New encapsulated bis-cyclometalated Ir( iii ) complexes with very potent anticancer PDT activity

{"full_text": " INORGANIC CHEMISTRY\n FRONTIERS\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n View Article Online\n RESEARCH ARTICLE View Journal | View Issue\n\n\n\n\n New encapsulated bis-cyclometalated Ir(III)\n Cite this: Inorg. Chem. Front., 2025,\n complexes with very potent anticancer PDT\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n 12, 7304 activity\u2020\n Cristina Bermejo-Casades\u00fas, \u2021a Carlos Gonzalo-Navarro, \u2021b\n Juan Angel Organero,c Ana Mar\u00eda Rodr\u00edguez,d Luc\u00eda Santos,e Elisenda Zafon,a\n Joao Carlos Lima, f Artur J. Moro,f M\u00f2nica Iglesias, g Pedro Tavares, h,i\n Daniel Mart\u00ednez,b Blanca R. Manzano, *b Anna Massaguer *a and\n Gema Dur\u00e1 *b\n\n Photodynamic therapy (PDT) has emerged as a promising approach for cancer treatment, due to its ability\n to reduce side e\ufb00ects. In the search for luminescent iridium [Ir(C^N)2(N^N)]+ complexes with high ability\n to generate ROS (reactive oxygen species) under irradiation, we employed C^N ligands with high\n \u03c0-expansion ( pbpz (4,9,14-triazadibenzo[a,c]anthracene), 1, or pbpn (4,9,16-triazadibenzo[a,c]naphtha-\n cene), 3) that should lead to long excited state lifetimes. The photophysical properties were signi\ufb01cantly\n in\ufb02uenced by the degree of C^N ligand \u03c0-expansion. Complex 1 exhibited a long \ufb02uorescence lifetime,\n matching the triplet lifetime observed in TAS, suggesting delayed \ufb02uorescence. In contrast, the additional\n ring in complex 3 generated two near-HOMO orbitals, increasing the excited state\u2019s LC character and\n reducing spin\u2013orbit coupling (SOC) and intersystem crossing (ISC). They exhibited a notable ability to\n generate 1O2 and O2\u2022\u2212. TD-DFT studies nicely explained the di\ufb00erentiated photophysical properties. Both\n complexes exhibited signi\ufb01cant phototoxicity against human cancer cells in both monolayer and multicel-\n lular spheroids models, with complex 1 exhibiting a higher e\ufb00ect. They e\ufb00ectively photogenerated intra-\n cellular ROS, including O2\u2022\u2212. The mitochondrial accumulation of 1 and its disruption of mitochondrial\n functions were veri\ufb01ed. Wound healing and clonogenic assays demonstrated their potential as antimeta-\n Received 18th March 2025, static agents. In general, complexes\u2019 encapsulation signi\ufb01cantly facilitated their cellular accumulation and\n Accepted 1st July 2025\n increased photocytotoxic indexes, with NP1 achieving one of the lowest IC50 values reported in iridium\n DOI: 10.1039/d5qi00775e chemistry. Furthermore, the nanoparticles showed good anticancer activity even in 3D models. Thus, 1\n rsc.li/frontiers-inorganic and 3 and especially NP1 show great promise as type I and II PDT agents with theragnostic potential.\n\n\n\n\n a g\n Universitat de Girona, Departament de Biologia, Facultat de Ci\u00e8ncies, Maria Aurelia Universitat de Girona, Departament de Qu\u00edmica, Facultat de Ci\u00e8ncies, Maria\n Capmany 40, 17003 Girona, Spain. E-mail: anna.massaguer@udg.edu Aurelia Capmany 40, 17003 Girona, Spain\n b\n Universidad de Castilla-La Mancha, Departamento de Qu\u00edmica Inorg\u00e1nica, h\n Associate Laboratory i4HB \u2013 Institute for Health and Bioeconomy, NOVA School of\n Org\u00e1nica y Bioqu\u00edmica- IRICA, Facultad de Ciencias y Tecnolog\u00edas Qu\u00edmicas, Avda. Science and Technology, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal\n Camilo Jos\u00e9 Cela, 10, 13071 Ciudad Real, Spain. E-mail: gema.dura@uclm.es, i\n UCIBIO \u2013 Applied Molecular Biosciences Unit, Department of Chemistry, NOVA\n blanca.manzano@uclm.es School of Science and Technology, Universidade NOVA de Lisboa, 2829-516\n c\n Universidad de Castilla-La Mancha, Departamento de Qu\u00edmica F\u00edsica, Caparica, Portugal\n Facultad de Ciencias Ambientales y Bioqu\u00edmicas and INAMOL, 45071 Toledo, \u2020 Electronic supplementary information (ESI) available: Experimental section,\n Spain NMR spectra, ESI+ MS spectra, HPLC traces, X-ray di\ufb00raction, analysis of \u03c0\u2013\u03c0\n d\n Universidad de Castilla-La Mancha, Departamento de Qu\u00edmica Inorg\u00e1nica, stacking by 1H NMR spectroscopy, stability and photostability, photophysical\n Org\u00e1nica y Bioqu\u00edmica- IRICA, Escuela T\u00e9cnica Superior de Ingenieros Industriales, properties, transient absorption spectroscopy (TAS) measurements, ROS gene-\n Avda. Camilo Jos\u00e9 Cela, 3, 13071 Ciudad Real, Spain ration, computational studies, synthesis and characterization of nanoparticles,\n e\n Universidad de Castilla-La Mancha, Departamento de Qu\u00edmica F\u00edsica, Facultad de hemolytic activity, NADH oxidation, internalization analyses. Biological Studies\n Ciencias y Tecnolog\u00edas Qu\u00edmicas, Avda. Camilo Jos\u00e9 Cela, s/n, 13071 Ciudad Real, Raw Data. CCDC 2431327\u20132431329 for complexes 1, 2 and 4. For ESI and crystal-\n Spain lographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/\n f\n Universidade NOVA de Lisboa, LAQV-REQUIMTE, Departamento de Qu\u00edmica, d5qi00775e\n Faculdade de Ci\u00eancias e Tecnologia, 2829-516 Caparica, Portugal \u2021 Both authors have contributed equally to the work.\n\n\n\n 7304 | Inorg. Chem. Front., 2025, 12, 7304\u20137332 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n\n 1. Introduction make Ir(III) complexes potent candidates for enhancing the\n e\ufb03cacy of PDT. Besides, octahedral bis(cyclometalated) com-\n Photodynamic therapy (PDT)1\u20134 has emerged as a promising plexes of Ir(III) are remarkable for their intrinsic luminescence\n approach for cancer treatment because of its ability to reduce properties and are used as cellular imaging reagents and bio-\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n side e\ufb00ects, circumventing one of the main problems limiting molecular probes.27\n the e\ufb00ectiveness of chemotherapy. This technique is a mini- Studies have shown that compounds with long excited state\n mally invasive therapeutic modality that has gained significant lifetimes are more e\ufb03cient at producing ROS, exhibit high\n attention for its ability to selectively target and eradicate singlet oxygen (1O2) quantum yields, and have demonstrated\n cancer cells with spatio-temporal control. PDT relies on the significant photocytotoxicity in vitro.28,29 It is known that the\n use of a photosensitizer (PS) that, upon light activation, greater the 3\u03c0\u03c0* character of the triplet excited state, the longer\n increases the cytotoxic e\ufb00ect in cancer cells through the gene- the T1 lifetime.30 Interestingly, utilizing ligands with extended\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n ration of reactive oxygen species (ROS). This ROS generation is \u03c0-systems reduces the energy of the ligand-centered (LC) 3\u03c0\u03c0*\n divided into two pathways depending on the type of inter- state to below or near that of the 3MLCT state, significantly\n action between the PS and the molecules of the medium. Type increasing the 3\u03c0\u03c0* character and lifetime of the T1\n I PDT in which an electron transfer mechanism to O2 or other state.28,31\u201333 The use of \u03c0-extended ligands also favors absorp-\n oxygen-containing species renders di\ufb00erent ROS, among them tion at longer wavelengths in the visible and near-infrared\n the superoxide anion radical (O2\u2022\u2212), which not only acts as an regions, which is advantageous for deeper tissue penetration\n oxidant to destroy cancer cells, but can also su\ufb00er a series of and minimizing damage to surrounding healthy tissue. This\n cascade bioreactions leading to species such as hydroxyl rad- approach has been successfully used, reaching PDT activity in\n icals (HO\u2022) or H2O2, which are also cytotoxic. In type II PDT, an ruthenium chemistry28,34\u201339 and also in iridium derivatives of\n energy transfer mechanism to the fundamental oxygen (3O2) N6 type,40,41 half-sandwich42 and derivatives of stoichiometry\n generates singlet oxygen (1O2), a very reactive and toxic [IrCl(C^N)(N^N^N)]+.43 The use of \u03c0-extending ligands has\n species.5,6 Type II processes are more dependent on O2 concen- also been applied to bis(cyclometalated) species of the type\n tration, and thus PSs acting by type I or both are more adequate [Ir(C^N)2(N^N)]+ where the \u03c0-extension has mainly focused on\n for hypoxic tumours. One measure of the potential of a PS is the N^N ligand,44\u201354 but it has also been explored for the C^N\n the phototoxic index (PI), which is the ratio of the cytotoxicity in ligand25,55,56 or both.57\u201364 Some complexes demonstrated\n the dark and after irradiation (PI = IC50,dark/IC50,light). superior ROS generation capabilities, making them e\ufb00ective\n There are a number of PSs in clinical use that have been at inducing phototoxicity in cancer cells. With some\n approved either globally or in specific countries for di\ufb00erent exceptions,55,65,66 in general, the \u03c0-extension used in C^N\n types of cancer.7\u20139 The majority are organic agents with a tetra- ligands consists of the incorporation of a fused benzene to the\n pyrrolic sca\ufb00old, which su\ufb00er, among other problems, pro- phenylpyridinate ( ppy) ligand, usually in the pyridine frag-\n longed retention in tissues, photobleaching and a small Stokes ment. In their excellent work, Vilar et al. have studied,60 by a\n shift that greatly increases the interference between excitation high-throughput approach, the photocytotoxicity of a wide\n and emission.10 Metal-based tetrapyrrolic derivatives are under library of iridium compounds. From DFT calculations, they\n clinical trials or approved, as in the case of TOOKAT, a palla- concluded that, although the N^N ligand also influenced the\n dium complex.8 In the last decades attention has been paid to outcome of the derivatives, the C^N ligand dominated the elec-\n d6 transition metal complexes, which o\ufb00er easy ligand modifi- tronic structure properties of the complexes. In her research\n cation, potential redox features and versatile photochemical on bis(cyclometalated) iridium complexes, McFarland discov-\n and photophysical properties with intense absorptions in the ered that the degree of \u03c0-conjugation in the diimine ligand\n visible region.11\u201314 Specifically, the triplet state, which has a predominantly influences the 1\u03c0,\u03c0* transitions observed in\n longer lifetime than the singlet state and facilitates PSs reac- their UV-vis absorption spectra and that the \u03c0-conjugation of\n tions with O2, is typically more prevalent in metal complexes.13 the cyclometalating ligand primarily determines the nature\n Interesting results have been obtained with osmium,15 and energies of the lowest singlet and emitting triplet excited\n rhenium16 and ruthenium.17,18 It is noteworthy that the ruthe- states.67 Recently, we have reported the first case of half-sand-\n nium derivative TLD1433, developed by McFarland, is under- wich cyclometalated iridium complexes with PDT activity\n going clinical trials.19 using the C^N ligands pbpz (4,9,14-triazadibenzo[a,c]anthra-\n Among the various PSs reviewed in the literature, cyclome- cene) or pbpn (4,9,16-triazadibenzo[a,c]naphthacene)\n talated Ir(III) complexes have shown exceptional promise due (Fig. 1).42 Considering all these precedents, we decided to\n to their unique and tunable photophysical properties, favour- focus on studying the \u03c0-extension of the C^N ligand and syn-\n able absorption characteristics, large Stokes shifts and robust thesize derivatives of the type [Ir(C^N)2(bpy)]+ (bpy = 2,2\u2032-bipyr-\n stability and photostability under physiological conditions. idine) using the pbpz and pbpn C^N ligands, which exhibit a\n Additionally, the triplet excited states of Ir(III) complexes are remarkable \u03c0-extension. A potential advantage of the targeted\n highly sensitive to molecular oxygen and high quantum yields derivatives over the half-sandwich complexes with the same\n for ROS generation are observed.20\u201324 Moreover, it is common ligands is the expected luminescence, which enables a therag-\n for these derivatives to target mitochondria, giving them high nosis e\ufb00ect, combining both therapeutic and diagnostic func-\n potential in PDT cancer therapy.25,26 These characteristics tionalities in cancer treatment.\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 7304\u20137332 | 7305\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n (Scheme 1).18 These ligands were previously used in octahedral\n polypyridine Ru(II) complexes,18 [IrCl(C^N)(N^N^N)]+ deriva-\n tives81 and half-sandwich Ir(III) complexes42 exhibiting excel-\n lent PDT behaviour.\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n The synthesis of the biscyclometalated Ir(III) dimer D1 and\n the equivalent with the pbpn ligand was attempted by the con-\n ventional method, that implies the use of a ethylene glycol\n monomethyl ether : water (3 : 1) mixture.82,83 However, this\n approach was unsuccessful, possibly because the proligands\n are not su\ufb03ciently nucleophilic to react with the Ir(III) center\n Fig. 1 Structure of the proligands Hpbpz and Hpbpn.\n due to electron delocalization. To improve the reaction con-\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n ditions, microwave techniques were employed. However, only\n Hpbpz was successfully cyclometalated in these conditions\n Despite their potential, the clinical application of Ir(III) and D1 was obtained. Subsequently, the cationic complex 1\n complexes in PDT faces significant challenges related to their was prepared by abstraction of the chloride using AgOSO2CF3\n delivery and bioavailability.68 To overcome these limitations, and coordination of the N^N ligand, followed by anion\n encapsulation of PSs within nanoparticles has been proposed exchange with NH4PF6.