# Monomer Versus Dimer of Cationic Ir(III) Complexes for Photodynamic Therapy by Two-Photon Activation: A Comparative Study

Agoston Barta, Laetitia Vanwonterghem, Matéo Lavaud, Florian Molton, Guillaume Micouin, Anne-Laure Bulin, A. Banyasz, Jean-Luc Coll, Frédérique Loiseau, Amandine Hurbin, et al.

► To cite this version:

Agoston Barta, Laetitia Vanwonterghem, Matéo Lavaud, Florian Molton, Guillaume Micouin, et al. Monomer Versus Dimer of Cationic Ir(III) Complexes for Photodynamic Therapy by Two-Photon Activation: A Comparative Study. ACS Applied Bio Materials, 2025, 8 (5), pp.4272-4284. 10.1021/acsbm.5c00393. http://dx.doi.org/10.1021/acsbm.5c00393

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# Monomer *versus* dimer of cationic Ir(III) complexes for Photodynamic therapy by two-photon activation: A comparative study

Agoston Barta,ª Laetitia Vanwonterghem,ᵇ Matéo Lavaud,ᵇ Florian Molton,ª Guillaume Micouin,ᶜ Anne-Laure Bulin,ᵇ Akos Banyasz,ᶜ Jean-Luc Coll,ᵇ Frédérique Loiseau,ª Amandine Hurbin,ᵇ* and Pierre-Henri Lanoëª*

## Abstract

Iridium(III) complexes have been recognized as promising candidates for two-photon sensitized photodynamic therapy (PDT). In this context, we report on the study of two complexes, a monomer (IrL¹) and a dimer (Ir₂L²). Both complexes possess 2-phenylpyridine cyclometallating ligands and a pyridylbenzimidazole derivative as ancillary ligand. In the dimer, the two Ir(III) centers are connected by a non-conjugated bridged bis(pyridylbenzimidazole). We compare the photophysical properties of these complexes. Both display phosphorescent emission in the orange-red part of the visible spectrum, with emissions centered at 610 nm for IrL¹ and 625 nm for Ir₂L², both exhibiting quantum yields of ~24%. However, Ir₂L² turns to be much brighter than the monomer making the dimer four times brighter than IrL¹. This trend is consistent under two-photon excitation (TPE) and the singlet oxygen generation quantum yields, indeed the dimer displaying a *figure of merit* ($\sigma_{TPA} \times \phi_\Delta$) of 40, compared to only 5 for the monomer. Both complexes generate intracellular ROS and exhibit strong phototoxicity upon blue light activation ($\lambda=420$ nm), achieving sub-micromolar IC₅₀ values in HT29 and A549 cell lines after 24h of incubation. Moreover, with TPE ($\lambda=800$ nm), both complexes also generate intracellular ROS and induce cancer cell death.

**Keywords:** Photodynamic therapy, cancer, Ir complexes, Two-photon excitation, photosensitizers, Reactive Oxygen Species

## Introduction

Photodynamic therapy (PDT) represents a powerful therapeutic technique against cancer, bacterial growth and age-related macular degeneration (AMD). It is believed to be the next-generation cancer treatment due to its non-invasiveness, high specificity, and low systemic toxicity.¹⁻⁸ PDT involves administering a molecule, called a photosensitizer (PS), to a patient via topical application, tablet, or injection. This compound should ideally accumulate preferentially in the

<footnote>ª Univ. Grenoble Alpes, CNRS, DCM, 38000 Grenoble, France, E-mail: pierre-henri.lanoe@univ-grenoble-alpes.fr</footnote>

<footnote>ᵇ Univ. Grenoble Alpes, INSERM U1209, CNRS UMR5309, Institute for Advanced Biosciences, 38000 Grenoble, France, E-mail: amandine.hurbin@inserm.fr</footnote>

<footnote>ᶜ Laboratoire de Chimie École Normale Supérieure de Lyon, ENS, CNRS, UCBL UMR 5182, 46 Allée d'Italie, Lyon, 69364 France</footnote>

<footnote>* Both authors contributed equally</footnote>
targeted area and, once a peak concentration is reached, the treatment is initiated by shining light on the area. Upon light excitation, the photosensitizers generate highly cytotoxic singlet oxygen (¹O₂), causing cell death to targeted cancer. Despite recent advances in PDT, its clinical application remains limited to early-stage pulmonary, gastric, and esophageal carcinoma, as well as retinal diseases,⁹,¹⁰ indicating the need for further development of this technique.¹,³,⁶,¹¹,¹² For instance, light penetration is influenced by both the tissue's optical properties and the wavelength used, which in turn affects the amount of light available for absorption by the PS.¹³ Intensive research is currently underway to advance PDT and expand its applications, by developing new light sources for example.¹⁴ A key challenge in this effort is the development of new PSs with enhanced properties, such as improved light absorption and higher ¹O₂ generation quantum yields.

Phosphorescent transition metal complexes are potent candidates as photosensitizers in PDT due to their desirable characteristics, particularly those based on iridium(III): (i) low toxicity,³,⁴,¹² (ii) ability to penetrate cells (cytoplasm and/or organelles)³,¹²,¹⁵,¹⁶ (iii) efficient absorption of light in the visible range or even in the tissue transparency window (roughly 700-1200 nm), (iv) presence of the heavy atom allowing quasi-quantitative intersystem crossing (ISC) to the triplet state,¹⁷ generally associated with high ¹O₂ generation, (v) resistance to photobleaching,¹⁷ and (vi) tendency for cancer cells to accumulate lipophilic cations more efficiently compared to healthy cells due to their more negative potential;¹⁸ (vii) chemical versatility of the molecular structure, especially when dealing with the heteroleptic derivatives.²,¹⁹

One can consider the optimization of a PS for PDT, a fortiori an iridium(III) complex, for PDT, and especially in type II, where the ¹O₂ generation ability of PSs is critical to the PDT effect. This involves the simultaneous improvement of the amount of light absorbed by the molecule (ε at a given wavelength) and the quantum yield of singlet oxygen generation (φΔ).²⁰ This leads to a new coefficient ζΔ = ε x φΔ,²¹,²² which allow to estimate the highest molecular singlet oxygen generation efficiency. ε can be increased by tinkering with the number of chromophores, for example, and φΔ is directly dependent on the efficiency of the PS intersystem crossing to achieve a maximum triplet state for sensitizing molecular oxygen.⁴,²⁰

Currently, most of the efforts focus on mononuclear complexes³,¹²,²³⁻²⁵ where the modifications of the cyclometallating or ancillary ligands allow for tuning cell penetration and both ε and φΔ. Notably, ε can be significantly enhanced by incorporating organic chromophores into the ligand backbone²⁶⁻³⁰ For example, using xanthene in the ancillary ligand backbone results in a mitochondria-targeting PS with high molar absorptivity.²⁵ Under light irradiation (400-800 nm, 100 mW cm⁻²), the conjugate induces apoptotic cell death by generating ¹O₂ with IC₅₀ of the order of 5 µM.

