Liang Tian

Ivane Bottala-Gambetta

Victor Marchetto

Manoël Jacquemin

Alexandre Crisci

Roman Reboud

Arnaud Mantoux

Frédéric J Mercier

Gregory Berthomé

André Sulpice

Gregory Berthome

Laetitia Rapenne

François Weiss

Carmen Jimenez

Elisabeth Blanquet

Improved critical temperature of superconducting plasma-enhanced atomic layer deposition of niobium nitride thin films by thermal annealing

Keywords:

Introduction

Niobium nitride (NbN) thin films exhibit excellent physical, chemical, and electrical properties that are adaptable to applications such as hard surface coatings [START_REF] Benkahoul | Structural and mechanical properties of sputtered cubic and hexagonal NbN x thin films[END_REF][START_REF] Chen | Hard superconducting nitrides[END_REF][START_REF] Delacour | Superconducting single photon detectors made by local oxidation with an atomic force microscope[END_REF][START_REF] Hazra | Superconducting properties of very high quality NbN thin films grown by high temperature chemical vapor deposition[END_REF] or superconducting radio frequency cavities. NbN thin films can be deposited by physical vapor deposition (PVD), chemical vapor deposition (CVD), and atomic layer deposition (ALD). ALD, which is based on a surface reaction leading to a self-limiting, layer-by-layer growth, is the most favorable method for growing conformal films on high-aspect-ratio structures at relatively low temperatures [START_REF] Suntola | Atomic layer epitaxy[END_REF]. The importance of the ALD process for obtaining NbN film layers depends on the potential improvement of the efficiency of Nb-based superconducting radio frequency cavities by coating the internal surfaces with a multilayer coating of superconductor-insulator-superconductor [START_REF] Gurevich | Enhancement of rf breakdown field of superconductors by multilayer coating[END_REF][START_REF] Antoine | Study of nanometric superconducting multilayers for RF field screening applications[END_REF]. This application requires a controlled preparation of an atomically precise thickness of NbN on a complex surface.

The deposition of NbN by thermal ALD has been generally performed by alternatively pulsing NbCl 5 and NH 3 [START_REF] Hiltunen | Nitrides of titanium, niobium, tantalum and molybdenum grown as thin films by the atomic layer epitaxy method[END_REF]. In some cases, an intermediate Zn pulse, which is used as an additional reducing agent to increase the Nb reduction from niobium(V) chloride (NbCl 5 ) to niobium(III) nitride (NbN), was also introduced between the NbCl 5 and NH 3 pulses. The resulting NbN films exhibited a resistivity reduction by a factor of 2 [START_REF] Elers | NbCl 5 as a precursor in atomic layer epitaxy[END_REF][START_REF] Ritala | Effects of intermediate zinc pulses on properties of TiN and NbN films deposited by atomic layer epitaxy[END_REF]. The intermediate Zn pulse improved the superconducting critical temperature (Tc) from 4 K to 7.5 K [START_REF] Proslier | Atomic layer deposition of superconductors[END_REF]. Nevertheless, the reducing power of the reactive gases like NH 3 used in thermal ALD is insufficient to obtain stoichiometric nitrides from a metal precursor [START_REF] Ritala | Controlled Growth of TaN, Ta 3 N 5 , and TaO x N y Thin Films by Atomic Layer Deposition[END_REF]. Therefore, plasma-enhanced ALD (PEALD) is used to greatly improve the quality and the density of the films [START_REF] Heil | Deposition of TiN and HfO 2 in a commercial 200 mm remote plasma atomic layer deposition reactor[END_REF]. Furthermore, organometallic and coordination complex precursors are often preferred because halide precursors are susceptible to equipment corrosion and environmental issues.

A prospective molecular precursor for NbN deposition is (tert-butylimido)-tris (diethylamino)-niobium(V) (TBTDEN), which can be combined with H 2 plasma [START_REF] Ziegler | Superconducting niobium nitride thin films deposited by metal organic plasma-enhanced atomic layer deposition[END_REF][START_REF] Ziegler | Effects of Plasma Parameter on Morphological and Electrical Properties of Superconducting Nb-N Deposited by MO-PEALD[END_REF]. However, in this case, the nitrogen originates from the precursor and it is difficult to prevent carbon contamination in the films. In other cases, the TBTDEN is combined with N 2 [START_REF] Deguns | Plasma-Enabled ALD of niobium nitride using an organometallic Nb precursor[END_REF] or H 2 /N 2 [START_REF] Sowa | Plasma-Enabled ALD of niobium nitride using an organometallic Nb precursor[END_REF] plasma as the reducing agent to form NbN. Efforts have been focused on the evolution of film properties with the process parameters (plasma duration, temperature) and film thickness.

Extremely interesting results (Tc: ~14 K for the ~30 nm thick film) have been reported using TBTDEN/H 2 [START_REF] Ziegler | Effects of Plasma Parameter on Morphological and Electrical Properties of Superconducting Nb-N Deposited by MO-PEALD[END_REF][START_REF] Linzen | Structural and electrical properties of ultrathin niobium nitride films grown by atomic layer deposition[END_REF] or TBTDEN/H 2 /N 2 [START_REF] Sowa | Plasma-Enabled ALD of niobium nitride using an organometallic Nb precursor[END_REF][START_REF] Ukibe | Superconducting characteristics of NbN films deposited by atomic layer deposition[END_REF]. Several authors [START_REF] Ziegler | Superconducting niobium nitride thin films deposited by metal organic plasma-enhanced atomic layer deposition[END_REF][START_REF] Linzen | Structural and electrical properties of ultrathin niobium nitride films grown by atomic layer deposition[END_REF] reported that the films may contain relatively high amounts of oxygen and carbon, up to 50 at%. However, high impurity levels make it difficult to make definite conclusions on the relationship between material properties and the superconductivity. Nitrogen atoms can be substituted by carbon to form a solid solution of NbCN in the full composition range. In this case, the structure remains cubic with an improved Tc value for 15-40 at% C [START_REF] Pessall | A study of superconductivity in interstitial compounds[END_REF][START_REF] Shoji | Fabrication of all-NbN Josephson tunnel junctions using single crystal NbN films for the base electrodes[END_REF][START_REF] Francavilla | Superconducting properties of reactively sputtered NbCN thin films[END_REF]. Nitrogen atoms can be also substituted by oxygen.

The oxynitride NbON remained cubic and the Tc increased as the oxygen concentration approached 20 at% [START_REF] Roy | The effect of composition on the superconducting transition temperature in NbNxOy[END_REF][START_REF] Motohashi | Crystal Structure and Superconducting Properties of Hexagonal Lithium-Niobium Oxynitride[END_REF][START_REF] Ohashi | Local structure around the flux pinning centers in superconducting niobium silicon oxynitride[END_REF]. In this study, we used a non-conventional approach (termed supercycle), which consists of a pulse of TBTDEN, followed by a pulse of H 2 plasma. In addition, we added another pulse of NH 3 . The objective of the supercycle was to break the TBTDEN ligands due to the H 2 plasma to facilitate the surface chemical reaction with NH 3 in order to limit the incorporation of carbon and oxygen in the film. This approach was already verified in the PEALD process of the AlN thin films [START_REF] Tian | Aluminum nitride thin films deposited by hydrogen plasma enhanced and thermal atomic layer deposition[END_REF]. After testing the superconducting properties of the asdeposited PEALD films, a post-deposition annealing in Ar was also performed to investigate its influence on the quality of the films and on the Tc. In this paper, the superconducting properties of the films were evaluated using the value of the Tc, with the objective of obtaining a Tc above 9 K, which is the Tc of the pure niobium.

Experimental

NbN films ALD growth

NbN thin films were grown on the (100) silicon substrates in a Picosun R-200 Advanced ALD system, which features thermal ALD and PEALD equipped with an inductively coupled remote plasma (ICP) source. TBTDEN (99.99%, STREM Chemicals) was used as the Nb precursor. The Nb precursor container was maintained at 90 °C and delivered to the system with a precursor Picohot TM boosting system. The boosting system used a Venturi effect generated in the precursor source by the carrier gas to increase or boost the amount of precursor injected into the ALD reactor, substantially improving the transfer of the low vapor pressure material from the cylinder to the substrate surface. Each of the substrates were previously cleaned in an ultrasonic bath in acetone and isopropanol for 15 min before the deposition.

We used N 2 (99.9999%, Air Liquide) as carrier gas to dilute the NH 3 (99.999%, Air Liquide).

The NH 3 and N 2 flow rates were maintained at 100 and 120 mL/min, respectively. The pulse time of the niobium precursors was controlled between 0.5 and 4 s with the boosting function. The NbN growth was obtained by sequentially exposing the heated substrate to the precursor gases in the following order: TBTDEN, N 2 purge/4 s, remote H 2 plasma/variable time, N 2 purge/4 s, NH 3 /5 s, and N 2 purge/4 s. This reacting gas steps plasma H 2 /thermal NH 3 listed in Table 1 as process 1 enable the separation of the surface activation by H 2 from the reaction step with NH 3 . The substrate temperature was varied between 200 and 400 °C to determine the ALD window. Herein, 2000 cycles were performed in all the experiments. In normal operation, the plasma is subjected to an inductive regime; however, a capacitive regime could appear when the injected gas flow is large. These capacitive discharges could damage our reactor; therefore, the gas flow rate was selected within the limit of our reactor [START_REF] Tian | Aluminum nitride thin films deposited by hydrogen plasma enhanced and thermal atomic layer deposition[END_REF].

The maximum gas flow rate in the induction mode is a function of the electrical power delivered by the generator. In this study, the plasma power was fixed at 2800 W, which allows a wide range of flow variation. The Ar flow (80 mL/min) was used as a carrier gas and the H 2 flow rate used to generate the reducing plasma was varied from 40 to 170 mL/min. In this setup, the plasma is functioned in an inductive regime [START_REF] Tian | Aluminum nitride thin films deposited by hydrogen plasma enhanced and thermal atomic layer deposition[END_REF].

NbN films were also grown by a simpler ALD cycle consisting of an NH 3 plasma (170 mL/min) and an Ar carrier gas (80 mL/min). This alternative ALD route constitutes process 2 in Table 1. * The boosting function is performed with two N 2 flow rates: half the pulse time at 80 mL/min then 600 mL/min After the deposition, the films were annealed in a tubular furnace in a controlled Ar atmosphere (99.9999 %, Air Liquide) between 800 and 1200 °C inside an alumina tube with a heating rate of 20 °C/min. The annealing duration was varied between 10 and 60 min. The gas flow rate and base pressure during annealing were 200 mL/min and 800 Pa, respectively.

NbN films characterization

The composition of the NbN films was determined by an Electron Probe Micro-Analyzer (EPMA) using a Cameca SX50 controlled by an SAMx software. Niobium, nitrogen, carbon, and oxygen were analyzed at 3, 5, 7 and 10 kV acceleration voltage, respectively. The thickness and composition of the thin films were extracted from the measurements using the Stratagem software.

The XPS (X-ray Photoelectron Spectroscopy) analyses were conducted using a Thermo Scientific™ K-Alpha™ XPS spectrometer. The sample surfaces were irradiated with Al Kα radiation at 15 kV for a current of 20 mA. To correct any charging effect, all the binding energies were calibrated to the C-C component of the C 1s core spectrum at 285.0 eV. Analyses were conducted at 90° angles between the sample surface and the analyzer, corresponding to an analyzing depth of 8-9 nm. An Ar ion gun (3 keV) was used to etch the surface for 5 min to remove the surface contamination and probe the in-depth composition of the NbN films. Peak deconvolutions were performed using Thermo Scientific Avantage software.