\n as a viable strategy.69\u201371 Nanoparticles o\ufb00er several advantages To achieve the analogous complex with the pbpn ligand, a\n as drug delivery systems, including improved solubility, protec- di\ufb00erent approach was employed. In this case, benzo[h]quino-\n tion from premature degradation and the enhanced per- line-5,6-dione was first cyclometalated to the Ir(III) center using\n meability and retention (EPR) e\ufb00ect,72,73 thus increasing their 2-methoxyethanol : water (3 : 1) mixture under microwave\n accumulation in cancerous tissues74 and minimizing systemic irradiation leading to the formation of the dimer D2.\n toxicity. Cross-linked polymeric nanoparticles have shown Subsequently, the N^N ligand was coordinated mediated by\n enormous potential as carriers due to their low toxicity and the chloride abstraction with AgOSO2CF3, followed by anion\n biocompatibility.75 Since these nanoparticles do not establish exchange with NH4PF6 (formation of 2). Finally, 2,3-diamino-\n dynamic equilibria, they retain their structure even at low con- naphtalene was condensed to the cyclometalated benzo[h]qui-\n centrations, and they are preserved in the bloodstream longer noline-5,6-dione to obtain 3.\n than self-assembled systems. This favours their accumulation The complexes were obtained in moderate yields (38\u201341%)\n in tumours.76 A wide variety of molecules such as proteins, as black, yellow or red solids. The complexes 1\u20133 were fully\n DNA, drugs, hydrophobic and hydrophilic molecules, have characterized by elemental analysis, 1H, 13C{1H} and 19F{1H}\n been encapsulated in this type of system.77\u201380 NMR spectroscopy, including 1H\u20131H gCOSY, 1H\u201313C gHSQC\n In this study, we report the synthesis and characterization and 1H\u201313C gHMBC and mass spectrometry (Fig. S1\u2013S27\u2020).\n of two new biscyclometalated Ir(III) complexes with \u03c0-extended Moreover, the crystal structures of 1, 2 and the adduct formed\n C^N ligands and their encapsulation into polymeric nano- after reaction of D2 with DMSO, 4 (Scheme 1), were also solved\n particles. The use of this type of ligand has allowed long life- by single crystal X-ray di\ufb00raction. The HPLC traces were also\n times of the excited states to be obtained, and a remarkable obtained for complexes 1 and 3 (Fig. S28 and S29\u2020).\n generation not only of 1O2 but also of O2\u2022\u2212. The photophysical Complexes 1 and 2 exhibited good solubility in polar sol-\n properties were highly dependent on the type of ligand, a fact vents such as dimethyl sulfoxide (DMSO) and chlorinated sol-\n that was excellently explained by TD-DFT studies. The com- vents as well as in N,N-dimethylformamide (DMF), methanol\n plexes exhibited photodynamic e\ufb03cacy under blue light or acetonitrile. Complex 3 exhibited solubility in DMSO and\n irradiation, resulting in mitochondrial damage and an antime- DMF. Complexes 1\u20133 were poorly soluble in aqueous media.\n tastatic e\ufb00ect. Encapsulation of the complexes significantly However, the cationic complexes were soluble in water pro-\n enhanced their cellular uptake and photodynamic activity. vided that a small amount of another solvent, such as DMSO\n Notably, one of the nanoparticles exhibited, under irradiation, or DMF was added. Thus, nontoxic amounts of DMSO were\n one of the highest values of cytotoxicity found in iridium used in the biological experiments to assist dissolution.\n chemistry (IC50 = 0.86 nM). In the 1H NMR spectra, the lack of a signal for the proton\n of the metalated carbon (H7) and the shift of the resonances\n for the protons of pbpz and pbpn in the complexes with\n 2. Results and discussion respect to those for the corresponding proligands (Hpbpz and\n Hpbpn) corroborates the coordination of the C^N ligands to\n 2.1. Synthesis and characterization of complexes the metal centre. The resonances were fully assigned by means\n Two new biscyclometalated Ir(III) complexes of general formula of 1H\u20131H COSY experiments and considering the di\ufb00erent\n [Ir(C^N)2(N^N)]+ (C^N = pbpz, 4,9,14-triazadibenzo[a,c]anthra- coupling constants expected in the pyridyl ring.42,84 The strong\n cene; or pbpn, 4,9,16-triazadibenzo[a,c]naphthacene; N^N = shielding of the proton adjacent to the metallated carbon (H12)\n 2,2\u2032-bipyridine) have been prepared (Scheme 1). The proli- of both the pbpz or pbpn ligands due to the e\ufb00ect of the ring\n gands were synthesized using a literature procedure current anisotropy of the pyridine ring of the other C^N ligand\n\n\n 7306 | Inorg. Chem. Front., 2025, 12, 7304\u20137332 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n Scheme 1 Synthesis of ligands and complexes presented in this paper. (i) EtOH, re\ufb02ux, 4 h. (ii) IrCl3\u00b73H2O, diglyme/H2O (3 : 1), MW, 220 \u00b0C, 10 min.\n (iii) bpy, AgOSO2CF3, DCM/MeOH (2 : 1), re\ufb02ux, 24 h and NH4PF6, room temperature, 1 h. (iv) IrCl3\u00b73H2O, 2-methoxyethanol/H2O (3 : 1), MW, 150 \u00b0C,\n 10 min. (v) bpy, AgOSO2CF3, DCM/MeOH (2 : 1), re\ufb02ux, 36 h and NH4PF6, room temperature, 1 h. (vi) 2,3-Diaminonaphtalene, EtOH, re\ufb02ux, 4 h. (vii)\n DMSO, room temperature.\n\n\n\n\n is noticeable, as it is clearly observed in the structure of 1\n determined by X-ray di\ufb00raction described below.85,86 It was\n also possible to assign most of the 13C{1H} NMR resonances\n through 1H\u201313C gHSQC and 1H\u201313C gHMBC experiments.\n\n 2.2. Solid-state characterization by X-ray di\ufb00raction and\n analysis of \u03c0\u2013\u03c0 stacking\n The molecular and crystal structures of complexes 1, 2\n [OSO2CF3] and 4 were determined by X-ray di\ufb00raction. The\n corresponding ORTEP diagram of complex 1 is shown in\n Fig. 2. The ORTEP diagrams of complexes 2[OSO2CF3] and 4\n are shown in Fig. S30.\u2020 The methods used to obtain single\n crystals are detailed in the Experimental section. Selected\n bond distances and angles are gathered in Table S1.\u2020 The crys-\n tallographic data are provided in Table S2.\u2020 The three com- Fig. 2 ORTEP diagram of cation of complex \u0394 1. Ellipsoids are at the\n 30% probability level. Hydrogen atoms and PF6\u2212 anion have been\n pounds exhibit a distorted octahedral geometry with the two omitted for clarity.\n nitrogen atoms of the C^N ligands in a relative trans disposi-\n tion, as expected.87\u201389 The Ir\u2013C and Ir\u2013N distances of the C^N\n ligand are about 2 \u00c5 while those involving the N atoms of the\n N^N ligand are a bit longer because of the trans influence of into trimers by weak \u03c0\u2013\u03c0 interactions (Fig. S31\u2020). There are two\n the carbon atoms.26,90,91 The iridium atoms are chiral but trimers in the asymmetric unit; one contains three \u0394 enantio-\n both enantiomers are observed in a single crystal. mers (trimer \u0394) and the other three \u039b enantiomers (trimer \u039b)\n Concerning non-covalent interactions, besides the cation\u2013 (Fig. S32\u2020). Molecules of di\ufb00erent trimers are further packaged\n anion coulombic attractions and hydrogen bonding, it is by other \u03c0\u2212\u03c0 interactions (Fig. S33\u2020). Other examples of \u03c0\u2013\u03c0\n worth noting the presence of \u03c0\u2013\u03c0 interactions involving the interactions in cationic complexes involving a N^N ligand\n C^N ligands. In the case of 1, the molecules are assembled similar to pbpz49,92\u201395 and in neutral and cationic complexes\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 7304\u20137332 | 7307\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n of formula [Cp*Ir(C^N)L]0/+ with pbpz and pbpn42 have been been previously observed for other compounds with this type\n reported. of \u03c0-expansive ligand. It was proposed that light favours an\n Considering the \u03c0\u2013\u03c0 stacking interactions observed in the aggregation process.42\n solid state, it was decided to ascertain whether this interaction In order to ascertain if the compounds aggregate spon-\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n was also present in solution. Thus, complexes 1 and 3 were taneously and confirm the light-induced aggregation of the\n studied by 1H NMR spectroscopy at di\ufb00erent concentrations. complexes, a Dynamic Light Scattering (DLS) experiment was\n The existence of the \u03c0\u2013\u03c0 stacking interaction on increasing the performed for solutions of both complexes at 1.0 \u00d7 10\u22124 M in\n concentration could be proved by observing the shielding of water (10% DMSO) mixture before and after 1 h of blue light\n specific ring protons due to the influence of the ring current exposure. The average hydrodynamic diameter of particles of 1\n from the adjacent aromatic moieties.96,97 In addition, this and 3 before irradiation was 79 and 117 nm, respectively\n study could provide information about the regions that are (Fig. S43\u2020). These results indicate that the compounds form\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n mostly involved in the interaction. The di\ufb00erences in chemical nanoaggregates in the presence of a high percentage of water,\n shifts of the C^N aromatic protons for the most diluted and which is favoured by the hydrophobicity of the \u03c0-extended\n concentrated solutions in DMSO-d6 are provided in Table S3.\u2020 ligands and the polarity of water, as has been seen for other\n The corresponding sets of spectra are given in Fig. S34 and Ir(III) complexes in the literature.98 In addition, after 1 h of blue\n S35.\u2020 Although nearly all C^N aromatic protons su\ufb00er concen- light irradiation, the average hydrodynamic diameter of both\n tration-dependent changes in chemical shift, the e\ufb00ect of compounds increased. The values for 1 and 3 were 378 and\n shielding when the concentration is increased depends on the 323 nm, respectively (Fig. S43\u2020). Therefore, light irradiation\n specific compound and on the position of the proton on the induces an increase in the aggregation of the particles,\n ligand. Higher \u0394\u03b4 values were found for complex 3 that con- resulting in larger aggregates. Although it is not a common\n tains pbpn, the more \u03c0-expansive ligand, probably due to a process, other examples of light-induced aggregation have\n more e\ufb03cient \u03c0-stacking interaction. Besides, the protons been reported.99\u2013101\n mostly a\ufb00ected are those situated in the central region of the\n ligands (see Fig. S36\u2020). 2.3. Photophysical properties\n Stability, photostability and aggregation studies (NMR, UV- UV-vis absorption spectra. The UV-vis absorption spectra of\n vis and DLS). The stability of the new Ir(III) complexes was complexes 1 and 3 were recorded in degassed acetonitrile at\n studied by 1H NMR spectroscopy in the dark and under blue 1.0 \u00d7 10\u22125 M at 25 \u00b0C (Fig. S44,\u2020 and Table 1). Their spectra are\n light irradiation (470 nm, 51.4 mW cm\u22122) in DMSO-d6 due to relatively similar. Both spectra show two intense bands below\n the poor water solubility of the complexes (Fig. S37\u2013S40, and 350 nm with a red shift of about 30 nm for 3 with respect to 1.\n Table S4\u2020). Both compounds were highly stable during the They are assigned to \u03c0\u2212\u03c0* transitions of the ligands (see the\n irradiation time used in the biological studies. To simulate the spectra of the proligands in Fig. S45\u2020). Moreover, the spectra\n conditions in the biological experiments, the stability in bio- exhibit two additional bands of lower intensity at longer wave-\n logical medium (DMEM) was studied by UV-vis spectroscopy lengths, centred at around 390 and 440 nm for 1 and at 414\n in the dark and under blue light irradiation (Fig. S41 and and 480 nm for 3. The simulated TD-DFT absorption spectra\n S42\u2020). As can be seen, the UV-vis spectra of both complexes for both complexes (Fig. S77 and S78\u2020) and the calculated elec-\n were unchanged in the dark after 48 h, revealing the stability tronic transitions (see below) indicate that these bands primar-\n of the complexes in these conditions. Under blue light ily correspond to a mixture of metal-to-C^N ligand charge\n irradiation, some changes were observed for both complexes. transfer (d \u2192 \u03c0*, 1MLCT) and C^N ligand-to-ligand charge\n The UV-vis spectra seem to evolve into a new species, as has transfer (\u03c0 \u2192 \u03c0*, 1LLCT) transitions in both series. There are\n\n\n Table 1 Photophysical properties of complexes 1 and 3 and proligands Hpbpz and Hpbpn in acetonitrile, unless otherwise stated\n\n \u03b5a (M\u22121 cm\u22121) \u03bbem \u03c4em/ns kr knr \u03c4em/ns \u03c4em/ns\n Comp. \u03bb/nm (\u03b5 \u00d7 10\u22124/M\u22121 cm\u22121) (460, 515, 635 nm) (\u03bbexc)/nm \u03a6em b (contrib.)c (s\u22121 \u00d7 10\u22126)d (s\u22121 \u00d7 10\u22127)d (aerat.)e (degas.)e \u03c6\u0394 f\n\n 1 272 (15.9), 302 (6.5), 358 6290 596 0.32 150 (14%) 0.80 0.17 360 3450 0.59\n (2.8), 390 (2.0), 438 (1.2) 920 (440) 440 (86%)\n 10\n 3 246 (8.2), 300 (13.2), 320 8510 550 0.02 0.60 (25%) 2.0 9.8 180 2160 0.49\n (8.7), 340 (7.1), 394 (2.2), 5790 (420) 13.1 (75%)\n 414 (2.5), 478 (1.5) 190\n Hpbpz 308 (1.8), 350 (1.2), 366 n.m. 508 (412) n.m. n.m. n.m. n.m. n.m. n.m. n.m.\n (2.0), 388 (2.3)\n Hpbpn 302 (3.4), 376 (0.5), 396 n.m. 544 (400) n.m. n.m. n.m. n.m. n.m. n.m. n.m.