The performance of a PS can also be enhanced by using doped nanoparticles³¹,³² by synthesizing self-assembled nanoparticles.³³⁻³⁵ For instance, a series of amphiphilic iridium(III) complexes bearing quaternary ammonium groups were able to self-assemble in vesicle in water buffer. These nanovesicles displayed low dark cytotoxicity, mitochondria-targeted imaging capabilities and induced efficient PDT in tumor cells.³⁵

Finally, a limitation of PDT is the use of visible light for photo-activation, which falls out the therapeutic window in the range 600-1300 nm. Working within this range is important as it allows deeper tissues penetration. Two-photon absorption (TPA) involves simultaneous absorption of two photons of the same energy (i.e. same wavelength) and requires an intense light source (focused short pulse laser). TPA enables to work in the tissues transparent zone and provides spatial resolution.³⁶,³⁷ Ir(III) complexes are suitable for TPA without special molecular engineering.³⁸
Therefore, the ideal PS should have a large two-photon cross section ($\sigma_{TPA}$) and a high $\phi_{\Delta}$, leading to the concept of *figure of merit*, defined as $\sigma_{TPA} \times \phi_{\Delta}$.$^{39}$

In this contribution, we have chosen a molecular approach using a multinuclear iridium(III) complex to increase the light absorption. This approach has proven to be promising to enhance the phosphorescence rate through the enhanced spin-orbit coupling (SOC), especially when both iridium(III) cores shared a common arene, benzene or heterocycle moiety such as pyrimidine, through coordination or cyclometallation.$^{40-43}$ Recently, Shafikov *et al* have reported a dinuclear Ir(III) complex, with a rigid bridging chromophoric ligand 2,6-diphenyl[1,3]thiazolo[5,4-f][1,3]benzothiazole displaying a high $\varepsilon$ (24 300 L.mol$^{-1}$.cm$^{-1}$ at 462 nm) and $\phi_{\Delta}$ of 71% in $CH_2Cl_2$.41 Our approach is different, as we do not seek to modify the photophysical properties in the multinuclear complex in comparison to the mononuclear. However, a fistful of multimetallic assemblies has been reported for PDT application.$^{31,33,34,44,45}$ Recently, we reported the synthesis and characterization of two chiral binuclear iridium(III) complexes ($\Lambda\Lambda$ and $\Delta\Delta$) with their diastereomeric mixture ($Ir_2L^2$) and a corresponding racemic monomer.$^{46}$ The two complexes $\Lambda\Lambda$ and $\Delta\Delta$ were optically active, by both the circular dichroism (CD) spectra and their chiral polarized luminescence (CPL). The dimers ($Ir_2L^2$, $\Lambda\Lambda$ and $\Delta\Delta$) demonstrate a four-times higher brightness with respect to the monomer ($IrL^1$) and the CPL brightness ($B_{CPL}$) of the binuclear complexes exhibiting among the highest reported to date for chiral iridium(III) complexes. Phosphorescent dyes allow increased imaging resolution by using a delay between excitation and acquisition, which permits the background fluorescence to be significantly decreased.$^{47,48}$ These compounds could be exploited as theragnostic agent for image-guided surgery and cancer PDT,$^{49,50}$ we investigated the potential of $IrL^1$ and $Ir_2L^2$ complexes in PDT. Thus, we present an in vitro study of the binuclear $Ir_2L^2$ complex and its mononuclear counterpart $IrL^1$ - corresponding to half of $Ir_2L^2$ – as photosensitizers for PDT, evaluating their $^1O_2$ generation efficiency (Figure 1).

## Synthesis and characterization

<figure><img src="image_2.png" /><figcaption>Figure 1: Cationic Ir(III) complexes isolated with $PF_6^-$ counter ions.</figcaption></figure>

All the compounds were characterized by NMR ($^1$H and $^{13}$C), mass spectroscopy and elemental analysis. The synthesis of both ligand $L^1$ and ditopic ligand $L^2$, as well as the final complexes, $IrL^1$ and $Ir_2L^2$, was described elsewhere.$^{46}$ However, the synthesis schemes, the NMR ($^1$H and $^{13}$C), and mass spectroscopy are displayed in the supporting information.
## Photophysical properties

<figure><img src="image_3.png" /><figcaption>**Figure 2:** Absorption and deaerated normalized emission spectra in CH₂Cl₂ of complexes IrL¹ and Ir₂L².</figcaption></figure>

Both the emission and absorption spectra have been recorded in dilute CH₂Cl₂ solution under air equilibrated and deaerated conditions (Figure 2 and Table 1). The absorption spectra of iridium complexes could be divided into three spectral domains. In the UV region (250-340 nm), intense absorption bands can be assigned to the spin-allowed π-π* ligand centered transitions (¹LC) in both cyclometallating and chelating ligand (i.e. L¹ and L²). In the second region ranging from 320 nm to 440 nm moderately intense to low absorption bands can be ascribed to mixing of spin allowed metal-to-ligand charge transfer (MLCT) admixed with ligand-to-ligand from the cyclometallating ligand to the π*(L¹/L²) charge transfer (¹ILCT) transitions. The third region (see SI) is above 440 nm with very low absorption tail, which can be assigned to the direct spin forbidden absorption from ground state to the ³MLCT/³LLCT excited states, partially allowed thanks to the high spin-orbit coupling constant of the iridium core (ζ = 3909 cm⁻¹).⁵¹ In addition, the bimetallic complex displays a bit more than the double of the absorbance of complex IrL¹. For example, the highest absorption band has a molar extinction coefficient of 46.3 × 10³ M⁻¹ cm⁻¹ for IrL¹ and 98.7 × 10³ M⁻¹ cm⁻¹ for Ir₂L². The bimetallic complex displays higher molar absorptivity in the visible region of the spectrum, and the bands corresponding the (M/L)LCT are red-shifted (ΔE = 1853 cm⁻¹), from 355 nm to roughly 380 nm, for IrL¹ and Ir₂L², respectively. Such a feature has been previously observed.⁵² Finally, the absorption tail above 440 nm displays a molar absorptivity that corresponds almost exactly to the addition of the absorbance spectra of two IrL¹ units, pointing out that there is no electronic coupling in the ground state between the two parts of the dimer, which would be the case with a rigid bridging ligand.⁵³,⁵⁴

Both complexes display an orange-red color, with an emission maximum in the range 610-625 nm, which can be ascribed to phosphorescence considering the lifetimes of hundreds of ns and the sensitivity to oxygen. In comparison with previously reported complexes bearing 2-2'-pyridylbenzimidazole, the introduction of bromide atoms in position 5' of the pyridyl moiety leads to a somewhat strong bathochromic shift of 30 nm and 45 nm, for the monometallic and bimetallic complexes respectively.⁵⁵⁻⁵⁷ The photoluminescence quantum yields remain high with respect to the energy gap law (i.e. nonradiative transitions become increasingly important with red emitters).⁵⁸ In comparison to IrL¹, Ir₂L² displays a slight redshift (λ<sub>max</sub> = 610 nm and 625 nm, respectively). However,
considering the emission energy difference of only 0.05 eV, this redshift is negligible, suggesting that the ground state of L² may deviate slightly from the expected planar structure. The emission spectra obtained in rigid matrix at low temperature (77 K, Figure S7) display a rigidochromism, which is typical of transition metal complexes submitted to a rigidification of the environment by a decrease of the temperature. Together, the rigidochromism at low temperature and the broad emission spectra at room temperature observed for both complexes allow to conclude the phosphorescence emanates from the radiative deactivation of the ³MLCT* state to the ground state. The brightness (B₃₈₀ nm = ε x φ)⁵⁹ takes in account both absorption of light and φ, since a high quantum yield coupled with low absorption of light would yield a poorly luminescent active material.²⁰,⁶⁰ The bimetallic derivative Ir₂L² displays a four times higher brightness value than the monometallic, with values of B = 13.0 x 10³ and B = 3.0 x 10³, respectively. This demonstrates that Ir₂L² displays advantageous emission properties in comparison with IrL¹.