Field emission gun scanning electron microscopy (ZEISS Ultra-55) was used to characterize the surface morphology of the NbN layers. High-resolution transmission electron microscopy (HRTEM) was performed on samples with a JEOL 2010 microscope operating at 200 kV. The samples were prepared for TEM observation by the grating method. X-ray diffraction (XRD) patterns were collected with a Siemens (Bruker) D5000 diffractometer equipped with a Cu source (= 1.5406Å) according to the Bragg-Brentano configuration. The θ-2θ scans were performed between 20° and 75°. X-Ray Reflectivity (XRR) was performed with the same diffractometer operating in the reflectivity mode. Fitting with X-Pert Reflectivity software enables evaluation of the film thickness and density.

Magnetic measurements were performed in the field perpendicular to the largest plane of the sample using a commercial Metronique Ingenierie Superconducting Quantum Interference Device Device (SQUID) magnetometer with a sensitivity of of 10 -10 A.m 2 at low field. After cooling the sample at zero field, the magnetization was first measured at 2 K by increasing the field to to 795.6 A/m. The sample was then cooled at constant field by increasing (Zero Field Cooled) and decreasing (Field Cooled) the temperature. The deviation of these curves with the normal state at high temperature was used to determine Tc.

Results and discussion

ALD window

To optimize the NbN growth process under the conditions described in process 1, two steps were conducted: 1) the TBTDEN pulse duration, and 2) the substrate temperature were varied.

Only these two parameters were considered because they strongly depend on the precursor nature and chemistry. The other step durations (purge, H 2 plasma, and NH 3 step) were optimized in a previous study using the same reactor [START_REF] Tian | Aluminum nitride thin films deposited by hydrogen plasma enhanced and thermal atomic layer deposition[END_REF]. We varied the TBTDEN pulse duration between 0.5 and 4 s using the Picohot TM boosting function. All other parameters were fixed at the values specified for process 1 in Table 1. The optimal pulse duration corresponds to the precursor volume required to react with the entire active surface, characterized by a growth per cycle (GPC) value independent of the pulse duration. The deposition temperature was fixed at 350 °C to optimize this parameter. We postulated that 1 s of the TBTDEN using the boost function is the minimum time required to obtain a reproducible and constant GPC of ~0.01 nm/cycle. As expected, a further increase in the TBTDEN pulse duration does not alter the GPC in a selflimited ALD process. Therefore, 1 s is sufficient for the reactions to occur in the ALD mode.

In the second step, the deposition temperature range was adjusted to maintain a constant GPC independent of the substrate temperature, also called the ALD window. We varied the substrate temperature between 240 and 400 °C kept all the other parameters for process 1 constant, as

shown in Table 1. The GPC and film density as functions of the substrate temperature are shown in Fig. 1. As the substrate temperature increased, the GPC decreased initially, then stabilized to 0.015 nm/cycle between 350 and 380 °C. This GPC value could be defined as the ALD window in our process. A further increase in temperature leads to a decrease in the GPC. The GPC obtained by our process is lower than the value obtained by Ziegler et al. [START_REF] Ziegler | Effects of Plasma Parameter on Morphological and Electrical Properties of Superconducting Nb-N Deposited by MO-PEALD[END_REF], i.e., a saturated GPC of 0.045 nm/cycle for films deposited at 350 °C, using TBTDEN/H 2 plasma. This difference in the GPC might be due to the different growth conditions (such as additional step of NH 3 ) and consequent different film compositions (low amount of C and/or O impurities). For instance, Sowa [START_REF] Sowa | Plasma-Enabled ALD of niobium nitride using an organometallic Nb precursor[END_REF] demonstrated that using TBTDEN and plasma N 2 /H 2 , the ALD window was not observed and an increase in GPC as a function of the substrate temperature was found. Hinz et al.

[27] reported a slight increase in GPC from 0.065 to 0.072 nm/cycle between 200 and 350 °C when using another molecular precursor in the same family as the niobium precursor, i.e., (tertbutylimido)-tris (dimethylamino)-niobium Nb(N-t-Bu)(NMeEt) 3 (TBTMEN).

Importantly, the film density increased continuously as the substrate temperature increased to 350 °C (Fig. 1); further increase in the substrate temperature does not significantly alter the film density. This relationship with temperature may be associated with a low concentration of incorporated impurities like C and O above 350 °C. Another possible explanation is that an increase in the substrate temperature thermally accelerates the chemical reactions. Therefore, denser films are formed, leading to a decrease in GPC. The GPC and film density values in the ALD window for process 1 are 0.015 nm/cycle and 7 g/cm 3 , respectively. For comparison, the bulk NbN density is 8.47 g/cm 3 [START_REF]Handbook of Inorganic Compounds[END_REF].

The ALD growth using process 2 was performed at a deposition temperature of 350 °C to evaluate the effect of the ammonia plasma. The GPC and the NbN thin film density obtained in these conditions are 0.0245 nm/cycle and 7.2 g/cm 3 , respectively. The PEALD deposition of NbN using an NH 3 plasma has a higher growth rate and a slightly higher density, which might be due to the higher reducing power of NH 3 as compared to that of the H 2 plasma.

The films deposited using process 1 (H 2 plasma/NH 3 ) and process 2 (NH 3 plasma) were characterized by XPS to assess the complete decomposition of the precursor. Fig. 2 shows the C 1s peak of the NbN samples deposited at 330 and 350 °C by process 1, and at 350° C by process 2. The peak located at ~282.8 eV was assigned to the presence of C-Nb bonds because only the metal-carbon bonds could be assigned to such a low binding energy [START_REF] Kovács | X-ray photoelectron spectroscopic study of magnetron sputtered carbon-nickel composite films[END_REF]. Fig. 2 shows that the area of the carbon peak is most pronounced in the sample deposited at a lower temperature (330 °C) by process 1. The presence of the C-Nb bonds suggests an incomplete reaction between TBTDEN and H 2 plasma, NH 3 reactive. At 350 °C, the intensity of this peak decreases, whereas no C-Nb bonds exist in the sample deposited by the NH 3 plasma (process 2) at the same temperature. Therefore, we fixed the substrate temperature at 350 °C for the remainder of the study to have carbon-free NbN samples.

Based on the literature, deposition was only obtained with plasma assistance. In thermal ALD, the NH 3 step itself is not sufficiently reducing to ensure deposition from TBTDEN, which has previously been reported by Ritala et al. as Nb chloride precursors [START_REF] Ritala | Controlled Growth of TaN, Ta 3 N 5 , and TaO x N y Thin Films by Atomic Layer Deposition[END_REF].

After parameter optimization, to study the influence of the different plasmas on the superconducting properties of the NbN films, four samples were deposited for two plasma durations using processes 1 and 2 for 2000 ALD cycles. The measured thickness of all samples is between 30 and 50 nm.

 Samples S1 and S2 were deposited using process 1 with H 2 plasma pulse durations of 12 and 24 s, respectively, followed by an NH 3 step of 5 s.

 Samples S3 and S4 were deposited using process 2 with NH 3 plasma pulse durations of 12 s and 24 s, respectively.

We studied these four different experimental conditions and repeated each experiment five times.

No differences were observed among the samples deposited under identical conditions.

Henceforth, we used the same label for the same deposition conditions.

Crystallographic phase

θ-2θ scans were performed on samples S1 to S4 and results are shown in Fig. 3. The samples were tilted slightly with 2° to decrease the contribution of the Si substrates. The peak centered at 70° is due to Si substrates after tilting. Peaks were ascribed to the (111), ( 200), (220), and (222) diffraction planes of the δ-NbN cubic fcc structure (ICDD: 04-004-2895). This reported phase is associated with a chemical composition of NbN 0.98 with a lattice parameter of a = 0.439 m [START_REF] Christensen | Preparation and structure of stoichiometric -niobium nitride[END_REF].

From the HRTEM images and selected area electron diffraction (SAED) of sample S2 shown in Fig. 4a and4c, the films were not completely crystallized, whereas the NbN cubic phase with an Fm-3m structure was embedded in a matrix of the amorphous phase. The estimated mean size of the crystallites is 10 nm.

Critical temperature

The superconducting behavior was tested for all the samples and a superconducting transition at 3 K was only detected for sample S2. All other samples had a Tc below 2 K. To understand the origin of these low values of Tc and to increase the superconducting properties of the films, the thermal treatments in the Ar atmosphere were conducted at different temperatures ranging from 800-1200 °C for a duration between 10 and 60 min. The samples annealed in the Ar atmosphere at 1000 °C for 30 min exhibited the optimum Tc; Fig. 5 shows the measurements (SQUID method) for the same set of samples, which are renamed with an "A" to distinguish the asdeposited samples. The Tc of all the samples significantly improves and sample S2-A has the highest Tc (13.8 K), which is the same as the highest value reported in the literature for samples prepared by a thermal or a plasma-enhanced ALD process. This value was validated by R(T) measurements. S3-A reveals a Tc of 11.6 K, which is similar to that of S1-A (11.1 K). S4-A exhibits the lowest Tc value (10.1 K). The evolution of the Tc of all our samples with annealing differs from that of several previously published results, where it is shown that annealing lowers the Tc [START_REF] Ukibe | Superconducting characteristics of NbN films deposited by atomic layer deposition[END_REF][START_REF] Hatano | Effects of thermal annealing on superconducting Nb and NbN films[END_REF]. Other publications show that annealing between 800 and 1000 ° C increases the Tc of the NbN samples [START_REF] Oya | Transition temperatures and crystal structures of single-crystal and polycrystalline NbNx films[END_REF][START_REF] Shy | Superconducting properties, electrical resistivities, and structure of NbN thin films[END_REF][START_REF] Guziewicz | Technology of ultrathin NbN and NbTiN films for superconducting photodetectors[END_REF].

To understand why Tc improved significantly after annealing and to establish the contribution of film crystallinity, density, or composition, we characterized the as-deposited and annealed samples by SEM, XRD, XPS, and EPMA.

Effect of annealing in argon

SEM was used to examine the surface morphology of the samples. No cracks were observed in any sample after thermal annealing. Fig. 6 shows the SEM images of sample S2 before and after annealing. Sample S2 consisted of small round grains and no difference in the surface morphology was observed before and after annealing. The same result was observed for all the samples. However, we noticed an increase in the nanocrystal size (lateral size) from 10 nm (Fig. 6a) before annealing to ~30 nm after annealing (Fig. 6b). This result was confirmed by HRTEM Å). However, the crystallinity of all the samples increased significantly, as indicated by the strong (111) peak compared to sample before annealing. This result is consistent with the increase in the signal intensity in SAED (Fig. 4d), as previously discussed. All the samples have a (111) preferred orientation. The lattice parameters of samples S1 to S4 before and after annealing were calculated and plotted in Fig. 8.

The lattice parameters are smaller than the theoretical ones (a = 4.39 Å), as recorded by selected ICDD 04-004-2895 crystallographic data. The lattice parameter of the as-grown samples is extremely small for S1, S2, and S3, and is similar to the theoretical value for S4. The peak shift observed in Fig. 3 was attributed to the composition difference rather than to the stress in the film.

