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

Abstract

Photodynamic therapy (PDT) has emerged as a promising approach for cancer treatment, due to its ability to reduce side effects. In the search for luminescent iridium [Ir(C^N)₂(N^N)]⁺ complexes with high ability to generate ROS (reactive oxygen species) under irradiation, we employed C^N ligands with high π-expansion (pbpz (4,9,14-triazadibenzo[a,c]anthracene), 1, or pbpn (4,9,16-triazadibenzo[a,c]naphthacene), 3) that should lead to long excited state lifetimes. The photophysical properties were significantly influenced by the degree of C^N ligand π-expansion. Complex 1 exhibited a long fluorescence lifetime, matching the triplet lifetime observed in TAS, suggesting delayed fluorescence. In contrast, the additional ring in complex 3 generated two near-HOMO orbitals, increasing the excited state's LC character and reducing spin–orbit coupling (SOC) and intersystem crossing (ISC). They exhibited a notable ability to generate ¹O₂ and O₂˙⁻. TD-DFT studies nicely explained the differentiated photophysical properties. Both complexes exhibited significant phototoxicity against human cancer cells in both monolayer and multicellular spheroids models, with complex 1 exhibiting a higher effect. They effectively photogenerated intracellular ROS, including O₂˙⁻. The mitochondrial accumulation of 1 and its disruption of mitochondrial functions were verified. Wound healing and clonogenic assays demonstrated their potential as antimetastatic agents. In general, complexes’ encapsulation significantly facilitated their cellular accumulation and increased photocytotoxic indexes, with NP1 achieving one of the lowest IC₅₀ values reported in iridium chemistry. Furthermore, the nanoparticles showed good anticancer activity even in 3D models. Thus, 1 and 3 and especially NP1 show great promise as type I and II PDT agents with theragnostic potential.

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1. Introduction

Photodynamic therapy (PDT)^1–4 has emerged as a promising approach for cancer treatment because of its ability to reduce side effects, circumventing one of the main problems limiting the effectiveness of chemotherapy. This technique is a minimally invasive therapeutic modality that has gained significant attention for its ability to selectively target and eradicate cancer cells with spatio-temporal control. PDT relies on the use of a photosensitizer (PS) that, upon light activation, increases the cytotoxic effect in cancer cells through the generation of reactive oxygen species (ROS). This ROS generation is divided into two pathways depending on the type of interaction between the PS and the molecules of the medium. Type I PDT in which an electron transfer mechanism to O₂ or other oxygen-containing species renders different ROS, among them the superoxide anion radical (O₂˙⁻), which not only acts as an oxidant to destroy cancer cells, but can also suffer a series of cascade bioreactions leading to species such as hydroxyl radicals (HO˙) or H₂O₂, which are also cytotoxic. In type II PDT, an energy transfer mechanism to the fundamental oxygen (³O₂) generates singlet oxygen (¹O₂), a very reactive and toxic species.^5,6 Type II processes are more dependent on O₂ concentration, and thus PSs acting by type I or both are more adequate for hypoxic tumours. One measure of the potential of a PS is the phototoxic index (PI), which is the ratio of the cytotoxicity in the dark and after irradiation (PI = IC_50,dark/IC_50,light).

There are a number of PSs in clinical use that have been approved either globally or in specific countries for different types of cancer.^7–9 The majority are organic agents with a tetrapyrrolic scaffold, which suffer, among other problems, prolonged retention in tissues, photobleaching and a small Stokes shift that greatly increases the interference between excitation and emission.¹⁰ Metal-based tetrapyrrolic derivatives are under clinical trials or approved, as in the case of TOOKAT, a palladium complex.⁸ In the last decades attention has been paid to d⁶ transition metal complexes, which offer easy ligand modification, potential redox features and versatile photochemical and photophysical properties with intense absorptions in the visible region.^11–14 Specifically, the triplet state, which has a longer lifetime than the singlet state and facilitates PSs reactions with O₂, is typically more prevalent in metal complexes.¹³ Interesting results have been obtained with osmium,¹⁵ rhenium¹⁶ and ruthenium.^17,18 It is noteworthy that the ruthenium derivative TLD1433, developed by McFarland, is undergoing clinical trials.¹⁹

Among the various PSs reviewed in the literature, cyclometalated Ir(III) complexes have shown exceptional promise due to their unique and tunable photophysical properties, favourable absorption characteristics, large Stokes shifts and robust stability and photostability under physiological conditions. Additionally, the triplet excited states of Ir(III) complexes are highly sensitive to molecular oxygen and high quantum yields for ROS generation are observed.^20–24 Moreover, it is common for these derivatives to target mitochondria, giving them high potential in PDT cancer therapy.^25,26 These characteristics make Ir(III) complexes potent candidates for enhancing the efficacy of PDT. Besides, octahedral bis(cyclometalated) complexes of Ir(III) are remarkable for their intrinsic luminescence properties and are used as cellular imaging reagents and biomolecular probes.²⁷

Studies have shown that compounds with long excited state lifetimes are more efficient at producing ROS, exhibit high singlet oxygen (¹O₂) quantum yields, and have demonstrated significant photocytotoxicity in vitro.^28,29 It is known that the greater the ³ππ* character of the triplet excited state, the longer the T₁ lifetime.³⁰ Interestingly, utilizing ligands with extended π-systems reduces the energy of the ligand-centered (LC) ³ππ* state to below or near that of the ³MLCT state, significantly increasing the ³ππ* character and lifetime of the T₁ state.^28,31–33 The use of π-extended ligands also favors absorption at longer wavelengths in the visible and near-infrared regions, which is advantageous for deeper tissue penetration and minimizing damage to surrounding healthy tissue. This approach has been successfully used, reaching PDT activity in ruthenium chemistry^28,34–39 and also in iridium derivatives of N₆ type,^40,41 half-sandwich⁴² and derivatives of stoichiometry [IrCl(C^N)(N^N^N)]⁺.⁴³ The use of π-extending ligands has also been applied to bis(cyclometalated) species of the type [Ir(C^N)₂(N^N)]⁺ where the π-extension has mainly focused on the N^N ligand,^44–54 but it has also been explored for the C^N ligand^25,55,56 or both.^57–64 Some complexes demonstrated superior ROS generation capabilities, making them effective at inducing phototoxicity in cancer cells. With some exceptions,^55,65,66 in general, the π-extension used in C^N ligands consists of the incorporation of a fused benzene to the phenylpyridinate (ppy) ligand, usually in the pyridine fragment. In their excellent work, Vilar et al. have studied,⁶⁰ by a high-throughput approach, the photocytotoxicity of a wide library of iridium compounds. From DFT calculations, they concluded that, although the N^N ligand also influenced the outcome of the derivatives, the C^N ligand dominated the electronic structure properties of the complexes. In her research on bis(cyclometalated) iridium complexes, McFarland discovered that the degree of π-conjugation in the diimine ligand predominantly influences the ¹π,π* transitions observed in their UV-vis absorption spectra and that the π-conjugation of the cyclometalating ligand primarily determines the nature and energies of the lowest singlet and emitting triplet excited states.⁶⁷ Recently, we have reported the first case of half-sandwich cyclometalated iridium complexes with PDT activity using the C^N ligands pbpz (4,9,14-triazadibenzo[a,c]anthracene) or pbpn (4,9,16-triazadibenzo[a,c]naphthacene) (Fig. 1).⁴² Considering all these precedents, we decided to focus on studying the π-extension of the C^N ligand and synthesize derivatives of the type [Ir(C^N)₂(bpy)]⁺ (bpy = 2,2′-bipyridine) using the pbpz and pbpn C^N ligands, which exhibit a remarkable π-extension. A potential advantage of the targeted derivatives over the half-sandwich complexes with the same ligands is the expected luminescence, which enables a theragnosis effect, combining both therapeutic and diagnostic functionalities in cancer treatment.

Fig. 1 Structure of the proligands Hpbpz and Hpbpn.

Despite their potential, the clinical application of Ir(III) complexes in PDT faces significant challenges related to their delivery and bioavailability.⁶⁸ To overcome these limitations, encapsulation of PSs within nanoparticles has been proposed as a viable strategy.^69–71 Nanoparticles offer several advantages as drug delivery systems, including improved solubility, protection from premature degradation and the enhanced permeability and retention (EPR) effect,^72,73 thus increasing their accumulation in cancerous tissues⁷⁴ and minimizing systemic toxicity. Cross-linked polymeric nanoparticles have shown enormous potential as carriers due to their low toxicity and biocompatibility.⁷⁵ Since these nanoparticles do not establish dynamic equilibria, they retain their structure even at low concentrations, and they are preserved in the bloodstream longer than self-assembled systems. This favours their accumulation in tumours.⁷⁶ A wide variety of molecules such as proteins, DNA, drugs, hydrophobic and hydrophilic molecules, have been encapsulated in this type of system.^77–80

In this study, we report the synthesis and characterization of two new biscyclometalated Ir(III) complexes with π-extended C^N ligands and their encapsulation into polymeric nanoparticles. The use of this type of ligand has allowed long lifetimes of the excited states to be obtained, and a remarkable generation not only of ¹O₂ but also of O₂˙⁻. The photophysical properties were highly dependent on the type of ligand, a fact that was excellently explained by TD-DFT studies. The complexes exhibited photodynamic efficacy under blue light irradiation, resulting in mitochondrial damage and an antimetastatic effect. Encapsulation of the complexes significantly enhanced their cellular uptake and photodynamic activity. Notably, one of the nanoparticles exhibited, under irradiation, one of the highest values of cytotoxicity found in iridium chemistry (IC₅₀ = 0.86 nM).

2. Results and discussion

2.1. Synthesis and characterization of complexes

Two new biscyclometalated Ir(III) complexes of general formula [Ir(C^N)₂(N^N)]⁺ (C^N = pbpz, 4,9,14-triazadibenzo[a,c]anthracene; or pbpn, 4,9,16-triazadibenzo[a,c]naphthacene; N^N = 2,2′-bipyridine) have been prepared (Scheme 1). The proligands were synthesized using a literature procedure (Scheme 1).¹⁸ These ligands were previously used in octahedral polypyridine Ru(II) complexes,¹⁸ [IrCl(C^N)(N^N^N)]⁺ derivatives⁸¹ and half-sandwich Ir(III) complexes⁴² exhibiting excellent PDT behaviour.

Scheme 1 Synthesis of ligands and complexes presented in this paper. (i) EtOH, reflux, 4 h. (ii) IrCl₃·3H₂O, diglyme/H₂O (3 : 1), MW, 220 °C, 10 min. (iii) bpy, AgOSO₂CF₃, DCM/MeOH (2 : 1), reflux, 24 h and NH₄PF₆, room temperature, 1 h. (iv) IrCl₃·3H₂O, 2-methoxyethanol/H₂O (3 : 1), MW, 150 °C, 10 min. (v) bpy, AgOSO₂CF₃, DCM/MeOH (2 : 1), reflux, 36 h and NH₄PF₆, room temperature, 1 h. (vi) 2,3-Diaminonaphtalene, EtOH, reflux, 4 h. (vii) DMSO, room temperature.

The synthesis of the biscyclometalated Ir(III) dimer D1 and the equivalent with the pbpn ligand was attempted by the conventional method, that implies the use of a ethylene glycol monomethyl ether : water (3 : 1) mixture.^82,83 However, this approach was unsuccessful, possibly because the proligands are not sufficiently nucleophilic to react with the Ir(III) center due to electron delocalization. To improve the reaction conditions, microwave techniques were employed. However, only Hpbpz was successfully cyclometalated in these conditions and D1 was obtained. Subsequently, the cationic complex 1 was prepared by abstraction of the chloride using AgOSO₂CF₃ and coordination of the N^N ligand, followed by anion exchange with NH₄PF₆.