<table><caption>**Table 1:** Absorption and emission data recorded in CH₂Cl₂, singlet oxygen generation quantum yields in CHCl₃ and molecular singlet oxygen generation efficiency.</caption><thead><tr><th>Complex</th><th>Absorption<br>(x10<sup>1</sup><br>.cm<sup>-1</sup>)</th><th>L.mol<sup>-1</sup></th><th>λ<sub>em</sub><br>/nm</th><th>φ<sup>[a], [b]</sup><br>[air]</th><th>τ<sup>[b]</sup>/ns<br>[air]</th><th>k[O<sub>2</sub>]/L.mol<sup>-1</sup>.s<sup>-1</sup><br>(x 10<sup>9</sup>)</th><th>Φ<sub>Δ</sub><sup>[c]</sup></th><th>B<sup>[d]</sup><br>x 10<sup>3</sup><br>/mol<sup>-1</sup>.cm<sup>-1</sup></th><th>ζ<sub>Δ</sub><sup>[e]</sup><br>(ε x Φ<sub>Δ</sub>) x<br>10<sup>3</sup></th><th>Figure<br>of<br>merit<sup>[f]</sup><br>(σ<sub>TPA</sub> x<br>Φ<sub>Δ</sub>)</th><th>Electrochemical<br>properties<sup>[g]</sup> (V)</th></tr><tr><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th></th><th>E<sup>red</sup></th><th>E<sup>ox</sup></th></tr></thead><tbody><tr><td>IrL<sup>1</sup></td><td>257 (4.7), 302<br>(2.6), 340 (2.6),<br>355 (2.7), 388<br>(1.1), 416 (0.5),<br>470 (0.1),</td><td>610</td><td></td><td>0.23<br>[0.08]</td><td>918<br>[228]</td><td>0.8</td><td>0.66</td><td>3.0</td><td>5</td><td>5</td><td>-1.56<sup>irr</sup></td><td>0.95</td></tr><tr><td>Ir₂L<sup>2</sup></td><td>267 (9.8), 303<br>(5.1), 335 (3.3),<br>378 (5.5), 470<br>(0.2)</td><td>625</td><td></td><td>0.24<br>[0.07]</td><td>783<br>[242]</td><td>1.4</td><td>0.78</td><td>13.0</td><td>24</td><td>40</td><td>-1.55<sup>irr</sup></td><td>0.96</td></tr></tbody></table>

[a] [Ru(bpy)₃]²⁺ in H₂O was used as reference, [b] dearated, [c] singlet oxygen generation quantum yield obtained in CHCl₃ from Stern-Volmer plot using phenalenone as standard with ΦΔ = 0.98 in CHCl₃, [d] molecular brilliance B = ε x Φ (eq. 1), ε at 380 nm [e] ζΔ, molecular singlet oxygen generation efficiency with ε at 420 nm. [f] *Figure of merit* is calculated at 780 nm as Ti:sapphire laser operating at 780 nm is the most commonly used laser for *in vivo* applications. [g] Potentials obtained by cyclic voltammetry using Bu₄NPF₆ (0.1 M) as the supporting electrolyte in CH₃CN. Potentials are given relative to the Ag⁺/Ag couple (Reference electrode ANE2, AgNO₃ = 10⁻² M) measured under the same conditions.
## Two-photons absorption

The two-photons absorption cross-sections, $\sigma_{TPA}$ of IrL¹ and Ir₂L² were measured by the two-photons excited fluorescence (TPEF) technique. Figure 3 presents the results of the TPEF measurement for IrL¹ and Ir₂L². First, we assessed the emission intensity as a function of the excitation power (Figure 3a). Subsequently, a nonlinear regression analysis was performed to ascertain the order of the power dependence. Notably, the slope values were determined to be 1.97 for IrL¹ and 1.91 for Ir₂L², indicating that in both cases two-photons absorption is the origin of the observed emission. Figure 3b illustrates the one- and two-photons absorption spectra of IrL¹ and Ir₂L². For comparison the two-photons absorption cross-section values are normalized by the number of metal centers ($n_{Ir}$), the one-photon absorption spectra are normalized so that their maxima match with their two-photon counterparts. The shapes of the one-and two-photons absorption spectra show a significant overlap for both compounds. Like the linear absorption, the two-photons absorption spectrum of Ir₂L² exhibits a pronounced red shift in comparison to IrL¹, peaking at approximately 740 nm. A relatively weak though significant cross section is measured for IrL¹ with $\sigma_{TPA}$ maximum of roughly 18 G.M at 700 nm, and in stark contrast Ir₂L² exhibits a higher $\sigma_{TPA}$ maximum of 80 G.M. at 740 nm. These absolute values of $\sigma_{TPA}$ are comparable with those observed for similar Ir(III) complexes, especially those without specific ligand engineering for TPE.⁴,¹²,⁶¹⁻⁶³ Intriguingly, the red shift is accompanied by a remarkable more-than-doubled enhancement in the two-photon absorption cross section per Ir-center for Ir₂L² as compared to IrL¹. Once again, the use of binuclear complex displays advantageous properties, that surpass the mononuclear counterpart.

<figure><img src="image_4.png" /><figcaption>Figure 3: (a) The luminescence intensity as a function of the excitation power at 800 nm, (b) two-photons (symbols) absorption spectra normalized per Ir-centers ($n_{Ir}$) along with the arbitrarily normalized one-absorption spectra (solid) for IrL¹ (black) and Ir₂L² (red). The two-photon absorption spectra were measured at 40 mW excitation power.</figcaption></figure>

## Singlet oxygen generation

As mentioned above, the phosphorescence of both complexes is highly sensitive to molecular oxygen, indeed, as triplet energy of the emissive excited state is higher than 0.97 eV (~1270 nm), which corresponds to the energy difference of the triplet oxygen ground state and its lowest singlet excited state. In short, there is a competition between the relaxation of the triplet excited state of the PS and the Dexter energy transfer to the ground state oxygen generating ¹O₂. The singlet oxygen generation quantum yields of both complexes were measured by the relative method in aerated CHCl₃ solution with phenalenone as standard using the Stern-Volmer plot for analysis (figure S8). IrL¹
and Ir₂L² can generate singlet oxygen with high yields $\phi_{\Delta} = 0.66$ and 0.78, respectively. In view of the molecular singlet oxygen generation efficiency coefficient ($\zeta_{\Delta}$),²¹,²² the bimetallic Ir₂L² complex which exhibits both the highest $\phi_{\Delta}$ and $\epsilon_{420nm}$ values, proves to be a more effective PS than IrL¹, nearly five times greater $\zeta_{\Delta}$ than IrL¹. In addition, Ir₂L² displays a higher figure of merit at 780 nm than its monometallic counterpart.