The lattice parameters of the annealed films exhibit similar values. From this perspective, no differences among the four samples could be established. Previous reports [START_REF] Linzen | Structural and electrical properties of ultrathin niobium nitride films grown by atomic layer deposition[END_REF][START_REF] Wang | Superconducting properties and crystal structures of single-crystal niobium nitride thin films deposited at ambient substrate temperature[END_REF][START_REF] Kang | Fabrication and characterization of NbN, AlN and NbN/AlN/NbN on MgO substrates[END_REF][START_REF] Kidszun | Ion-beam-assisted deposition of textured NbN thin films[END_REF] on NbN thin films obtained by PVD or ALD processes demonstrated that the superconducting properties of the NbN thin films are strongly dependent on its lattice parameter; moreover, a larger value leads to a higher Tc. The lattice parameter depends on the chemical composition and vacancies of Nb or N, and the presence of O reduces the lattice parameter [START_REF] Hatano | Effects of thermal annealing on superconducting Nb and NbN films[END_REF]. In our study, the four annealed samples exhibited extremely similar lattice parameters after annealing. Therefore, there is no correlation between Tc and the lattice parameter, which contradicts other reports. We can conclude that, apart from the lattice parameter, there are other more significant factors that influence the Tc of the films.

The film density was measured by XRR for samples S1-S4 before and after annealing. Fig. 9,

shows that the density of the samples increased after annealing and S2 exhibited the highest density among all of the samples.

The stoichiometry and composition of the films were also directly associated with the superconducting properties of NbN. It is important to achieve an optimal Nb/N ratio for the highest Tc, and to understand the contribution of carbon and oxygen. The chemical compositions of the NbN samples before and after annealing were measured by the electron probe microanalyzer (EPMA). The atomic ratios of Nb/N and the at% O atomic concentrations are shown in Fig. 10a andb. Annealing in Ar improved the stoichiometry of the NbN, approaching values close to 1. Importantly, annealing did not increase the oxygen content in the films, which remained low (from 14 at% to 5 at%). From this composition analysis, the origin of the low Tc (< 3 K) before annealing could be correlated with the absence of carbon in our films. This correlates with the results obtained by Proslier et al. where the NbN films grown from NbCl 5 (i.e., carbonfree films) also exhibit a Tc value below 3 K [START_REF] Proslier | Atomic layer deposition of superconductors[END_REF]. The carbon enhanced the Tc and, based on existing information in previous studies on the ALD synthesis of NbN [START_REF] Ziegler | Superconducting niobium nitride thin films deposited by metal organic plasma-enhanced atomic layer deposition[END_REF][START_REF] Ziegler | Effects of Plasma Parameter on Morphological and Electrical Properties of Superconducting Nb-N Deposited by MO-PEALD[END_REF][START_REF] Deguns | Plasma-Enabled ALD of niobium nitride using an organometallic Nb precursor[END_REF][START_REF] Sowa | Plasma-Enabled ALD of niobium nitride using an organometallic Nb precursor[END_REF][START_REF] Linzen | Structural and electrical properties of ultrathin niobium nitride films grown by atomic layer deposition[END_REF][START_REF] Ukibe | Superconducting characteristics of NbN films deposited by atomic layer deposition[END_REF], the presence of carbon as an impurity in the classical ALD process could significantly impact the superconductivity. Therefore, the improvement of the Tc of our carbon-free samples after annealing is related to another parameter. Hence, XPS was performed on sample S2 before and after annealing, which demonstrated the optimum superconducting properties. The samples were first etched by argon ions for 5 min. The spectra were fitted by Gaussian-Lorentzian functions.

The fitting for the Nb 3d state took into account a spin orbital splitting of 2.72 eV of the 3/2 and 5/2 components with an area ratio of 2:3. The Nb 3d spectrum was fitted with the three chemical states of Nb according to a report by Halbritter et al. [START_REF] Halbritter | Angle-resolved XPS studies of oxides at NbN, NbC, and Nb surfaces[END_REF] and the values of the binding energies of Nb 3d5/2 are indicated in Fig. 11. These three components were assigned to Nb-N (204 eV), Nboxynitride (205.3 eV), and Nb-oxide (207.1 eV), which were also retrieved in N 1s and O 1s spectra. Similar results were observed by Ziegler for the optimized conditions and they fitted the Nb 3d spectra with four contributions; the additional one was attributed to Nb-carboxide, which was not observed in our case [START_REF] Ziegler | Effects of Plasma Parameter on Morphological and Electrical Properties of Superconducting Nb-N Deposited by MO-PEALD[END_REF]. In the O 1s spectra, the contributions of niobium oxide and niobium oxynitride are observed. Notably, a third contribution (blue line in the XPS spectra) was not clearly identified. For this contribution, the signature of Nb-carbonitride might be considered in the O 1s spectra, but no carbon (C 1s) was observed.

It can be concluded that the annealing step did not alter the sample composition; in particular, the O contamination was the same before and after annealing, which is in agreement with the EPMA results. Nevertheless, the improvement of the superconducting properties could be clearly related to the transformation of niobium oxide into niobium oxynitride [START_REF] Brauer | Oxidnitride als Katalysatoren zur Methanaktivierung[END_REF], as deduced from the XPS results. Additionally, as previously discussed, the grain growth induced by annealing could also contribute to the enhanced Tc. The understanding of each contribution requires further investigation.

With respect to the comparison of the plasma nature and duration, the H 2 plasma for 24 s is presumably the best compromise because it yields the optimal result before and after annealing.

The oxynitride formation and resulting excellent Tc result may have originated from the formation of Nb in the metallic state during the hydrogen pulse (followed by the TBTDEN pulse), which is easily oxidized to form oxynitride. This latter point is supported by thermodynamic considerations using Factsage 7.2 thermodynamic software; the FactPS database demonstrates the strongest affinity for oxygen of niobium compared to that for niobium nitride:

ΔG reaction (Nb->Nb 2 O 5 ) = -944 kJ/at (@ 500 K) ΔG reaction (NbN->Nb 2 O 5 ) = -711 kJ/at (@ 500 K).

Conclusions

In this study, we presented an ALD approach which consists of combining TBTDEN as the niobium precursor with a supercycle of plasma H 2 followed by a pulse of NH 3 . The objective of this step was to break the TBTDEN ligands due to the H 2 plasma and to facilitate the surface chemical reaction with NH 3 . The ALD window of this was obtained between 350 and 380 °C with a GPC of 0.015 nm/cycle. Under these conditions, the NbN film density was 7 g/cm 3 . At lower temperatures, the decomposition of TBTDEN did not complete as presumed from the XPS contribution in C 1s associated with the binding of Nb-C. The NbN films were also grown at 350

°C by a PEALD process of TBTDEN and NH 3 plasma and the effect of plasma duration was performed to enable comparison.

The superconducting properties of the as-deposited carbon-free NbN films were not satisfactory; hence, an annealing treatment in argon at 1000 °C was conducted. All the annealed samples exhibited a superconducting behavior with a Tc above 10 K. The optimum Tc (13.8 K)

was obtained for a sample grown with the supercycle approach.

The structural characterization and variation in the chemical state of the elements in the film indicated that the improved Tc was due to a partial transformation of the niobium oxide into niobium oxynitride in the NbN film, with no change in the impurity level. However, the reduction of the grain boundaries induced by the grain growth cannot be excluded. Finally, the ability of an RF cavity to sustain thermal annealing should be investigated. The technical aspects of this heat treatment require consideration. We expected the NbN layer to function as a diffusion barrier to prevent the diffusion of Ar in Nb, which degrades the performance of the cavities [START_REF] Grassellino | Nitrogen and argon doping of niobium for superconducting radio frequency cavities: a pathway to highly efficient accelerating structures[END_REF].

Figure captions

Fig

(

  Fig. 7 depicts the θ/2θ diffraction patterns of samples S1-S4 after annealing. Diffraction

Fig. 3 θ

 3 Fig. 3 θ/2θ X-ray diffraction pattern of samples S1, S2, S3, and S4. Samples were tilted by 2° to decrease the silicon contribution.

Fig. 4 (

 4 Fig. 4 (a) Bright-field HRTEM image of the as-grown S2 sample. (b) Bright-field HRTEM image of the S2 sample annealed at 1000 °C for 30 min in Ar. (c) SAED pattern of the as grown sample sample (same sample than a)) compared to the theoretical pattern for the NbN cubic structure. (d) SAED pattern of the S2 annealed at 1000°C for 30 min in Ar sample (same sample than b)).

Fig. 5 Fig. 8

 58 Fig. 5 Tc measurement of samples S1 to S4 after annealing in argon at 1000 °C for 30 min Fig. 6 SEM morphology of the sample S2: (a) as-grown and (b) after annealing in argon at 1000 °C for 30 min Fig. 7 θ/2θ X-ray diffraction pattern using a 2° tilt of samples S1-S4 after annealing in argon at 1000 °C for 30 min Fig. 8 Lattice parameters calculated from the XRD patterns of samples S1-S4 before and after annealing in argon at 1000 °C for 30 min.

Fig. 9

 9 Fig. 9 Density of samples S1-S4 before and after annealing measured by XRR Fig. 10 EPMA results of samples S1-S4 before and after annealing in argon at 1000 °C for 30 min: a) atomic ratio between Nb and N+O, b) oxygen atomic percentage.

Fig. 11

 11 Fig. 11 Core-level XPS spectrum of the Nb 3d and O 1s states of sample S2: a) as-grown and b) after annealing in argon at 1000 °C for 30 min. The Nb 3d spectra were fitted by three chemical states of Nb and the binding energies of Nb 3d5/2 are indicated.

  Figure 1

Table 1 :

 1 Parameter values for ALD processes 1 and 2.

	ALD Steps	T (°C)

  1 Growth per cycle (GPC) and density of the NbN films deposited on the Si substrates by PEALD as a function of the substrate temperature. Plasma power: 2800 W, H 2 flow rate: 80 mL/min. The other growth conditions are listed in Table 1 process 1.Fig 2 C 1s peak of the as-grown NbN samples deposited at 330 and 350 °C by process 1 (with H 2 and NH 3 plasma.), and at 350 °C by process 2 (with NH 3 plasma). The XPS characterization was performed on the samples etched by Ar ions for 5 min to remove the oxide and contamination on the top surface.

Acknowledgements

The authors wish to thank S. Coindeau, H. Roussel, and T. Encinas for detailed discussions of the XRD results and C. Antoine and T. Proslier for discussions on the superconducting radio frequency device criteria. This work was supported by the Investments for the Future Program and was conducted within the framework of the Centre of Excellence of Multifunctional Architectured Materials [CEMAM, grant number ANR-10-LABX-44-01]. This work was also partly supported by EuCARD-2 (312453) project (Enhanced European Coordination for Accelerator Research & Development).