\n (1.1), 420 (1.5)\n a\n In water (1% DMSO). b The \u03a6em in degassed acetonitrile was determined using [Ir(ppy)2(bpy)]PF6 as reference (\u03c6em = 0.0707).102 c In aerated\n acetonitrile solutions. d Radiative decay rate kr = \u03a6/\u03c4 and nonradiative decay rate knr = (1 \u2212 \u03a6)/\u03c4. e Excited-state lifetimes in acetonitrile measured\n by TAS. f The \u03d5\u0394 (1O2 generation quantum yield) in water (10% DMSO) under blue light irradiation (470 nm) was determined using [Ru(bpy)3]2+\n as reference (\u03d5\u0394 = 0.18).103 n.m. = not measured.\n\n\n\n 7308 | Inorg. Chem. Front., 2025, 12, 7304\u20137332 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n also absorption tails that extend until 500 and 550 nm for 1 in the values of \u03c6\u0394 of 1 and 3 (Table 1) indicate close quantum\n and 3, respectively. The red-shift observed in the absorption yields of triplet formation for 1 and 3.\n spectrum of 3 reflects the strong electron-withdrawing ability The emission properties of the complexes were also studied\n and extended \u03c0-conjugation of the benzo-quinoxaline ligand, in aerated water (1% DMSO, 1.0 \u00d7 10\u22125 M) at 25 \u00b0C upon exci-\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n as was observed before.42 This fact enhances its light-harvest- tation at \u03bb = 422\u2013470 nm (Fig. 3, and Table S5\u2020). In aqueous\n ing capability and increases its sensitivity to light sources that solution, complex 1 remains highly emissive, which makes it a\n can penetrate deeper into tissues. good candidate as a bioimaging agent. However, complex 3 is\n In order to better understand the photophysical properties hardly emissive. In both cases the emission bands undergo a\n of the complexes in biological media, the UV-vis spectra were red shift with respect to the results in acetonitrile, with a more\n also recorded in water (1% DMSO) (Fig. 3). Comparing with pronounced displacement for complex 1 (\u03bbmax = 632 nm). This\n the spectra recorded in acetonitrile, all the absorption bands may be related to the stated aggregation process104 as con-\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n in water were red-shifted (Table S5\u2020). In water, the absorption firmed in the following experiment.\n tails extended until 540 and 580 nm for 1 and 3, respectively, The possible aggregation e\ufb00ect in the emission properties\n promoting absorption of longer wavelengths in biological was evaluated in H2O : DMSO mixtures with di\ufb00erent water\n experiments, which is desirable for a PS. See in Table 1 the fractions ( fw) (Fig. S47\u2020). As can be seen in Fig. S48,\u2020 both\n values of absorptivity at the wavelengths used in the biological complexes showed an aggregation-caused quenching (ACQ)\n studies. process. The e\ufb00ect is stronger in the case of 3, probably due to\n Emission spectra. The photoluminescence spectra of both the larger \u03c0-conjugation of the C^N ligand. Interestingly, the\n complexes were initially recorded in solutions of dry deoxyge- quenching process is only partial in the case of 1. Concerning\n nated acetonitrile (1.0 \u00d7 10\u22125 M) at 25 \u00b0C upon excitation at \u03bb = the position of the emission bands, the red-shifting is small in\n 420\u2013440 nm (Fig. S44\u2020 and Table 1). Both compounds emitted the case of complex 3 but for 1 there is a notable red-shifting\n at ca. 550\u2013600 nm upon excitation at the lower energy band of upon aggregation.\n the absorption spectrum. The emission spectrum for complex The weak fluorescence of 3 may be due to its aggregation in\n 1 exhibited one band with \u03bbmax at 596 nm and a shoulder at aqueous media, driven by the greater hydrophobicity and\n around 550 nm. The emission spectrum for complex 3 showed stronger \u03c0\u2013\u03c0 interactions of the pbpn ligand. In addition, the\n one emission band centred at around 550 nm, similar to the expected reduction in spin\u2013orbit coupling (SOC) in this com-\n emission spectrum of Hpbpn (Fig. S46\u2020). However, the emis- pound (see below) favours fluorescence as the predominant\n sion spectrum of 1 was quite di\ufb00erent to the spectrum of radiative deactivation pathway.\n Hpbpz (Fig. S46\u2020), with the main band red-shifted with respect Luminescence lifetimes. Time-correlated single-photon\n to the proligand, and also red-shifted with respect to the emis- counting (TC-SPC) was performed to analyse the decay times\n sion of 3. The photoluminescence quantum yields (\u03a6em) associated with the luminescence. Iridium complexes are\n recorded for these complexes were also quite di\ufb00erent known to present multiple exponential decays due to the decay\n (Table 1). Complex 1 exhibited a good quantum yield (0.32) of singlet and triplet states at the same emission wave-\n while the value for complex 3 was very low (0.02), which indi- length.105 The multiexponential profiles of the luminescence\n cates that an additional non-radiative pathway is in action for decays are also observed in this case (Fig. S49\u2020), but the decay\n 3, while absent in 1. This additional non-radiative pathway times of complexes 1 and 3 present clear di\ufb00erences in time\n does not seem to be intersystem crossing, since the proximity scale. 1 presents luminescence decays that are best fitted with\n a sum of two exponentials. The longer decay time, 440 ns, is\n responsible for 86% of the observed emission. 3 also presents\n luminescence decays that are best described as a sum of two\n exponentials. However, the longest decay time, 13.1 ns, which\n accounts for 75% of the luminescence, is much shorter (see\n below for the lifetimes obtained from TAS).\n Transient absorption spectroscopy (TAS) measurements.\n Transient absorption spectra of both compounds measured at\n room temperature in acetonitrile solutions (Fig. S50\u2020) between\n 280 and 600 nm display negative and positive optical density\n values yielding the same lifetime for both the negative regions\n (ground state depletion) and positive regions (transient state\n formation) of the transient absorption spectrum, which con-\n firms that the disappearance of the transient state and recovery\n of the ground state are connected (Table 1). The optical\n density variations at both wavelengths decay to zero at longer\n times, indicating that no other species is being formed in sig-\n Fig. 3 UV-vis absorption and emission spectra of complexes 1 and 3 in nificant amounts throughout the experiments (Fig. S51\u2020), i.e.,\n water (1% DMSO). there is no evidence for photochemistry arising from the\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 7304\u20137332 | 7309\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n observed transients. Furthermore, the solutions were degassed of its triplet state. Therefore, complex 1 is expected to be a\n by bubbling Argon until saturation (\u223c20 minutes), and the good photosensitizer.\n transients were once more acquired, yielding excited state life- Singlet oxygen generation was studied using the ABDA\n times around one order of magnitude higher (Table 1). These assay in water (10% DMSO) to assess its performance under\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n results suggest that the observed transients are of triplet conditions more representative of those used in bioassays. In\n nature, which are strongly quenched in the presence of O2. UV- this experiment, 1O2 generation quantum yields (\u03c6\u0394) were\n vis spectra acquired before and after laser flash photolysis on quantified monitoring the decrease in the ABDA absorbance\n both complexes are identical, confirming that there is no by UV-vis spectroscopy with the irradiation for 3.5 minutes.\n photochemistry occurring in these conditions (Fig. S52\u2020). The experiment was performed in dark conditions and under\n The long luminescence lifetimes, similar to the triplet life- blue light irradiation (470 nm, 51.4 mW cm\u22122). [Ru(bpy)3]2+\n times observed in TAS, point to a contribution of delayed fluo- was used as reference (\u03c6\u0394 = 0.18 in water).109 First, the stability\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n rescence in the fluorescence emission of 1. The luminescence of ABDA was confirmed under blue light irradiation\n lifetime values obtained for 3 are much shorter than the ones (Fig. S55\u2020). In the presence of the Ir(III) complexes and [Ru\n obtained from TAS and we conclude that the contribution of (bpy)3]2+, a decrease in the absorption bands of the probe (360,\n delayed fluorescence is thus considerably less in this 378 and 400 nm) over time was only observed upon blue light\n compound. irradiation (Fig. S56\u201358\u2020). In dark conditions, the spectrum of\n TAS was also performed in aqueous solution (1% DMSO) of ABDA remained unaltered. The \u03c6\u0394 values of the two new com-\n 1 (15 \u00b5M). Transients at selected wavelengths, namely 270 nm plexes were calculated from the slopes obtained for ABDA con-\n (for monitoring ground-state depletion) and 560 nm (for sumption (Fig. S59\u2020). Although there is not much di\ufb00erence, 1\n monitoring transient absorption) were acquired (Fig. S53\u2020) exhibited the highest \u03c6\u0394 (0.59) while 3 exhibited a slightly\n and compared with the transients obtained in acetonitrile. It lower value (0.49) (Table 1). This result indicates that the\n can be seen that the transients of 1 are greatly diminished in quantum yields for triplet formation and the e\ufb03ciency of\n water, when compared with acetonitrile (around ten-fold energy transfer to O2 are comparable for both complexes.\n decrease in the initial absorbance of the transient), while the Despite the luminescence of complex 3 being less a\ufb00ected by\n lifetime of the transient is not significantly changed (Table 2). the presence of O2, it is still a good photosensitizer.\n These findings indicate that the initial concentration of T1 is Generation of O2\u2022\u2212 was studied by the DHR123 assay in\n greatly diminished in water, because either S1 or higher triplet water (0.2% DMSO) monitoring the emission change of the\n states are undergoing photochemistry that competes e\ufb03ciently probe by fluorescence spectroscopy with the irradiation for 30\n with the formation of T1 in aqueous solution. seconds. The non-emissive DHR123 probe is oxidized by the\n ROS generation. The ability of Ir(III) complexes to generate photogenerated O2\u2022\u2212 into the fluorescent rhodamine 123, with\n ROS was investigated through four di\ufb00erent methods: quench- a maximum emission at 526 nm. As for 1O2 studies, the experi-\n ing of the triplet state by O2, the 9,10-anthracenediyl-bis ment was carried out in dark conditions and upon blue light\n (methylene) malonic acid (ABDA) test for 1O2 production, the irradiation (470 nm, 51.4 mW cm\u22122) using [Ru(bpy)3]2+ as\n dihydrorhodamine 123 (DHR123) assay for O2\u2022\u2212 production reference. In dark conditions (Fig. S60\u2020) or under blue light\n and EPR spectroscopy with specific radical spin-traps. without any complex (Fig. S61\u2020), no oxidation of the probe was\n The relationship between the triplet state quenching of detected. Upon blue light irradiation, with complexes 1, 3 or\n Ir(III) complexes by O2 bubbling and ROS formation is signifi- [Ru(bpy)3]2+, O2\u2022\u2212 generation was confirmed by the increased\n cant because it largely relies on energy and/or electron transfer emission of the oxidized rhodamine 123 (Fig. S62 and S63\u2020).\n processes, as has been previously described.106\u2013108 The result The relative rates are in order of 1 \u226b 3 > [Ru(bpy)3]2+.\n of these transfer processes is the generation of singlet oxygen Moreover, ascorbic acid and sodium azide were used as scaven-\n and di\ufb00erent oxygen radicals. As can be seen in Fig. S54,\u2020 in gers for O2\u2022\u2212 and 1O2, respectively, to determine their relative\n acetonitrile, the quenching of emission by O2 is several times contributions to the oxidation of DHR123 (Fig. S64 and S65\u2020).\n higher in complex 1 than in complex 3. In part, this is related As expected, the oxidation of DHR123 was inhibited, and the\n to the triplet contribution to the emission (delayed fluo- increase in its fluorescent signal was significantly reduced in\n rescence) of complex 1, which is much smaller in the case of the presence of ascorbic acid, while sodium azide had no mea-\n complex 3. The significant decrease in the emission of surable e\ufb00ect. These results confirm that both complexes\n complex 1 in the presence of oxygen confirms the quenching generate O2\u2022\u2212 under light irradiation in aqueous solution.\n Notably, the triplet state of complex 1 exhibits a greater ability\n to act as an O2\u2022\u2212 photosensitizer, in contrast to 1O2 photosen-\n Table 2 Excited-state lifetimes and initial optical densities for 1 in sitization where the \u03c6\u0394 values were comparable for both\n water (1% DMSO) and acetonitrile, measured with laser \ufb02ash photolysis\n compounds.\n OD (t = 0)\n The generation of O2\u2022\u2212 for 1 was also verified in acetonitrile\n with the DHR123 assay (Fig. S66\u2020). The superoxide production\n Solvent Lifetime (ns) 270 nm 560 nm was higher in water than in acetonitrile, as is clearly seen in\n Water (1% DMSO) 371 \u22120.0071 0.0031 Fig. S67.\u2020 This is aligned with the TAS results, which showed\n Acetonitrile 367 \u22120.0727 0.0335 that the transients of complex 1 were significantly reduced in\n\n\n 7310 | Inorg. Chem. Front., 2025, 12, 7304\u20137332 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n water compared with acetonitrile (Table 2). The initial concen- resulting EPR spectrum confirmed the presence of O2\u2022\u2212 in\n tration of the lowest triplet state T1 is greatly diminished in solution (Fig. S68B\u2020).