To achieve the analogous complex with the pbpn ligand, a different approach was employed. In this case, benzo[h]quinoline-5,6-dione was first cyclometalated to the Ir(III) center using 2-methoxyethanol : water (3 : 1) mixture under microwave irradiation leading to the formation of the dimer D2. Subsequently, the N^N ligand was coordinated mediated by the chloride abstraction with AgOSO₂CF₃, followed by anion exchange with NH₄PF₆ (formation of 2). Finally, 2,3-diaminonaphtalene was condensed to the cyclometalated benzo[h]quinoline-5,6-dione to obtain 3.

The complexes were obtained in moderate yields (38–41%) as black, yellow or red solids. The complexes 1–3 were fully characterized by elemental analysis, ¹H, ¹³C{¹H} and ¹⁹F{¹H} NMR spectroscopy, including ¹H–¹H gCOSY, ¹H–¹³C gHSQC and ¹H–¹³C gHMBC and mass spectrometry (Fig. S1–S27†). Moreover, the crystal structures of 1, 2 and the adduct formed after reaction of D2 with DMSO, 4 (Scheme 1), were also solved by single crystal X-ray diffraction. The HPLC traces were also obtained for complexes 1 and 3 (Fig. S28 and S29†).

Complexes 1 and 2 exhibited good solubility in polar solvents such as dimethyl sulfoxide (DMSO) and chlorinated solvents as well as in N,N-dimethylformamide (DMF), methanol or acetonitrile. Complex 3 exhibited solubility in DMSO and DMF. Complexes 1–3 were poorly soluble in aqueous media. However, the cationic complexes were soluble in water provided that a small amount of another solvent, such as DMSO or DMF was added. Thus, nontoxic amounts of DMSO were used in the biological experiments to assist dissolution.

In the ¹H NMR spectra, the lack of a signal for the proton of the metalated carbon (H⁷) and the shift of the resonances for the protons of pbpz and pbpn in the complexes with respect to those for the corresponding proligands (Hpbpz and Hpbpn) corroborates the coordination of the C^N ligands to the metal centre. The resonances were fully assigned by means of ¹H–¹H COSY experiments and considering the different coupling constants expected in the pyridyl ring.^42,84 The strong shielding of the proton adjacent to the metallated carbon (H¹²) of both the pbpz or pbpn ligands due to the effect of the ring current anisotropy of the pyridine ring of the other C^N ligand is noticeable, as it is clearly observed in the structure of 1 determined by X-ray diffraction described below.^85,86 It was also possible to assign most of the ¹³C{¹H} NMR resonances through ¹H–¹³C gHSQC and ¹H–¹³C gHMBC experiments.

2.2. Solid-state characterization by X-ray diffraction and analysis of π–π stacking

The molecular and crystal structures of complexes 1, 2[OSO₂CF₃] and 4 were determined by X-ray diffraction. The corresponding ORTEP diagram of complex 1 is shown in Fig. 2. The ORTEP diagrams of complexes 2[OSO₂CF₃] and 4 are shown in Fig. S30.† The methods used to obtain single crystals are detailed in the Experimental section. Selected bond distances and angles are gathered in Table S1.† The crystallographic data are provided in Table S2.† The three compounds exhibit a distorted octahedral geometry with the two nitrogen atoms of the C^N ligands in a relative trans disposition, as expected.^87–89 The Ir–C and Ir–N distances of the C^N ligand are about 2 Å while those involving the N atoms of the N^N ligand are a bit longer because of the trans influence of the carbon atoms.^26,90,91 The iridium atoms are chiral but both enantiomers are observed in a single crystal.

Fig. 2 ORTEP diagram of cation of complex Δ1. Ellipsoids are at the 30% probability level. Hydrogen atoms and PF₆⁻ anion have been omitted for clarity.

Concerning non-covalent interactions, besides the cation–anion coulombic attractions and hydrogen bonding, it is worth noting the presence of π–π interactions involving the C^N ligands. In the case of 1, the molecules are assembled into trimers by weak π–π interactions (Fig. S31†). There are two trimers in the asymmetric unit; one contains three Δ enantiomers (trimer Δ) and the other three Λ enantiomers (trimer Λ) (Fig. S32†). Molecules of different trimers are further packaged by other π−π interactions (Fig. S33†). Other examples of π–π interactions in cationic complexes involving a N^N ligand similar to pbpz^49,92–95 and in neutral and cationic complexes of formula [Cp*Ir(C^N)L]^0/+ with pbpz and pbpn⁴² have been reported.

Considering the π–π stacking interactions observed in the solid state, it was decided to ascertain whether this interaction was also present in solution. Thus, complexes 1 and 3 were studied by ¹H NMR spectroscopy at different concentrations. The existence of the π–π stacking interaction on increasing the concentration could be proved by observing the shielding of specific ring protons due to the influence of the ring current from the adjacent aromatic moieties.^96,97 In addition, this study could provide information about the regions that are mostly involved in the interaction. The differences in chemical shifts of the C^N aromatic protons for the most diluted and concentrated solutions in DMSO-d₆ are provided in Table S3.† The corresponding sets of spectra are given in Fig. S34 and S35.† Although nearly all C^N aromatic protons suffer concentration-dependent changes in chemical shift, the effect of shielding when the concentration is increased depends on the specific compound and on the position of the proton on the ligand. Higher Δδ values were found for complex 3 that contains pbpn, the more π-expansive ligand, probably due to a more efficient π-stacking interaction. Besides, the protons mostly affected are those situated in the central region of the ligands (see Fig. S36†).

Stability, photostability and aggregation studies (NMR, UV-vis and DLS). The stability of the new Ir(III) complexes was studied by ¹H NMR spectroscopy in the dark and under blue light irradiation (470 nm, 51.4 mW cm⁻²) in DMSO-d₆ due to the poor water solubility of the complexes (Fig. S37–S40, and Table S4†). Both compounds were highly stable during the irradiation time used in the biological studies. To simulate the conditions in the biological experiments, the stability in biological medium (DMEM) was studied by UV-vis spectroscopy in the dark and under blue light irradiation (Fig. S41 and S42†). As can be seen, the UV-vis spectra of both complexes were unchanged in the dark after 48 h, revealing the stability of the complexes in these conditions. Under blue light irradiation, some changes were observed for both complexes. The UV-vis spectra seem to evolve into a new species, as has been previously observed for other compounds with this type of π-expansive ligand. It was proposed that light favours an aggregation process.⁴²

In order to ascertain if the compounds aggregate spontaneously and confirm the light-induced aggregation of the complexes, a Dynamic Light Scattering (DLS) experiment was performed for solutions of both complexes at 1.0 × 10⁻⁴ M in water (10% DMSO) mixture before and after 1 h of blue light exposure. The average hydrodynamic diameter of particles of 1 and 3 before irradiation was 79 and 117 nm, respectively (Fig. S43†). These results indicate that the compounds form nanoaggregates in the presence of a high percentage of water, which is favoured by the hydrophobicity of the π-extended ligands and the polarity of water, as has been seen for other Ir(III) complexes in the literature.⁹⁸ In addition, after 1 h of blue light irradiation, the average hydrodynamic diameter of both compounds increased. The values for 1 and 3 were 378 and 323 nm, respectively (Fig. S43†). Therefore, light irradiation induces an increase in the aggregation of the particles, resulting in larger aggregates. Although it is not a common process, other examples of light-induced aggregation have been reported.^99–101

2.3. Photophysical properties

UV-vis absorption spectra. The UV-vis absorption spectra of complexes 1 and 3 were recorded in degassed acetonitrile at 1.0 × 10⁻⁵ M at 25 °C (Fig. S44,† and Table 1). Their spectra are relatively similar. Both spectra show two intense bands below 350 nm with a red shift of about 30 nm for 3 with respect to 1. They are assigned to π−π* transitions of the ligands (see the spectra of the proligands in Fig. S45†). Moreover, the spectra exhibit two additional bands of lower intensity at longer wavelengths, centred at around 390 and 440 nm for 1 and at 414 and 480 nm for 3. The simulated TD-DFT absorption spectra for both complexes (Fig. S77 and S78†) and the calculated electronic transitions (see below) indicate that these bands primarily correspond to a mixture of metal-to-C^N ligand charge transfer (d → π*, ¹MLCT) and C^N ligand-to-ligand charge transfer (π → π*, ¹LLCT) transitions in both series. There are also absorption tails that extend until 500 and 550 nm for 1 and 3, respectively. The red-shift observed in the absorption spectrum of 3 reflects the strong electron-withdrawing ability and extended π-conjugation of the benzo-quinoxaline ligand, as was observed before.⁴² This fact enhances its light-harvesting capability and increases its sensitivity to light sources that can penetrate deeper into tissues.

Table 1 Photophysical properties of complexes 1 and 3 and proligands Hpbpz and Hpbpn in acetonitrile, unless otherwise stated

Comp.	λ/nm (ε × 10^−4/M^−1 cm^−1)	ε (M^−1 cm^−1) (460, 515, 635 nm)	λ em (λexc)/nm	Φ em ^b	τ em/ns (contrib.)^c	k r (s^−1 × 10^−6)^d	k nr (s^−1 × 10^−7)^d	τ em/ns (aerat.)^e	τ em/ns (degas.)^e	φ Δ ^f
a In water (1% DMSO). b The Φem in degassed acetonitrile was determined using [Ir(ppy)2(bpy)]PF6 as reference (φem = 0.0707).^102 c In aerated acetonitrile solutions. d Radiative decay rate kr = Φ/τ and nonradiative decay rate knr = (1 − Φ)/τ. e Excited-state lifetimes in acetonitrile measured by TAS. f The ϕΔ (^1O2 generation quantum yield) in water (10% DMSO) under blue light irradiation (470 nm) was determined using [Ru(bpy)3]^2+ as reference (ϕΔ = 0.18).^103 n.m. = not measured.
1	272 (15.9), 302 (6.5), 358 (2.8), 390 (2.0), 438 (1.2)	6290	596	0.32	150 (14%)	0.80	0.17	360	3450	0.59
		920	(440)		440 (86%)
		10
3	246 (8.2), 300 (13.2), 320 (8.7), 340 (7.1), 394 (2.2), 414 (2.5), 478 (1.5)	8510	550	0.02	0.60 (25%)	2.0	9.8	180	2160	0.49
		5790	(420)		13.1 (75%)
		190
Hpbpz	308 (1.8), 350 (1.2), 366 (2.0), 388 (2.3)	n.m.	508 (412)	n.m.	n.m.	n.m.	n.m.	n.m.	n.m.	n.m.
Hpbpn	302 (3.4), 376 (0.5), 396 (1.1), 420 (1.5)	n.m.	544 (400)	n.m.	n.m.	n.m.	n.m.	n.m.	n.m.	n.m.

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In order to better understand the photophysical properties of the complexes in biological media, the UV-vis spectra were also recorded in water (1% DMSO) (Fig. 3). Comparing with the spectra recorded in acetonitrile, all the absorption bands in water were red-shifted (Table S5†). In water, the absorption tails extended until 540 and 580 nm for 1 and 3, respectively, promoting absorption of longer wavelengths in biological experiments, which is desirable for a PS. See in Table 1 the values of absorptivity at the wavelengths used in the biological studies.

Fig. 3 UV-vis absorption and emission spectra of complexes 1 and 3 in water (1% DMSO).