Binding energy (eV)


Johannes Karges

Franz Heinemann

Marta Jakubaszek

Federica Maschietto

Chloé Subecz

Mazzarine Dotou

Robin Vinck

Olivier Blacque

Mickaël F Tharaud

Bruno Goud

Emilio Viñuelas Zahínos

Bernhard Spingler

Ilaria Ciofini

Gilles Gasser

Rationally Designed Long-Wavelength Absorbing Ru(II) Polypyridyl Complexes as Photosensitizers for Photodynamic Therapy

Keywords: Anticancer, Bioinorganic Chemistry, Medicinal Inorganic Chemistry, Metals in Medicine, Photodynamic Therapy

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Photodynamic Therapy (PDT) is a non-invasive medical technique for the treatment of various types of cancer (e.g., lung, bladder, esophageal, and brain cancer) as well as bacterial, fungal or viral infections. The effect of PDT relies on the combination of an ideally non-toxic molecule, a so-called photosensitizer (PS), oxygen, and light. The PS is injected either systemically or locally. Upon light irradiation, the PS is uplifted to an excited singlet state from which the PS can undergo an intersystem crossing process to reach an excited triplet state. This state can influence the biological environment either by a Type I or Type II pathway. A Type I mechanism is characterized by an electron or proton transfer from or to the PS, which leads to the formation of reactive oxygen species (ROS) or other highly reactive radicals. In a Type II mechanism, the energy is transferred to triplet oxygen ( 3 O 2 ) to generate singlet oxygen ( 1 O 2 ). Due to their high reactivity, ROS and 1 O 2 can cause oxidative stress and damage in different cellular compartments (i.e., membrane, nucleus, endoplasmic reticulum, lysosome, mitochondria). Both of these mechanisms can happen simultaneously upon light irradiation even so Type II pathway is the predominant one for most approved PSs. [START_REF] Dolmans | Photodynamic Therapy for Cancer[END_REF][START_REF] Plaetzer | Photophysics and photochemistry of photodynamic therapy: fundamental aspects[END_REF][START_REF] Bonnet | Why develop photoactivated chemotherapy[END_REF][START_REF] Bonnett | Photosensitizers of the porphyrin and phthalocyanine series for photodynamic therapy[END_REF][START_REF] Dougherty | Photodynamic therapy[END_REF][START_REF] Henderson | How does Photodynamic Therapy work?[END_REF] Photofrin is the most commonly used PS in PDT. It has been approved for the treatment of bladder cancer, early-stage lung cancer, esophageal cancer, and early non-small cell lung cancer. However, based on its low solubility and low absorption in the therapeutic window (i.e., 600-900 nm), high concentrations, as well as high light doses are required for an adequate tumor treatment making Photofrin not an ideal PS. Additionally, it was shown that this PS has an exceptionally long half-life excretion time leading to severe photosensitivity for the patients. Since the majority of investigated and approved PS are based on a tetrapyrrolic scaff old (i.e., porphyrins, chlorins, phthalocyanines), these PSs are likely to have similar drawbacks which include 1) poor water solubility; 2) tedious synthesis and purification; 3) low cancer selectivity; 4) photobleaching effect and 5) slow clearance from the body causing photosensitivity. Therefore, a need for modification of existing PSs or the development of new classes of PSs is needed. [START_REF] O'connor | Porphyrin and Nonporphyrin Photosensitizers in Oncology: Preclinical and Clinical Advances in Photodynamic Therapy[END_REF][START_REF] Naik | Visible-Light-Induced Annihilation of Tumor Cells with Platinum-Porphyrin Conjugates[END_REF][START_REF] Heinemann | Critical Overview of the Use of Ru(II) Polypyridyl Complexes as Photosensitizers in One-Photon and Two-Photon Photodynamic Therapy[END_REF][START_REF] Karges | Polymeric Encapsulation of Novel Homoleptic Bis(dipyrrinato) Zinc(II) Complexes with Long Lifetimes for Applications as Photodynamic Therapy Photosensitisers[END_REF][START_REF] Antoni | Metallo)porphyrins as Potent Phototoxic Anti-Cancer Agents after Irradiation with Red Light[END_REF][START_REF] Agostinis | Photodynamic therapy of cancer: an update[END_REF][START_REF] Callaghan | The good, the bad, and the uglycontrolling singlet oxygen through design of photosensitizers and delivery systems for photodynamic therapy[END_REF][START_REF] Schneider | Exocyclically metallated tetrapyridinoporphyrazine as a potential photosensitizer for photodynamic therapy[END_REF] Among the new classes of PSs investigated the development of Ru(II) polypyridyl complexes as PDT PSs has received much attention due to their ideal photophysical and photochemical properties (i.e., high water solubility, high chemical stability and photostability, intense luminescence, large Stokes shifts, high 1 O 2 production). [START_REF] Mari | Combination of Ru(II) complexes and light: new frontiers in cancer therapy[END_REF][START_REF] Monro | Transition Metal Complexes and Photodynamic Therapy from a Tumor-Centered Approach: Challenges, Opportunities, and Highlights from the Development of TLD1433[END_REF][START_REF] Zeng | The development of anticancer ruthenium(II) complexes: from single molecule compounds to nanomaterials[END_REF][START_REF] Mckenzie | Transition metal complexes as photosensitisers in oneand two-photon photodynamic therapy[END_REF][START_REF] Li | Ru(II) Polypyridyl Complexes Derived from Tetradentate Ancillary Ligands for Effective Photocaging[END_REF][START_REF] Liu | Harnessing ruthenium(II) as photodynamic agents: Encouraging advances in cancer therapy[END_REF][START_REF] Poynton | The development of ruthenium(II) polypyridyl complexes and conjugates for in vitro cellular and in vivo applications[END_REF][START_REF] Jakubaszek | Evaluation of the Potential of Cobalamin Derivatives Bearing Ru(II) Polypyridyl Complexes as Photosensitisers for Photodynamic Therapy[END_REF][START_REF] Shum | Luminescent Ruthenium(II) Polypyridine Complexes for a Wide Variety of Biomolecular and Cellular Applications[END_REF][START_REF] Qiu | Organelle-targeting metal complexes: From molecular design to bio-applications[END_REF][START_REF] Karges | Synthesis and Characterization of an Epidermal Growth Factor Receptor-Selective RuII Polypyridyl-Nanobody Conjugate as a Photosensitizer for Photodynamic Therapy[END_REF][START_REF] Lincoln | Exploitation of Long-Lived 3 IL Excited States for Metal-Organic Photodynamic Therapy: Verification in a Metastatic Melanoma Model[END_REF][START_REF] Ellahioui | Mesoporous silica nanoparticles functionalised with a photoactive ruthenium(II) complex: exploring the formulation of a metal-based photodynamic therapy photosensitiser[END_REF][START_REF] Howerton | Strained Ruthenium Complexes Are Potent Light-Activated Anticancer Agents[END_REF][START_REF] Knoll | Control and utilization of ruthenium and rhodium metal complex excited states for photoactivated cancer therapy[END_REF][START_REF] Palmer | Cytotoxicity of cyclometallated ruthenium complexes: the role of ligand exchange on the activity[END_REF] Worthy of note, the complex TLD-1433 [Ru(dmb) 2 (IP-TT)] 2+ (dmb=4,4′-dimethyl-2,2′bipyridine, IP-TT=2-(2′,2″:5″,2′ ′′-terthiophene)-imidazol [4,5-f][1,10]phenanthroline) has just entered phase II clinical trial as a PDT PS for the treatment of non-muscle invasive bladder cancer in Canada. [START_REF]Theralase Annouces First Patient Treated in Phase II Non-Muscle Invasive Bladder Cancer Clinical Study[END_REF][START_REF] Fong | A novel class of ruthenium-based photosensitizers effectively kills in vitro cancer cells and in vivo tumors[END_REF][START_REF] Kaspler | A ruthenium(II) based photosensitizer and transferrin complexes enhance photo-physical properties, cell uptake, and photodynamic therapy safety and efficacy[END_REF][START_REF] Mcfarland | Metal-based photosensitizers for photodynamic therapy: the future of multimodal oncology?[END_REF] Despite these remarkable properties, the majority of Ru(II)-based PS are typically excited using blue or UV-A light and therefore suffer from a lack of absorption in the biological spectral window (600-900 nm). [START_REF] Cloonan | Detailed Biological Profiling of a Photoactivated and Apoptosis Inducing pdppz Ruthenium(II) Polypyridyl Complex in Cancer Cells[END_REF][START_REF] Karges | Systematic investigation of the antiproliferative activity of a series of ruthenium terpyridine complexes[END_REF][START_REF] Yin | In Vitro Multiwavelength PDT with 3 IL States: Teaching Old Molecules New Tricks[END_REF][START_REF] Wachter | Light-activated ruthenium complexes photobind DNA and are cytotoxic in the photodynamic therapy window[END_REF][START_REF] Wang | π-Expansive Heteroleptic Ruthenium(II) Complexes as Reverse Saturable Absorbers and Photosensitizers for Photodynamic Therapy[END_REF] Based on absorption and light scattering eff ects in the biological environment, the light penetration depth into the tissue is low at this wavelength, which limits their application to treat deep tumors or large tumors. [START_REF] Ogawa | Recent advances in twophoton photodynamic therapy[END_REF][START_REF] Wilson | In vivo and post mortem measurements of the attenuation spectra of light in mammalian tissues[END_REF] To overcome this limitation, there is a need for optimization of the absorption properties of Ru(II)-based PSs. It has been well-established that the photophysical properties, including absorption, emission as well as excited-state lifetimes of Ru(II) polypyridyl complexes depend on the ligands bound to the Ru center. This variable can, therefore, be tuned. In this context, we applied a combined experimental and theoretical approach to design new suitable Ru-based PDT PSs. Based on the already wellestablished biological activity of the complex [Ru(phen) 3 ] 2+ (phen = 1,10-phenanthroline) as a minor groove binder [START_REF] Eriksson | Minor groove binding of [Ru(phen) 3 ] 2+ to [d(CGCGATCGCG)] 2 evidenced by two-dimensional NMR[END_REF] and [Ru(bphen) 3 ] 2+ (bphen = 4,7-diphenyl-1,10-phenanthroline) as a mitochondria and lysosome targeting agent [START_REF] Dickerson | Modifying charge and hydrophilicity of simple Ru(II) polypyridyl complexes radically alters biological activities: old complexes, surprising new tricks[END_REF] and their ability to be effective PDT PSs [START_REF] Audi | Cytotoxicity Modulation of Ruthenium (II) trisbathophenantholine Complexes With Systematically Varied Charge[END_REF][START_REF] Mehanna | Enhanced cellular uptake and photochemotherapeutic potential of a lipophilic strained Ru(ii) polypyridyl complex[END_REF][START_REF] Mansour | Photoactivatable RuII Complex Bearing 2,9-Diphenyl-1,10-phenanthroline: Unusual Photochemistry and Significant Potency on Cisplatin-Resistant Cell Lines[END_REF] , we decided to use [Ru(phen) 2 (bipy)] 2+ and [Ru(bphen) 2 (bipy)] 2+ (bipy = 2,2'bipyridine) derivatives as basic scaffolds. In this investigation, the electronic properties, the origin, and the magnitude of red shift towards the biologic spectral window are disclosed. The resulting complexes (1-7, Figure 1) were synthesized, characterized, and biologically evaluated in-depth. Thanks to this combined study, a highly active Ru(II)-based PDT PS that can be excited up to 595 nm could be unveiled.