The voltammetry cyclic was performed in acetonitrile using Bu₄NPF₆ (0.1 M) as the supporting electrolyte against Ag⁺/Ag (ANE2, Table 1 and Table S1).⁴⁶ The redox potentials of IrL¹ and Ir₂L² are almost identical, differing by only 10 mV both in reduction and oxidation. The one e⁻ potential reduction of O₂ to O₂⁻ is -0.33 V vs SHE and considering the excited state potentials vs SHE (Table S1) of IrL¹ (E<sup>red*</sup> = 1.34 V and E<sup>ox*</sup> = 0.87 V) and Ir₂L² (E<sup>ox*</sup> = -0.84 V and E<sup>red*</sup> = 1.33 V), it comes out that the complexes could be able to proceed to the reduction of ground state oxygen (Figure S9) leading to superoxide radical ion O₂⁻. However, the complexes demonstrate high $\phi_{\Delta}$ which is in contradiction. Indeed, the energy transfer to ³O₂ to produce ¹O₂ competes kinetically with the single electron transfer reaction and the letter is less likely because of the biradical nature of ground state oxygen.⁶⁴ That being said, the electron transfer is favored in polar solvent and the presence of O₂⁻ could be considered in the cells, in addition of ¹O₂.⁶⁴ The electron transfer could be assessed by electron paramagnetic resonance spectroscopy (EPR) using 5,5-dimethyl-1-pyrroline N-oxide (DMPO), a known for trapping O₂⁻ upon light illumination.⁶⁵ Unfortunately, we were unable to observed the EPR signal corresponding the DMPO radical despite our attempts.

To conclude on the photophysical properties of the two complexes, our results clearly demonstrate that the binuclear complex, Ir₂L², is highly promising for imaging applications, exhibiting a brightness four times greater than that of the mononuclear complex, IrL¹, with B values of 13.3 x 10³ and 3 x 10³, respectively. Considering the two-photon absorption, once again Ir₂L² displays better properties compared to IrL¹, with an enhancement in the two-photon absorption cross section maximum, $\sigma_{TPA} = 18$ G.M at 700 nm, $\sigma_{TPA}$ 80 G.M. at 740 nm, for IrL¹ and Ir₂L² respectively. Finally, the ability of the complexes to sensitize oxygen for singlet oxygen generation confirms that Ir₂L² is a significantly more effective photosensitizer than IrL¹, especially when considering the $\zeta_{\Delta}$ coefficient and the figure of merit parameters. With these results in hand, we decided to undertake *in vitro* studies to evaluate both complexes as PS in PDT.

## Cellular uptake

The cellular uptake of IrL¹ and Ir₂L² was first evaluated in two different human cancer cell lines and was found to be slower for Ir₂L² than for IrL¹. Indeed, confocal microscopy showed that both IrL¹ and Ir₂L² were mostly found in the cytoplasm of A549 (Figure 4A and 4B) and HT29 cells (Figure S10), confirming their internalization. IrL¹ showed a diffuse labelling from 2 to 24 h incubation at 37 C. In contrast, Ir₂L² showed a punctate labelling that increased over time, which is a hallmark of its intracytoplasmic trafficking after internalization. These results were confirmed by flow cytometry analyses: peak and mean fluorescence intensities were high for IrL¹ and similar after 4h or 24h, while they were higher for Ir₂L² after 24h than after 4h (Figure 4C and 4D). These differences between IrL¹ and Ir₂L² staining suggested that the IrL¹ may enter cells by passive diffusion, whereas Ir₂L² may use an active internalization process. However, internalization of both IrL¹ and Ir₂L² was prevented at 4°C (Figure 4C, 4D, and S11), suggesting that both IrL¹ and Ir₂L² entered cells through active internalization processes. Colocalization experiments with organelle-specific fluorescent dyes showed that the red fluorescent signal of IrL¹ strongly overlapped that corresponding to the mitochondria (Figure 4E). In contrast, the red fluorescent signal of Ir₂L² did not colocalize with either lysosomes or mitochondria (Figure 4E). These results indicate that IrL¹ accumulates in mitochondria, as expected with most Iridium(III) complexes,⁶⁶ while Ir₂L² may be retained in the cytosol or another organelle.
<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /><figcaption>Figure 4: IrL¹ and Ir₂L² are internalized in A549 cells. (A-B) Representatives confocal microscopy images of A549 cells incubated with 1 µmol.L⁻¹ IrL¹ (A) or Ir2L2 (B) for 2 h, 4 h or 24 h. Nuclei are stained with Hoechst 33342 (in blue). IrL¹ and Ir₂L² fluorescence is observed in red. Scale bars, 10 µm. (C-D) Flow cytometry analysis (C) and corresponding quantitative results (D) of the cellular uptake of 1 µmol.L⁻¹ IrL¹ or Ir₂L² after 4 h at 4°C or 37°C or after 24 h at 37°C. Data are expressed as mean ± standard deviation (n=2). (E) Colocalization of IrL¹ or Ir₂L² and organelle-specific probes in A549 cells. IrL¹ or Ir₂L² (red), MitoTracker orange (yellow), LysoTracker blue (cyan). Scale bars, 10 µm.</figcaption></figure>
## Phototoxicity in cells

We investigated the phototoxicity of IrL¹ and Ir₂L² *in vitro*. First, we assessed that illumination at 420 nm, in the absence of the compounds, induced only a moderate level of phototoxicity (Figure S12). Increasing concentrations of IrL¹ and Ir₂L² exposed to light reduced the viability of A549 and HT29 cells, whereas they had less toxic effect on the proliferation in the dark (Figure S13). Both A549 and HT29 cells appeared to be equally highly sensitive to IrL¹ treatments, with similar IC₅₀ values in the nanomolar range (Table 2 and Figure S13 and S14). In contrast, A549 and HT29 cells were less sensitive to Ir₂L² treatments, with IC₅₀ values in the range of several hundred nanomolar (Table 2 and Figure S13 and S15).