Emission spectra. The photoluminescence spectra of both complexes were initially recorded in solutions of dry deoxygenated acetonitrile (1.0 × 10⁻⁵ M) at 25 °C upon excitation at λ = 420–440 nm (Fig. S44† and Table 1). Both compounds emitted at ca. 550–600 nm upon excitation at the lower energy band of the absorption spectrum. The emission spectrum for complex 1 exhibited one band with λ_max at 596 nm and a shoulder at around 550 nm. The emission spectrum for complex 3 showed one emission band centred at around 550 nm, similar to the emission spectrum of Hpbpn (Fig. S46†). However, the emission spectrum of 1 was quite different to the spectrum of Hpbpz (Fig. S46†), with the main band red-shifted with respect to the proligand, and also red-shifted with respect to the emission of 3. The photoluminescence quantum yields (Φ_em) recorded for these complexes were also quite different (Table 1). Complex 1 exhibited a good quantum yield (0.32) while the value for complex 3 was very low (0.02), which indicates that an additional non-radiative pathway is in action for 3, while absent in 1. This additional non-radiative pathway does not seem to be intersystem crossing, since the proximity in the values of φ_Δ of 1 and 3 (Table 1) indicate close quantum yields of triplet formation for 1 and 3.

The emission properties of the complexes were also studied in aerated water (1% DMSO, 1.0 × 10⁻⁵ M) at 25 °C upon excitation at λ = 422–470 nm (Fig. 3, and Table S5†). In aqueous solution, complex 1 remains highly emissive, which makes it a good candidate as a bioimaging agent. However, complex 3 is hardly emissive. In both cases the emission bands undergo a red shift with respect to the results in acetonitrile, with a more pronounced displacement for complex 1 (λ_max = 632 nm). This may be related to the stated aggregation process¹⁰⁴ as confirmed in the following experiment.

The possible aggregation effect in the emission properties was evaluated in H₂O : DMSO mixtures with different water fractions (f_w) (Fig. S47†). As can be seen in Fig. S48,† both complexes showed an aggregation-caused quenching (ACQ) process. The effect is stronger in the case of 3, probably due to the larger π-conjugation of the C^N ligand. Interestingly, the quenching process is only partial in the case of 1. Concerning the position of the emission bands, the red-shifting is small in the case of complex 3 but for 1 there is a notable red-shifting upon aggregation.

The weak fluorescence of 3 may be due to its aggregation in aqueous media, driven by the greater hydrophobicity and stronger π–π interactions of the pbpn ligand. In addition, the expected reduction in spin–orbit coupling (SOC) in this compound (see below) favours fluorescence as the predominant radiative deactivation pathway.

Luminescence lifetimes. Time-correlated single-photon counting (TC-SPC) was performed to analyse the decay times associated with the luminescence. Iridium complexes are known to present multiple exponential decays due to the decay of singlet and triplet states at the same emission wavelength.¹⁰⁵ The multiexponential profiles of the luminescence decays are also observed in this case (Fig. S49†), but the decay times of complexes 1 and 3 present clear differences in time scale. 1 presents luminescence decays that are best fitted with a sum of two exponentials. The longer decay time, 440 ns, is responsible for 86% of the observed emission. 3 also presents luminescence decays that are best described as a sum of two exponentials. However, the longest decay time, 13.1 ns, which accounts for 75% of the luminescence, is much shorter (see below for the lifetimes obtained from TAS).

Transient absorption spectroscopy (TAS) measurements. Transient absorption spectra of both compounds measured at room temperature in acetonitrile solutions (Fig. S50†) between 280 and 600 nm display negative and positive optical density values yielding the same lifetime for both the negative regions (ground state depletion) and positive regions (transient state formation) of the transient absorption spectrum, which confirms that the disappearance of the transient state and recovery of the ground state are connected (Table 1). The optical density variations at both wavelengths decay to zero at longer times, indicating that no other species is being formed in significant amounts throughout the experiments (Fig. S51†), i.e., there is no evidence for photochemistry arising from the observed transients. Furthermore, the solutions were degassed by bubbling Argon until saturation (∼20 minutes), and the transients were once more acquired, yielding excited state lifetimes around one order of magnitude higher (Table 1). These results suggest that the observed transients are of triplet nature, which are strongly quenched in the presence of O₂. UV-vis spectra acquired before and after laser flash photolysis on both complexes are identical, confirming that there is no photochemistry occurring in these conditions (Fig. S52†).

The long luminescence lifetimes, similar to the triplet lifetimes observed in TAS, point to a contribution of delayed fluorescence in the fluorescence emission of 1. The luminescence lifetime values obtained for 3 are much shorter than the ones obtained from TAS and we conclude that the contribution of delayed fluorescence is thus considerably less in this compound.

TAS was also performed in aqueous solution (1% DMSO) of 1 (15 μM). Transients at selected wavelengths, namely 270 nm (for monitoring ground-state depletion) and 560 nm (for monitoring transient absorption) were acquired (Fig. S53†) and compared with the transients obtained in acetonitrile. It can be seen that the transients of 1 are greatly diminished in water, when compared with acetonitrile (around ten-fold decrease in the initial absorbance of the transient), while the lifetime of the transient is not significantly changed (Table 2). These findings indicate that the initial concentration of T₁ is greatly diminished in water, because either S₁ or higher triplet states are undergoing photochemistry that competes efficiently with the formation of T₁ in aqueous solution.

Table 2 Excited-state lifetimes and initial optical densities for 1 in water (1% DMSO) and acetonitrile, measured with laser flash photolysis

Solvent	Lifetime (ns)	OD (t = 0)
		270 nm	560 nm
Water (1% DMSO)	371	−0.0071	0.0031
Acetonitrile	367	−0.0727	0.0335

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ROS generation. The ability of Ir(III) complexes to generate ROS was investigated through four different methods: quenching of the triplet state by O₂, the 9,10-anthracenediyl-bis(methylene) malonic acid (ABDA) test for ¹O₂ production, the dihydrorhodamine 123 (DHR123) assay for O₂˙⁻ production and EPR spectroscopy with specific radical spin-traps.

The relationship between the triplet state quenching of Ir(III) complexes by O₂ bubbling and ROS formation is significant because it largely relies on energy and/or electron transfer processes, as has been previously described.^106–108 The result of these transfer processes is the generation of singlet oxygen and different oxygen radicals. As can be seen in Fig. S54,† in acetonitrile, the quenching of emission by O₂ is several times higher in complex 1 than in complex 3. In part, this is related to the triplet contribution to the emission (delayed fluorescence) of complex 1, which is much smaller in the case of complex 3. The significant decrease in the emission of complex 1 in the presence of oxygen confirms the quenching of its triplet state. Therefore, complex 1 is expected to be a good photosensitizer.

Singlet oxygen generation was studied using the ABDA assay in water (10% DMSO) to assess its performance under conditions more representative of those used in bioassays. In this experiment, ¹O₂ generation quantum yields (φ_Δ) were quantified monitoring the decrease in the ABDA absorbance by UV-vis spectroscopy with the irradiation for 3.5 minutes. The experiment was performed in dark conditions and under blue light irradiation (470 nm, 51.4 mW cm⁻²). [Ru(bpy)₃]²⁺ was used as reference (φ_Δ = 0.18 in water).¹⁰⁹ First, the stability of ABDA was confirmed under blue light irradiation (Fig. S55†). In the presence of the Ir(III) complexes and [Ru(bpy)₃]²⁺, a decrease in the absorption bands of the probe (360, 378 and 400 nm) over time was only observed upon blue light irradiation (Fig. S56–58†). In dark conditions, the spectrum of ABDA remained unaltered. The φ_Δ values of the two new complexes were calculated from the slopes obtained for ABDA consumption (Fig. S59†). Although there is not much difference, 1 exhibited the highest φ_Δ (0.59) while 3 exhibited a slightly lower value (0.49) (Table 1). This result indicates that the quantum yields for triplet formation and the efficiency of energy transfer to O₂ are comparable for both complexes. Despite the luminescence of complex 3 being less affected by the presence of O₂, it is still a good photosensitizer.

Generation of O₂˙⁻ was studied by the DHR123 assay in water (0.2% DMSO) monitoring the emission change of the probe by fluorescence spectroscopy with the irradiation for 30 seconds. The non-emissive DHR123 probe is oxidized by the photogenerated O₂˙⁻ into the fluorescent rhodamine 123, with a maximum emission at 526 nm. As for ¹O₂ studies, the experiment was carried out in dark conditions and upon blue light irradiation (470 nm, 51.4 mW cm⁻²) using [Ru(bpy)₃]²⁺ as reference. In dark conditions (Fig. S60†) or under blue light without any complex (Fig. S61†), no oxidation of the probe was detected. Upon blue light irradiation, with complexes 1, 3 or [Ru(bpy)₃]²⁺, O₂˙⁻ generation was confirmed by the increased emission of the oxidized rhodamine 123 (Fig. S62 and S63†). The relative rates are in order of 1 ≫ 3 > [Ru(bpy)₃]²⁺. Moreover, ascorbic acid and sodium azide were used as scavengers for O₂˙⁻ and ¹O₂, respectively, to determine their relative contributions to the oxidation of DHR123 (Fig. S64 and S65†). As expected, the oxidation of DHR123 was inhibited, and the increase in its fluorescent signal was significantly reduced in the presence of ascorbic acid, while sodium azide had no measurable effect. These results confirm that both complexes generate O₂˙⁻ under light irradiation in aqueous solution. Notably, the triplet state of complex 1 exhibits a greater ability to act as an O₂˙⁻ photosensitizer, in contrast to ¹O₂ photosensitization where the φ_Δ values were comparable for both compounds.

The generation of O₂˙⁻ for 1 was also verified in acetonitrile with the DHR123 assay (Fig. S66†). The superoxide production was higher in water than in acetonitrile, as is clearly seen in Fig. S67.† This is aligned with the TAS results, which showed that the transients of complex 1 were significantly reduced in water compared with acetonitrile (Table 2). The initial concentration of the lowest triplet state T₁ is greatly diminished in aqueous solution, which accounts for the reduced contribution of O₂˙⁻ to the oxidation of DHR123 in this solvent mixture. Moreover, this provides strong evidence for an additional photochemical process involving higher triplet states that compete with T₁ formation (see below), specifically leading to O₂˙⁻ generation. These results underscore complex 1 as a particularly promising PDT agent, as it implies a type I mechanism that, while enhanced by oxygen, can also function independently of it. This makes it especially useful against cells in hypoxic microenvironments.¹¹⁰

Finally, EPR experiments on 1 under dark and irradiated (365 nm) conditions were conducted in acetonitrile, in the presence of two radical specific spin-traps: 2,2,6,6-tetramethylpiperidine (TEMP) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In the case of TEMP, it reacts with ¹O₂ to yield 2,2,6,6-tetramethylpiperidine-N-oxide radical (TEMPO), characterized by a distinct three-line signal.^111–113 This radical is thermodynamically and kinetically stabilized and easily accumulates in solution. The signal was readily detected after irradiation (Fig. S68A†), supporting the presence of ¹O₂ in the sample. The reaction of O₂˙⁻ with the nitrone DMPO leads to the formation of the DMPO–OOH spin adduct, which gives rise to a characteristic EPR signal consisting of a quartet due to hyperfine splitting from the nitroxide nitrogen and the adjacent β-hydrogen.¹¹⁴ Contrary to TEMPO, this spin adduct radical presents a rapid decay due to spontaneous rearrangement and degradation. The short lifetime limits the detection window. As such, in situ irradiation was carried out, and the resulting EPR spectrum confirmed the presence of O₂˙⁻ in solution (Fig. S68B†).

2.4. Polymeric nanoparticles

We decided to explore the possibility of encapsulating the complexes into crosslinked polymeric nanoparticles with the aim of improving the cellular internalization and evaluating their behaviour as anticancer agents in PDT. The preparation of the nanoparticles involved two main steps: the synthesis of copolymers (Scheme S1†) and the crosslinked step to produce the nanoparticles via covalent bonds (Fig. 4).