RESULTS AND DISCUSSION

Rational Design. As the basis of the design of Ru(II) polypyridine complexes as PDT PSs with red-shifted absorption near or in the biological spectral window, the [Ru(phen) 2 (bipy)] 2+ scaffold was used due to its synthetic accessibility and generally high physical stability. To pursue this aim, systematic modification on the bipyridine moiety was investigated, and these effects studied by a theoretical and experimental approach. It is well known that, in a simplified picture, the highest occupied orbitals in a pseudo-octahedral Ru(II) polypyridyl complex are mainly consisting of the Ruthenium t 2g -d-orbitals while the lowest occupied orbitals typically correspond to π*-orbitals localized on the ligands. [START_REF] Finck | Structural and optical properties of new cyclometalated Ru(II) derived compounds[END_REF][START_REF] Campagna | Photochemistry and Photophysics of Coordination Compounds I[END_REF] Therefore, the lowest intense absorption band is expected to be of metal to ligand charge transfer (MLCT) character stemming from electronic transitions from the t 2g manifold to the empty ligands lowestlying orbitals and leading to the population of a singlet state of MLCT nature under light irradiation. A simple way to red shift the MLCT absorption energy is, therefore, to decrease the HOMO-LUMO gap by an ad-hoc functionalization of the ligands. In particular, functionalization of the ligands with electron-donating and electron-withdrawing (EDG/EWG) groups is expected to increase the occupied MOs and lower the LUMO energy, respectively. In the case of the unsubstituted compound 1, the ligands are not strictly equivalent (two phen and one bipy ligand) so that the t 2g orbitals are not expected to be strictly degenerate. Nonetheless, from the computed MOs energies of 1, it can be seen that the difference in energy between the t 2g orbitals is very tiny (roughly 0.04 eV) and the same holds for the LUMOs of π* character (roughly 0.08 eV) with contributions arising both from the phen and the bipy ligands. As a consequence, the functionalization of any of the two ligands shall induce a shift of the gap but is indeed expected to be easier in the case of the bipy, due to the reduced steric congestion around this ligand. For this reason, EDGs and EWGs were exclusively introduced only on the bipy ligand (1-5, Figure 1, optimized cartesian coordinates Table S1-S5). For this purpose, the energies of the frontier molecular orbitals were computed (Figure 2). Indeed, functionalization with -Me (2) (a weakly EDG) only negligibly affects the gap (reducing from 3.99 eV for 1 to 3.97 eV for 2). A slightly more significant effect is obtained by weak EWGs such as -Br (3) and -CONH 2 (4, 3.83 eV, and 3.77 eV, respectively), which induce a small stabilization of the LUMO. These observations are in line with the results previously obtained by some of us [START_REF] Karges | A Ru(II) polypyridyl complex bearing aldehyde functions as a versatile synthetic precursor for long-wavelength absorbing photodynamic therapy photosensitizers[END_REF] when functionalizing with a -CHO group, a better EWG for which the computed gap is indeed 3.47 eV. On the other hand, functionalization with the vinyl dimethylamine-EDG (5) results in a substantial reduction of the HOMO-LUMO gap (to 3.24 eV, roughly 0.7 eV lower than the native compound (1) due to a sizable increase of the HOMO energy. Nonetheless, it should be underlined that the gap is reduced here due to the presence of occupied orbitals centered on the vinyl dimethylamine group in the gap. Therefore, although de facto the gap is substantially reduced, there is no destabilization of the t 2g manifold so that the bright MLCT transition (occurring from the t 2g orbitals to the π* ligand orbitals) is expected not to be affected (that is red-shifted).

To capitalize on this theoretical insight, we additionally examined the functionalization with a methyl and vinyl dimethylamine group on the [Ru(bphen) 2 (bipy)] 2+ scaffold 6-7 (optimized cartesian coordinates Table S6-S7). Interestingly, changing the ligand scaffold from phen to bphen does not significantly affect the gapas expected due to the small electronic effect induced by the presence of the four phenyl groups on the phen ligands. Indeed comparing compounds 2 and 6 or 5 and 7 (that are the analogous in the two series), a difference of only 0.1 and 0.07 eV in the gap, respectively, can be observed. Overall the theoretical insight revealed that the vinyl dimethylamine functionalized (5,7) and bphen coordinated (6-7) complexes showed the strongest spectral red shift.

Synthesis and Characterization.

Based on the theoretical design, the Ru(II) polypyridine complexes 1-7 (Figure 1) were synthesized. To date, the synthesis of complexes 3-5 and 7 has not been yet reported, while complexes 1 [START_REF] Crosby | Excited states of mixed ligand chelates of ruthenium (II) and rhodium (III)[END_REF] , 2 [START_REF] Jones | Photochemistry of heterotris-chelated ruthenium (II) polypyridine complexes in dichloromethane[END_REF] , and 6 [START_REF] Mazuryk | Multifaceted interplay between lipophilicity, protein interaction and luminescence parameters of non-intercalative ruthenium (II) polypyridyl complexes controlling cellular imaging and cytotoxic properties[END_REF] are known. However, in this study, slightly different experimental procedures than the previously described were employed to obtain these compounds (for experimental protocols see supporting information). The identity of all complexes was confirmed by 1 H, [START_REF] Callaghan | The good, the bad, and the uglycontrolling singlet oxygen through design of photosensitizers and delivery systems for photodynamic therapy[END_REF] C-NMR (Scheme S1, Figures S1-S14), HRMS, and the purity by elemental analysis. In addition, the molecular structures of complexes 1-3 (Figure S15-17, Table S8-S9) were confirmed by single-crystal X-ray diffraction studies. The crystal structure of compound 1 has already been characterized by Huang and Ogawa [START_REF] Huang | Spontaneous resolution of Δ and Λ enantiomeric pair of [Ru(phen)(bpy) 2 ](PF 6 ) 2 (phen= 1, 10-phenanthroline, bpy= 2, 2′-bipyridine) by conglomerate crystallization[END_REF] , with the exception that the crystal structure presented here contains one solvent molecule of acetonitrile per ruthenium complex. In all molecular structures, the Ru(II) central atom adopts a distorted octahedral geometry chelated by two 1,10-phenanthroline ligands and one 2,2'-bipyridine ligand with Ru -N bond lengths ranging from 2.046(3) to 2.078(3) Å, N phen -Ru -N phen angles from 79.48 (12) to 80.1(2)°, and N bipy -Ru -N bipy angles from 78.55 (10) to 78.98(13)°. It is worth to note that the substitution of the bipyridine ligand by methyl groups in 2 and bromo ligands in 3 has no significant influence on the Ru -N bond distances.

Photophysical properties. The absorption spectra of the compounds 1-7 were measured in CH 3 CN (Figure S19,Table 1) and compared with the computed spectra (Figure 3). Although in the simulated spectra the energy of the MLCT band (around 450 nm) is systematically overestimated while the higher energy ligand centered (LE) band (around 300 nm) is better reproduced, small shifts towards the spectral windows of interest and a rise in intensity for the lowest energy band is indeed observed for the compounds 5, 6 and 7. Of note, in the case of 5 and 7, several electronic transitions are computed to contribute to the first -lowest energy-absorption band. The most intense has still an MLCT character while the one occurring at lower energy (less intense than those of MLCT character) and responsible for the tail and red-shift of the band are predicted to be essentially ligand centered. These transitions are indeed of HOMO-LUMO type and, as discussed above, corresponding essentially to a transition from the vinyl dimethylamine group to the π* orbitals of the ligand. This can be visualized and understood from the maps of the difference in density between ground (GS) and excited state (ES) for two representative transitions of compound 6 (Figure S18a) and 7 (Figure S18b). For both complexes, the lowest energy transition (first electronic transition ES1) and the most intense one contributing to the first band (ES 4 and ES 9 for 6 and 7, respectively) were analyzed.