\n aqueous solution, which accounts for the reduced contri-\n bution of O2\u2022\u2212 to the oxidation of DHR123 in this solvent 2.4. Polymeric nanoparticles\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n mixture. Moreover, this provides strong evidence for an We decided to explore the possibility of encapsulating the\n additional photochemical process involving higher triplet complexes into crosslinked polymeric nanoparticles with the\n states that compete with T1 formation (see below), specifically aim of improving the cellular internalization and evaluating\n leading to O2\u2022\u2212 generation. These results underscore complex their behaviour as anticancer agents in PDT. The preparation\n 1 as a particularly promising PDT agent, as it implies a type I of the nanoparticles involved two main steps: the synthesis of\n mechanism that, while enhanced by oxygen, can also function copolymers (Scheme S1\u2020) and the crosslinked step to produce\n independently of it. This makes it especially useful against the nanoparticles via covalent bonds (Fig. 4).\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n cells in hypoxic microenvironments.110 Synthesis of copolymers. The copolymers used in this work\n Finally, EPR experiments on 1 under dark and irradiated were prepared by RAFT (Reversible Addition Fragmentation chain\n (365 nm) conditions were conducted in acetonitrile, in the Transfer) polymerization, which was used on account of its fea-\n presence of two radical specific spin-traps: 2,2,6,6-tetramethyl- tures such as versatility, high compatibility with di\ufb00erent solvents\n piperidine (TEMP) and 5,5-dimethyl-1-pyrroline-N-oxide and monomers and experimental simplicity to obtain polymers\n (DMPO). In the case of TEMP, it reacts with 1O2 to yield with narrow Mw.115 Polyethyleneglycol (PEG)-based monomers\n 2,2,6,6-tetramethylpiperidine-N-oxide radical (TEMPO), charac- were selected in order to obtain polymers with higher solubility\n terized by a distinct three-line signal.111\u2013113 This radical is in protic solvents. In addition, it protects against non-specific\n thermodynamically and kinetically stabilized and easily interactions with the proteins in blood, avoiding aggregation.116\n accumulates in solution. The signal was readily detected after The chain transfer agent (CTA) used in both polymeriz-\n irradiation (Fig. S68A\u2020), supporting the presence of 1O2 in the ations was 2-cyano-2-propyl dodecyl trithiocarbonate due to its\n sample. The reaction of O2\u2022\u2212 with the nitrone DMPO leads to suitability to acrylates and methacrylates. The polymers were\n the formation of the DMPO\u2013OOH spin adduct, which gives purified by dialysis. Two polymers were obtained containing\n rise to a characteristic EPR signal consisting of a quartet due amine and carboxylic groups, which were used to generate\n to hyperfine splitting from the nitroxide nitrogen and the adja- cross-linked network via amide bonds (Scheme S1\u2020). Random\n cent \u03b2-hydrogen.114 Contrary to TEMPO, this spin adduct copolymer P1 was obtained via copolymerization of poly(ethy-\n radical presents a rapid decay due to spontaneous rearrange- leneglycol) methyl ether methacrylate (PEGMA) and 2-amino-\n ment and degradation. The short lifetime limits the detection ethyl methacrylate, which was used with the BOC protecting\n window. As such, in situ irradiation was carried out, and the group as monomer (BOC-aminoethylMA). This monomer was\n\n\n\n\n Fig. 4 Schematic formation of the NPs presented in this work with Ir(III) complexes encapsulated.\n\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 7304\u20137332 | 7311\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n prepared according to the literature.117 In a second step, the 2.5. Theoretical calculations\n protecting group was removed with TFA (trifluoroacetic acid),\n to obtain P1b with the amine groups. Random copolymer P2 In order to gain a better understanding of the significant\n was obtained via copolymerization of PEGMA and 2-carbox- di\ufb00erences in the photophysical properties of the complexes 1\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n yethyl acrylate (CEA) (Scheme S1\u2020). The conversion of the poly- and 3, time-dependent density functional theory (TD-DFT) cal-\n merizations was calculated by 1H NMR using trioxane as stan- culations were performed for both the singlet and triplet\n dard and was found to be around 87 and 90% for P1 and P2, excited states of these compounds. A comprehensive method\n respectively (Table S6\u2020). The three polymers were characterized for comparing the X-ray crystallography and theoretical geome-\n by 1H NMR (Fig. S69\u2013S71\u2020). tries involves superimposing the two structures and calculat-\n Synthesis and characterization of nanoparticles. Crosslink ing the root-mean-square deviation (RMSD) of atomic posi-\n experiments were performed between copolymers P1b and P2 tions (Fig. S76\u2020). For compound 3, the X-ray structure was not\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n in the presence of the coupling agent 1-ethyl-3-(3-dimethyl- obtained. The corresponding RMSD value corresponding to\n aminopropyl)carbodiimide (EDC) to yield polymeric nano- the overlap for those structures of 1 is 0.131 \u00c5. This indicates\n particles via covalent amide bond formation, as a result of that the experimental and the obtained theoretical geometries\n coupling of amine and carboxylic groups (Fig. 4). Equimolar are quite similar. No significant di\ufb00erences were observed in\n dilute solutions of copolymers were mixed in water, and the the length values of both structures. The primary di\ufb00erence\n corresponding amount of coupling agent was added dropwise arises from slight distinctions in the orientation of the planes\n while stirring. It was expected that these nanoparticles could of the aromatic fragments, which are influenced by the crystal\n encapsulate drug payloads, and thus, encapsulate the Ir com- packing e\ufb00ect in the structure determined by X-ray di\ufb00raction\n plexes. A similar procedure was performed to obtain the nano- but not in the obtained theoretical structures.\n particles in the presence of the complexes (Table S7\u2020). The corresponding fragmental contributions of each mole-\n The nanoparticles were characterised by DLS, a\ufb00ording cular fragment to the molecular orbitals (Fig. S79\u2020) corres-\n hydrodynamic diameters between 150\u2013200 nm with a good ponding to complexes 1 and 3 were obtained by a Milliken\n polydispersity index (PDI) (Table S7 and Fig. S72\u2020). This struc- population analysis (Table 3 and Fig. 5). The energy of the\n ture provides a stable aqueous suspension of the nanoparticles HOMO orbitals is comparable between the two compounds\n with good biological properties, such as long circulation in the because their main contributions are similarly derived from\n bloodstream and specific accumulation in the acidic tumour the atomic orbitals of the iridium atom (45%), and the\n microenvironment. The size of the nanoparticle without benzene fragment (38% and 40%) as well as the pyridine\n encapsulated drug was found to be 146 nm, showing that the moiety (9% and 8%) in the pbpz and pbpn ligands, respect-\n size of the nanoparticle does not change significantly with the ively. It is important to highlight that for 3, both HOMO\u22121\n encapsulation of drugs. SEM images of the nanoparticles and HOMO\u22122 orbitals are almost isoenergetic with the HOMO\n showed their spherical morphology (Fig. S73\u2020). orbital. Notably, the majority of their contribution (88%) orig-\n Nanoparticles NP1 and NP3 (containing compound 1 or 3, inates from the benzo-quinoxaline moiety, and these orbitals\n respectively) were analysed by UV-vis spectroscopy (Fig. S74\u2020) are characterized by their \u03c0 nature. The formation of a near-\n in water. The spectra are relatively similar to those of com- HOMO orbital centred on the benzo-quinoxaline fragment due\n plexes 1 and 3 in aqueous solution. The encapsulation of the to the introduction of the pbpn ligand has also been observed\n drugs into the nanoparticles improved the solubility of the in our previous work with [Cp*Ir(C^N)L]+ complexes.42 In the\n complexes but did not a\ufb00ect the UV-vis absorption spectra. case of compound 3, the introduction of two pbpn ligands\n The emission properties of the nanoparticles were also studied results in the presence of two near-HOMO orbitals of this\n in water at 25 \u00b0C upon excitation at \u03bbexc = 422\u2013470 nm. As nature. The nearly isoenergetic character of these orbitals indi-\n observed for the free complex, NP1 showed a highly emissive cates that the electrons in the HOMO orbital can be deloca-\n behaviour, which makes it a good candidate for a bioimaging lized across these three orbitals, thereby enhancing their delo-\n agent (Fig. S75\u2020). However, similar to complex 3, NP3 was calization over the \u03c0 system of the ligand and further contri-\n hardly emissive. This confirmed that encapsulation of com- buting to its stability. Interestingly, such type of orbital is\n plexes did not a\ufb00ect their photophysical properties. missing in compound 1, where the HOMO\u22121 closely\n The amount of complex encapsulated in each nanoparticle resembles and shares energy equivalence with the HOMO\u22123 of\n was confirmed by ICP-MS, with values of drug loading of compound 3, with a major contribution, in both compounds,\n 23.319 and 27.560 \u00b5M for NP1 and NP3, respectively of fused benzene and minor contributions of pyridine and qui-\n (Table S7\u2020). These values of drug loading could be considered noxaline(1)/benzo-quinoxaline(3) moieties. Regarding the\n low compared with other nanocarriers of Ir complexes,74 poss- LUMO and LUMO+1, they are predominantly isoenergetic in\n ibly on account of the high lipophilicity of the Ir(III) complexes both compounds, with their primary locations being on the\n and the absence of any surfactant in the synthesis method. benzo-quinoxaline fragment in compound 3, resembling\n The drug uploading values of these nanoparticles were 5.8 and HOMO\u22121 and HOMO\u22122 orbitals, and on the quinoxaline\n 7.7%, respectively. Despite the low Ir content, we decided to moiety in compound 1. Besides, the addition of an extra fused\n evaluate these nanoparticles and their e\ufb00ect on cytotoxicity benzene ring in 3 has the e\ufb00ect of lowering the LUMO level by\n and cellular uptake. extending the \u03c0-conjugation system, which leads to greater\n\n\n 7312 | Inorg. Chem. Front., 2025, 12, 7304\u20137332 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n Table 3 Energies in electron volts (E/eV) and main fragmental contributions ( percentage values within brackets) to some molecular orbitals of 1\n and 3 obtained at the TD-DFT (SMD, acetonitrile)/6-31G(d,p)//SDD level\n\n E/eV 1 3\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n Orbital 1 3 Fragmental contributions (%) Fragmental contributions (%)\n\n LUMO+5 \u22121.27 \u22121.29 Bpy (52) Py (15) Q (14) Bz (13) Ir (5) Bpy (53) Py (17) Bq (13) Bz (11)\n LUMO+4 \u22121.80 \u22121.78 Py (60) Bz (17) Q (17) Ir (5) Py (62) Bz (16) Bq (15) Ir (6)\n LUMO+3 \u22121.83 \u22121.81 Py (63) Bz (17) Q (13) Py (64) Bz (17) Bq (12)\n LUMO+2 \u22122.17 \u22122.17 Bpy (93) Bpy (93)\n LUMO+1 \u22122.41 \u22122.69 Q (76) Py (16) Bz (8) Bq (80) Py (12) Bz (7)\n LUMO \u22122.47 \u22122.73 Q (73) Py (16) Bz (10) Bq (77) Py (13) Bz (9)\n HOMO \u22125.59 \u22125.60 Ir (45) Bz (40) Py (8) Ir (45) Bz (38) Py (9) Bq (6)\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n HOMO\u22121 \u22126.17 \u22125.65 Bz (57) Py (22) Q (17) Bq (88) Bz (7)\n HOMO\u22122 \u22126.22 \u22125.65 Ir (32) Bz (27) Q (25) Py (13) Bq (88) Bz (7)\n HOMO\u22123 \u22126.25 \u22126.19 Q (34) Bz (31) Ir (22) Py (11) Bz (58) Bq (23) Py (15)\n HOMO\u22124 \u22126.39 \u22126.29 Ir (44) Q (29) Py (14) Bz (9) Ir (58) Py (14) Bz (12) Bq (10) Bpy (6)\n HOMO\u22125 \u22126.45 \u22126.35 Ir (52) Q (24) Py (12) Bz (7) Bpy (6) Ir (56) Py (17) Bz (14) Bq (8) Bpy (6)\n\n Ir: iridium, Q: quinoxaline, Bq: benzo-quinoxaline, Bz: fused benzene, Py: pyridine and Bpy: bipyridine.\n\n\n\n singlet and triplet excited states (Table 4). In compound 3 the\n di\ufb00erence in energy between S0 \u2192 S1\u20134 was \u2264 0.07 eV. This\n indicates that the excitations to these singlet excited states are\n very close in energy due to excitations involving multiple closely\n spaced occupied orbitals (HOMO, HOMO\u22121, and HOMO\u22122) to\n a pair of nearly degenerate unoccupied orbitals (LUMO and\n LUMO+1), as shown in Table 4. The transitions S0 \u2192 S1, S0 \u2192\n S2, and S0 \u2192 S4 exhibit a combination of metal-to-ligand\n charge transfer (MLCT), ligand-to-ligand charge transfer\n (LLCT), and ligand-centred (LC) characteristics. Conversely,\n the transition S0 \u2192 S3 is primarily characterized as ligand-\n centred (LC). The close energy spacing between these excited\n states enhances the probability of internal conversion (IC), as\n the excitation energy can be e\ufb03ciently transferred to vibrational\n modes and it could lead to strong vibronic coupling between\n states, giving a broad absorption spectrum where the electronic\n transitions can borrow intensity from each other.118,119\n As far as compound 1 is concerned, the electronic states S1\u2013\n S4 exhibit mixed MLCT/LLCT characteristics. This arises as\n Fig. 5 Optimized ground state structures for 1 (left) and 3 (right) each state is formed by excitations from HOMO to successive\n obtained from TD-DFT [(B3LYP/SDD for Ir(III)) and (6-31g** for C,H,N)] higher-energy unoccupied orbitals. Specifically, S1 involves\n with SMD (CH3CN). For the sake of clarity, the hydrogen atoms are not excitation to LUMO, S2 to LUMO+1, S3 to LUMO+2, and S4 to\n shown, and each molecular fragment has been assigned a colour code. LUMO+3. Additionally, these unoccupied orbitals predomi-\n The length of each colour bar is proportional to the percentage contri-\n bution of the corresponding coloured moiety to each molecular orbital.