<table><caption>Table 2: Sensitivity of cancer cells to IrL¹ and Ir₂L²</caption><thead><tr><th rowspan="2">Cell lines</th><th rowspan="2">Treatment conditions</th><th>IrL<sup>1</sup></th><th>Ir<sub>2</sub>L<sup>2</sup></th></tr><tr><th>IC<sub>50</sub> (nmol L<sup>-1</sup>)</th><th>IC<sub>50</sub> (µmol L<sup>-1</sup>)</th></tr></thead><tbody><tr><td rowspan="6">A549</td><td rowspan="3">4h</td><td>Dark</td><td>490 ± 30</td><td>5.12 ± 0.49</td></tr><tr><td>10 J cm<sup>-2</sup></td><td>27 ± 3</td><td>2.22 ± 0.29</td></tr><tr><td>25 J cm<sup>-2</sup></td><td>3 ± 1</td><td>0.23 ± 0.03</td></tr><tr><td rowspan="3">24h</td><td>Dark</td><td>164 ± 11</td><td>3.17 ± 0.29</td></tr><tr><td>10 J cm<sup>-2</sup></td><td>25 ± 0</td><td>0.66 ± 0.04</td></tr><tr><td>25 J cm<sup>-2</sup></td><td>6 ± 1</td><td>0.29 ± 0.01</td></tr><tr><td rowspan="6">HT29</td><td rowspan="3">4h</td><td>Dark</td><td>356 ± 37</td><td>6.76 ± 0.50</td></tr><tr><td>10 J cm<sup>-2</sup></td><td>34 ± 2</td><td>4.15 ± 0.28</td></tr><tr><td>25 J cm<sup>-2</sup></td><td>15 ± 1</td><td>1.85 ± 0.12</td></tr><tr><td rowspan="3">24h</td><td>Dark</td><td>229 ± 18</td><td>5.41 ± 0.45</td></tr><tr><td>10 J cm<sup>-2</sup></td><td>14 ± 1</td><td>0.91 ± 0.05</td></tr><tr><td>25 J cm<sup>-2</sup></td><td>7 ± 1</td><td>0.50 ± 0.03</td></tr></tbody></table>

The drug concentrations required to inhibit cell growth by 50% (IC₅₀) at 4 h or 24 h in A549 or HT29 cells after light exposure (420 nm; 10 and 25 J cm⁻²). Data represent the mean ± SEM of 3 independent experiments, each performed in triplicate.

Interestingly, the phototoxicity of IrL¹ and Ir₂L² was light-dose dependent (Figure 5A, 5B and Figure S13). Furthermore, in agreement with our cellular uptake experiments, the 4 h drug-light interval (exposure to light 4 h after treatment) allowed IrL¹ to induce the same high phototoxicity in A549 and HT29 cancer cells *in vitro* (Figure 5A) than the 24 h drug-light interval (Figure S14). In contrast, Ir₂L² required a 24 h drug-light interval to induce significant phototoxicity (Figure 5B) as compared with the 4 h drug-light interval, especially in HT29 cells (Figure S15).
<figure><img src="image_9.png" /><figcaption>Figure 5: IrL¹ and Ir₂L² induced phototoxicity in A549 and HT29 cells. A549 cells (upper panels) and HT29 cells (lower panels) were treated with increasing concentrations of IrL¹ for 4 h (A) or Ir2L2 for 24 h (B) before light exposure at 420 nm (fluency 10 J.cm⁻² and 25 J.cm⁻²). In parallel, cells were maintained in the dark. Cell viability was assessed 72 h following light exposure. Data are expressed as the mean ± SD of 3 independent experiments in triplicate. ** p < 0.01; *** p < 0.001; significantly different from the Dark; ANOVA with Dunnett's post hoc tests.</figcaption></figure>

A similar complex to IrL¹ featuring a 2-(pyridinyl)-1-H-benzimidazole ancillary ligand instead of the ligand 1-butyl-5-methyl-2-(pyridinyl)-1-H-benzimidazole, displays lower dark toxicity on both A549 and HeLa.⁶⁷ In addition, with an almost identical fluence of irradiation at 425 nm (LED light array with 1.2 J cm⁻², 4 mW cm⁻², 300 s); the compound displays a phototoxicity of 1.39 ± 0.05 µM and 0.95 ± 0.04 µM for HeLa and A549, respectively, which are lower than those observed with IrL¹. Other dinuclear Ir complexes studied as molecular PS displayed similar phototoxicity properties in the µmol range than Ir₂L².⁴⁰,⁴⁴ A shared properties could be the relative long incubation time for this type of complexes, at least 12h.

## ROS generation in cells

We next assessed the generation of intracellular reactive oxygen species (ROS) by IrL¹ and Ir₂L² in A549 cells using the widely adopted nonspecific ROS probe, 2',7'-dichlorofluorescein diacetate (DCFH-DA). As shown in Figure 6A, following incubation with IrL¹ and Ir₂L², A549 cells displayed green fluorescence signal when exposed to light (420 nm), especially after light dose of 10 J cm⁻², whereas only weak green fluorescence signal was observed in the control cells. The cellular fluorescence signal of DCFH-DA is strongly enhanced after illumination (Figure 6B). Cellular ROS production increased with Ir₂L² concentration in a dose-dependent manner, while IrL¹ induced high intracellular ROS production from the lowest concentrations studied.
<figure><img src="image_10.png" /></figure>

Figure 6: IrL¹ and Ir₂L² generate cellular ROS in A549 cells after light exposure. (A) Confocal images of A549 cells incubated with 100 nmol.L⁻¹ IrL¹ or 1 µmol.L⁻¹ Ir₂L² (in red) for 24 h before light exposure at 420 nm (fluency 5 J.cm⁻² and 10 J.cm⁻²) and intracellular ROS detection by DCFH-DA (in green). Nuclei are stained with Hoechst 33342 (in blue). Scale bar = 10 µm. Images are representatives of 2 independent experiments. (B) ROS production in A549 cells treated with increasing concentrations of IrL¹ or Ir₂L² and 5 or 10 J.cm⁻² light exposure at 420 nm. Bars: mean ± SEM (n=2). 100 µmol/L⁻¹ hydrogen peroxide is used as positive control.

A similar phenomenon was observed with 800 nm fs laser irradiation. After incubation with IrL¹ and Ir₂L², A549 cells showed a DCFH-DA green fluorescence signal in the area exposed to laser light, whereas only a weak green fluorescence signal was observed in control cells (Figure 7A). The fluorescence signal of DCFH-DA strongly increased after two-photon illumination, at the same time as the fluorescence of the compounds decreased particularly for Ir₂L². This ability to generate ROS suggested that IrL¹ and Ir₂L² could induce cell death after two-photon excitation. The complexes display a slight bleaching upon TPA illumination in cells, Ir₂L² being the most impacted. The in vitro PDT efficacy of IrL¹ and Ir₂L² with two-photon irradiation was then assessed. Interestingly, cell morphological changes were observed a few hours after two-photon irradiation. As shown in Figure 7B, before laser illumination, A549 cells were well adhered with normal morphological characteristics. However, after illumination with 800 nm fs laser, the cells were rounded or detached, darkened, and with blebbing, all typical morphological features of cell death. By using live/dead staining, in which green calcein-AM and red propidium iodide (PI) stained live and dead cells, respectively, we showed that IrL¹ and Ir₂L² combined with 800 nm fs light irradiation efficiently killed A549 cells in the irradiation area (Figure 7B). Laser irradiation alone did not induce cell death. These results suggested that IrL¹ and Ir₂L² could be effective PS for TPE PDT.
<figure><img src="image_11.png" /><figcaption>Figure 7: IrL¹ and Ir₂L² generate cellular ROS and cell death in A549 cells after two-photon irradiation. (A) Generation of intracellular ROS as detected by DCFH-DA (in green) in A549 cells incubated with or without IrL¹ or Ir₂L² (in red) for 24 h before and following irradiation of a square with 800 nm fs laser for 2 min. (B) Confocal microscopy images showing A549 cells incubated with or without IrL¹ or Ir₂L² (in red) at irradiation time and cell viability using calcein-AM (in green) and propidium iodide (in red) 15 h after *following irradiation of a square with 800 nm fs laser for 2 min.* Scale bars, 50 µm and 10 µm in zoom sections.</figcaption></figure>