In Figure S18, density depletion/increase regions upon excitation for each are represented by blue/yellow zones, and the barycenters of these regions -depicted as blue/yellow dotscan be interpreted as the position of the electron and hole upon excitation. It can be clearly seen that while for complex 6, both transitions have a clear MLCT character though not necessarily involving the same ligand, in the case of 7, in agreement with the MO diagram, the lowest energy transition is of interligand type and mostly involving the vinyl dimethylamine-part. Therefore, even if a redshift of the first absorption band is predicted and indeed experimentally observed, it is not necessarily expected to correlate with an improvement of the phototherapeutic properties that are indeed linked to the population of a MLCT state. Following this, the luminescence of the Ru(II) polypyridine complexes upon excitation at 355 nm was investigated. The maxima of the emission signals (Figure S20) were measured and found to be between 600-710 nm. Interestingly, complexes5 and 7, which showed the highest red shift in absorption, also demonstrated the strongest red shift in their emission maximum. The comparison of the excitation and absorption spectra of all compounds showed no significant differences. All complexes demonstrated a large Stokes shift implying minimal inference between excitation and emission. The luminescence quantum yields were found with values between 5.0% -1.4% (Table S10) for 1-4, 6 and are therefore in the same range then other Ru(II) polypyridine complexes. [START_REF] Cook | Luminescent metal complexes. Part 1. Tris-chelates of substituted 2, 2′-bipyridyls with ruthenium (II) as dyes for luminescent solar collectors[END_REF][START_REF] Balzani | Photochemistry and photophysics of Ru(II)polypyridine complexes in the Bologna group. From early studies to recent developments[END_REF] On the contrary, the luminescence of 5 and 7 were barely measurable -with luminescence quantum yields >0.1%. This is consistent with the computed vertical absorption (see before) highlighting that for these two complexes the lowest lying excited states are of LC character. This result is also in agreement with the exceptionally low luminescence quantum yield of (E,E')-4,4'-bis(N,N'dimethylaminovinyl)-2,2'-bipyridine in dichloromethane (1.5%) in comparison to other substituted 2,2'-bipyridines [START_REF] Maury | Design and synthesis of 4, 4′-π-conjugated [2, 2′]-bipyridines: a versatile class of tunable chromophores and fluorophores[END_REF] and of the [Ru((E,E')-4,4'-bis(N,N'-dimethylaminovinyl)-2,2'-bipyri-dine) 3 ] 2+ complex in CH 3 CN (>0.1%), [START_REF] Karges | Towards Long Wavelength Absorbing Photodynamic Therapy Photosensitizers via the Extension of a [Ru(bipy) 3 ] 2+ Core[END_REF] which were recently reported. The excited-state lifetimes were determined in degassed and air-saturated CH 3 CN solution to investigate the influence of the presence of oxygen. The obtained values (Figure S21-S27, Table 1) were found to be in the nanosecond scale in a degassed (312 -1387 ns) and air saturated (55 -326 ns) solution. All measured lifetimes were found to be in the same range as for other Ru(II) polypyridine complexes. [START_REF] Cook | Luminescent metal complexes. Part 1. Tris-chelates of substituted 2, 2′-bipyridyls with ruthenium (II) as dyes for luminescent solar collectors[END_REF][START_REF] Balzani | Photochemistry and photophysics of Ru(II)polypyridine complexes in the Bologna group. From early studies to recent developments[END_REF] Importantly, the data shows that the presence of oxygen has a significant influence on the lifetime of the excited state for all complexes indicating that molecular oxygen can interact with the triplet state of the complex.The gen-eration of singlet oxygen ( 1 O 2 ) was quantitatively measured upon excitation at 450 nm by two complementary methods: (i) direct by measurement of the phosphorescence of 1 O 2 , (ii) indirect by temporal monitoring the change of absorption of a 1 O 2 scavenger. [START_REF] Karges | Synthesis, Characterisation and Biological Evaluation of π-Extended Fe(II) Bipyridine Complexes as Potential Photosensitizers for Photodynamic Therapy[END_REF][START_REF] Karges | Synthesis, Characterization, and Biological Evaluation of Red-Absorbing Fe (II) Polypyridine Complexes[END_REF] Complexes 1-4, 6 were found to have 1 O 2 quantum yields (Table 1) between 53-69% in CH 3 CN and 5-36% in an aqueous solution, suggesting an application as a PDT agent. These values are comparable with those previously reported for related compounds. [START_REF] Abdel-Shafi | Photosensitized generation of singlet oxygen from (substituted bipyridine) ruthenium (II) complexes[END_REF][START_REF] Garcìa-Fresnadillo | Singlet-Oxygen (1Δg) Production by Ruthenium (II) complexes containing polyazaheterocyclic ligands in methanol and in water[END_REF] In comparison, the 1 O 2 quantum yields of the (E,E')-4,4'-bis(N,N'dimethylaminovinyl)-2,2'-bipyridine coordinated complexes 5 and 7 were found to be drastically lower with values of 22-35% in CH 3 CN and 7-21% in an aqueous solution. This was expected as these compounds show an untypical excited state behavior (emission, luminescence, lifetime) which is also explained by DFT calculations. Stability. The stability of a compounds is an essential parameter for their use as a PDT agent. As a first experiment, the stability of the complexes was investigated in a DMSO solution since this solvent was shown to be problematic for certain drug (candidates). [START_REF] Patra | DMSO-Mediated Ligand Dissociation: Renaissance for Biological Activity of N-Heterocyclic-[Ru (η 6 -arene)Cl 2 ] Drug Candidates[END_REF][START_REF] Hall | Say no to DMSO: dimethylsulfoxide inactivates cisplatin, carboplatin, and other platinum complexes[END_REF][START_REF] Keller | A tutorial for the assessment of the stability of organometallic complexes in biological media[END_REF] For this purpose, solutions of the complexes in DMSO-d 6 were prepared and stored in a NMR tube in the dark at room temperature. A 1 H-NMR spectrum was measured directly after preparing the solution after one, two and seven days. For complexes 1-4 and 6 (Figures S28-S31, S33), no significant change in the spectra were observed, indicating that no decomposition occurred. In contrast to this, small changes in the spectra for compounds 5 and 7 could be observed. For both compounds, changes in the signals could be detected after 7 days (Figures S32,S34). This indicates that these compounds are not stable in DMSO. To assess the compatibility of the compounds under biological conditions, the stability of the complexes was tested in pooled human plasma. The complexes were incubated for 48 h in the dark with coffeine as an internal standard, which was previously shown to be stable under these conditions [START_REF] Bruce | Investigation of human blood plasma sample preparation for performing metabolomics using ultrahigh performance liquid chromatography/mass spectrometry[END_REF] and then analysed by HPLC (Figures S35-41). The stability of complexes 1-4 and 6 and the previously mentioned decomposition of compounds 5 and 7 were confirmed. Based on these findings, the stability of compounds 5 and 7 has been investigated more in detail by incubation of these complexes with shorter time intervals (0 h, 4 h, 12 h, 24 h, 48 h) in the dark. 5 shows first sign of degradation after 12 h and compound 7 after 24 h. The degradation of both complexes advanced in the investigated time interval, but still show unreacted complex even after 48 h incubation. Following this, the potential decomposition of the complexes upon light irradiation was also tested as previous studied have shown that the stability of metal complexes could be influenced upon light exposure. [START_REF] Basu | Investigation of photo-activation on Ruthenium(II)arene complexes for the discovery of potential selective cytotoxic agents[END_REF][START_REF] Renfrew | Towards Light Activated Ruthenium-Arene (RAPTA-type) Prodrug Candidates[END_REF] This is crucially important as some of the currently approved PDT agents are associated with a strong photobleaching effect.The complexes were exposed to a continuous LED irradiation at 450 nm and the absorption spectra monitored. As a positive control [Ru(bipy) 3 ]Cl 2 and as a negative control Protoporphyrin IX was used. The comparison of the spectra shows that complexes 1-4 and 6 (Figure S42-46, 48) have a photobleaching effect in a similar range than [Ru(bipy) 3 ]Cl 2 (Figure S41). However, compounds 5 and 7 were found to be strongly affected by light irradiation with a loss of about half of their absorbance after one minute (Figure S47,49). This effect is even stronger than that observed for Protoporphyrin IX (Figure S50).

Biological Evaluation.

The lipophilicity/hydrophilicity of the compounds was determined by measuring the distribution coefficient (logP) between an organic octanol and aqueous phosphate buffer saline phase (Table S10). The complexes based on a [Ru(phen) 2 (bipy)] 2+ scaffold (1-5) were found with logP values between +0.2 -+0.7 and the complexes based on a [Ru(bphen) 2 (bipy)] 2+ scaffold (6-7) between +1.4 -+1.7. As all complexes were found majorly in the organic phase, their lipophilicity is indicated.

Following this, the cellular uptake of the compounds was investigated. This property is important as a PS with good photophysical properties could be still inactive due to poor cellular uptake. Amount of Ru metal accumulated inside the human cervical carcinoma (HeLa) cells upon incubation for 4 h at 25 µM concentration was determined by inductively coupled plasma mass spectrometry (ICP-MS). As expected, the compounds 6-7 which are based on a [Ru(bphen) 2 (bipy)] 2+ scaffold showed a 2.1-5.8 times higher cellular accumulation (Figure S51) in comparison to compounds 1-5, in agreement with their logP values. To determine the potential of the complexes to act as PDT agents, mouse colon carcinoma (CT-26), human glioblastoma (U87) human glioblastoma astrocytoma (U373), human cervical carcinoma (HeLa) as well as noncancerous retina pigmented epithelial (RPE-1) cell lines were treated with the complexes. Their cytotoxicity in the dark and upon light exposure was investigated using fluorometric cell viability assay (Table 2). Worthy of note, the light doses used during our experiments at different wavelengths were first optimised to the survival of the cells treated purely by light exposure. At the same irradiation wavelength, all cell lines were tested with the same light dose to investigate the influence on the different type of cells used and therefore explore the PS potential on different cancer types. Ideally, a PDT PS should be non-toxic in the dark and highly toxic upon light exposure. Promisingly, complexes 1-5 and 7 were found to be Table 2. IC 50 values (µM) for the complexes 1-7 and Protoporphyrin IX (PpIX) in mouse colon carcinoma (CT-26), human glioblastoma (U87), human glioblastoma astrocytoma (U373), human cervical carcinoma (HeLa) and non-cancerous retina pigmented epithelial (RPE-1) cell lines in the dark and upon light irradiation (480 nm, 10 min, 3.21 J cm -2 ). non-cytotoxic in the dark in all chosen cell lines (IC 50 >100 µM), while compound 6 showed a cytotoxic profile in the range from 3.09 to 28.77 µM in all investigated cell lines. Upon irradiation at 480 nm (10 min, 3.21 J cm -2 ), no or only poor toxicity (IC 50 range from >100 to 52.54 µM) was observed for complexes based on a [Ru(phen) 2 (bipy)] 2+ scaffold (1-5). In contrast, compounds based on the [Ru(bphen) 2 (bipy)] 2+ scaffold (6-7) showed a notable phototoxicity upon light irradiation (Phototoxic index (PI)-IC 50 in the dark/IC 50 in upon irradiation, ranges from 6.5 to 42.5). This effect can be attributed to the significantly higher uptake of 6 and 7. Overall, considering the instability of complex 7 in DMSO and human plasma and the absence/low phototoxicity of complexes 1-5, complex 6 was further studied. Following this preliminary examination, the ability to cause a phototoxic effect at longer wavelengths towards the biological spectral window was further evaluated. CT-26 cell line which was previously shown to be the strongest affected by this compound was chosen for subsequent studies. Importantly, light irradiation of the treated cells at 510 nm (40 min, 10.00 J cm -2 ) or 540 nm (60 min, 14.25 J cm -2 ) caused a phototoxic effect (Table 3). Strikingly, even irradiation at 595 nm (2 h, 22.47 J cm -2 ) generated a phototoxic effect in cells. It has to be noted that the lack of CO 2 atmosphere during irradiation also contributed to the obtained results. Nevertheless, the calculated PI values are reliable, as cells used as dark control were also incubated for the same amount of time at 37 °C in non-CO 2 atmosphere. Worthy of note, the compound lost its phototoxic effect upon irradiation at 620 nm (30 min, 3.3 J cm - 2 ). Interestingly, previous studies have shown that photosensi-tizers can show a phototoxic effect although its extinction coefficient is below 100 M -1 cm -1 . [START_REF] Monro | Transition Metal Complexes and Photodynamic Therapy from a Tumor-Centered Approach: Challenges, Opportunities, and Highlights from the Development of TLD1433[END_REF][START_REF] Yin | In Vitro Multiwavelength PDT with 3 IL States: Teaching Old Molecules New Tricks[END_REF] Overall, these results make compound 6 an impressive candidate as a PDT agent. To have a deeper insight in the mechanism of action of compound 6, its cellular localisation in HeLa cell line was determined by confocal microscopy experiments. Even after only 5 min incubation (14 µM), the complex could be detected in the cytoplasm (see Figure S52). Immunofluorescence studies with GM130 (cis-Golgy protein), TGN46 (trans-Golgy protein), KDEL (endoplasmic reticulum protein retention receptor) and LAMP (lysosome-associated membrane glycoprotein) antibodies demonstrated that compound 6 did not colocalize with any of them (Figure 4a). Correlation analysis including Pearson's R value as well as Manders' M1 and Manders's M2 values confirmed the lack of colocalisation of the tested probes with complex 6 (Figure 4b). It is possible that the cytosolic localisation of our complex could be explained by its binding to the cytoskeleton as recently reported for structurally similar complex by the group of MacDonnell. [START_REF] Alatrash | Disruption of microtubule function in cultured human cells by a cytotoxic ruthenium(ii) polypyridyl complex[END_REF] To further study the mechanism of action of complex 6, its influence on cellular metabolism was studied. For that purpose Seahorse XF instrument was used, it allows for real time measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in cells. To observe the effect of the compound 6 on oxidative phosphorylation (ATP production in mitochondria through electron transport chain), the Mito Stress test (Figure 5a) was performed. In this test, sequential injections of specific inhibitors of the electron transport chain proteins allows for determination of the effect that the compound has on the mitochondrial metabolism of the tested cells. Briefly, the cells were treated (4 h, 1µM) with complex 6 as well as with cisplatin and 5-ALA (5aminolevulinic acid) -precursor of protoporphyrin IX (PpIX), a known photosensitizer, as controls. [START_REF] Mahmoudi | 5-aminolevulinic acid photodynamic therapy for the treatment of high-grade gliomas[END_REF] After the incubation time, the cells were irradiated at 595 nm (2 h, 22.47 J cm -2 , see Figure S53) and the Mito Stress Test was performed. Strikingly, the data shows that only the cells, which were treated with complex 6 and irradiated, had their metabolism impaired right after the irradiation process. Injection of oligomycin (a specific inhibitor of ATP synthase) or FCCP (an uncoupling agent) did not affect their oxygen consumption rates. The mitochondrial membrane of these cells lost the capacity to restore the proton balance. ATP production was inhibited and spare respiratory capacity (difference between OCR values of maximal respiration and basal respiration) was strongly reduced, contrary to the cells treated with complex 6 that were not irradiated (Figure 5a and Figure S54). Additional tests investigating whether the glycolysis is also affected were performed. Indeed, the glycolysis process is also severely impaired in the cells that are treated with complex 6 and irradiated (Figure 5b and Figure S55). It is known that the glycolysis process is significantly reduced during apoptosis. [START_REF] Pradelli | Glucose metabolism is inhibited by caspases upon the induction of apoptosis[END_REF] Additionally, mitochondria are important compartment, which are responsible for triggering an intrinsic cell death. [START_REF] Mason | Cell metabolism: An essential link between cell growth and apoptosis[END_REF] It is then likely that the start of apoptosis is responsible for the initial effect in the cellular metabolism observed. A similar metabolic response could not be noticed for the cisplatin. This phenomenon could be explained by the short incubation time (only 4 h) and the very low concentration tested (1 µM) that is not sufficient to trigger apoptosis by this drug in CT-26 cell line. Overall, compound 6 has an immediate effect on irradiated cells but not in the ones kept in the dark, resulting in disturbed mitochondrial respiration and glycolysis processes.