\n nantly receive contributions from various aromatic fragments\n Colour codes for bars and molecular fragments: iridium atom (green), within the molecule (Fig. 5 and Table 4). Therefore, we can\n quinoxaline or benzo-quinoxaline ( pink), fused benzene (navy blue), pyr- observe that the absence of transitions of LC nature in com-\n idine (blue), bipyridine (brown). pound 1 causes the excited states of this compound to have a\n greater MLCT character than those of compound 3. This\n enhances the \u2018heavy atom e\ufb00ect\u2019 in 1, which is expected to\n delocalization of the LUMO and LUMO+1. This extended con- amplify its spin orbit coupling (SOC) and intersystem crossing\n jugation leads to a greater delocalization of the \u03c0 and \u03c0* orbi- (ISC) rates.\n tals, resulting in a narrowing of the energy gaps between the The excitation energies for the T1 and T2 states of 3, at 1.50\n frontier molecular orbitals. This extension results in a red shift eV, are notably low. These energies primarily arise from elec-\n in the absorption compared with compound 1. tron excitations moving from HOMO\u22121 and HOMO\u22122 to\n To gain a better understanding about the behaviour of the LUMO and LUMO+1, which are mainly centred on the benzo-\n studied compounds in their electronic excited states, the verti- quinoxaline moiety as mentioned above (Fig. 5 and Table 4).\n cal excitation energies were calculated along with the orbitals Aromatic ligands can better delocalize the unpaired electrons\n involved in these excitations and their relative contributions to in the triplet state, resulting in greater stabilization of the\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 7304\u20137332 | 7313\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n Table 4 Vertical excitation energies (eV) of S1\u2013S4, T1\u2013T4 states and percentages (values in brackets) of dominant contributions to the calculated\n transitions for 1 and 3 obtained at the TD-DFT (SMD, acetonitrile)/6-31G(d,p)//SDD level\n\n Energy/eV Electronic structure\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n State 1 3 1 3\n\n S1 2.61 2.38 d/\u03c0H \u2192 \u03c0*L (98) (MLCT/LLCT) d/\u03c0H \u2192 \u03c0*L (95), \u03c0H\u22122 \u2192 \u03c0*L (4) (MLCT/LC)\n S2 2.67 2.42 d/\u03c0H \u2192 \u03c0*L+1 (98) (MLCT/LLCT) d/\u03c0H \u2192 \u03c0*L+1 (48), \u03c0H\u22121 \u2192 \u03c0*L (35), \u03c0H\u22122 \u2192\n \u03c0*L+1 (16) (MLCT/LC)\n S3 2.74 2.43 d/\u03c0H \u2192 \u03c0*L+2 (97) (MLCT/LLCT) \u03c0H\u22122 \u2192 \u03c0*L (58), \u03c0H\u22121 \u2192 \u03c0*L+1 (39) (LC)\n S4 3.06 2.45 d/\u03c0H \u2192 \u03c0*L+3 (94) (MLCT/LLCT) d/\u03c0H \u2192 \u03c0*L+1 (48), \u03c0H\u22122 \u2192 \u03c0*L+1 (27), \u03c0H\u22121 \u2192\n \u03c0*L (23) (MLCT/LC)\n T1 2.31 1.50 \u03c0/dH\u22123 \u2192 \u03c0*L (23), dH\u22124 \u2192 \u03c0L+1 (19), \u03c0/dH\u22122 \u2192 \u03c0H\u22121 \u2192 \u03c0*L (48), \u03c0H\u22122 \u2192 \u03c0*L+1 (44) (LC)\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n \u03c0L+1 (17), dH\u22125 \u2192 \u03c0L (13) (MLCT/LLCT)\n T2 2.45 1.50 \u03c0/dH\u22123 \u2192 \u03c0*L+1 (21), dH\u22124 \u2192 \u03c0L (20), \u03c0/dH\u22122 \u2192 \u03c0H\u22122 \u2192 \u03c0*L (46), \u03c0H\u22121 \u2192 \u03c0*L+1 (46) (LC)\n \u03c0L (20), dH\u22125 \u2192 \u03c0L+1 (13) (MLCT/LLCT)\n T3 2.41 2.22 dH \u2192 \u03c0*L+1 (73), \u03c0H\u22121 \u2192 \u03c0*L (13), \u03c0H\u22127 \u2192 d/\u03c0H \u2192 \u03c0*L (66), \u03c0H\u22123 \u2192 \u03c0*L+1 (10) (MLCT/LLCT)\n \u03c0*L (4) (MLCT/LLCT)\n T4 2.48 2.26 dH \u2192 \u03c0*L (79), \u03c0H\u22121 \u2192 \u03c0*L+1 (9), \u03c0/dH\u22123 \u2192 d/\u03c0H \u2192 \u03c0*L+1 (77), \u03c0H\u22123 \u2192 \u03c0*L (14) (MLCT/LLCT)\n \u03c0*L (4) (MLCT/LLCT)\n\n\n\n excited state. This stabilization leads to the small excitation The triplet\u2013triplet energy transfer process is e\ufb03cient when\n energy observed in these states with a 3LC/3\u03c0\u03c0* nature. the triplet state energy exceeds the energy required to excite\n Regarding T3 and T4, they showed a MLCT/LLCT character molecular 3O2 to 1O2 (approximately 0.98 eV or 1270 nm).\n which involves transitions from HOMO and HOMO\u22123 to Although this calculated energy gap is below the threshold\n LUMO and LUMO+1, which implicates charge transfers from required for direct 1O2 generation, it is important to consider\n the metal, fused benzene and pyridine moieties to the benzo- that TD-DFT methodology is well known to underestimate the\n quinoxaline fragment. These states are much closer in energy energy of \u03c0\u03c0* singlet and triplet excitations states, which\n to S1 (1MLCT/1d\u03c0*) and S2 (1LC/1\u03c0\u03c0*) states than T1 and T2 suggests the energy for T1/T2 state is likely too low.121,122\n states (3LC/3\u03c0\u03c0*). Regarding 1, the excitation energies for T1\u2013T4 Indeed, several studies have found systematic \u201cred-shifts\u201d in\n states are closely matched, exhibiting MLCT/LLCT character- TD-DFT/B3LYP predicted triplet energies for states both large\n istics. These states exhibit a substantial multiconfigurational aromatic chromophores and organometallic complexes.123,124\n nature due to excitations across multiple orbitals. This con- Consequently, the actual adiabatic T1/T2 energy in our system,\n trasts with the singlet excited states, which only involve single which is not far from 0.98 eV, is plausibly higher than the\n excitations (Table 4). TD-DFT result, likely in the range necessary to achieve 1O2 sen-\n The strength of SOC is influenced by the energy gap sitization. This alignment with the observed 1O2 generation\n between adiabatic excitation energies, which encompass the supports the photophysical conclusions of this study.\n coupling between electronic and nuclear motions. Therefore, Additionally, the long lifetime of this state may favour direct\n we calculated these adiabatic excitation energies in electron electron transfer from the triplet state to molecular oxygen,\n volts (eV) for the optimized excited states (Fig. 6 and leading to the formation of O2\u2022\u2212, either through direct inter-\n Table S8\u2020). For compound 3, the substantial energy gap action or via an intermediate electron donor or substrate.125\n between the S1 and T1/T2 states (1.56 eV) significantly restricts In compound 1, similar energy gaps were observed between\n intersystem crossing (ISC) between these states, consistent the S1 and T3/T4 states, specifically 0.178 eV and 0.082 eV,\n with the Fermi golden rule.120 Moreover, the almost complete respectively, as compared with 3. The absence of isoenergetic \u03c0\n absence of d orbitals in the metal configurations of the T1/T2 orbitals (HOMO\u22121 and HOMO\u22122) relative to the HOMO\n states due to their 3\u03c0\u03c0* character contributes to weaker SOC, increases the involvement of metal-centred orbitals in the tran-\n further inhibiting ISC between these states. In contrast, the sitions of these singlet excited states. Consequently, as stated,\n smaller energy gaps between the S1 and T3/T4 states (0.160 eV spin\u2013orbit coupling (SOC) is expected to be more e\ufb00ective in 1\n and 0.07 eV, respectively) are expected to promote ISC. than in 3. Additionally, di\ufb00erences in the orbital geometries\n Additionally, the adiabatic excitation energies of the T3 and T4 involved in these singlet and triplet excited states (see Table 4)\n states (2.074 eV and 2.160 eV, respectively) are relatively close further enhance SOC, in accordance with El Sayed\u2019s rule.126\n to the vertical excitation energies of the T1 and T2 states (1.50 Moreover, a key di\ufb00erence observed in 1 compared with 3 is\n eV). This proximity suggests potential overlap of vibrational the significantly smaller energy gaps between the S1 and T1/T2\n levels, which could facilitate internal conversion (IC) as a relax- states (approximately 0.414 eV). These observations suggest\n ation pathway from higher triplet states through vibronic coup- the presence of two possible competing ISC channels. The first\n ling mechanisms. The small energy gap observed between the is a thermally activated ISC between S1 and T2/T3 states. Due to\n adiabatic energies of S0 and T1/T2 states (0.69 eV) suggests a the very small energy gaps and the di\ufb00erent orbital geometries,\n non-emissive character for these states, along with a long-lived the SOC will be very e\ufb03cient, leading to rapid ISC. IC from T2/\n nature due to their \u03c0\u03c0* configuration. T3 to T1/T2 is also fast, quickly populating the lowest excited\n\n\n 7314 | Inorg. Chem. Front., 2025, 12, 7304\u20137332 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n Fig. 6 Proposed Jablonski diagram for 1 and 3 and their interaction with molecular oxygen. Triplet excited states can undergo electron transfer\n generating O2\u2022\u2212 or energy transfer to molecular oxygen producing 1O2.\n\n\n\n\n triplet states. The second channel is a slow ISC between S1 and bromide) assays under dark and light conditions. Under the\n T1/T2, as the orbitals of S1 and T1/T2 are almost identical, redu- experimental conditions, light exposure was confirmed to exert\n cing the SOC rate, and the energy gap between them is signifi- a minimal e\ufb00ect on cell viability. However, to ensure accurate\n cantly larger. Furthermore, the small energy gaps between T1 assessment of cytotoxicity, the viability of cells subjected to\n and T4 states cause the vibrational levels of the higher triplet light or dark treatments was analyzed relative to untreated\n states to overlap with those of the lower triplet states, facilitat- control cells that were exposed to the same irradiation con-\n ing vibronic coupling and enabling reverse internal conversion ditions as the treated cells. As summarized in Table 5, after\n (IC).127 This overlap increases the probability of rapid electron 48 h of treatment in the dark, both complexes exhibited intrin-\n exchange among the T1\u2013T4 states, allowing for a large number sic cytotoxicity, with IC50,dark values ranging from 0.89 to\n of electron configurations and enhancing the return to the 3.65 \u03bcM. These values were comparable to that of the che-\n singlet manifold via reverse intersystem crossing (RISC).128 motherapeutic agent cisplatin. In all cases, complex 1 dis-\n Therefore, the expected larger SOC of 1, due to the greater played moderately higher cytotoxicity than complex 3. Notably,\n MLCT character of the S1 state and the multiconfigurational upon photoactivation with blue light (460 nm, 24.1 J cm\u22122),\n character of the T1\u2013T4 states, as well as the existence of several the activity of both complexes significantly increased, resulting\n channels to produce ISC in this compound, suggests the exist- in IC50,light values in the nanomolar range and phototoxic\n ence of a more e\ufb03cient population of triplet states where the indexes (PIs) between 11 and 32 for complex 1 and from 6 to\n electron can undergo rapid exchange between these states due 124 for complex 3. Again, complex 1 exhibited higher photocy-\n to their close energy levels. This spreads the population over totoxicity than complex 3, except in A549 lung cancer cells,\n various triplet states, prolonging the overall triplet-state life- where complex 3 displayed an outstanding photodynamic\n time. The longer lifetime of the triplet states in 1, compared activity, resulting in a PI of 124 and an IC50,light of 0.016 \u00b5M.\n with 3, should contribute to an enhanced production of ROS Given the favorable photodynamic behavior of the complexes,\n from the former compound. Further experiments on the emis- their photocytotoxic activity was also examined in A549 cells\n sion profile of 1 in acetonitrile were also performed, both at after activation with green (515 nm) and red (635 nm) light,\n room temperature and at 77 K. These spectra allowed us to which possess deeper tissue penetration properties.129\n e\ufb00ectively determine the energy gap through the di\ufb00erence of Following green light irradiation, IC50,light values of 0.18 \u00b1\n the energy of the onset from the emission bands at RT and 0.03 \u03bcM were obtained for complex 1 and 0.204 \u00b1 0.01 \u03bcM for\n 77 K (Fig. S80\u2020). These results are consistent with TD-DFT cal- complex 3, representing PI values of 5 and 11. However,\n culations, as well as the observed delayed fluorescence and the irradiation with red light only produced a 2-fold increase in\n higher values obtained in the case of 1 for the quantum yields the cytotoxicity of complex 1 and a 4-fold increase in the cyto-\n of emission and ROS generation. toxicity of complex 3 compared with treatments in dark con-\n ditions, respectively, with IC50,light values of 0.49 \u00b1 0.1 \u03bcM and\n 2.6 Biological studies 0.502 \u00b1 0.04 \u03bcM. These results confirm that optimal photoacti-\n Anticancer activity. The phototoxic activity of complexes 1 vation for both complexes is achieved with blue light\n and 3 against di\ufb00erent cancer cells was first evaluated using irradiation, which agrees with their absorption spectra.