Conclusion

We report two Ir(III) complexes for PDT applications, a monomer (IrL¹) and a dimer (Ir₂L²). These complexes have been fully characterized and display phosphorescent emission in the orange-red region of the visible spectrum with high photoluminescence quantum yields. The spectroscopic investigations allow to attribute the emission to the radiative deactivation of the ³MLCT excited state to the ground state. Both complexes can generate ¹O₂ in good quantum yield, especially Ir₂L² of which ΦΔ is nearly 80%. The TPE spectra have been determined for both complexes and the σ<sub>TPA</sub> maximum is dramatically increased for the dimer and has a concomitant bathochromic shift, which is also observed in absorption. Thus, we believe that our results may have an impact in the design of a new
generation of Ir(III) complexes for TPE. Indeed, considering both the molecular ¹O₂ generation efficiency coefficient ζΔ and the figure of merit highlight the advantageous properties of Ir₂L², which dramatically surpasses IrL¹. However, this advantage is not observed in cells, which internalize Ir₂L² more slowly than IrL¹. The latter displays high phototoxicity, with IC₅₀ of 27-34 nmol after 4h of incubation and low illumination power for both cell lines. In addition, TPE appears to generate ROS and cell death with both IrL¹ and Ir₂L². Our results put in light the advantages of designing multinuclear metal complexes for PDT applications by a molecular approach and may be used as guidelines in the field. Further work is still needed on the cell delivery aspects to take full advantage of the multimetallic's photophysical properties.

## Experimental details: general consideration

**Synthesis:** Commercially available reagents were purchased from Sigma-Aldrich, Alfa Aesar, Acros Organics, TCI Chemical, Merck, Strem, Fluorochem, or bldpharma and used as received unless otherwise specified. Solvents were obtained from the same commercial sources and used without further purification. For moisture sensitive reactions, glassware was oven-dried prior to use. ¹H NMR spectra were recorded on a 400 MHz and on a 500 MHz in deuterated solvent (CDCl₃, DMSO-d₆ or CD₂Cl₂) and data are reported as follows: chemical shift in ppm from tetramethylsilane with the solvent as an internal indicator (CDCl₃ 7.26 ppm, CD₂Cl₂ 5.32 ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, p = pentet, hept = heptuplet, m = multiplet or overlap of non-equivalent resonances), J-coupling constants (Hz), and proton integration. ¹³C{¹H} NMR spectra were recorded either at 101 MHz or at 126 MHz in suitable deuterated solvent and data are reported as follows: chemical shift in ppm from tetramethylsilane with the solvent as an internal indicator (CDCl₃ 77.16 ppm, DMSO-d₆ 39.52 ppm, CD₂Cl₂ 53.84 ppm. The ligands, L¹ and L², and the complexes, IrL¹ and Ir₂L², were synthesized according to our reported procedure.⁴⁶

**Electrochemistry:** Cyclic voltammograms were recorded with a BioLogic SP-300 potentiostat. All electrochemical measurements were performed in a standard three-compartment electrochemical cell under an argon atmosphere using tetra-n-butylammonium hexafluorophosphate (TBAPF₆, Aldrich) as supporting electrolyte (0.1 M) in dry CH₃CN. Silver/silver nitrate in CH₃CN (Ag/AgNO₃ 10 mM in CH₃CN, ANE2) was used as reference, counter electrodes was a Pt wire, and the working electrode a 3 mm diameter glassy carbon disk.

**Absorption and emission spectroscopies:** Absorption spectra were recorded on a Cary 300 UV-visible spectrophotometer (Varian) and emission spectra (in solution at room temperature and in rigid matrix at 77 K) were recorded on a Horiba Fluoromax 4 or on FLS-1000 Edinburgh Instrument equipped with a photomultiplier (PMT) detector cooled by a Peltier. Quartz cuvettes with 1 cm optical path were used. Lifetimes were measured using LP900 spectrometer with a Flashlamp pumped by Q-switched Nd:Yag laser operating at 355 nm and with a photomultiplier (PMT) detector, or with a picosecond laser diode operating at 405 nm and using a time-correlated single photon counting detection (TCSPC, PicoHarp 300). Singlet oxygen emission was recorded using FLS-1000 Edinburgh Instrument equipped with a InGaAs detector, with quartz cuvettes with 1 cm optical path were used.

Emission quantum yields in solution were determined in deaerated CH₂Cl₂ solution at room temperature and calculated with the following equation.

$$QY_s = QY_{\text{ref}} \times \frac{I_s}{I_{\text{ref}}} \times \frac{A_{\text{ref}}}{A_s} \times \left( \frac{\eta_s}{\eta_{\text{ref}}} \right)^2$$

QYs corresponds to the quantum yield of the sample to be analysed, QYref is the quantum yield of the reference compound (in this work is Ru(bpy)₃²⁺ in air equilibrated CH₃CN solution with value of 0.018±0.002)⁶⁸, I corresponds to the intensity of the emission (area of the spectrum), A is the absorption at the excitation wavelength, η corresponds to the refractive index of the solvents.
The two properties that provide information about the excited state beside the shape of the emission are the QY (Φ) and lifetime (τ), represented in the following equations.

$$ \Phi = \frac{k_r}{k_r+k_{nr}} \quad \tau = \frac{1}{k_r+k_{nr}} $$

The radiative (k<sub>r</sub>) and non-radiative (k<sub>nr</sub>) constants can be obtained from combination and rearrangement of the QY (Φ) and τ equations to give equations:

$$ k_r = \frac{\Phi}{\tau} \quad k_{nr} = \frac{1}{\tau} - k_r $$

Singlet oxygen generation yields in solution were determined using the Stern-Vollmer equation in deaerated CH<sub>3</sub>Cl solution at room temperature and calculated with the following equation.

$$ f\Delta_x = f_{\Delta ST} \times \frac{\text{Gradx}}{\text{GradST}} \times \left( \frac{\eta_s}{\eta_{\text{ref}}} \right)^2 $$

$\Phi_{\Delta x}$ corresponds to the singlet oxygen generation quantum yield of the sample to be analysed, $\Phi_{\Delta ST}$ is the singlet oxygen generation quantum yield of the standard compound (in this work is phenalenone in air equilibrated CH<sub>3</sub>Cl solution with value of 0.98), Grad the gradient from the plot of integrated fluorescence intensity vs absorbance η corresponds to the refractive index of the solvents.