CT

b) Glycolysis Stress

Test profile in CT-26 cells after 4 h treatment and 2 h irradiation at 595 nm; extracellular acidification rate that corresponds to the glycolysis process changes after treatment with glucose (basal level of glycolysis in cells), oligomycin (inhibitor of ATP synthase (complex V)-mitochondria inhibition), 2-deoxyglucose (analog of glucose that inhibits glycolytic pathway).

After evaluation of the (photo-)cytotoxicity on 2D monolayer cells, the effect of complex 6 on multicellular tumor spheroids (MCTS) was investigated. This is of special interest as many anticancer drug candidates have failed the translation from monolayer cells to an in vivo model due to compromised drug delivery. MCTS are small spherical cell aggregates that mimic cell tumors. They can simulate the gradient of nutrients availability from upper cell layers, that are highly exposed, to lower layers and are able to model the potential penetration of a drug inside a 3D struture. [START_REF] Friedrich | Spheroid-based drug screen: considerations and practical approach[END_REF][START_REF] Goodman | 3-D Tissue Culture Systems for the Evaluation and Optimization of Nanoparticle-Based Drug Carriers[END_REF] Therefore, compound 6 was incubated for 24 h in HeLa MCTS and its cytotoxic effect determined by measurement of the ATP concentration. Importantly, upon irradiation at 595 nm (2 h, 22.47 J cm -2 ), compound 6 showed a phototoxic effect (IC 50,dark = 29.42 ± 4.60 μM, IC 50,595nm = 20.07 ± 4.15 μM, PI 595nm = 1.5), indicating that the compound is able to exert its action inside the 3D MCTS and act as a PDT agent. Worthy of note, the critical concentration to trigger cell death in 3D MCTS was found to be significantly larger than in 2D monolayer cells. The need for higher concentrations to cause an eradication of the MCTS was previously described in the literature, [START_REF] Liu | Ruthenium(II) polypyridyl complexes as mitochondria-targeted two-photon photodynamic anticancer agents[END_REF] including for structurally related Ru(II) polypyridine complexes, and is assumingly caused by a poor light penetration, potential diffusion problems and/or the hypoxic center of the MCTS.

CONCLUSION

In summary, we have combined the theoretical understanding provided by DFT calculations with the photophysical and biological experimental evaluation of Ru(II) polypyridine complexes as PSs for PDT. Thanks to this rational design, ruthenium complexes with a strong red shift in their absorption profile could be successfully prepared. While the (E,E')-4,4'-bis(N,N'-dimethylaminovinyl)-2,2'-bipyridine coordinated complexes showed the desired red shift, they were however found to have poor photophysical properties (luminescence, 1 O 2 production) and poor stability. In contrast, the [Ru(bphen) 2 (bmb)] 2+ complex was found to have an absorption tail towards the biological spectral window. While being stable in human plasma as well as upon light irradiation, it was found to localize in the cytoplasm of HeLa cells. Upon irradiation at clinically relevant 595 nm it led to the disturbance of mitochondrial respiration and glycolysis process in 2D monolayer cells as well as 3D MCTS. We strongly believe that the approach presented here for the rational design of compounds holds great potential for the development of new PDT agents. The prediction of the photophysical properties of potential PSs enables a directed search for efficient compounds. We are planning to investigate the in vivo efficiency of compound 6 in the future.

ASSOCIATED CONTENT

The Supporting Information is available free of charge via the Internet at http://pubs.acs.org.

Detailed experimental protocols, supporting figures and tables.

Figure 1 .

 1 Figure 1. Chemical structures of the Ru(II) polypyridyl complexes investigated in this work. The complexes 1-7 were isolated as PF 6 -salts.

Figure 2 .

 2 Figure 2. Computed frontier orbitals' energies and HOMO-LUMO gaps (in eV). Occupied/virtual orbitals energies are represented as black/blue line. Blue background: Ru(phen) 2 (bipy) 2+ skeleton. Purple background: Ru(bphen) 2 (bipy) 2+ skeleton.

Figure 3 .

 3 Figure 3. Simulated (blue) and experimental (black) spectra of compounds 1-7 in CH 3 CN (c = 7.5 µM). Computed vertical electronic transitions are depicted as vertical blue bars. Corresponding oscillator strength (f) is given in a.u.

Figure 4 .

 4 Figure 4. a) Immunofluorescent images of HeLa cells treated with complex 6 (14 µM, 30 min). DNA visualised by NucBlue staining, immunofluorescence for GM130, TGN46, KDEL and LAMP proteins shown in green, complex 6 shown in red. Scale bar, 20 µm. b) Person's R, Manders' M1 and Manders' M2 colocalisation values obtained for complex 6 and fluorescent probes.

Figure 5 .

 5 Figure 5. a) Mito Stress Test profile in CT-26 cells after 4 h treatment and 2 h irradiation at 595 nm; oxygen consumption rate changes after treatment with specific electron transport chain inhibitors, namely oligomycin (inhibitor of ATP synthase (complex V)), FCCP (uncoupling agent), antimycin-A (complex III inhibitor) and rotenone (complex I inhibitor). b) Glycolysis Stress Test profile in CT-26 cells after 4 h treatment and 2 h irradiation at 595 nm; extracellular acidification rate that corresponds to the

Table 1 .

 1 Spectroscopic properties of compounds 1-7 in CH 3 CN. Singlet oxygen quantum yields of compounds 1-7 in CH 3 CN and aqueous solution determined by direct and indirect methods by excitation at 450 nm. Average of three independent measurements, ±10%.

		Spectroscopic properties				Singlet oxygen quantum yield
		UV/Vis λ / nm (ε / M -1 cm -1 × 10 3 ) Absorption	λ em / nm Φ em nm	/	τ saturat-air	τ gassed de-	CH 3 CN direct	CH 3 CN indirect	D 2 O direct	PBS indirect
						ed
	1	200 (73.2), 225 (64.3), 264 (86.5), 284 (44.1), 446 (15.0), 480 (5.5)	600	0.027		130	766	0.57	0.54	0.27	0.36
		202 (77.9), 222 (61.5), 264 (81.7),
	2	280 (43.9), 421 (12.8), 449 (13.9),	606	0.050		110	918	0.69	0.53	0.31	0.34
		480 (5.8)
		201 (72.9), 223 (91.0), 263 (95.2),
	3	289 (45.1), 388 (11.5), 441 (14.8),	645	0.014		207	617	0.55	0.56	n.d.	0.21
		480 (10.0)
		201 (100.1), 223 (91.3), 263 (105.8),
	4	308 (28.2), 386 (13.8), 438 (16.7),	654	0.020		326	1387	0.62	0.59	0.25	0.26
		441 (16.8), 480 (13.1)
	5	201 (89.3), 224 (81.2), 265 (91.1), 379 (25.6), 458 (23.1), 480 (20.0)	703	<0.001		75	339	0.24	0.30	n.d.	0.21
		192 (183.4), 279 (126.3), 441 (23.2),
	6	457 (23.2), 480 (18.9), 510 (5.3), 540	623	0.021		161	1096	0.61	0.63	n.d.	0.05
		(1.3), 595 (0.014)
	7	192 (168.8), 280 (102.5), 371 (35.0), 465 (30.1), 480 (28.8)	694	<0.001		55	312	0.22	0.35	n.d.	0.07

Table 3 .

 3 IC 50 values (µM) for 6 in mouse colon carcinoma (CT-26) cells in the dark and upon light irradiation at 510 nm (40 min, 10.00 J cm -2 ), 540 nm (60 min, 14.25 J cm -2 ) and 595 nm (2 h, 22.47 J cm -2 ).

										Wavelength	Dark		Light		PI
										/nm
									510 nm		4.18 ± 0.56	0.20 ± 0.005	20.6
									540 nm		3.27 ± 0.64	0.34 ± 0.005	9.6
									595 nm		1.41 ± 0.003 0.06 ± 0.004	23.5
			-26			U87			U373			HeLa			RPE-1
		Dark Light PI	Dark Light PI	Dark Light PI	Dark Light PI	Dark Light PI
	1	>100	>100	-	>100	93.68	>1	>100	>100	-	>100	>100	-	>100	>100	-
						±
						2.50
	2	>100	91.24	>1	>100	71.40	>1	>100	>100	-	>100	>100	-	>100	>100	-
			± 7.54			±
						5.67
	3	>100	85.71	>1	>100	>100	-	>100	>100	-	>100	>100	-	>100	>100	-
			± 9.47
	4	>100	72.59	>1	>100	>100	-	>100	>100	-	>100	>100	-	>100	>100	-
			± 7.44
	5	>100	52.54	>2	>100	>100	-	>100	>100	-	>100	>100	-	>100	>100	-
			± 6.04
	6	3.09	0.19 ±	16.3 28.45	0.67	42.5	23.37	1.89	12.4 13.57	0.61	22.2 28.77	0.83	34.9
		±	0.04		±	±		±	±		±	±		±	±
		0.30			1.97	0.13		0.53	0.07		1.30	0.06		0.94	0.03
	7	94.47	6.62 ±	14.3 >100	7.90	>12.7 >100	14.85	>6.7 >100	15.21	>6.5 >100	8.95	>11.2
		±	0.07			±			±			±			±
		7.38				0.54			0.81			1.29			0.50

ACKNOWLEDGMENT

We thank Dr. Philippe Goldner for access to state-of-the-art laser apparatus. This work was financially supported by an ERC Consolidator Grant PhotoMedMet to G.G. (GA 681679) and has received support under the program "Investissements d' Avenir" launched by the French Government and implemented by the ANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.). I.C and F.M. gratefully acknowledge the European Research Council (ERC) for funding (ERC Consolidator Grant STRIGES to I.C., GA No 648558).