Fig. 4 Schematic formation of the NPs presented in this work with Ir(III) complexes encapsulated.

Synthesis of copolymers. The copolymers used in this work were prepared by RAFT (Reversible Addition Fragmentation chain Transfer) polymerization, which was used on account of its features such as versatility, high compatibility with different solvents and monomers and experimental simplicity to obtain polymers with narrow M_w.¹¹⁵ Polyethyleneglycol (PEG)-based monomers were selected in order to obtain polymers with higher solubility in protic solvents. In addition, it protects against non-specific interactions with the proteins in blood, avoiding aggregation.¹¹⁶

The chain transfer agent (CTA) used in both polymerizations was 2-cyano-2-propyl dodecyl trithiocarbonate due to its suitability to acrylates and methacrylates. The polymers were purified by dialysis. Two polymers were obtained containing amine and carboxylic groups, which were used to generate cross-linked network via amide bonds (Scheme S1†). Random copolymer P1 was obtained via copolymerization of poly(ethyleneglycol) methyl ether methacrylate (PEGMA) and 2-aminoethyl methacrylate, which was used with the BOC protecting group as monomer (BOC-aminoethylMA). This monomer was prepared according to the literature.¹¹⁷ In a second step, the protecting group was removed with TFA (trifluoroacetic acid), to obtain P1b with the amine groups. Random copolymer P2 was obtained via copolymerization of PEGMA and 2-carboxyethyl acrylate (CEA) (Scheme S1†). The conversion of the polymerizations was calculated by ¹H NMR using trioxane as standard and was found to be around 87 and 90% for P1 and P2, respectively (Table S6†). The three polymers were characterized by ¹H NMR (Fig. S69–S71†).

Synthesis and characterization of nanoparticles. Crosslink experiments were performed between copolymers P1b and P2 in the presence of the coupling agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to yield polymeric nanoparticles via covalent amide bond formation, as a result of coupling of amine and carboxylic groups (Fig. 4). Equimolar dilute solutions of copolymers were mixed in water, and the corresponding amount of coupling agent was added dropwise while stirring. It was expected that these nanoparticles could encapsulate drug payloads, and thus, encapsulate the Ir complexes. A similar procedure was performed to obtain the nanoparticles in the presence of the complexes (Table S7†).

The nanoparticles were characterised by DLS, affording hydrodynamic diameters between 150–200 nm with a good polydispersity index (PDI) (Table S7 and Fig. S72†). This structure provides a stable aqueous suspension of the nanoparticles with good biological properties, such as long circulation in the bloodstream and specific accumulation in the acidic tumour microenvironment. The size of the nanoparticle without encapsulated drug was found to be 146 nm, showing that the size of the nanoparticle does not change significantly with the encapsulation of drugs. SEM images of the nanoparticles showed their spherical morphology (Fig. S73†).

Nanoparticles NP1 and NP3 (containing compound 1 or 3, respectively) were analysed by UV-vis spectroscopy (Fig. S74†) in water. The spectra are relatively similar to those of complexes 1 and 3 in aqueous solution. The encapsulation of the drugs into the nanoparticles improved the solubility of the complexes but did not affect the UV-vis absorption spectra. The emission properties of the nanoparticles were also studied in water at 25 °C upon excitation at λ_exc = 422–470 nm. As observed for the free complex, NP1 showed a highly emissive behaviour, which makes it a good candidate for a bioimaging agent (Fig. S75†). However, similar to complex 3, NP3 was hardly emissive. This confirmed that encapsulation of complexes did not affect their photophysical properties.

The amount of complex encapsulated in each nanoparticle was confirmed by ICP-MS, with values of drug loading of 23.319 and 27.560 μM for NP1 and NP3, respectively (Table S7†). These values of drug loading could be considered low compared with other nanocarriers of Ir complexes,⁷⁴ possibly on account of the high lipophilicity of the Ir(III) complexes and the absence of any surfactant in the synthesis method. The drug uploading values of these nanoparticles were 5.8 and 7.7%, respectively. Despite the low Ir content, we decided to evaluate these nanoparticles and their effect on cytotoxicity and cellular uptake.

2.5. Theoretical calculations

In order to gain a better understanding of the significant differences in the photophysical properties of the complexes 1 and 3, time-dependent density functional theory (TD-DFT) calculations were performed for both the singlet and triplet excited states of these compounds. A comprehensive method for comparing the X-ray crystallography and theoretical geometries involves superimposing the two structures and calculating the root-mean-square deviation (RMSD) of atomic positions (Fig. S76†). For compound 3, the X-ray structure was not obtained. The corresponding RMSD value corresponding to the overlap for those structures of 1 is 0.131 Å. This indicates that the experimental and the obtained theoretical geometries are quite similar. No significant differences were observed in the length values of both structures. The primary difference arises from slight distinctions in the orientation of the planes of the aromatic fragments, which are influenced by the crystal packing effect in the structure determined by X-ray diffraction but not in the obtained theoretical structures.

The corresponding fragmental contributions of each molecular fragment to the molecular orbitals (Fig. S79†) corresponding to complexes 1 and 3 were obtained by a Milliken population analysis (Table 3 and Fig. 5). The energy of the HOMO orbitals is comparable between the two compounds because their main contributions are similarly derived from the atomic orbitals of the iridium atom (45%), and the benzene fragment (38% and 40%) as well as the pyridine moiety (9% and 8%) in the pbpz and pbpn ligands, respectively. It is important to highlight that for 3, both HOMO−1 and HOMO−2 orbitals are almost isoenergetic with the HOMO orbital. Notably, the majority of their contribution (88%) originates from the benzo-quinoxaline moiety, and these orbitals are characterized by their π nature. The formation of a near-HOMO orbital centred on the benzo-quinoxaline fragment due to the introduction of the pbpn ligand has also been observed in our previous work with [Cp*Ir(C^N)L]⁺ complexes.⁴² In the case of compound 3, the introduction of two pbpn ligands results in the presence of two near-HOMO orbitals of this nature. The nearly isoenergetic character of these orbitals indicates that the electrons in the HOMO orbital can be delocalized across these three orbitals, thereby enhancing their delocalization over the π system of the ligand and further contributing to its stability. Interestingly, such type of orbital is missing in compound 1, where the HOMO−1 closely resembles and shares energy equivalence with the HOMO−3 of compound 3, with a major contribution, in both compounds, of fused benzene and minor contributions of pyridine and quinoxaline(1)/benzo-quinoxaline(3) moieties. Regarding the LUMO and LUMO+1, they are predominantly isoenergetic in both compounds, with their primary locations being on the benzo-quinoxaline fragment in compound 3, resembling HOMO−1 and HOMO−2 orbitals, and on the quinoxaline moiety in compound 1. Besides, the addition of an extra fused benzene ring in 3 has the effect of lowering the LUMO level by extending the π-conjugation system, which leads to greater delocalization of the LUMO and LUMO+1. This extended conjugation leads to a greater delocalization of the π and π* orbitals, resulting in a narrowing of the energy gaps between the frontier molecular orbitals. This extension results in a red shift in the absorption compared with compound 1.

Fig. 5 Optimized ground state structures for 1 (left) and 3 (right) obtained from TD-DFT [(B3LYP/SDD for Ir(III)) and (6-31g** for C,H,N)] with SMD (CH₃CN). For the sake of clarity, the hydrogen atoms are not shown, and each molecular fragment has been assigned a colour code. The length of each colour bar is proportional to the percentage contribution of the corresponding coloured moiety to each molecular orbital. Colour codes for bars and molecular fragments: iridium atom (green), quinoxaline or benzo-quinoxaline (pink), fused benzene (navy blue), pyridine (blue), bipyridine (brown).

Table 3 Energies in electron volts (E/eV) and main fragmental contributions (percentage values within brackets) to some molecular orbitals of 1 and 3 obtained at the TD-DFT (SMD, acetonitrile)/6-31G(d,p)//SDD level

Orbital	E/eV		1	3
	1	3	Fragmental contributions (%)	Fragmental contributions (%)
Ir: iridium, Q: quinoxaline, Bq: benzo-quinoxaline, Bz: fused benzene, Py: pyridine and Bpy: bipyridine.
LUMO+5	−1.27	−1.29	Bpy (52) Py (15) Q (14) Bz (13) Ir (5)	Bpy (53) Py (17) Bq (13) Bz (11)
LUMO+4	−1.80	−1.78	Py (60) Bz (17) Q (17) Ir (5)	Py (62) Bz (16) Bq (15) Ir (6)
LUMO+3	−1.83	−1.81	Py (63) Bz (17) Q (13)	Py (64) Bz (17) Bq (12)
LUMO+2	−2.17	−2.17	Bpy (93)	Bpy (93)
LUMO+1	−2.41	−2.69	Q (76) Py (16) Bz (8)	Bq (80) Py (12) Bz (7)
LUMO	−2.47	−2.73	Q (73) Py (16) Bz (10)	Bq (77) Py (13) Bz (9)
HOMO	−5.59	−5.60	Ir (45) Bz (40) Py (8)	Ir (45) Bz (38) Py (9) Bq (6)
HOMO−1	−6.17	−5.65	Bz (57) Py (22) Q (17)	Bq (88) Bz (7)
HOMO−2	−6.22	−5.65	Ir (32) Bz (27) Q (25) Py (13)	Bq (88) Bz (7)
HOMO−3	−6.25	−6.19	Q (34) Bz (31) Ir (22) Py (11)	Bz (58) Bq (23) Py (15)
HOMO−4	−6.39	−6.29	Ir (44) Q (29) Py (14) Bz (9)	Ir (58) Py (14) Bz (12) Bq (10) Bpy (6)
HOMO−5	−6.45	−6.35	Ir (52) Q (24) Py (12) Bz (7) Bpy (6)	Ir (56) Py (17) Bz (14) Bq (8) Bpy (6)

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To gain a better understanding about the behaviour of the studied compounds in their electronic excited states, the vertical excitation energies were calculated along with the orbitals involved in these excitations and their relative contributions to singlet and triplet excited states (Table 4). In compound 3 the difference in energy between S₀ → S_1–4 was ≤ 0.07 eV. This indicates that the excitations to these singlet excited states are very close in energy due to excitations involving multiple closely spaced occupied orbitals (HOMO, HOMO−1, and HOMO−2) to a pair of nearly degenerate unoccupied orbitals (LUMO and LUMO+1), as shown in Table 4. The transitions S₀ → S₁, S₀ → S₂, and S₀ → S₄ exhibit a combination of metal-to-ligand charge transfer (MLCT), ligand-to-ligand charge transfer (LLCT), and ligand-centred (LC) characteristics. Conversely, the transition S₀ → S₃ is primarily characterized as ligand-centred (LC). The close energy spacing between these excited states enhances the probability of internal conversion (IC), as the excitation energy can be efficiently transferred to vibrational modes and it could lead to strong vibronic coupling between states, giving a broad absorption spectrum where the electronic transitions can borrow intensity from each other.^118,119

Table 4 Vertical excitation energies (eV) of S₁–S₄, T₁–T₄ states and percentages (values in brackets) of dominant contributions to the calculated transitions for 1 and 3 obtained at the TD-DFT (SMD, acetonitrile)/6-31G(d,p)//SDD level