## Two-photon experiments

The two-photons absorption cross-sections, $\sigma_{TPA}$ of IrL<sup>1</sup> and Ir<sub>2</sub>L<sup>2</sup> were measured by the two-photons excited fluorescence (TPEF) technique. This method consists of measuring the two-photons absorption induced emission and comparing it to that of reference compounds with known $\sigma_{TPA}$.⁶⁹ The TPEF experiments were performed using a home-made setup. The excitation source was a tunable, femtosecond Ti-sapphire laser (Chameleon Ultra II, Coherent, 80 MHz), which was focused on a quartz spectroscopic cell containing the sample. The emitted light was collected at right-angle relative to the excitation beam and after removal of the scattered laser light with a low-pass filter, it was coupled to an optical fiber connected to CCD-spectrometer. The cell position was adjusted to reduce the optical path length of the exciting and emitted light in the cell reducing absorption by the solvent and inner-filter effect respectively.

The samples were dissolved in dichloromethane and continuously stirred during the experiment to avoid eventual photodegradation. The measurements were performed in aerated conditions.

The two-photons cross section was evaluated at several excitation wavelengths by tuning the excitation laser. The measured luminescence spectra were spectrally corrected. The two-photons cross section values were calculated in comparison to a reference compound as follows:

$$ \sigma_{TPA, X} = \frac{\Phi_R \cdot P^2_R \cdot c_R \cdot A_X \cdot I_X^m}{\sigma} \quad (1) $$

where I<sup>m</sup> corresponds to the maximum of the luminescence intensity, A is the normalized and integrated fluorescence spectrum, φ corresponds to the fluorescence quantum yield, c is the fluorophore concentration and P is the incident average laser power. The indices X and R denote the unknown and reference compounds respectively. Fluorescein (5x10<sup>-5</sup> mol·L<sup>-1</sup>) dissolved in deuterated water at pH 13 was used as a two-photons absorption cross-section reference.⁷⁰
## Synthesis of the complexes IrL¹ and Ir₂L²:⁴⁶

General procedure: [μ-Cl₂(Ir(ppy)₂)]⁴⁶ (ppy = 2-phenylpyridine) was suspended in a mixture of CH₂Cl₂/MeOH (1:1) and the ligand (2 eq. in the case of IrL¹ and 1-0.9 eq. for Ir₂L² was added. The yellow solution became rapidly orange after a few minutes and the reaction was refluxed overnight under Ar. At r.t., the solvent was concentrated to the 1/3rd, an excess of aqueous KPF₆ (sat.) was added drop-by-drop and an orange precipitate appeared rapidly, and the suspension was stirred an additional 10 min. The solution was filtrated off with a millipor® apparatus. The solid was washed with water, ice-cold methanol (5 mL) and finally with ice cold diethylether. The reaction gave clean complexes which were isolated as a dark yellowish to orange-red solid.

**IrL¹:** [μ-Cl₂(Ir(ppy)₂)] (50 mg, 0.05 mmol) was suspended in a mixture of CH₂Cl₂/MeOH (10 mL, 1:1) and the ligand L¹ (32 mg, 0.09 mmol) was added. The refluxing yellow solution became rapidly orange after few minutes and the reaction was stirred overnight under Ar. At r.t., the solvent was concentrated to the 1/3rd, an excess of aqueous KPF₆ (sat.) was added drop-by-drop and an orange precipitate appeared rapidly, and the suspension was stirred an additional 10 min. The solution was filtrated off with a Millipor® apparatus. The solid was washed with water (10 mL), ice-cold methanol (5 mL) and finally with ice cold diethylether (15 mL). IrL¹ was obtained pure after recrystallization by vapor diffusion of diethylether to a concentrated CH₂Cl₂ solution as a yellow powder. (87 mg, 93%). ¹H NMR (500 MHz, CDCl₃) δ 8.34 (dd, J = 8.8 Hz, J = 2.2 Hz, 1H), 8.26 (d, J = 8.8 Hz, 1H), 8.06 (d, J = 2.1 Hz, 1H), 7.98 (d, J = 8.2 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.81 – 7.70 (m, 4H), 7.62 (dd, J = 5.7 Hz, J = 1.6 Hz, 1H), 7.52 (dd, J = 5.7 Hz, J = 1.8 Hz, 1H), 7.35 (s, 1H), 7.13-7.09 (m, J = 7.6 Hz, J = 4.5 Hz, 1.2 Hz, 2H), 7.05 – 6.91 (m, 4H), 6.89 (dd, J = 8.7 Hz, J = 1.5 Hz, 1H), 6.37 (dd, J = 7.7, 1.2 Hz, 1H), 6.33 (dd, J = 7.6 Hz, J = 1.2 Hz, 1H), 6.23 (d, J = 8.6 Hz, 1H), 4.81 – 4.67 (m, 2H), 2.45 (s, 3H), 2.03 – 1.90 (m, J = 6.7 Hz, 2H), 1.47 (h, J = 7.5 Hz, 2H), 0.99 (t, J = 7.3 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 168.2, 168.1, 153.5, 151.0, 150.6, 149.8, 149.0, 146.8, 146.4, 144.5, 144.4, 142.8, 138.6, 138.3, 138.1, 138.0, 137.3, 132.2, 132.0, 131.3, 130.5, 128.0, 125.6, 125.5, 125.0, 124.7, 123.8, 123.6, 123.3, 123.0, 120.3, 119.8, 118.3, 111.4, 46.3, 32.2, 22.2, 20.4, 13.9.

**Ir₂L²:** [μ-Cl₂(Ir(ppy)₂)] (50 mg, 47 mmol), ligand L² (31 mg, 47 mmol) and solvent 10 mL. The Ir₂L² mixture was isolated after recrystallisation by vapor diffusion of diethylether to a CH₂Cl₂ solution, as a dark yellowish solid (72 mg, 79%). ¹H NMR (400 MHz, CD₃)₂CO) δ 8.76 (d, J = 8.7 Hz, 2H), 8.57 (d, J = 8.9 Hz, 3H), 8.28 (t, 1H J = 8.0 Hz), 8.21 (s, 1H), 8.16 (dd, J = 8.2 Hz, 2H), 8.10 – 8.03 (m, 3H), 8.02 – 7.93 (m, 8H), 7.90 (t, J = 7.7 Hz, 2H), 7.26 (dd, J = 7.4 Hz, 2H), 7.19 – 6.95 (m, 10H), 6.83 (d, J = 8.2 Hz, 3H), 6.52 (t, J = 6.0 Hz, 2H), 6.41 (d, J = 7.7 Hz, 2H), 5.10 (d, J = 5.1 Hz, 2H), 1.56-1.40 (m, 4H), 0.98 (t, J = 7.4 Hz, 6H). ¹³C NMR (101 MHz, CD₃)₂CO) δ 172.9, 172.7, 157.8, 157.5, 156.1, 155.3, 155.1, 155.0, 152.3, 151.4, 150.1, 149.6, 147.6, 145.3, 143.9, 143.6, 141.8, 141.5, 137.7, 136.8, 135.7, 135.1, 131.7, 130.7, 130.3, 130.0, 129.4, 128.8, 128.0, 127.8, 125.1, 124.7, 117.8, 51.1, 36.9, 24.8, 18.3.

