Milad Mirabedin

Hugues Vergnes

Nicolas Caussé

Constantin Vahlas

Brigitte Caussat

Liquid antimony pentachloride as oxidant for robust oxidative chemical vapor deposition of poly(3,4-ethylenedioxythiophene) films

Keywords:

Thin films

EDOT

Introduction

The growing development of elect: ronic and optoelectronic devices such as organic light-emitting diodes (OLEDs) and organic solar cells require scalable and transparent electrodes, composed of thin films that sinrnltaneously provide electt•ical conductivity, flexibility and optical n•ansparency [1-3). Conducting polymers present unique advantages over more common inorganic materials, nan1ely cost effectiveness, lightweight and resource abundance, while ensuring satisfacto1y elec ttical conductivity. They can be conveniently processed, even on flexible substrates [3]. Nowadays, they are considered as "a new generation of materials which exhibit the electt• ical prope1ties of metals or senti conductors and retain the attractive mechanical properties and pro cessing advantages of polymers" [START_REF] Matĕjka | Hybrid nanocomposites for nanotechnology: electronic, optical, magnetic and biomedical applications[END_REF]. Poly(3,4 ethylenedioxythiophene) (PEDOT) is an1ong the most chentically and thermally stable conducting polymers. lt is commercialized in its dispersion form mixed with poly styrene sulfonate (PSS) polymer [START_REF] Elschner | PEDOT: principles and applications of an intrinsically conductive polymer[END_REF]. Thin films of PEDOT:PSS are most often processed by spin coating from aqueous ntixtures of PSS and PEDOT and are now present in OLEDs, supercapacitors, solar cells, electrochromic devices and other devices [START_REF] Zabihi | Morphology, conductivity, and wetting characteristics of PEDOT: PSS thin films deposited by spin and spray coating[END_REF].

• Corresponding author.

E-mail address: brigitte.caussat@ensiacet.fr (B. caussat).

Since two decades, vapor phase deposition processes, in pa1ticular vapor phase polymerization (VPP) [START_REF] Kim | The preparation and characteristics of conductive poly (3, 4-ethylenedioxythiophene) thin film by vapor-phase polymerization[END_REF] and oxidative chentical vapor deposition (oCVD) [START_REF] Lock | Oxidative chemical vapor deposition of electrically conducting poly (3, 4-ethylenedioxythiophene) films[END_REF] have been developed to avoid the substrate solvent incompatibility of liquid solution technologies and to provide confo1mal coatings on complex porous subsn• ates. In VPP and oCVD, the oxidant se1ves both to promote monomer polymerization and to sub sequently oxidize the neutral polymer to fotm the conductive, doped polymer. According to Lock et al. [START_REF] Lock | Oxidative chemical vapor deposition of electrically conducting poly (3, 4-ethylenedioxythiophene) films[END_REF], the formation of PEDOT follows a

Step Growth Polymerization (SGP) mechanism, requiting the simulta neous presence of monomer and oxidant for continuous polymerization. VPP still involves a first liquid step to deposit the oxidant layer, followed by the introduction of EDOT vapors at a second step. The polymerization stops when the oxidant is no more available, even if EDOT vapor is still present [START_REF] Ali | Translated structural morphology of conductive polymer nanofilms synthesized by vapor phase polymerization[END_REF]. In contrast, oCVD is a one-step purely gas phase technique, allowing to control the film thickness by vaiying the deposition time. Provided the vaporized oxidant flow rate is fully controlled, it yields films whose characteristics and prope1ties can be finely tuned.

The nature of the oxidant plays an in1po1tant role in the chai• acter istics of oCVD PEDOT films [START_REF] Ali | Translated structural morphology of conductive polymer nanofilms synthesized by vapor phase polymerization[END_REF][START_REF] Im | Conformal coverage of poly (3, 4-ethylenedioxythiophene) films with tunable nanoporosity via oxidative chemical vapor deposition[END_REF]. Oxidants are mostly ionic com pounds with one of their atoms belonging to the halogen group, mainly https:/ /doi.org/10.1 0l 6/j.apsusc.2021.149501 and electrical conductivity of the films, without any post deposition rinsing step. We finally investigate in details the film uniformity over a large scale inside the reactor chamber.

Materials and methods

oCVD experiments are performed in a custom-built stainless steel reactor (Mageva, Neyco®), as illustrated in Fig. 1a and already presented in [START_REF] Mirabedin | An out of the box vision over oxidative chemical vapor deposition of PEDOT involving sublimed iron trichloride[END_REF]. The reactor (total effective volume of 17 l) is equipped in its top part with a ceramic substrate holder heated by Joule effect fixed horizontally at the lid. A thermocouple is placed over the back of the ceramic and is connected to a PID controller to monitor the substrate temperature (T sub ) between 25 and 130 • C with an accuracy of 0.8%. The total pressure is controlled within the range 1-133 Pa by a system composed of a Leybold Ruta 501 pump, a gate valve and a Baratron pressure gauge. Unless otherwise mentioned, the total pressure is fixed at 75 Pa and the deposition time is kept at 30 min.