State	Energy/eV		Electronic structure
	1	3	1	3
S1	2.61	2.38	d/πH → π*L (98) (MLCT/LLCT)	d/πH → π*L (95), πH−2 → π*L (4) (MLCT/LC)
S2	2.67	2.42	d/πH → π*L+1 (98) (MLCT/LLCT)	d/πH → π*L+1 (48), πH−1 → π*L (35), πH−2 → π*L+1 (16) (MLCT/LC)
S3	2.74	2.43	d/πH → π*L+2 (97) (MLCT/LLCT)	πH−2 → π*L (58), πH−1 → π*L+1 (39) (LC)
S4	3.06	2.45	d/πH → π*L+3 (94) (MLCT/LLCT)	d/πH → π*L+1 (48), πH−2 → π*L+1 (27), πH−1 → π*L (23) (MLCT/LC)
T1	2.31	1.50	π/dH−3 → π*L (23), dH−4 → πL+1 (19), π/dH−2 → πL+1 (17), dH−5 → πL (13) (MLCT/LLCT)	πH−1 → π*L (48), πH−2 → π*L+1 (44) (LC)
T2	2.45	1.50	π/dH−3 → π*L+1 (21), dH−4 → πL (20), π/dH−2 → πL (20), dH−5 → πL+1 (13) (MLCT/LLCT)	πH−2 → π*L (46), πH−1 → π*L+1 (46) (LC)
T3	2.41	2.22	dH → π*L+1 (73), πH−1 → π*L (13), πH−7 → π*L (4) (MLCT/LLCT)	d/πH → π*L (66), πH−3 → π*L+1 (10) (MLCT/LLCT)
T4	2.48	2.26	dH → π*L (79), πH−1 → π*L+1 (9), π/dH−3 → π*L (4) (MLCT/LLCT)	d/πH → π*L+1 (77), πH−3 → π*L (14) (MLCT/LLCT)

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As far as compound 1 is concerned, the electronic states S₁–S₄ exhibit mixed MLCT/LLCT characteristics. This arises as each state is formed by excitations from HOMO to successive higher-energy unoccupied orbitals. Specifically, S₁ involves excitation to LUMO, S₂ to LUMO+1, S₃ to LUMO+2, and S₄ to LUMO+3. Additionally, these unoccupied orbitals predominantly receive contributions from various aromatic fragments within the molecule (Fig. 5 and Table 4). Therefore, we can observe that the absence of transitions of LC nature in compound 1 causes the excited states of this compound to have a greater MLCT character than those of compound 3. This enhances the ‘heavy atom effect’ in 1, which is expected to amplify its spin orbit coupling (SOC) and intersystem crossing (ISC) rates.

The excitation energies for the T₁ and T₂ states of 3, at 1.50 eV, are notably low. These energies primarily arise from electron excitations moving from HOMO−1 and HOMO−2 to LUMO and LUMO+1, which are mainly centred on the benzo-quinoxaline moiety as mentioned above (Fig. 5 and Table 4). Aromatic ligands can better delocalize the unpaired electrons in the triplet state, resulting in greater stabilization of the excited state. This stabilization leads to the small excitation energy observed in these states with a ³LC/³ππ* nature. Regarding T₃ and T₄, they showed a MLCT/LLCT character which involves transitions from HOMO and HOMO−3 to LUMO and LUMO+1, which implicates charge transfers from the metal, fused benzene and pyridine moieties to the benzo-quinoxaline fragment. These states are much closer in energy to S₁ (¹MLCT/¹dπ*) and S₂ (¹LC/¹ππ*) states than T₁ and T₂ states (³LC/³ππ*). Regarding 1, the excitation energies for T₁–T₄ states are closely matched, exhibiting MLCT/LLCT characteristics. These states exhibit a substantial multiconfigurational nature due to excitations across multiple orbitals. This contrasts with the singlet excited states, which only involve single excitations (Table 4).

The strength of SOC is influenced by the energy gap between adiabatic excitation energies, which encompass the coupling between electronic and nuclear motions. Therefore, we calculated these adiabatic excitation energies in electron volts (eV) for the optimized excited states (Fig. 6 and Table S8†). For compound 3, the substantial energy gap between the S₁ and T₁/T₂ states (1.56 eV) significantly restricts intersystem crossing (ISC) between these states, consistent with the Fermi golden rule.¹²⁰ Moreover, the almost complete absence of d orbitals in the metal configurations of the T₁/T₂ states due to their ³ππ* character contributes to weaker SOC, further inhibiting ISC between these states. In contrast, the smaller energy gaps between the S₁ and T₃/T₄ states (0.160 eV and 0.07 eV, respectively) are expected to promote ISC. Additionally, the adiabatic excitation energies of the T₃ and T₄ states (2.074 eV and 2.160 eV, respectively) are relatively close to the vertical excitation energies of the T₁ and T₂ states (1.50 eV). This proximity suggests potential overlap of vibrational levels, which could facilitate internal conversion (IC) as a relaxation pathway from higher triplet states through vibronic coupling mechanisms. The small energy gap observed between the adiabatic energies of S₀ and T₁/T₂ states (0.69 eV) suggests a non-emissive character for these states, along with a long-lived nature due to their ππ* configuration.

Fig. 6 Proposed Jablonski diagram for 1 and 3 and their interaction with molecular oxygen. Triplet excited states can undergo electron transfer generating O₂˙⁻ or energy transfer to molecular oxygen producing ¹O₂.

The triplet–triplet energy transfer process is efficient when the triplet state energy exceeds the energy required to excite molecular ³O₂ to ¹O₂ (approximately 0.98 eV or 1270 nm). Although this calculated energy gap is below the threshold required for direct ¹O₂ generation, it is important to consider that TD-DFT methodology is well known to underestimate the energy of ππ* singlet and triplet excitations states, which suggests the energy for T₁/T₂ state is likely too low.^121,122 Indeed, several studies have found systematic “red-shifts” in TD-DFT/B3LYP predicted triplet energies for states both large aromatic chromophores and organometallic complexes.^123,124 Consequently, the actual adiabatic T₁/T₂ energy in our system, which is not far from 0.98 eV, is plausibly higher than the TD-DFT result, likely in the range necessary to achieve ¹O₂ sensitization. This alignment with the observed ¹O₂ generation supports the photophysical conclusions of this study. Additionally, the long lifetime of this state may favour direct electron transfer from the triplet state to molecular oxygen, leading to the formation of O₂˙⁻, either through direct interaction or via an intermediate electron donor or substrate.¹²⁵

In compound 1, similar energy gaps were observed between the S₁ and T₃/T₄ states, specifically 0.178 eV and 0.082 eV, respectively, as compared with 3. The absence of isoenergetic π orbitals (HOMO−1 and HOMO−2) relative to the HOMO increases the involvement of metal-centred orbitals in the transitions of these singlet excited states. Consequently, as stated, spin–orbit coupling (SOC) is expected to be more effective in 1 than in 3. Additionally, differences in the orbital geometries involved in these singlet and triplet excited states (see Table 4) further enhance SOC, in accordance with El Sayed's rule.¹²⁶

Moreover, a key difference observed in 1 compared with 3 is the significantly smaller energy gaps between the S₁ and T₁/T₂ states (approximately 0.414 eV). These observations suggest the presence of two possible competing ISC channels. The first is a thermally activated ISC between S₁ and T₂/T₃ states. Due to the very small energy gaps and the different orbital geometries, the SOC will be very efficient, leading to rapid ISC. IC from T₂/T₃ to T₁/T₂ is also fast, quickly populating the lowest excited triplet states. The second channel is a slow ISC between S₁ and T₁/T₂, as the orbitals of S₁ and T₁/T₂ are almost identical, reducing the SOC rate, and the energy gap between them is significantly larger. Furthermore, the small energy gaps between T₁ and T₄ states cause the vibrational levels of the higher triplet states to overlap with those of the lower triplet states, facilitating vibronic coupling and enabling reverse internal conversion (IC).¹²⁷ This overlap increases the probability of rapid electron exchange among the T₁–T₄ states, allowing for a large number of electron configurations and enhancing the return to the singlet manifold via reverse intersystem crossing (RISC).¹²⁸ Therefore, the expected larger SOC of 1, due to the greater MLCT character of the S₁ state and the multiconfigurational character of the T₁–T₄ states, as well as the existence of several channels to produce ISC in this compound, suggests the existence of a more efficient population of triplet states where the electron can undergo rapid exchange between these states due to their close energy levels. This spreads the population over various triplet states, prolonging the overall triplet-state lifetime. The longer lifetime of the triplet states in 1, compared with 3, should contribute to an enhanced production of ROS from the former compound. Further experiments on the emission profile of 1 in acetonitrile were also performed, both at room temperature and at 77 K. These spectra allowed us to effectively determine the energy gap through the difference of the energy of the onset from the emission bands at RT and 77 K (Fig. S80†). These results are consistent with TD-DFT calculations, as well as the observed delayed fluorescence and the higher values obtained in the case of 1 for the quantum yields of emission and ROS generation.

2.6 Biological studies

Anticancer activity. The phototoxic activity of complexes 1 and 3 against different cancer cells was first evaluated using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assays under dark and light conditions. Under the experimental conditions, light exposure was confirmed to exert a minimal effect on cell viability. However, to ensure accurate assessment of cytotoxicity, the viability of cells subjected to light or dark treatments was analyzed relative to untreated control cells that were exposed to the same irradiation conditions as the treated cells. As summarized in Table 5, after 48 h of treatment in the dark, both complexes exhibited intrinsic cytotoxicity, with IC_50,dark values ranging from 0.89 to 3.65 μM. These values were comparable to that of the chemotherapeutic agent cisplatin. In all cases, complex 1 displayed moderately higher cytotoxicity than complex 3. Notably, upon photoactivation with blue light (460 nm, 24.1 J cm⁻²), the activity of both complexes significantly increased, resulting in IC_50,light values in the nanomolar range and phototoxic indexes (PIs) between 11 and 32 for complex 1 and from 6 to 124 for complex 3. Again, complex 1 exhibited higher photocytotoxicity than complex 3, except in A549 lung cancer cells, where complex 3 displayed an outstanding photodynamic activity, resulting in a PI of 124 and an IC_50,light of 0.016 μM. Given the favorable photodynamic behavior of the complexes, their photocytotoxic activity was also examined in A549 cells after activation with green (515 nm) and red (635 nm) light, which possess deeper tissue penetration properties.¹²⁹ Following green light irradiation, IC_50,light values of 0.18 ± 0.03 μM were obtained for complex 1 and 0.204 ± 0.01 μM for complex 3, representing PI values of 5 and 11. However, irradiation with red light only produced a 2-fold increase in the cytotoxicity of complex 1 and a 4-fold increase in the cytotoxicity of complex 3 compared with treatments in dark conditions, respectively, with IC_50,light values of 0.49 ± 0.1 μM and 0.502 ± 0.04 μM. These results confirm that optimal photoactivation for both complexes is achieved with blue light irradiation, which agrees with their absorption spectra. However, green light could also induce a significant phototoxic response. These results can be compared with those obtained with analogous Ir complexes with bpy and C^N ligands with less π-extension using MCF-7 breast cancer cells. With ppy, there was no increase in cytotoxicity by light activation.¹⁰⁸ With C^N ligands with moderate π-expansion as 2-phenylquinolinate (2pq), and 1-phenylisoquinolinate (1pq), the obtained PI values (up to 5.9) were lower and the IC_50,light values (0.83 or 0.63 μM) were 8 or 6 times higher than those obtained with our compounds, reflecting the beneficial effect of the π-expansion of our ligands.¹⁰⁸

Table 5 Photocytotoxic activity of the complexes in 2D cell cultures

Complex	1			3			Cisplatin
	IC50 (μM)		PI	IC50 (μM)		PI	IC50 (μM)
	Dark	Light		Dark	Light		Dark
Cells were treated with the compounds for 4 h at 37 °C to ensure their maximum internalization and then kept in the dark or irradiated with blue light for 1 h (460 nm, 24.1 J cm^−2). Cell viability was assessed 43 h later by MTT assays. Data represent the mean ± SD of at least three independent experiments, each performed in triplicate. PI: phototoxicity index = IC50,dark/IC50,light.
A549	0.890 ± 0.204	0.028 ± 0.019	32	2.028 ± 0.784	0.016 ± 0.003	124	5.998 ± 1.185
HeLa	1.048 ± 0.253	0.063 ± 0.014	17	2.515 ± 0.173	0.252 ± 0.059	10	1.528 ± 0.326
MCF-7	1.142 ± 0.164	0.100 ± 0.024	11	3.310 ± 0.093	0.535 ± 0.059	6	5.750 ± 0.070
BxPC3	0.984 ± 0.062	0.078 ± 0.018	13	3.652 ± 0.261	0.140 ± 0.055	26	1.008 ± 0.009
MRC-5	2.587 ± 0.199	0.125 ± 0.032	21	3.067 ± 0.409	0.292 ± 0.008	10	5.327 ± 0.377

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It should be noted that, in general, the complexes exerted comparable cytotoxicity against the cancer cell lines and non-malignant MRC-5 fibroblasts in dark conditions, revealing a lack of intrinsic selectivity towards malignant cells. However, a remarkable photoselectivity was observed when comparing the effects of the photoactivated complexes on A549 lung cancer cells to the dark toxicity against MRC-5 lung fibroblasts. Upon irradiation, the IC_50,light values of complexes 1 and 3 in A549 cells were 92.4 and 191.7 times lower, respectively, than the IC_50,dark values determined in MRC-5 cells. These findings show the potential of these complexes to exert a selective light-activated action against cancer cells with minimal effects on the non-irradiated healthy tissue.