## Electrochemistry

Cyclic voltammograms were recorded with a BioLogic SP-300 potentiostat. All electrochemical measurements were performed in a standard three-compartment electrochemical cell under an argon atmosphere using tetra-n-butylammonium hexafluorophosphate (TBAPF₆, Aldrich) as supporting electrolyte (0.1 M) in dry CH3CN. Silver/silver nitrate in CH3CN (Ag/AgNO₃ 10 mM in CH3CN, ANE2) was used as reference, counter electrodes was a Pt wire, and the working electrode a 3 mm diameter glassy carbon disk.

## Cell Lines and culture
The human lung adenocarcinoma A549 and colon carcinoma HT29 cell lines were obtained from LGC standard (Molsheim, France). All cells were routinely tested for the presence of mycoplasma (Mycoalert® Mycoplasma Detection Kit, Lonza, Rockland, ME, USA) and used within three months after thawing. Cells were maintained in culture at 37 °C in RPMI-1640 medium (A549) or in DMEM medium (HT29), with 10% FBS in a 5% CO2 humidified atmosphere. The cell morphology was routinely checked.

## Cellular uptake

Cells were plated on labtek® and treated with 1 µmol.L⁻¹ IrL¹ or Ir₂L² for 2 h, 4 h, or 24 h in the dark at 37 °C, or for 4 h at 4 °C, before confocal imaging. Hoechst (5 µmol.L⁻¹) was used to counterstain the cell nuclei. Fluorescence microscopy was carried out using a confocal microscope (LSM 710; Carl Zeiss, Jena, Germany). An objective Plan Apochromat 20x/0.8 NA in air and an objective Plan Apochromat 63x/1.4 NA in oil were used. The excitation was at 405 nm and emission filter between 600 and 760 nm for IrL¹ and Ir₂L², and between 415 and 500 nm for Hoechst. The cells were also collected and quantified by flow cytometry (Attune Nxt, Thermo Fisher Scientific, New York city, NY, US). Flow cytometric analysis was performed using FCS Express 7 software (De Novo Software, Dotmatics, Pasadena, CA, US).

## Subcellular localization

Cells were plated on labtek® and treated with 1 µmol.L⁻¹ IrL¹ or Ir₂L² for 24 h in the dark at 37 °C. The cells were then stained with 25 nmol.L⁻¹ MitoTracker Orange and 120 nmol.L⁻¹ LysoTracker blue (Invitrogen, ThermoFisher Scientific) for 30 min at 37°C. The cells were then washed with PBS to eliminate any excess dye and were immediately examined using a Stellaris confocal microscope. A 63x objective (HC PL APO CS2, 1.2 water) was used. The fluorescence images from dyes were collected in the range λ<sub>em</sub> = 410-455 nm (λ<sub>exc</sub> = 405 nm) for LysoTracker, and in the range λ<sub>em</sub> = 556-642 nm (λ<sub>exc</sub> = 551 nm) for MitoTracker. Both IrL¹ and Ir₂L² were imaged in the range λ<sub>em</sub> = 670-800 nm (λ<sub>exc</sub> = 405 nm).

## Irradiation of the cells

Illumination at 420 nm with power output at 78.125 mW.cm⁻² (1 min and 4 s, fluency 5 J.cm⁻²; 2 min and 8 s, fluency 10 J.cm⁻²; and 5 min and 20 s, fluency 25 J.cm⁻²) was performed after treatment using Lumidox® II 96-position LED Array (Anaytical Sales and Services, Flanders, NJ, US). Two-photon illumination was performed at λ= 800 nm with a Ti:Sapphire laser (Chameleon Vision II, Coherent, > 3 W, 140 fs pulses, 80MHz repetition rate).

## ROS generation in cells

A549 cells were seeded overnight for adherence and then incubated with IrL¹ and Ir₂L² for 24 h. Twenty-five µmol.L⁻¹ 2',7'-dichlorofluorescein diacetate (H2DCFH-DA, Sigma-Aldrich, France) were then incubated for 30 min. Hoechst (5 µmol.L⁻¹) was used to counterstain the cell nuclei. The fluorescence images before and after 420 nm irradiation were acquired using a Stellaris 8 confocal microscope equipped with a white laser (Leica Microsystems). A 10x objective (HC PL APO, NA=0.40) was used to collect the light. The emission from the Hoechst dye was collected in the range λ<sub>em</sub> = 412-464 nm (HyD detector), upon excitation with the 405 nm laser diode. Both photosensitizers were imaged using the same excitation diode (λ<sub>exc</sub> = 405 nm) and the emission was collected within the range λ<sub>em</sub> = 670-800 nm (HyD detector). The emission of the DCF dye was measured upon excitation at λ<sub>exc</sub> = 491 nm and the light was collected in the range λ<sub>em</sub> = 500-547 nm (HyD detector). Image analysis was conducted using the “Analysis” module within the LasX software of the Stellaris microscope. Each cell was individually identified and the mean intensity of emission of the DHF dye was quantified per cell. The fluorescence images before and after 800 nm irradiation were acquired using a confocal microscope (LSM 710, Zeiss) with a 40x/1.2 W Plan Apochromat objective.
## In vitro cytotoxicity assays

Cell proliferation assays were conducted in 96-well culture plates. Cells were cultured for 24 h prior to treatment with increasing concentration of IrL¹ or Ir₂L² for in complete medium. Illumination at 420 nm was performed 4 h or 24 h after treatment. The cell viability was quantified 72 h after light exposure with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT assay). Cell viability was normalized to control cells (no drug and un-illuminated). The drug concentrations required to inhibit cell growth by 50% (IC₅₀) were determined by interpolation from the dose to response curves.

For live/dead cell staining, A549 cells were incubated with IrL¹ or Ir₂L² for 24 h, irradiated at 800 nm, and stained with 2 µmol.L⁻¹ calcein-AM (Invitrogen, ThermoFisher Scientific) and 3 µmol.L⁻¹ PI (Sigma-Aldrich) after 15h.

## ASSOCIATED CONTENT

Supporting Information is material is available free of charge via the Internet at http://pubs.acs.org.
The reader will find synthesis scheme overviews, information about the Structural characterization,
and the NMR spectra.

## Author Contributions

The manuscript was written through contributions of all authors.

## ACKNOWLEDGMENT

This work is supported by the French National Research Agency in the framework of the "Investissements d'avenir" program (ANR-15-IDEX-02). This work has been partially supported by CBH-EUR-GS (ANR-17-EURE-0003). The authors would like to thank Kamilla Mammadzada and Dr. Maria V. Vlasova who participated with enthusiasm to the project during their internships. The Nano-Bio ICMG (UAR 2607), is acknowledged for providing facilities for mass spectrometry (A. Durand, L. Fort and R. Gueret) and single crystal X-ray diffraction (N. Altounian). A-L.B. acknowledges the support of the European Research Council (ERC STG, RADIANCE, 101116304). We acknowledge the MicroCell facility (GIS IBiSA, ISdV, IAB), member of the national infrastructure France-BioImaging supported by the French National Research Agency (ANR-24-INBS-0005 FBI BIOGEN) for optical microscopy. Views and opinions expressed are however those of the authors only and do not necessarily reflect those of the European Union or the European Research Council Executive Agency. Neither the European Union nor the granting authority can be held responsible for them.

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