ORCID-ID:

Johannes Karges: 0000-0001-5258-0260 Franz Heinemann: 0000-0001-9590-443X Federica Maschietto: 0000-0002-5995-2765 Marta Jakubaszek: 0000-0001-7590-2330 Chloé Subecz : 0000-0001-8510-4818 Mazzarine Dotou: 0000-0001-87814-6763 Olivier Blacque: 0000-0001-9857-4042 Bruno Goud: 0000-0003-1227-4159 Emilio Viñuelas Zahínos: 0000-0003-0634-1829 Bernhard Spingler: 0000-0003-3402-2016 Ilaria Ciofini: 0000-0002-5391-4522 Gilles Gasser: 0000-0002-4244-5097

ABBREVIATIONS

PDT, Photodynamic Therapy; PS, Photosensitiser; ROS, reactive oxygen species; 1 O 2 , singlet oxygen; dmb, 4,4′-dimethyl-2,2′bipyridine; IP-TT, 2-(2′,2″:5″,2′ ′′-terthiophene)-imidazol[4,5f][1,10]phenanthroline; phen, 1,10-phenanthroline; bphen, 4,7diphenyl-1,10-phenanthroline; bipy, 2,2'-bipyridine; MLCT, metal to ligand charge transfer; EDG, electron donating group; EWG, electron withdrawing group; LE, ligand centered; GS, ground state; ES, excited state; 1 O 2 , singlet oxygen; logP, distribution coefficient; PI, phototoxic index; ICP-MS, inductively coupled plasma mass spectrometry; HeLa, human cervical carcinoma cell line; CT-26, mouse colon carcinoma cell line; U87 human glioblastoma cell line, U373, human glioblastoma astrocytoma cell line, RPE-1 retina pigmented epithelial cell line; MCTS, multicellular tumor spheroid.

AUTHOR INFORMATION

Corresponding Author *spingler@chem.uzh.ch *Ilaria.ciofini@chimieparistech.psl.eu *gilles.gasser@chimieparistech.psl.eu; www.gassergroup.com; Tel. +33 1 44 27 56 02.


MD, PhD Hervé Laborde-Castérot
email: herve.laborde-casterot@aphp.fr

Éric Darrouzet

PharmD Gaël Le Roux

MD Magali Labadie

PharmD Nicolas Delcourt

Luc De Haro

MD, PhD Dominique Vodovar

MD Jérôme Langrand

PhD Eric Darrouzet

MD, PhD ; Luc De Haro

Ocular Lesions Other Than Stings Following Yellow-Legged Hornet ( Vespa velutina nigrithorax ) Projections, as Reported to French Poison Control Centers

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Ocular injuries related to hymenopterans (bees, wasps, ants, and sawflies) are usually related to corneal stings. Complications threaten sight via different mechanisms (e.g., venom toxicity, foreign-body reaction, infection, immunological reaction). [START_REF] Nowroozzadeh | Corneal wasp sting: A case report and review of literature[END_REF] To date, liquid projection from any hymenopterous insect is not a known cause of ocular injury.

Commonly referred to as the "Asian hornet," the yellow-legged hornet Vespa velutina nigrithorax was introduced from China to southwestern France around 2004. [START_REF] Arca | Reconstructing the invasion and the demographic history of the yellow-legged hornet, Vespa velutina, in Europe[END_REF] It is not very aggressive toward humans and attacks only when it feels threatened. [START_REF] Darrouzet | Un Redoutable Prédateur[END_REF] Similar to the European hornet (Vespa crabro), it may be responsible for severe medical complications, but typically only in cases of multiple stings or anaphylaxis. [START_REF] De Haro | Medical consequences of the Asian black hornet (Vespa velutina) invasion in Southwestern France[END_REF][START_REF] Vidal | Anaphylaxis to Vespa velutina nigrithorax: pattern of sensitization for an emerging problem in Western countries[END_REF] Over the past decade, French poison control centers (PCCs) have received several unusual calls about patients experiencing ocular symptoms related to the yellow-legged hornet, without having been stung.

That particular behavior from V velutina and its clinical consequences have not yet been reported. This study aimed to describe the characteristics and outcomes of yellow-legged hornet ocular projections recorded by French PCCs.

Methods

All cases of exposure to xenobiotics collected by France's eight PCCs are registered anonymously in the French National Database of Poisonings (FNDP; French Ministry of Health). The FNDP's use in this study was authorized by the French data protection authorities, and informed consent was waived.

We carried out a retrospective study to analyze all ocular exposure cases caused by the yellow-legged hornet (excluding stings in the eyes) collected in the FNDP between January 1, 2004 (the estimated year the yellow-legged hornet appeared in France), and December 31, 2019. V velutina was identified based on the victim's description during a phone call to the PCC. [START_REF] Darrouzet | Un Redoutable Prédateur[END_REF] A clinical toxicologist reviewed all cases and collected the following data: sex and age of the victim, geographical location (eFigure 1), date and circumstances of exposure (occupational or not), symptoms, and outcome.

Results

The FNDP presented 29 ocular exposure cases caused by a yellow-legged hornet, excluding stings in the eye (Table 1). These cases represent about 6% of all accidental exposure to the yellow-legged hornet cases recorded in the FNDP during the same period (516 cases). There was one case per year in 2009, 2010, 2012, and 2013, three or four cases between 2014 and 2017, eight in 2018, and two in 2019. Except for two cases, all occurred between July and November. Exposures were initially predominant in the southwest area.

Circumstances of Exposure

About two-thirds of cases involved professionals dealing with nests, mostly firefighters and wasp exterminators, who were wearing the usual protective clothing, including a woven mesh in front of the face. The remaining patients were individuals exposed while they were trying to, and/or succeeding in, squashing a yellow-legged hornet, while they were resting outdoors, or while gardening. Two patients wore vision glasses that had slipped because of head movements (cases 9, 20).

Symptoms

All patients experienced unilateral eye redness and pain. In addition, five patients developed a periorbital edema, three reported blurred vision, and one suffered from rhinitis. Two patients experienced persistent radiating pains from the affected eye to the ipsilateral hemicranium although other ocular symptoms had resolved. Two patients had a history of anaphylaxis to the European hornet but developed no allergic manifestation during their call to the PCC (i.e., after 60 minutes for case 5 and 90 minutes for case 20).

Outcome

Eight patients were lost to follow-up. Nineteen patients visited a physician. Among 13 fluorescein eye stain tests performed, two revealed corneal lesions. Ocular symptoms resolved quickly, usually within a few hours of decontamination, with water or saline solution or sometimes after a few days of symptomatic treatment. One patient reported a persistent periorbital edema on day 6, and one presented with hemicranial neuropathic pain for 20 days.

Discussion

French PCCs recorded a series of 29 cases of ocular symptoms after a projection from a yellow-legged hornet, which were associated with minor injuries and favorable outcomes. After immediate ocular irritation, characterized by eye redness and pain, symptoms normally resolved quickly. However, two patients suffered from keratitis and two others experienced neuropathic pain. No allergic reaction was observed in this series.

The yellow-legged hornet has a surprising ability to project liquid, [START_REF]Drone Attacked by Asian hornets[END_REF] which was commonly thought to be venom; however, there are no scientific data on its nature or anatomic origin. Indeed, like all Vespidae species, the yellow-legged hornet attempts to sting or exude venom from its stinger if it feels threatened. Moreover, individuals can use sting venom volatiles as an alarm pheromone to attract multiple hornets for an attack. [START_REF] Cheng | Poison and alarm: the Asian hornet Vespa velutina uses sting venom volatiles as an alarm pheromone[END_REF] The venom's composition could explain the symptoms observed in these cases. Nearly 300 putative toxins have been identified in V velutina venom, some of which could be involved as metalloproteases in corneal damage. [START_REF] Liu | Deciphering the venomic transcriptome of killer-wasp Vespa velutina[END_REF] Others are neurotoxins that could explain the prolonged neuropathic pain observed in two patients. [START_REF] De Haro | Medical consequences of the Asian black hornet (Vespa velutina) invasion in Southwestern France[END_REF] However, because the venom gland's volume is largely inferior to the volume of liquid projected (i.e., the venom reservoir is about 1 mm in length), [START_REF] Le | Asian hornet Vespa velutina nigrithorax venom: Evaluation and identification of the bioactive compound responsible for human keratinocyte protection against oxidative stress[END_REF] this liquid might originate from the intestinal tract, with or without venom.

Moreover, the projection mechanism is also unexplained; it may be voluntary or a reflex during flight. This phenomenon has also been observed in other hymenoptera species, such as ants, [START_REF] Faith | Chemical analysis of the defensive secretion from the Western Thatching Ant, Formica obscuripes[END_REF] or coleoptera species, such as carabid beetles, [START_REF] Will | Quantification and evidence for mechanically metered release of pygidial secretions in formic acid-producing Carabid Beetles[END_REF] which project a liquid containing formic acid. This projection occurs when the insect feels threatened. However, to our knowledge, this behavior has not been observed in Vespidae species. One hypothesis is that insects with a sting present venom without formic acid and use different behavior for defense (sting vs. liquid projection). Our findings in V velutina could indicate defense behaviors, stinging, and liquid projection. In addition, in the domestic setting, exposures occurred mainly when killing the insect; in these cases, the projection could be a result of being threatened or a mechanical effect of abdomen compression from crushing the insect.

When dealing with a yellow-legged hornet nest, professionals are at risk of being sprayed in the face; wearing a mesh over the face is insufficient protection. In fact, this risk could be increased by the mesh if hornets indeed release their venom when stinging the mesh. Wearing safety glasses is highly recommended. Our case series shows that wearing vision glasses is ineffective protection. Therefore, prompt ocular decontamination with water or saline solution is strongly suggested in cases of ocular exposure.

Limitations

The identification of a yellow-legged hornet based on the information provided by callers to PCCs was compatible with its temporal and geographical evolution. The first case was reported in 2009, and the number of annual cases increased gradually, spreading from southwestern France to the rest of the country.

Cases increased each year until 2018, but fewer cases were recorded in 2019, consistent with field observations that found fewer nests that year, probably due to preventive measures and/or weather conditions. Moreover, no case of ocular exposure to a projection from a hornet was found in the FNDP from 2000 to 2003, before the yellow-legged hornet appeared in France. Furthermore, our data are limited by the retrospective design of the study and should be confirmed by further research.

Conclusions

This original case series shows that the yellow-legged hornet V velutina can extrude and project an unidentified liquid in the eyes, causing ocular lesions. Periorbital edema, neuropathic pain, and keratitis were reported, which had favorable outcomes. This study highlights a new occupational risk for professionals dealing with yellow-legged hornet nests that requires the usual preventive measures.

Funding/Support: No specific funding.

Additional Contributions:

We thank Ali Saïd (Centre antipoison de Paris) for his help in collecting the data and Claire Villemant and Quentin Rome (Muséum National d'Histoire Naturelle, Paris) for their expert advice. None of these individuals received compensation for their contributions. We would like to thank Enago for the English language review.

Acknowledgments

Collaborators: the French Poison Control Centers Research Group members: Emmanuel Puskarczyk and Christine Tournoud (Nancy), Wenico Caré and Laurène Dufayet (Paris), Fanny Pelissier and Nicolas Franchitto (Toulouse), Camille Paradis (Bordeaux), Nicolas Simon (Marseille), Patrick Nisse (Lille), Nathalie Paret and Anne-Marie Patat (Lyon), Marie Deguigne and Marion Legeay (Angers).

Conclusions and Relevance: The projection of a liquid into the eyes by the yellow-legged hornet presents a new risk to human health, but its precise nature remains to be determined. Ocular lesions had a favorable outcome. For professionals who deal with these insects, adaptation of the usual protections designed for native hymenopterans is required.

Author Contributions: Drs Laborde-Castérot and Langrand had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Concept and design: Laborde-Castérot, Langrand.

Acquisition, analysis, or interpretation of data: All authors.

Drafting of the manuscript: Laborde-Castérot, Darrouzet, Vodovar.

Critical revision of the manuscript for important intellectual content: All authors.

Administrative, technical, or material support: Laborde-Castérot, Langrand. Supervision: Laborde-Castérot, Langrand.

Conflict of Interest Disclosures: None reported.