To further assess the toxicity of the complexes, hemolytic experiments were conducted.¹³⁰ Porcine red blood cells (RBC) were incubated with 1 and 3 in the dark or under blue light irradiation at concentrations close to the IC_50,dark (1 μM) or IC_50,light (0.2 μM and 0.02 μM). Hemoglobin release was measured as an indicator of the capacity of the complexes to destabilize RBC membranes. Both complexes exhibited minimal hemolytic activity (≤5% hemolysis) under these conditions, indicating a favorable compatibility with blood cells in the context of potential intravenous administration (Table S9†).

The anticancer activity of 1 and 3 was further evaluated using clonogenic assays, which better assess the long-term survival and proliferative potential of cells following the treatment.¹³¹ HeLa cells were selected for this study due to their superior ability to form colonies in vitro. Cells were exposed to complexes 1 and 3 at the respective IC_50,light values for 4 h, followed by incubation in dark conditions or under blue light irradiation. Ten days later, the number of colonies formed in the absence of light irradiation was similar to that of the corresponding untreated control, indicating the absence of a cytotoxic effect. In contrast, photoactivation of the complexes significantly reduced colony numbers to 41.0 ± 4.9% for complex 1 and 52.2 ± 2.9% for complex 3 (Fig. 7). No significant differences were observed between dark and light conditions for both untreated cells and cells treated with cisplatin, consistent with the non-photoactivatable nature of cisplatin. These results support the light-dependent anticancer activity of the complexes at the IC_50,light values determined in MTT assays. Furthermore, they demonstrated the capability of the complexes to inhibit the clonogenic potential of the cells, a critical factor in preventing metastasis, as proliferation in distant tissues is essential for the formation of secondary tumors.

Fig. 7 Clonogenic assays. (A) Images of the colonies generated after exposure of HeLa cells to complexes 1 and 3 at the corresponding IC_50,light, or medium alone as a control, both in the dark or with blue light irradiation (1 h, 460 nm, 24.1 J cm⁻²). Cisplatin at 5 μM was used as the positive control. (B) Bar charts represent the percentage of colonies after each treatment relative to the control cells (mean ± SD of three experiments). *** p < 0.001.

Intracellular ROS generation. To elucidate the mechanism underlying the photocytotoxicity of the complexes, their capacity to generate ROS at the cellular level was investigated. This is a primary characteristic for PSs to be effective in photodynamic therapy (PDT). The cellular ROS levels were quantified using the dichlorodihydrofluorescein diacetate (H2DCFDA) probe, which is converted to the green fluorescent derivative dichlorofluorescein (DCF) upon oxidation. A549 and HeLa cells were treated with 1 and 3 at their respective IC_50,light and changes in cellular fluorescence were monitored by flow cytometry. As shown in Fig. 8A, no significant differences in cell fluorescence were observed in the absence of light irradiation compared with untreated control cells. However, blue light irradiation induced a marked increase in the green fluorescent signal, indicating ROS generation within the cells. Specifically, treatment with complex 1 resulted in a 10.4 ± 0.7-fold increase in fluorescence intensity in A549 cells and an 8.51 ± 2.8-fold increase in HeLa cells. Complex 3 also induced a strong fluorescence rise, with a 14.61 ± 0.9-fold increase in A549 cells and a 16.95 ± 3.5-fold increase in HeLa cells. As was demonstrated, these complexes were able to generate singlet oxygen under light irradiation (Fig. S56 and S57†). This type II reaction is the primary cytotoxic mechanism for most PSs in PDT.¹³² Furthermore, by the DHR123 test, it was shown that the complexes showed the ability to produce superoxide anions (O₂˙⁻) (Fig. S62†) and, thus, undergo type I PDT processes. O₂˙⁻ is a transient but highly reactive species that originates oxidative cascades, causing substantial and potentially irreversible damage to cellular components.¹³³ Consequently, the contribution of this radical to the photocytotoxic activity of the complexes was investigated. Generation of O₂˙⁻ was quantified using the Superoxide Detection Reagent (Enzo Life Sciences), which emits an orange signal upon interaction with this radical. As expected, no significant differences in fluorescence emission were observed when treatments were conducted in dark conditions compared with control cells. Conversely, treatment with photoactivated complex 1 resulted in a 3.57 ± 0.78-fold and 4.49 ± 1.38-fold increase in fluorescence emission in A549 and HeLa cells, respectively. Similarly, complex 3 induced a 3.14 ± 0.52-fold and 4.73 ± 1.26-fold increase in fluorescence in A549 and HeLa cells (Fig. 8B), confirming O₂˙⁻ generation within cells, with a similar activity for both complexes. It should be noted that these results differed from those previously obtained with the DHR123 test, where a higher superoxide anion production was observed for complex 1. This discrepancy may be attributed to the detection of endogenous mitochondrial superoxide radicals. Mitochondrial electron transport chain (ETC) leakage is a well-established source of superoxide radicals.¹³⁴ Thus, the activity of complexes 1 and 3 in mitochondria could potentially disrupt ECT which may result in further intrinsic O₂˙⁻ generation. Finally, it should be noted that no clear correlation was observed between the ROS levels and the PI of complexes 1 and 3 in A549 and HeLa cells, which suggests that additional factors may contribute to the photocytotoxic activity of these complexes.

Fig. 8 Intracellular ROS generation and effect of specific ROS on cell viability. A549 and HeLa cells were treated with complexes 1 and 3 at their respective IC_50,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⁻²). The elevation of general ROS (A) and superoxide anion (B) levels were determined with specific probes and cell fluorescence was measured by flow cytometry. Bars represent the mean fold increase (±standard deviation) relative to untreated control cells from three independent experiments. *p < 0.05; **p < 0.01 versus non-irradiated cells. (C) HeLa cells were treated at their IC_50,light and exposed to blue light irradiation either without (∅) or with specific ROS scavengers (DMSO for ˙OH, sodium azide for ¹O₂, or tiron for O₂˙⁻). Bars indicate the mean percentage of viable cells (±standard deviation) 48 hours post-treatment relative to untreated control cells incubated with medium alone or with the corresponding scavenger. * p < 0.05; ** p < 0.01 compared with treated cells without scavengers. Each condition was tested in triplicate across three independent experiments.

Finally, to validate the contribution of individual ROS to the photocytotoxic activity of the complexes, HeLa cells were incubated with complexes 1 and 3 at their respective IC_50,light and irradiated with blue light in the presence of specific ROS scavengers (dimethyl sulfoxide (DMSO) for ˙OH, sodium azide for ¹O₂ or tiron for O₂˙⁻) or with medium alone as a control. Cell viability was determined 48 h post-treatment. As shown in Fig. 8C, the percentage of viable cells significantly increased when treatments were conducted in the presence of sodium azide and tiron, confirming the involvement of ¹O₂ and O₂˙⁻ in the photocytotoxic mechanism of both complexes. Furthermore, the cytotoxic effect of complex 1 was also inhibited by DMSO, suggesting a contribution from ˙OH.

These results collectively demonstrate that type II photochemical processes play a predominant role in the photocytotoxicity of these complexes. Furthermore, the involvement of O₂˙⁻ and ˙OH supports the contribution of type I mechanisms, which exhibit reduced dependence on molecular oxygen. Thus, these complexes hold potential for treating cancer cells in hypoxic microenvironment, where resistance to conventional photodynamic therapy is often observed.

Mitochondrial damage. Cationic iridium-based complexes bearing lipophilic ligands have a high propensity to accumulate in the mitochondria, driven by the negative potential across the inner mitochondrial membrane. The lipophilicity of our complexes was quantified by determining their octanol/water partition coefficients (log P_o/w) using the traditional shake-flask method. Complexes 1 and 3 displayed log P_o/w values of 1.15 ± 0.27 and 1.58 ± 0.23, respectively, indicating their lipophilic character and a high probability of mitochondrial localization.⁵² This cellular distribution is particularly significant because mitochondria are involved in essential cellular functions, including the production of adenosine triphosphate (ATP), the regulation of apoptosis, and the maintenance of the redox homeostasis. The disruption of these processes ultimately results in cell death, thereby establishing mitochondria as a primary therapeutic target in cancer. As described below (Fig. 14), confocal microscopy experiments with complex 1 confirmed a high degree of co-localization of the complex with mitochondria.

Thus, the effect of the complexes on mitochondrial function was investigated as a potential cytotoxic mechanism. HeLa cells were treated with complexes 1 and 3 under dark and light conditions and mitochondrial integrity was assessed by confocal microscopy using the fluorescent dye MitoTracker™ Red CMXRos, which accumulates within healthy mitochondria based on the mitochondrial membrane potential (MMP).¹³⁵ As shown in Fig. 9A, intense red mitochondrial staining was detected in both control cells and cells treated in the dark. However, upon photoactivation, the staining was significantly attenuated, revealing mitochondrial damage. To corroborate these results, changes in the MMP were assessed using the JC-1 dye, which emits red fluorescence when accumulated in healthy mitochondria and green fluorescence when the MMP is dissipated. As shown in Fig. 9B, 94.49% of untreated control cells exhibited red fluorescence emission from JC-1. This percentage was similar for cells treated with complexes 1 (91.64%) and 3 (88.42%) in the dark, while it decreased to 44.04% for complex 1 and 43.16% for complex 3 upon blue light irradiation. Overall, these results corroborated the activity of the complexes at the mitochondrial level.

Fig. 9 Effect on mitochondria. HeLa cells were incubated with complexes 1 and 3 at the corresponding IC_50,light for 4 h at 37 °C and then kept in darkness or exposed to blue light for 1 h. (A) Confocal microscopy images of the cells. Cell nuclei were localized in blue with Hoescht (λ_ex: 400 nm; λ_em: 450 nm), and healthy mitochondria were labelled with MitoTracker™ Red CMXRos (λ_ex: 543 nm; λ_em: 595 nm). Attenuation of red fluorescence emission upon irradiation indicates mitochondrial dysfunction. (B) Percentage of cells exhibiting green and red JC-1 fluorescence after treatment in dark or irradiated conditions (mean ± SD of three independent experiments). Cells incubated with medium alone were used as control. Loss of MMP was determined by a decrease in the percentage of red fluorescent cells. * p < 0.05; ** p < 0.01.