\n MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium However, green light could also induce a significant phototoxic\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 7304\u20137332 | 7315\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n Table 5 Photocytotoxic activity of the complexes in 2D cell cultures\n\n 1 3 Cisplatin\n\n IC50 (\u03bcM) IC50 (\u03bcM) IC50 (\u03bcM)\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n PI PI\n Complex Dark Light Dark Light Dark\n\n A549 0.890 \u00b1 0.204 0.028 \u00b1 0.019 32 2.028 \u00b1 0.784 0.016 \u00b1 0.003 124 5.998 \u00b1 1.185\n HeLa 1.048 \u00b1 0.253 0.063 \u00b1 0.014 17 2.515 \u00b1 0.173 0.252 \u00b1 0.059 10 1.528 \u00b1 0.326\n MCF-7 1.142 \u00b1 0.164 0.100 \u00b1 0.024 11 3.310 \u00b1 0.093 0.535 \u00b1 0.059 6 5.750 \u00b1 0.070\n BxPC3 0.984 \u00b1 0.062 0.078 \u00b1 0.018 13 3.652 \u00b1 0.261 0.140 \u00b1 0.055 26 1.008 \u00b1 0.009\n MRC-5 2.587 \u00b1 0.199 0.125 \u00b1 0.032 21 3.067 \u00b1 0.409 0.292 \u00b1 0.008 10 5.327 \u00b1 0.377\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n Cells were treated with the compounds for 4 h at 37 \u00b0C to ensure their maximum internalization and then kept in the dark or irradiated with\n blue light for 1 h (460 nm, 24.1 J cm\u22122). Cell viability was assessed 43 h later by MTT assays. Data represent the mean \u00b1 SD of at least three inde-\n pendent experiments, each performed in triplicate. PI: phototoxicity index = IC50,dark/IC50,light.\n\n\n\n response. These results can be compared with those obtained irradiation. Ten days later, the number of colonies formed in\n with analogous Ir complexes with bpy and C^N ligands with the absence of light irradiation was similar to that of the\n less \u03c0-extension using MCF-7 breast cancer cells. With ppy, corresponding untreated control, indicating the absence of a\n there was no increase in cytotoxicity by light activation.108 cytotoxic e\ufb00ect. In contrast, photoactivation of the complexes\n With C^N ligands with moderate \u03c0-expansion as 2-phenylqui- significantly reduced colony numbers to 41.0 \u00b1 4.9% for\n nolinate (2pq), and 1-phenylisoquinolinate (1pq), the obtained complex 1 and 52.2 \u00b1 2.9% for complex 3 (Fig. 7). No signifi-\n PI values (up to 5.9) were lower and the IC50,light values (0.83 or cant di\ufb00erences were observed between dark and light con-\n 0.63 \u03bcM) were 8 or 6 times higher than those obtained with ditions for both untreated cells and cells treated with cisplatin,\n our compounds, reflecting the beneficial e\ufb00ect of the consistent with the non-photoactivatable nature of cisplatin.\n \u03c0-expansion of our ligands.108 These results support the light-dependent anticancer activity\n It should be noted that, in general, the complexes exerted of the complexes at the IC50,light values determined in MTT\n comparable cytotoxicity against the cancer cell lines and non- assays. Furthermore, they demonstrated the capability of the\n malignant MRC-5 fibroblasts in dark conditions, revealing a complexes to inhibit the clonogenic potential of the cells, a\n lack of intrinsic selectivity towards malignant cells. However, a critical factor in preventing metastasis, as proliferation in\n remarkable photoselectivity was observed when comparing the distant tissues is essential for the formation of secondary\n e\ufb00ects of the photoactivated complexes on A549 lung cancer tumors.\n cells to the dark toxicity against MRC-5 lung fibroblasts. Upon Intracellular ROS generation. To elucidate the mechanism\n irradiation, the IC50,light values of complexes 1 and 3 in A549 underlying the photocytotoxicity of the complexes, their\n cells were 92.4 and 191.7 times lower, respectively, than the capacity to generate ROS at the cellular level was investigated.\n IC50,dark values determined in MRC-5 cells. These findings This is a primary characteristic for PSs to be e\ufb00ective in photo-\n show the potential of these complexes to exert a selective light- dynamic therapy (PDT). The cellular ROS levels were quantified\n activated action against cancer cells with minimal e\ufb00ects on using the dichlorodihydrofluorescein diacetate (H2DCFDA)\n the non-irradiated healthy tissue. probe, which is converted to the green fluorescent derivative\n To further assess the toxicity of the complexes, hemolytic dichlorofluorescein (DCF) upon oxidation. A549 and HeLa\n experiments were conducted.130 Porcine red blood cells (RBC) cells were treated with 1 and 3 at their respective IC50,light and\n were incubated with 1 and 3 in the dark or under blue light changes in cellular fluorescence were monitored by flow cyto-\n irradiation at concentrations close to the IC50,dark (1 \u03bcM) or metry. As shown in Fig. 8A, no significant di\ufb00erences in cell\n IC50,light (0.2 \u03bcM and 0.02 \u03bcM). Hemoglobin release was fluorescence were observed in the absence of light irradiation\n measured as an indicator of the capacity of the complexes to compared with untreated control cells. However, blue light\n destabilize RBC membranes. Both complexes exhibited irradiation induced a marked increase in the green fluorescent\n minimal hemolytic activity (\u22645% hemolysis) under these con- signal, indicating ROS generation within the cells. Specifically,\n ditions, indicating a favorable compatibility with blood cells in treatment with complex 1 resulted in a 10.4 \u00b1 0.7-fold increase\n the context of potential intravenous administration in fluorescence intensity in A549 cells and an 8.51 \u00b1 2.8-fold\n (Table S9\u2020). increase in HeLa cells. Complex 3 also induced a strong fluo-\n The anticancer activity of 1 and 3 was further evaluated rescence rise, with a 14.61 \u00b1 0.9-fold increase in A549 cells and\n using clonogenic assays, which better assess the long-term sur- a 16.95 \u00b1 3.5-fold increase in HeLa cells. As was demonstrated,\n vival and proliferative potential of cells following the treat- these complexes were able to generate singlet oxygen under\n ment.131 HeLa cells were selected for this study due to their light irradiation (Fig. S56 and S57\u2020). This type II reaction is the\n superior ability to form colonies in vitro. Cells were exposed to primary cytotoxic mechanism for most PSs in PDT.132\n complexes 1 and 3 at the respective IC50,light values for 4 h, fol- Furthermore, by the DHR123 test, it was shown that the com-\n lowed by incubation in dark conditions or under blue light plexes showed the ability to produce superoxide anions (O2\u2022\u2212)\n\n\n 7316 | Inorg. Chem. Front., 2025, 12, 7304\u20137332 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n Fig. 7 Clonogenic assays. (A) Images of the colonies generated after exposure of HeLa cells to complexes 1 and 3 at the corresponding IC50,light, or\n medium alone as a control, both in the dark or with blue light irradiation (1 h, 460 nm, 24.1 J cm\u22122). Cisplatin at 5 \u03bcM was used as the positive\n control. (B) Bar charts represent the percentage of colonies after each treatment relative to the control cells (mean \u00b1 SD of three experiments). *** p\n < 0.001.\n\n\n\n\n Fig. 8 Intracellular ROS generation and e\ufb00ect of speci\ufb01c ROS on cell viability. A549 and HeLa cells were treated with complexes 1 and 3 at their\n respective IC50,light for 4 h, followed by incubation in the dark or exposure to blue light irradiation for 1 h (460 nm, 24.1 J cm\u22122). The elevation of\n general ROS (A) and superoxide anion (B) levels were determined with speci\ufb01c probes and cell \ufb02uorescence was measured by \ufb02ow cytometry. Bars\n represent the mean fold increase (\u00b1standard deviation) relative to untreated control cells from three independent experiments. *p < 0.05; **p < 0.01\n versus non-irradiated cells. (C) HeLa cells were treated at their IC50,light and exposed to blue light irradiation either without (\u2205) or with speci\ufb01c ROS\n scavengers (DMSO for \u2022OH, sodium azide for 1O2, or tiron for O2\u2022\u2212). Bars indicate the mean percentage of viable cells (\u00b1standard deviation) 48 hours\n post-treatment relative to untreated control cells incubated with medium alone or with the corresponding scavenger. * p < 0.05; ** p < 0.01 com-\n pared with treated cells without scavengers. Each condition was tested in triplicate across three independent experiments.\n\n\n\n (Fig. S62\u2020) and, thus, undergo type I PDT processes. O2\u2022\u2212 is a bution of this radical to the photocytotoxic activity of the com-\n transient but highly reactive species that originates oxidative plexes was investigated. Generation of O2\u2022\u2212 was quantified\n cascades, causing substantial and potentially irreversible using the Superoxide Detection Reagent (Enzo Life Sciences),\n damage to cellular components.133 Consequently, the contri- which emits an orange signal upon interaction with this\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 7304\u20137332 | 7317\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n radical. As expected, no significant di\ufb00erences in fluorescence lular functions, including the production of adenosine tripho-\n emission were observed when treatments were conducted in sphate (ATP), the regulation of apoptosis, and the mainten-\n dark conditions compared with control cells. Conversely, treat- ance of the redox homeostasis. The disruption of these pro-\n ment with photoactivated complex 1 resulted in a 3.57 \u00b1 0.78- cesses ultimately results in cell death, thereby establishing\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n fold and 4.49 \u00b1 1.38-fold increase in fluorescence emission in mitochondria as a primary therapeutic target in cancer. As\n A549 and HeLa cells, respectively. Similarly, complex 3 described below (Fig. 14), confocal microscopy experiments\n induced a 3.14 \u00b1 0.52-fold and 4.73 \u00b1 1.26-fold increase in fluo- with complex 1 confirmed a high degree of co-localization of\n rescence in A549 and HeLa cells (Fig. 8B), confirming O2\u2022\u2212 the complex with mitochondria.\n generation within cells, with a similar activity for both com- Thus, the e\ufb00ect of the complexes on mitochondrial func-\n plexes. It should be noted that these results di\ufb00ered from tion was investigated as a potential cytotoxic mechanism.\n those previously obtained with the DHR123 test, where a HeLa cells were treated with complexes 1 and 3 under dark\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n higher superoxide anion production was observed for complex and light conditions and mitochondrial integrity was assessed\n 1. This discrepancy may be attributed to the detection of by confocal microscopy using the fluorescent dye\n endogenous mitochondrial superoxide radicals. Mitochondrial MitoTracker\u2122 Red CMXRos, which accumulates within\n electron transport chain (ETC) leakage is a well-established healthy mitochondria based on the mitochondrial membrane\n source of superoxide radicals.134 Thus, the activity of com- potential (MMP).135 As shown in Fig. 9A, intense red mito-\n plexes 1 and 3 in mitochondria could potentially disrupt ECT chondrial staining was detected in both control cells and cells\n which may result in further intrinsic O2\u2022\u2212 generation. Finally, treated in the dark. However, upon photoactivation, the stain-\n it should be noted that no clear correlation was observed ing was significantly attenuated, revealing mitochondrial\n between the ROS levels and the PI of complexes 1 and 3 in damage. To corroborate these results, changes in the MMP\n A549 and HeLa cells, which suggests that additional factors were assessed using the JC-1 dye, which emits red fluorescence\n may contribute to the photocytotoxic activity of these when accumulated in healthy mitochondria and green fluo-\n complexes. rescence when the MMP is dissipated. As shown in Fig. 9B,\n Finally, to validate the contribution of individual ROS to 94.49% of untreated control cells exhibited red fluorescence\n the photocytotoxic activity of the complexes, HeLa cells were emission from JC-1. This percentage was similar for cells\n incubated with complexes 1 and 3 at their respective treated with complexes 1 (91.64%) and 3 (88.42%) in the dark,\n IC50,light and irradiated with blue light in the presence of while it decreased to 44.04% for complex 1 and 43.16% for\n specific ROS scavengers (dimethyl sulfoxide (DMSO) for \u2022OH, complex 3 upon blue light irradiation. Overall, these results\n sodium azide for 1O2 or tiron for O2\u2022\u2212) or with medium corroborated the activity of the complexes at the mitochondrial\n alone as a control. Cell viability was determined 48 h post- level.\n treatment. As shown in Fig. 8C, the percentage of viable cells Photocatalytic oxidation of NADH. Nicotinamide adenine\n significantly increased when treatments were conducted in dinucleotide (NAD) is a crucial coenzyme in cellular metab-\n the presence of sodium azide and tiron, confirming the olism that acts as an electron carrier in redox reactions.136\n involvement of 1O2 and O2\u2022\u2212 in the photocytotoxic mecha- NADH, which is the reduced form of NAD, transfers electrons\n nism of both complexes. Furthermore, the cytotoxic e\ufb00ect of to the mitochondrial ETC to generate ATP. This crucial role in\n complex 1 was also inhibited by DMSO, suggesting a contri- the cell energy generation has made NADH a promising target\n bution from \u2022OH. in anticancer therapy.107,137\u2013141 The oxidation of NADH by\n These results collectively demonstrate that type II photoche- anticancer agents disrupts ATP generation and contributes to\n mical processes play a predominant role in the photocytotoxi- mitochondrial dysfunction. In recent years, several papers\n city of these complexes. Furthermore, the involvement of O2\u2022\u2212 have been published on the photocatalytic oxidation of NADH\n and \u2022OH supports the contribution of type I mechanisms, by Ir(III) complexes via a single-electron transfer (SET) mecha-\n which exhibit reduced dependence on molecular oxygen. nism with generation of O2\u2022\u2212 and carbon-center radicals.107,137\n Thus, these complexes hold potential for treating cancer cells This suggests an additional potential mechanism of action for\n in hypoxic microenvironment, where resistance to convention- Ir(III) complexes in type I PDT, which we have investigated for\n al photodynamic therapy is often observed. our complexes.