, 2429\u20132438.\nSpectrometry (ICP-MS)\u201d through \u201cPromotion of University [22] S. K. Seth, P. Purkayastha, Eur. J. Inorg. Chem. 2020, 2020, 2990\u20132997.\nResearch and Scientific Excellence (PURSE)\u201d under Grant No. SR/ [23] A. K. Yadav, A. Upadhyay, A. Bera, R. Kushwaha, A.-A. Mandal, S.\n Acharjee, A. Kunwar, S. Banerjee, Inorg. Chem. Front. 2024, 11, 5435\u2013\nPURSE/2020/34 (TPN 56960) and carry out the work.\n 5448.\n [24] C. Reghukumar, S. Shamjith, V. P. Murali, P. K. Ramya, K.-V. Radhak-\n rishnan, K.-K. Maiti, J Photochem. Photobiology B: Biology 2024, 250,\n 112832.\nConflict of Interests [25] M. Mart\u00ednez\u2013Alonso, C. G. Jones, J. D. Shipp, D. Chekulaev, H. E. Bryant,\n J. A. Weinstein, J. Bio. Inorg. Chem. 2024, 29, 113\u2013125.\n\u201cThere are no conflicts to declare\u201d. [26] N. Roy, T. Dasgupta, S. Ghosh, M. Jayaprakash, M. Pal, S. Shanavas, S.\n Kanta Pal, V. Muthukumar, A. Senthil Kumar, R. Tamizhselvi, M. Roy, B.\n Bose, D. Panda, R. Chakrabarty, P. Paira, Langmuir 2024, 40, 25390\u2013\n 25404.\nData Availability Statement [27] N. Wu, T. Liu, M. Tian, C. Liu, S. Ma, H. Cao, H. Bian, L. Wang, Y. Feng, J.\n Qi, Molecular Medicine Reports 2023, 29, 24.\n [28] A. Merlino, Coord. Chem. Rev. 2023, 480, 215026.\nThe data that supports the findings of this study are available in [29] B. Kar, P. Paira, Chemistry\u2013A Euro. J. 2024, 202401720.\nthe supplementary material of this article. [30] R. Kushwaha, A. Upadhyay, S. Peters, A. K. Yadav, A. Mishra, A. Bera, T.\n Sadhukhan, S. Banerjee, Langmuir 2024, 40, 12226\u201312238.\n [31] R. Bresol\u00ed-Obach, M. Frattini, S. Abbruzzetti, C. Viappiani, M. Agut, S.\nKeywords: Triple Negative Breast Cancer \u00b7 Photodynamic Nonell, Sensors 2020, 20, 5952.\n [32] R. Nimal, A. Shah, M. Saddiq, J. Photochem. Photobiol. 2020, 2, 100006.\nTherapy \u00b7 ROS \u00b7 DNA Cleavage \u00b7 NADH Oxidation \u00b7 GSH [33] H. G\u00f6kce, N. \u00d6zt\u00fcrk, \u00dc. Ceylan, Y. B. Alpaslan, G. Alpaslan, Spectrochimica\nDepletion \u00b7 Cell Death Acta Part A: Mol. Biomol. Spect. 2016, 163, 170\u2013180.\n\n\n [1] B. Liu, H. Zhou, L. Tan, K. T. H. Siu, X.-Y. Guan, Signal Trans. Targeted\n Ther. 2024, 9, 175.\n [2] S. \u0141ukasiewicz, M. Czeczelewski, A. Forma, J. Baj, R. Sitarz, A. Stanis\u0142awek,\n Cancers 2021, 13, 4287. Manuscript received: November 22, 2024\n [3] P. Khongorzul, C. J. Ling, F. U. Khan, A. U. Ihsan, J. Zhang, Mol. Cancer Revised manuscript received: February 2, 2025\n Res. MCR 2020, 18, 3\u201319. Version of record online: February 18, 2025\n\n\n\n\nEur. J. Inorg. Chem. 2025, 28, e202400769 (15 of 15) \u00a9 2025 Wiley-VCH GmbH\n\f", "pages_extracted": 15, "text_length": 94266}