Photocatalytic oxidation of NADH. Nicotinamide adenine dinucleotide (NAD) is a crucial coenzyme in cellular metabolism that acts as an electron carrier in redox reactions.¹³⁶ NADH, which is the reduced form of NAD, transfers electrons to the mitochondrial ETC to generate ATP. This crucial role in the cell energy generation has made NADH a promising target in anticancer therapy.^{107,137–141} The oxidation of NADH by anticancer agents disrupts ATP generation and contributes to mitochondrial dysfunction. In recent years, several papers have been published on the photocatalytic oxidation of NADH by Ir(III) complexes via a single-electron transfer (SET) mechanism with generation of O₂˙⁻ and carbon-center radicals.^107,137 This suggests an additional potential mechanism of action for Ir(III) complexes in type I PDT, which we have investigated for our complexes.

The evolution of NADH in the presence of complexes 1 and 3 (complex/NADH ratio = 1/100) was monitored by UV–vis spectroscopy in aqueous solution over a period of 10 minutes at room temperature. In order to evaluate the light effect in the oxidation reaction, the experiment was carried out in dark conditions and upon blue light irradiation (470 nm, 51.4 mW cm⁻²). The evolution of the photocatalytic reaction was monitored by the decrease of the absorption band at 340 nm (Fig. S81–S83†). In dark conditions, a very low level of NADH oxidation was observed. However, the reaction under light irradiation was very fast for both complexes. The reaction proceeded with first-order kinetics with respect to NADH. The TONs (Turnover Numbers, measured after 5 minutes) of the complexes were up to 46 (Fig. S83 and Table S10†) and TOFs (Turnover Frequencies) were up to 555 h⁻¹, which make complexes 1 and 3 among the most active NADH photocatalysts.^107,138 As has been reported for other Ir(III) photosensitizers, H₂O₂ was detected (test strips) in the photocatalytic NADH oxidation with 1 and 3,¹⁰⁷ which indicates the generation of O₂˙⁻ and the conversion to H₂O₂.

Cell death mechanism. The depolarization of the mitochondrial membrane is a critical event that can initiate programmed cell death via apoptosis. To assess the apoptotic potential of complexes 1 and 3, flow cytometry experiments were conducted using annexin V-FITC and propidium iodide (PrI) staining. Viable cells are impermeable to both dyes, whereas early and late apoptotic cells exhibit annexin V binding due to phosphatidylserine exposure on the cell surface. In addition, late-apoptotic and necrotic cells are PrI-positive, due to the loss of membrane integrity. Treatments with cisplatin were included as a positive control for apoptosis. As shown in Fig. 10, treatments with photoactivated complexes 1 and 3 at the corresponding IC_50,light led to a significant increase in both the early and late apoptotic populations to 10.98 ± 2.03% and 6.26 ± 3.01%, respectively, for complex 1, and to 17.64 ± 10.48% and 6.63 ± 4.66% for complex 3. Treatments using five times the IC_50,light resulted in higher apoptotic rates, with early and late apoptotic populations, respectively, reaching 21.46 ± 15.31% and 35.78 ± 15.37%, for complex 1 and 21.48 ± 15.55% and 28.91 ± 20.54% for complex 3. Importantly, minimal necrotic cell populations were observed across all treatment conditions. These findings indicate that the complexes induce a form of programmed cell death involving apoptosis, thereby minimizing potential collateral tissue damage and inflammatory responses typically associated with necrosis.¹⁴²

Fig. 10 Cell death mechanism. HeLa cells were treated with complexes 1 and 3 for 4 h at the IC_50,light or five times IC_50,light. Cisplatin (5 μM) was used as positive control. Cells were double stained with propidium iodide and Annexin V-FITC. Bars represent the percentages of viable, early apoptotic, late apoptotic, and necrotic cells after each treatment (mean ± SD) determined in three independent experiments. Statistical analysis was performed compared with untreated cells. * p < 0.05; ** p < 0.01; *** p < 0.001.

However, the lower apoptotic population induced by complex 1 in comparison with the cisplatin control (Fig. 10) suggests that apoptosis may not be the exclusive primary death mechanism. Concurrent with this observation, the rapid photocatalytic oxidation of NADH induced by complex 1, coupled with the significant increase in cellular reactive oxygen species (ROS) levels and mitochondria shrinkage, suggest the involvement of ferroptosis. Ferroptosis is a distinct form of regulated cell death characterized by iron-dependent lipid peroxidation and is intrinsically linked to oxidative stress. Recent literature demonstrates the capacity of various metal-based complexes, including iridium(III) complexes, to induce ferroptosis or other non-apoptotic pathways, such as pyroptosis.^143–145 Therefore, while apoptosis remains a key cell death mechanism, other non-apoptotic pathways, potentially including ferroptosis, may also contribute to the overall toxicity of complex 1.

Cell migration. Mitochondrial energy production is crucial for modifying focal adhesions and cytoskeleton remodeling, which are essential processes for cell migration.¹⁴⁶ The migratory capacity is of particular relevance in the context of cancer, as it enables the invasion of surrounding tissues, which represents the initial step of the metastatic cascade that ultimately enables cancer cell dissemination from the primary tumor to distant organs.¹⁴⁷ Given the mitochondrial-targeted activity of complexes 1 and 3, their impact on cell migration was evaluated using wound healing assays. In the absence of light, A549 cells treated with complexes 1 and 3 at the IC_50,light demonstrated effective scratch closure in the cell monolayer, with migration rates comparable to the control cells (Fig. 11). However, following photoactivation, a significant reduction in the wound closure was observed, with an 88.4% inhibition in the cell migration rate for complex 1 and a 93.0% inhibition for complex 3, compared with control cells. In contrast, cisplatin treatment resulted in only a moderate reduction in cell migration. This substantial difference suggests that complexes 1 and 3 exhibit additional characteristics in their antitumor action that could potentially be exploited for the prevention of metastasis.

Fig. 11 Wound healing assays. A549 cells were incubated with complexes 1 and 3 at their respective IC_50,light, either in the dark or under blue light irradiation. Cisplatin (5 μM) was used as a control. (A) Representative images of the wound at time 0 and its closure after 24 h. (B) Migration rate (μm² h⁻¹) of cells exposed to the different treatments. Bars represent the mean ± SD of three independent experiments. * p < 0.01; *** p < 0.001 compared with the control or dark treatment.

Biological activity of NP1 and NP3. The encapsulation of compounds in nanoparticles (NPs) allows their selective accumulation in cancer cells, taking advantage of the EPR effect. In addition, NPs protect the complexes during their circulation in the blood system and improve their cellular uptake, especially if the non-encapsulated compounds tend to aggregate.¹⁴⁸ The influence of encapsulation on the photocytotoxic properties of the complexes was initially examined using two-dimensional (2D) monolayers of A549 and HeLa cells. Interestingly, compound 1 exhibited a marked increase in cytotoxicity when encapsulated in NP1. As shown in Table 6, the IC_50,dark values of NP1 in A549 and HeLa cells were, respectively, 14.6 and 15.2-fold lower than those of complex 1. To rule out the possibility that this effect was due to the intrinsic cytotoxicity of the empty nanoparticle or the constituent polymers (P1b polymer with amine groups, and P2, polymer with carboxylic groups), their impact on cell viability was assessed under the same experimental conditions (Table S11†). No detectable toxicity was observed across a concentration range of up to 200 μg mL⁻¹, which exceeds the nanoparticle concentrations corresponding to the IC_50,dark values of NP1 and NP3 in both cell lines.

Table 6 Photocytotoxic activity of 1, 3, NP1 and NP3 in 2D and 3D cell cultures

2D-cultures	A549			HeLa
	IC50 (μM)		PI	IC50 (μM)		PI
	Dark	Light		Dark	Light
NP1	0.061 ± 0.0002	0.00086 ± 0.0004	70	0.069 ± 0.016	0.0024 ± 0.0011	28
NP3	1.79 ± 0.28	0.0415 ± 0.009	45	1.908 ± 0.27	0.0302 ± 0.0045	65

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3D-cultures	A549
	IC50 (μM)		PI
	Dark	Light
Cells were treated with the indicated complexes and NPs for 4 h at 37 °C and then kept in the dark or irradiated with blue light for 1 h (460 nm, 24.1 J cm^−2). Cell viability was assessed 43 h later by MTT assays (2D-cultures) or CellTiter Glo (3D-cultures). Data represent the mean ± SD. PI: phototoxicity index = IC50,dark/IC50,light.
1	5.10 ± 2.64	0.21 ± 0.08	24.3
NP1	1.44 ± 0.39	0.10 ± 0.03	14.4
3	38.78 ± 15.65	5.53 ± 0.01	7.0
NP3	>50.00	1.71 ± 0.53	>29.2

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Following blue light irradiation, the IC_50,light values of NP1 were further reduced, by 32.5-fold in A549 cells and 26.2-fold in HeLa cells compared with complex 1 (Table 6), yielding PI values of 70 and 28. These represent 2.2- and 1.6-fold increases over the PI values of complexes 1 obtained in A549 and HeLa cells, respectively. These results revealed that NP1 exhibits excellent properties for PDT. Of particular relevance is the outstanding cytotoxic activity of NP1 upon irradiation, evidenced by an IC_50,light value of 0.86 nM in A549 cells. Compared with other iridium derivatives with IC₅₀ < 10 nM (Table S12†), the activity of NP1 is only surpassed by our recently described [Cp*Ir(C^N)L]⁺ derivatives with π-expansive ligands.⁴² An identical IC₅₀ value of 0.86 nM has been described for a complex of the type [Ir(C^N)₂(NHC^NHC)]⁺, although ultraviolet irradiation was used.¹⁴⁹ Conversely, the dark activity of complex 3 was minimally affected by encapsulation. However, upon irradiation, the cytotoxicity of NP3 was notably higher, leading to a remarkable increase in the PI value for NP3versus3 (from 10 to 65) in HeLa cells. In contrast, in A549 cells the high PI value obtained for 3 decreased in NP3 (from 124 to 45).

These results were validated using A549 multicellular spheroids (MCSs), which are 3D cell models that more closely reproduce tumor complexity and organization, including cell–cell interactions, nutrient distribution and oxygen gradients.^150,151 The IC₅₀ values obtained indicated that MCSs were significantly less sensitive to both the free and encapsulated complexes than cells growing in monolayers (Table 6). The reduced efficacy in MCSs is consistent with observations for other chemotherapeutic agents, such as doxorubicin, and could be attributed to a population of quiescent cells within the spheroids that are less susceptible to the antiproliferative effects of the drugs.¹⁵² Furthermore, the complex structure of MCSs has been described to hinder the diffusion of drugs and oxygen to the inner cell layers, further compromising the efficacy of the PSs. It is noteworthy that our complexes and NPs were able to exert a remarkable photocytotoxic effect against MCSs, resulting in a significant reduction in their size and a concomitant loss of refractivity, which is indicative of extensive cell death (Fig. 12A). Particularly, complex 1 and NP1 exhibited a notable activity, with IC_50,light values in the nanomolar range and PIs of 24.3 and 14.4, respectively (Table 6). As observed in the 2D cultures, the photocytotoxicity of NP1 was found to be higher than that of the free complex 1. The encapsulation of complex 3 also improved its photocytotoxic activity against MCSs. However, the IC_50,light values of 3 and NP3 were higher than those of 1 and NP1. It should be highlighted that NP3 demonstrated minimal activity in the dark, leading to the highest PI value (>29.2) in MCSs.