\n Mitochondrial damage. Cationic iridium-based complexes The evolution of NADH in the presence of complexes 1 and\n bearing lipophilic ligands have a high propensity to accumu- 3 (complex/NADH ratio = 1/100) was monitored by UV\u2013vis spec-\n late in the mitochondria, driven by the negative potential troscopy in aqueous solution over a period of 10 minutes at\n across the inner mitochondrial membrane. The lipophilicity of room temperature. In order to evaluate the light e\ufb00ect in the\n our complexes was quantified by determining their octanol/ oxidation reaction, the experiment was carried out in dark con-\n water partition coe\ufb03cients (log Po/w) using the traditional ditions and upon blue light irradiation (470 nm, 51.4 mW\n shake-flask method. Complexes 1 and 3 displayed log Po/w cm\u22122). The evolution of the photocatalytic reaction was moni-\n values of 1.15 \u00b1 0.27 and 1.58 \u00b1 0.23, respectively, indicating tored by the decrease of the absorption band at 340 nm\n their lipophilic character and a high probability of mitochon- (Fig. S81\u2013S83\u2020). In dark conditions, a very low level of NADH\n drial localization.52 This cellular distribution is particularly oxidation was observed. However, the reaction under light\n significant because mitochondria are involved in essential cel- irradiation was very fast for both complexes. The reaction pro-\n\n\n 7318 | Inorg. Chem. Front., 2025, 12, 7304\u20137332 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n Fig. 9 E\ufb00ect on mitochondria. HeLa cells were incubated with complexes 1 and 3 at the corresponding IC50,light for 4 h at 37 \u00b0C and then kept in\n darkness or exposed to blue light for 1 h. (A) Confocal microscopy images of the cells. Cell nuclei were localized in blue with Hoescht (\u03bbex: 400 nm;\n \u03bbem: 450 nm), and healthy mitochondria were labelled with MitoTracker\u2122 Red CMXRos (\u03bbex: 543 nm; \u03bbem: 595 nm). Attenuation of red \ufb02uorescence\n emission upon irradiation indicates mitochondrial dysfunction. (B) Percentage of cells exhibiting green and red JC-1 \ufb02uorescence after treatment in\n dark or irradiated conditions (mean \u00b1 SD of three independent experiments). Cells incubated with medium alone were used as control. Loss of MMP\n was determined by a decrease in the percentage of red \ufb02uorescent cells. * p < 0.05; ** p < 0.01.\n\n\n\n\n ceeded with first-order kinetics with respect to NADH. The Treatments using five times the IC50,light resulted in higher\n TONs (Turnover Numbers, measured after 5 minutes) of the apoptotic rates, with early and late apoptotic populations,\n complexes were up to 46 (Fig. S83 and Table S10\u2020) and respectively, reaching 21.46 \u00b1 15.31% and 35.78 \u00b1 15.37%, for\n TOFs (Turnover Frequencies) were up to 555 h\u22121, which complex 1 and 21.48 \u00b1 15.55% and 28.91 \u00b1 20.54% for complex\n make complexes 1 and 3 among the most active NADH 3. Importantly, minimal necrotic cell populations were\n photocatalysts.107,138 As has been reported for other Ir(III) observed across all treatment conditions. These findings indi-\n photosensitizers, H2O2 was detected (test strips) in the photo- cate that the complexes induce a form of programmed cell\n catalytic NADH oxidation with 1 and 3,107 which indicates the death involving apoptosis, thereby minimizing potential collat-\n generation of O2\u2022\u2212 and the conversion to H2O2. eral tissue damage and inflammatory responses typically\n Cell death mechanism. The depolarization of the mitochon- associated with necrosis.142\n drial membrane is a critical event that can initiate pro- However, the lower apoptotic population induced by\n grammed cell death via apoptosis. To assess the apoptotic complex 1 in comparison with the cisplatin control (Fig. 10)\n potential of complexes 1 and 3, flow cytometry experiments suggests that apoptosis may not be the exclusive primary death\n were conducted using annexin V-FITC and propidium iodide mechanism. Concurrent with this observation, the rapid\n (PrI) staining. Viable cells are impermeable to both dyes, photocatalytic oxidation of NADH induced by complex 1,\n whereas early and late apoptotic cells exhibit annexin V coupled with the significant increase in cellular reactive\n binding due to phosphatidylserine exposure on the cell oxygen species (ROS) levels and mitochondria shrinkage,\n surface. In addition, late-apoptotic and necrotic cells are PrI- suggest the involvement of ferroptosis. Ferroptosis is a distinct\n positive, due to the loss of membrane integrity. Treatments form of regulated cell death characterized by iron-dependent\n with cisplatin were included as a positive control for apoptosis. lipid peroxidation and is intrinsically linked to oxidative stress.\n As shown in Fig. 10, treatments with photoactivated complexes Recent literature demonstrates the capacity of various metal-\n 1 and 3 at the corresponding IC50,light led to a significant based complexes, including iridium(III) complexes, to induce\n increase in both the early and late apoptotic populations to ferroptosis or other non-apoptotic pathways, such as\n 10.98 \u00b1 2.03% and 6.26 \u00b1 3.01%, respectively, for complex 1, pyroptosis.143\u2013145 Therefore, while apoptosis remains a key cell\n and to 17.64 \u00b1 10.48% and 6.63 \u00b1 4.66% for complex 3. death mechanism, other non-apoptotic pathways, potentially\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 7304\u20137332 | 7319\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n Fig. 10 Cell death mechanism. HeLa cells were treated with complexes 1 and 3 for 4 h at the IC50,light or \ufb01ve times IC50,light. Cisplatin (5 \u03bcM) was\n used as positive control. Cells were double stained with propidium iodide and Annexin V-FITC. Bars represent the percentages of viable, early apop-\n totic, late apoptotic, and necrotic cells after each treatment (mean \u00b1 SD) determined in three independent experiments. Statistical analysis was per-\n formed compared with untreated cells. * p < 0.05; ** p < 0.01; *** p < 0.001.\n\n\n\n\n including ferroptosis, may also contribute to the overall tox- light, A549 cells treated with complexes 1 and 3 at the IC50,light\n icity of complex 1. demonstrated e\ufb00ective scratch closure in the cell monolayer,\n Cell migration. Mitochondrial energy production is crucial with migration rates comparable to the control cells (Fig. 11).\n for modifying focal adhesions and cytoskeleton remodeling, However, following photoactivation, a significant reduction in\n which are essential processes for cell migration.146 The the wound closure was observed, with an 88.4% inhibition in\n migratory capacity is of particular relevance in the context of the cell migration rate for complex 1 and a 93.0% inhibition\n cancer, as it enables the invasion of surrounding tissues, for complex 3, compared with control cells. In contrast, cispla-\n which represents the initial step of the metastatic cascade that tin treatment resulted in only a moderate reduction in cell\n ultimately enables cancer cell dissemination from the primary migration. This substantial di\ufb00erence suggests that complexes\n tumor to distant organs.147 Given the mitochondrial-targeted 1 and 3 exhibit additional characteristics in their antitumor\n activity of complexes 1 and 3, their impact on cell migration action that could potentially be exploited for the prevention of\n was evaluated using wound healing assays. In the absence of metastasis.\n\n\n\n\n Fig. 11 Wound healing assays. A549 cells were incubated with complexes 1 and 3 at their respective IC50,light, either in the dark or under blue light\n irradiation. Cisplatin (5 \u00b5M) was used as a control. (A) Representative images of the wound at time 0 and its closure after 24 h. (B) Migration rate (\u00b5m2\n h\u22121) of cells exposed to the di\ufb00erent treatments. Bars represent the mean \u00b1 SD of three independent experiments. * p < 0.01; *** p < 0.001 com-\n pared with the control or dark treatment.\n\n\n\n 7320 | Inorg. Chem. Front., 2025, 12, 7304\u20137332 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n\n Biological activity of NP1 and NP3. The encapsulation of ligands.42 An identical IC50 value of 0.86 nM has been\n compounds in nanoparticles (NPs) allows their selective described for a complex of the type [Ir(C^N)2(NHC^NHC)]+,\n accumulation in cancer cells, taking advantage of the EPR although ultraviolet irradiation was used.149 Conversely, the\n e\ufb00ect. In addition, NPs protect the complexes during their cir- dark activity of complex 3 was minimally a\ufb00ected by encapsula-\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n culation in the blood system and improve their cellular uptake, tion. However, upon irradiation, the cytotoxicity of NP3 was\n especially if the non-encapsulated compounds tend to aggre- notably higher, leading to a remarkable increase in the PI value\n gate.148 The influence of encapsulation on the photocytotoxic for NP3 versus 3 (from 10 to 65) in HeLa cells. In contrast, in\n properties of the complexes was initially examined using two- A549 cells the high PI value obtained for 3 decreased in NP3\n dimensional (2D) monolayers of A549 and HeLa cells. (from 124 to 45).\n Interestingly, compound 1 exhibited a marked increase in cyto- These results were validated using A549 multicellular spher-\n toxicity when encapsulated in NP1. As shown in Table 6, the oids (MCSs), which are 3D cell models that more closely repro-\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n IC50,dark values of NP1 in A549 and HeLa cells were, respect- duce tumor complexity and organization, including cell\u2013cell\n ively, 14.6 and 15.2-fold lower than those of complex 1. To rule interactions, nutrient distribution and oxygen gradients.150,151\n out the possibility that this e\ufb00ect was due to the intrinsic cyto- The IC50 values obtained indicated that MCSs were signifi-\n toxicity of the empty nanoparticle or the constituent polymers cantly less sensitive to both the free and encapsulated com-\n (P1b polymer with amine groups, and P2, polymer with car- plexes than cells growing in monolayers (Table 6). The reduced\n boxylic groups), their impact on cell viability was assessed e\ufb03cacy in MCSs is consistent with observations for other che-\n under the same experimental conditions (Table S11\u2020). No motherapeutic agents, such as doxorubicin, and could be\n detectable toxicity was observed across a concentration range attributed to a population of quiescent cells within the spher-\n of up to 200 \u00b5g mL\u22121, which exceeds the nanoparticle concen- oids that are less susceptible to the antiproliferative e\ufb00ects of\n trations corresponding to the IC50,dark values of NP1 and NP3 the drugs.152 Furthermore, the complex structure of MCSs has\n in both cell lines. been described to hinder the di\ufb00usion of drugs and oxygen to\n Following blue light irradiation, the IC50,light values of NP1 the inner cell layers, further compromising the e\ufb03cacy of the\n were further reduced, by 32.5-fold in A549 cells and 26.2-fold PSs. It is noteworthy that our complexes and NPs were able to\n in HeLa cells compared with complex 1 (Table 6), yielding exert a remarkable photocytotoxic e\ufb00ect against MCSs, result-\n PI values of 70 and 28. These represent 2.2- and 1.6-fold ing in a significant reduction in their size and a concomitant\n increases over the PI values of complexes 1 obtained in A549 loss of refractivity, which is indicative of extensive cell death\n and HeLa cells, respectively. These results revealed that NP1 (Fig. 12A). Particularly, complex 1 and NP1 exhibited a notable\n exhibits excellent properties for PDT. Of particular relevance activity, with IC50,light values in the nanomolar range and PIs\n is the outstanding cytotoxic activity of NP1 upon irradiation, of 24.3 and 14.4, respectively (Table 6). As observed in the 2D\n evidenced by an IC50,light value of 0.86 nM in A549 cells. cultures, the photocytotoxicity of NP1 was found to be higher\n Compared with other iridium derivatives with IC50 < 10 nM than that of the free complex 1. The encapsulation of complex\n (Table S12\u2020), the activity of NP1 is only surpassed by our 3 also improved its photocytotoxic activity against MCSs.\n recently described [Cp*Ir(C^N)L]+ derivatives with \u03c0-expansive However, the IC50,light values of 3 and NP3 were higher than\n\n\n\n\n Table 6 Photocytotoxic activity of 1, 3, NP1 and NP3 in 2D and 3D cell cultures\n\n A549 HeLa\n 2D-cultures\n IC50 (\u03bcM) IC50 (\u03bcM)\n\n Dark Light PI Dark Light PI\n\n NP1 0.061 \u00b1 0.0002 0.00086 \u00b1 0.0004 70 0.069 \u00b1 0.016 0.0024 \u00b1 0.0011 28\n NP3 1.79 \u00b1 0.28 0.0415 \u00b1 0.009 45 1.908 \u00b1 0.27 0.0302 \u00b1 0.0045 65\n\n A549\n 3D-cultures\n IC50 (\u03bcM)\n\n Dark Light PI\n\n 1 5.10 \u00b1 2.64 0.21 \u00b1 0.08 24.3\n NP1 1.44 \u00b1 0.39 0.10 \u00b1 0.03 14.4\n 3 38.78 \u00b1 15.65 5.53 \u00b1 0.01 7.0\n NP3 >50.00 1.71 \u00b1 0.53 >29.2\n\n Cells were treated with the indicated complexes and NPs for 4 h at 37 \u00b0C and then kept in the dark or irradiated with blue light for 1 h (460 nm,\n 24.1 J cm\u22122). Cell viability was assessed 43 h later by MTT assays (2D-cultures) or CellTiter Glo (3D-cultures). Data represent the mean \u00b1 SD. PI:\n phototoxicity index = IC50,dark/IC50,light.\n\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 7304\u20137332 | 7321\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n Fig. 12 Photocytotoxic activity against MCSs. (A) Representative microscopy images of A549 MCSs generated within the Geltrex extracellular\n matrix. Spheroids were treated under photoactivation conditions with complexes 1 and 3 at 0.3 \u00b5M and 10 \u00b5M, respectively, and nanoparticles NP1\n and NP3, at 0.2 \u00b5M and 2.7 \u00b5M, respectively. These concentrations are close to the IC50,light values for each compound. Untreated cells were used as\n a control. (B) Microscopy images of larger A549 MCSs generated in round-bottom wells with an ultra-low attachment surface. These MCSs were\n treated under photoactivation conditions with the complexes and nanoparticles at their IC50,light values ( previously determined in the 3D models) or\n \ufb01ve times the IC50,light. Images were acquired 43 h later (t = 48 h). Untreated spheroids served as a control (t = 0 h). Scale bars for all images:\n 100 \u00b5m.\n\n\n\n\n those of 1 and NP1. It should be highlighted that NP3 demon-\n strated minimal activity in the dark, leading to the highest PI\n value (>29.2) in MCSs.\n To further corroborate these findings, an alternative 3D\n model was established to produce larger A549 MCSs with\n diameters of approximately 400 \u00b5m (Fig. 12B). These MCSs\n were treated with either the previously determined IC50,light\n values from the 3D models or with fivefold higher concen-\n trations, followed by blue light irradiation. After 48 hours of\n treatment at the IC50,light, MCSs exhibited reduced structural\n integrity and compactness compared with the control Fig. 13 Cellular internalization of complexes 1 and 3 and nanoparticles\n MCS images obtained before the treatment. Moreover, all NP1 and NP3 in A549 and HeLa cells. The amount of iridium (ng) per\n million cells after 4 h of treatment was determined by ICP-MS. Each bar\n MCSs exposed to fivefold the IC50,light showed a marked\n in the graph represents the mean \u00b1 SD of three independent experi-\n reduction in size and a highly disrupted morphology. These ments. * p < 0.05; ** p < 0.01.\n results align with those obtained using the earlier 3D\n model, further supporting the antitumor potential of the\n compounds. NP3 exhibited lower cellular uptake. Encapsulation reduced\n To gain deeper insight into the impact of encapsulation on the uptake of 3 in A549 cells; however a 1.9-fold higher intern-\n the biologic behavior of the complexes, cellular uptake studies alization of NP3 was detected in HeLa cells, which is in line\n were conducted using inductively coupled plasma mass spec- with the relative values of photocytotoxicity observed for 3 and\n trometry (ICP-MS) to quantify the amount of iridium inside NP3 in this cell line (Table 6).\n the cells. The iridium levels were found to be 2.5- to 2.8 times Overall, these findings demonstrated a strong correlation\n higher in cells treated with NP1 than with the free complex 1 between cellular internalization of the complexes and their anti-\n (Fig. 13). These results aligned with the previously reported tumor e\ufb03cacy. Interestingly, the higher lipophilicity of complex\n higher photocytotoxicity of NP1. In contrast, complex 3 and 1 (log Po/w = 1.58 \u00b1 0.23) than complex 3 (log Po/w = 1.15 \u00b1 0.27)\n\n\n 7322 | Inorg. Chem. Front., 2025, 12, 7304\u20137332 This journal is \u00a9 the Partner Organisations 2025\n\f View Article Online\n\n Inorganic Chemistry Frontiers Research Article\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n Fig. 14 Confocal microscopy imaging of the subcellular distribution of 1 and NP1. HeLa cells were incubated with 1 or NP1 (\u03bbex/\u03bbem: 440/596 nm)\n at 50 \u03bcg mL\u22121 for 1 h at 37 \u00b0C. The commercial dyes MitoView\u2122 Green (Biotium) (\u03bbex/\u03bbem: 490/523 nm) and LysoTracker\u2122 Green DND-26\n (ThermoFisher Scienti\ufb01c) (\u03bbex/\u03bbem: 504/511 nm) and Hoechst (\u03bbex/\u03bbem: 352/454 nm) were used to localize the mitochondria, lysosomes, and cell\n nuclei, respectively. Images were captured using a Nikon A1R confocal microscope using the following acquisition settings: blue channel: \u03bbex/\u03bbem:\n 400/450 nm 33258, green channel: \u03bbex/\u03bbem: 488/525 nm and red channel: \u03bbex/\u03bbem: 488/595 nm. Merged images show the green and red \ufb02uor-\n escence overlapping in orange.\n\n\n\n correlated with an increased cellular accumulation. In negligible, as demonstrated by the absence of co-localization\n addition, the smaller size of the pbpz ligand in complex 1 and with the nuclear dye Hoechst 33258. It is noteworthy that\n its lower tendency to aggregate likely enhanced its cell pene- encapsulation did not alter the subcellular distribution of\n tration ability. Similar results have been reported with other Ir complex 1.\n (III) complexes bearing \u03c0-extended N^N ligands analogous to To confirm the mitochondrial localization of 1 and NP1,\n the C^N pbpz and pbpn ligands used in the present work.153 HeLa cells were incubated with each compound at 5 \u00b5M for\n Taking advantage of the luminescence properties of 4 hours, followed by isolation of mitochondrial and residual\n complex 1, the internalization mechanism of its free and cytosolic fractions using a commercial kit. Iridium content in\n encapsulated forms was further explored by flow cytometry. both fractions was then quantified by ICP-MS. Complex 1\n A549 and HeLa cells were incubated with 1 and NP1 for 4 h at showed preferential accumulation in the mitochondria\n either 37\u00b0 or at 4 \u00b0C, to di\ufb00erentiate between energy-depen- (0.964 \u00b5g Ir per mg protein) compared with the remaining\n dent and passive uptake processes. As illustrated in Fig. S84,\u2020 cytosolic fraction (0.029 \u00b5g Ir per mg protein). Similarly, NP1\n the fluorescence intensity was markedly reduced at 4 \u00b0C in exhibited a significantly higher mitochondrial concentration\n comparison with 37 \u00b0C in both cell lines, suggesting that (3.16 \u00b5g Ir per mg protein) relative to the cytosolic fraction\n both complex 1 and NP1 accumulate in the cells in an (0.014 \u00b5g Ir per mg protein). The increased mitochondrial\n energy-dependent manner that involves active transport or iridium content observed in NP1-treated cells compared with\n endocytosis, rather than passive di\ufb00usion through the cell cells treated with complex 1 is consistent with its higher\n membrane.154 overall cellular uptake, as illustrated in Fig. 13. Collectively,\n Subcellular distribution. Finally, the subcellular distribution these findings confirm a strong tendency of both 1 and NP1 to\n of complex 1 and NP1 was assessed using laser confocal accumulate in the mitochondria.\n microscopy. Microscopy images revealed a significant degree\n of co-localization between the red fluorescent signal of both\n complex 1 and NP1 with MitoView\u2122 Green, as shown in 3. Conclusions\n orange in the merged images (Fig. 14, and Fig. S85\u2020). The\n Pearson\u2019s correlation coe\ufb03cients (PCCs) were 0.75 for complex Through a molecular design strategy and using innovative pro-\n 1 and 0.76 for NP1. Mitochondrial accumulation is character- cedures we were able to obtain the new derivatives [Ir\n istic of these types of iridium complexes.26 In contrast, the (C^N)2(bpy)]+ which include the \u03c0-expansive C^N ligands pbpz\n minimal degree of co-localization observed for 1 and for NP1 (complex 1) or pbpn (complex 3). The \u03c0-expansion led to an\n with the lysosomal marker LysoTracker\u2122 Green, with PCCs of increase in the lifetimes of the excited states and a remarkable\n 0.36 and 0.46 respectively, excluded these organelles as generation not only of 1O2 but also of O2\u2022\u2212, especially in the\n primary cellular targets. Nuclear localization was found to be case of 1. This allowed the complexes to act both by type II and\n\n\n This journal is \u00a9 the Partner Organisations 2025 Inorg. Chem. Front., 2025, 12, 7304\u20137332 | 7323\n\f View Article Online\n\n Research Article Inorganic Chemistry Frontiers\n\n type I PDT processes. The generation of O2\u2022\u2212 allows the cir- agents that could also behave as theragnosis agents. Besides,\n cumvention of the problem of low O2 content in some tumors. there is also scope for further improving the properties by\n The photophysical properties were notably a\ufb00ected by the modifying the N^N ligand.\n degree of \u03c0-expansion of the C^N ligand, and TD-DFT studies,\n This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.\n\n\n\n\n which found very di\ufb00erent characteristics of the triplet excited\n states, nicely explained the divergences. In fact, 1 showed Con\ufb02icts of interest\n small energy di\ufb00erences among the four lowest triplet states\n enabling e\ufb03cient ISC and RISC, promoting rapid interconver- There are no conflicts to declare.\n sion within the triplet manifold. Consequently, compound 1\n displayed delayed fluorescence, where the intersystem crossing\n between singlet and triplet states can extend the excited-state Data availability\nOpen Access Article. Published on 04 July 2025. Downloaded on 5/12/2026 12:45:28 PM.\n\n\n\n\n lifetime, enhancing its e\ufb03ciency in reactive oxygen species\n The data supporting this article have been included in the\n (ROS) production. This phenomenon was not present in 3. In\n main text and as part of the ESI.\u2020\n this complex, the extra ring, in a similar way to that found in\n Crystallographic data (CIF-files) have been uploaded to the\n half-sandwich Ir(III) complexes with the pbpn ligand, causes\n CCDC and can be obtained via the CCDC homepage using the\n the generation of two near-HOMO orbitals leading to a higher\n CCDC numbers provided in the manuscript.\n LC character of the excited states. Thus, lower degrees of SOC\n and ISC are found in 3 with respect to 1. This rationale may be\n important in the design of new derivatives with improved\n properties.\n Acknowledgements\n Regarding anticancer activity, both complexes demon- This work was supported by the Spanish Ministerio de\n strated notable phototoxicity against human cancer cells, both Ciencia, Innovaci\u00f3n y Universidades (PID2021-127187OB-C21,\n in monolayer and MCS cultures. It is noteworthy that in all PID2021-127187OB-C22), Junta de Comunidades de Castilla-La\n cases, complex 1 demonstrated a markedly higher e\ufb00ect on Mancha-FEDER (JCCM) (grant SBPLY/23/180225/000192), and\n cell viability than complex 3. The complexes did not exhibit UCLM-FEDER (grants 2019-GRIN-27183, 2019-GRIN-27209 and\n intrinsic selectivity for malignant cells. However, they demon- 2022-GRIN-34193). C. G. acknowledges his fellowship to both\n strated a high capacity to exert a selective light-activated action the European Social Fund and Plan Propio de I + D + I of\n against cancer cells with minimal e\ufb00ects on non-irradiated UCLM (2022-PRED-20649). G. D. thanks the Junta de\n healthy cells and erythrocytes. Upon photoactivation, both Comunidades de Castilla-La Mancha and EU for financial\n complexes induced a substantial increase in intracellular ROS support through the European Regional Development Fund\n levels, including the generation of superoxide anions. Cell con- ( project SBPLY/19/180501/000191). J. C. L. and\n focal imaging revealed that complex 1 exhibited a marked A. M. acknowledge the Portuguese Foundation for Science and\n accumulation in mitochondria, which correlated with the dis- Technology for funding through LAQV-REQUIMTE (UIDB/\n ruption of mitochondrial physiological functions, as evidenced 50006/2020 and UIDP/50006/2020). C. B and E. Z. acknowledge\n by a decrease in mitochondrial membrane potential. Besides, their predoctoral grants University of Girona (IFUdG2021) and\n complexes 1 and 3 were found to be among the most active Generalitat de Catalunya (AGAUR; 2021 FI_B 01036). P. T.\n NADH photocatalysts described to date. Wound healing and acknowledges national funds from FCT \u2013 Funda\u00e7\u00e3o para a\n clonogenic assays demonstrated the capacity of the complexes Ci\u00eancia e a Tecnologia (FCT-MCTES), I. P., in the scope of the\n to inhibit the spread of malignant cells and their capacity to project UIDB/04378/2020 of the Research Unit on Applied\n generate secondary tumors, which are crucial steps in the Molecular Biosciences \u2013 UCIBIO and LA/P/0140/2020 of the\n metastatic process. These findings are promising, as meta- Associate Laboratory Institute for Health and Bioeconomy \u2013\n stasis is responsible for the greatest number of cancer-related i4HB. The collaboration of Prof. Antonio de la Hoz is acknowl-\n deaths. edged for the facilities to use the microwave system. The\n The encapsulation of the complexes into nanoparticles did authors would like to thank the IRICA services at UCLM and\n not a\ufb00ect their photophysical properties. Moreover, it Eduardo Prado Garc\u00eda-Consuegra of the SEM services team for\n improved some of the drawbacks of the complexes such as the technical assistance.\n aggregation and low solubility in water and low cellular\n uptake. Remarkably, the cellular uptake, even with low payload\n content, was improved by encapsulation and also the photo- References\n toxicity indexes were notably improved. Even in 3D cancer\n models this good behaviour was observed. NP1 led to one of 1 D. E. J. G. J. Dolmans, D. Fukumura and R. K. Jain,\n the lowest values of IC50 reported to date in iridium chemistry Photodynamic Therapy for Cancer, Cancer, 2003, 3(5),\n (0.86 nM). 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