Fig. 12 Photocytotoxic activity against MCSs. (A) Representative microscopy images of A549 MCSs generated within the Geltrex extracellular matrix. Spheroids were treated under photoactivation conditions with complexes 1 and 3 at 0.3 μM and 10 μM, respectively, and nanoparticles NP1 and NP3, at 0.2 μM and 2.7 μM, respectively. These concentrations are close to the IC_50,light values for each compound. Untreated cells were used as a control. (B) Microscopy images of larger A549 MCSs generated in round-bottom wells with an ultra-low attachment surface. These MCSs were treated under photoactivation conditions with the complexes and nanoparticles at their IC_50,light values (previously determined in the 3D models) or five times the IC_50,light. Images were acquired 43 h later (t = 48 h). Untreated spheroids served as a control (t = 0 h). Scale bars for all images: 100 μm.

To further corroborate these findings, an alternative 3D model was established to produce larger A549 MCSs with diameters of approximately 400 μm (Fig. 12B). These MCSs were treated with either the previously determined IC_50,light values from the 3D models or with fivefold higher concentrations, followed by blue light irradiation. After 48 hours of treatment at the IC_50,light, MCSs exhibited reduced structural integrity and compactness compared with the control MCS images obtained before the treatment. Moreover, all MCSs exposed to fivefold the IC_50,light showed a marked reduction in size and a highly disrupted morphology. These results align with those obtained using the earlier 3D model, further supporting the antitumor potential of the compounds.

To gain deeper insight into the impact of encapsulation on the biologic behavior of the complexes, cellular uptake studies were conducted using inductively coupled plasma mass spectrometry (ICP-MS) to quantify the amount of iridium inside the cells. The iridium levels were found to be 2.5- to 2.8 times higher in cells treated with NP1 than with the free complex 1 (Fig. 13). These results aligned with the previously reported higher photocytotoxicity of NP1. In contrast, complex 3 and NP3 exhibited lower cellular uptake. Encapsulation reduced the uptake of 3 in A549 cells; however a 1.9-fold higher internalization of NP3 was detected in HeLa cells, which is in line with the relative values of photocytotoxicity observed for 3 and NP3 in this cell line (Table 6).

Fig. 13 Cellular internalization of complexes 1 and 3 and nanoparticles NP1 and NP3 in A549 and HeLa cells. The amount of iridium (ng) per million cells after 4 h of treatment was determined by ICP-MS. Each bar in the graph represents the mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01.

Overall, these findings demonstrated a strong correlation between cellular internalization of the complexes and their antitumor efficacy. Interestingly, the higher lipophilicity of complex 1 (log P_o/w = 1.58 ± 0.23) than complex 3 (log P_o/w = 1.15 ± 0.27) correlated with an increased cellular accumulation. In addition, the smaller size of the pbpz ligand in complex 1 and its lower tendency to aggregate likely enhanced its cell penetration ability. Similar results have been reported with other Ir(III) complexes bearing π-extended N^N ligands analogous to the C^N pbpz and pbpn ligands used in the present work.¹⁵³

Taking advantage of the luminescence properties of complex 1, the internalization mechanism of its free and encapsulated forms was further explored by flow cytometry. A549 and HeLa cells were incubated with 1 and NP1 for 4 h at either 37° or at 4 °C, to differentiate between energy-dependent and passive uptake processes. As illustrated in Fig. S84,† the fluorescence intensity was markedly reduced at 4 °C in comparison with 37 °C in both cell lines, suggesting that both complex 1 and NP1 accumulate in the cells in an energy-dependent manner that involves active transport or endocytosis, rather than passive diffusion through the cell membrane.¹⁵⁴

Subcellular distribution. Finally, the subcellular distribution of complex 1 and NP1 was assessed using laser confocal microscopy. Microscopy images revealed a significant degree of co-localization between the red fluorescent signal of both complex 1 and NP1 with MitoView™ Green, as shown in orange in the merged images (Fig. 14, and Fig. S85†). The Pearson's correlation coefficients (PCCs) were 0.75 for complex 1 and 0.76 for NP1. Mitochondrial accumulation is characteristic of these types of iridium complexes.²⁶ In contrast, the minimal degree of co-localization observed for 1 and for NP1 with the lysosomal marker LysoTracker™ Green, with PCCs of 0.36 and 0.46 respectively, excluded these organelles as primary cellular targets. Nuclear localization was found to be negligible, as demonstrated by the absence of co-localization with the nuclear dye Hoechst 33258. It is noteworthy that encapsulation did not alter the subcellular distribution of complex 1.

Fig. 14 Confocal microscopy imaging of the subcellular distribution of 1 and NP1. HeLa cells were incubated with 1 or NP1 (λ_ex/λ_em: 440/596 nm) at 50 μg mL⁻¹ for 1 h at 37 °C. The commercial dyes MitoView™ Green (Biotium) (λ_ex/λ_em: 490/523 nm) and LysoTracker™ Green DND-26 (ThermoFisher Scientific) (λ_ex/λ_em: 504/511 nm) and Hoechst (λ_ex/λ_em: 352/454 nm) were used to localize the mitochondria, lysosomes, and cell nuclei, respectively. Images were captured using a Nikon A1R confocal microscope using the following acquisition settings: blue channel: λ_ex/λ_em: 400/450 nm 33258, green channel: λ_ex/λ_em: 488/525 nm and red channel: λ_ex/λ_em: 488/595 nm. Merged images show the green and red fluorescence overlapping in orange.

To confirm the mitochondrial localization of 1 and NP1, HeLa cells were incubated with each compound at 5 μM for 4 hours, followed by isolation of mitochondrial and residual cytosolic fractions using a commercial kit. Iridium content in both fractions was then quantified by ICP-MS. Complex 1 showed preferential accumulation in the mitochondria (0.964 μg Ir per mg protein) compared with the remaining cytosolic fraction (0.029 μg Ir per mg protein). Similarly, NP1 exhibited a significantly higher mitochondrial concentration (3.16 μg Ir per mg protein) relative to the cytosolic fraction (0.014 μg Ir per mg protein). The increased mitochondrial iridium content observed in NP1-treated cells compared with cells treated with complex 1 is consistent with its higher overall cellular uptake, as illustrated in Fig. 13. Collectively, these findings confirm a strong tendency of both 1 and NP1 to accumulate in the mitochondria.

3. Conclusions

Through a molecular design strategy and using innovative procedures we were able to obtain the new derivatives [Ir(C^N)₂(bpy)]⁺ which include the π-expansive C^N ligands pbpz (complex 1) or pbpn (complex 3). The π-expansion led to an increase in the lifetimes of the excited states and a remarkable generation not only of ¹O₂ but also of O₂˙⁻, especially in the case of 1. This allowed the complexes to act both by type II and type I PDT processes. The generation of O₂˙⁻ allows the circumvention of the problem of low O₂ content in some tumors.

The photophysical properties were notably affected by the degree of π-expansion of the C^N ligand, and TD-DFT studies, which found very different characteristics of the triplet excited states, nicely explained the divergences. In fact, 1 showed small energy differences among the four lowest triplet states enabling efficient ISC and RISC, promoting rapid interconversion within the triplet manifold. Consequently, compound 1 displayed delayed fluorescence, where the intersystem crossing between singlet and triplet states can extend the excited-state lifetime, enhancing its efficiency in reactive oxygen species (ROS) production. This phenomenon was not present in 3. In this complex, the extra ring, in a similar way to that found in half-sandwich Ir(III) complexes with the pbpn ligand, causes the generation of two near-HOMO orbitals leading to a higher LC character of the excited states. Thus, lower degrees of SOC and ISC are found in 3 with respect to 1. This rationale may be important in the design of new derivatives with improved properties.

Regarding anticancer activity, both complexes demonstrated notable phototoxicity against human cancer cells, both in monolayer and MCS cultures. It is noteworthy that in all cases, complex 1 demonstrated a markedly higher effect on cell viability than complex 3. The complexes did not exhibit intrinsic selectivity for malignant cells. However, they demonstrated a high capacity to exert a selective light-activated action against cancer cells with minimal effects on non-irradiated healthy cells and erythrocytes. Upon photoactivation, both complexes induced a substantial increase in intracellular ROS levels, including the generation of superoxide anions. Cell confocal imaging revealed that complex 1 exhibited a marked accumulation in mitochondria, which correlated with the disruption of mitochondrial physiological functions, as evidenced by a decrease in mitochondrial membrane potential. Besides, complexes 1 and 3 were found to be among the most active NADH photocatalysts described to date. Wound healing and clonogenic assays demonstrated the capacity of the complexes to inhibit the spread of malignant cells and their capacity to generate secondary tumors, which are crucial steps in the metastatic process. These findings are promising, as metastasis is responsible for the greatest number of cancer-related deaths.

The encapsulation of the complexes into nanoparticles did not affect their photophysical properties. Moreover, it improved some of the drawbacks of the complexes such as aggregation and low solubility in water and low cellular uptake. Remarkably, the cellular uptake, even with low payload content, was improved by encapsulation and also the phototoxicity indexes were notably improved. Even in 3D cancer models this good behaviour was observed. NP1 led to one of the lowest values of IC₅₀ reported to date in iridium chemistry (0.86 nM). Thus, our strategy was successful, and the study points to the high potential of the pbpz and pbpn ligands, at least in iridium derivatives active in PDT processes. Complexes 1 and 3, NP1 and NP3 show high promise as type I and II PDT agents that could also behave as theragnosis agents. Besides, there is also scope for further improving the properties by modifying the N^N ligand.

Conflicts of interest

There are no conflicts to declare.

Data availability

The data supporting this article have been included in the main text and as part of the ESI.†

Crystallographic data (CIF-files) have been uploaded to the CCDC and can be obtained via the CCDC homepage using the CCDC numbers provided in the manuscript.

Acknowledgements

This work was supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (PID2021-127187OB-C21, PID2021-127187OB-C22), Junta de Comunidades de Castilla-La Mancha-FEDER (JCCM) (grant SBPLY/23/180225/000192), and UCLM-FEDER (grants 2019-GRIN-27183, 2019-GRIN-27209 and 2022-GRIN-34193). C. G. acknowledges his fellowship to both the European Social Fund and Plan Propio de I + D + I of UCLM (2022-PRED-20649). G. D. thanks the Junta de Comunidades de Castilla-La Mancha and EU for financial support through the European Regional Development Fund (project SBPLY/19/180501/000191). J. C. L. and A. M. acknowledge the Portuguese Foundation for Science and Technology for funding through LAQV-REQUIMTE (UIDB/50006/2020 and UIDP/50006/2020). C. B and E. Z. acknowledge their predoctoral grants University of Girona (IFUdG2021) and Generalitat de Catalunya (AGAUR; 2021 FI_B 01036). P. T. acknowledges national funds from FCT – Fundação para a Ciência e a Tecnologia (FCT-MCTES), I. P., in the scope of the project UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences – UCIBIO and LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy – i4HB. The collaboration of Prof. Antonio de la Hoz is acknowledged for the facilities to use the microwave system. The authors would like to thank the IRICA services at UCLM and Eduardo Prado García-Consuegra of the SEM services team for the technical assistance.

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Footnotes

† Electronic supplementary information (ESI) available: Experimental section, NMR spectra, ESI+ MS spectra, HPLC traces, X-ray diffraction, analysis of π–π stacking by ¹H NMR spectroscopy, stability and photostability, photophysical properties, transient absorption spectroscopy (TAS) measurements, ROS generation, computational studies, synthesis and characterization of nanoparticles, hemolytic activity, NADH oxidation, internalization analyses. Biological Studies Raw Data. CCDC 2431327–2431329 for complexes 1, 2 and 4. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d5qi00775e

‡ Both authors have contributed equally to the work.

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