PEDOT films were produced from monomeric 3,4-ethylenedioxythiophene (EDOT, 97%, Sigma-Aldrich) and SbCl 5 (99%, Acros Organic). The liquid SbCl 5 was evaporated from a bubbler immersed in a thermally-regulated bath at 90 • C. At this temperature, the SbCl 5 saturation vapor pressure is close to 5000 Pa [START_REF] Stull | Vapor pressure of pure substances. Organic and inorganic compounds[END_REF]. The bubbler was loaded in a glove box under argon atmosphere. The flow rate of the SbCl 5 vapors was adjusted with a needle valve placed on the gas line at the exit of the bubbler. EDOT was vaporized in another bubbler, also immersed in a thermally-regulated bath in the range 65-90 • C. The EDOT saturation vapor pressure at 90 • C is 1000 Pa. All gas lines between the bubblers and the reactor inlets were heated at 100 • C to avoid condensation. 10 standard centimeters per minute (sccm) of nitrogen (99.999%, Air Liquide) were also sent into the reactor during the depositions. PEDOT films were deposited on 10 cm Si (1 0 0) wafers, used as received. Their sheet resistance was measured as close to 35 ± 5 kΩ/□, significantly high to avoid sheet resistance correction due to parallel conduction of the substrate. No post deposition treatment was applied.

Before and after each experiment, the mass difference of the SbCl 5 bubbler, of the EDOT jar and of the silicon wafer was systematically determined using a KERN ABS 320-4 N balance with a precision of 0.1 mg for the two bubblers, and a Sartorius R180D balance with a precision of 0.01 mg for the substrate. This procedure allowed to ascertain the flow rates of the vaporized EDOT and SbCl 5 , thus the inlet molar SbCl 5 / EDOT ratio, and the mass of the deposited film.

Film thickness was measured by profilometry with a DektakXT instrument with a 2 µm stylus tip radius and 3 mg stylus load. Measurements were performed on consecutive 1 cm distant points, along two perpendicular × and y diameters. Step heights were made on purpose by chosen for their oxidation potential. Common drawbacks are their sensitivity to oxygen and humidity, their corrosive nature and their toxicity. Since the introduction of oCVD of PEDOT by Gleason and coworkers [START_REF] Lock | Oxidative chemical vapor deposition of electrically conducting poly (3, 4-ethylenedioxythiophene) films[END_REF], several solid oxidants have been used to produce PEDOT films from EDOT monomer. Among them, iron trichloride (FeCl 3 ) is the most studied one [START_REF] Lock | Oxidative chemical vapor deposition of electrically conducting poly (3, 4-ethylenedioxythiophene) films[END_REF][START_REF] Mirabedin | An out of the box vision over oxidative chemical vapor deposition of PEDOT involving sublimed iron trichloride[END_REF][START_REF] Barr | Cathode buffer layers based on vacuum and solution deposited poly (3, 4-ethylenedioxythiophene) for efficient inverted organic solar cells[END_REF][START_REF] Im | Systematic control of the electrical conductivity of poly (3, 4-ethylenedioxythiophene) via oxidative chemical vapor deposition[END_REF][START_REF] Im | Doping level and work function control in oxidative chemical vapor deposited poly (3, 4-ethylenedioxythiophene)[END_REF], because of its availability and relatively low toxicity [START_REF] Arnold | Microstructuredependent electrochemical properties of chemical-vapor deposited poly (3, 4-ethylenedioxythiophene)(PEDOT) films[END_REF][START_REF] Drewelow | Factors controlling conductivity of PEDOT deposited using oxidative chemical vapor deposition[END_REF]. However, sublimation of solid oxidants is challenging as these compounds are highly hygroscopic and require manipulation under inert atmosphere [START_REF] Vahlas | Liquid and solid precursor delivery systems in gas phase processes[END_REF]. In a previous study, we demonstrated that the sublimation of FeCl 3 in a conventional crucible was only active for the first minutes of process, due to unavoidable oxidation and sintering of the powder and we concluded that the stability of the sublimation rate over time is a prerequisite for process implementation [START_REF] Mirabedin | An out of the box vision over oxidative chemical vapor deposition of PEDOT involving sublimed iron trichloride[END_REF]. Besides this process issue, FeCl 3 derivatives and residues are often present in oCVD processed PEDOT films due to oxidant condensation during deposition. These hydroscopic contaminants degrade the electrical conductivity and the durability of the films, and require additional steps of post deposition liquid rinsing to remove them [START_REF] Howden | oCVD poly (3, 4-ethylenedioxythiophene) conductivity and lifetime enhancement via acid rinse dopant exchange[END_REF].

For these reasons, solid oxidants have been progressively replaced by easier to vaporize liquid ones, such as bromine (Br 2 ) [START_REF] Chelawat | Conformal, conducting poly (3, 4-ethylenedioxythiophene) thin films deposited using bromine as the oxidant in a completely dry oxidative chemical vapor deposition process[END_REF], and more recently sulfuric acid (H 2 SO 4 ) [START_REF] Farka | Metallic conductivity beyond the Mott minimum in PEDOT: Sulphate at low temperatures[END_REF], molybdenum(V) chloride (MoCl 5 ) [START_REF] Atanasov | Highly conductive and conformal poly (3, 4-ethylenedioxythiophene) (PEDOT) thin films via oxidative molecular layer deposition[END_REF], vanadium oxytrichloride (VOCl 3 ) [START_REF] Xu | Building ultraconformal protective layers on both secondary and primary particles of layered lithium transition metal oxide cathodes[END_REF][START_REF] Gharahcheshmeh | Tuning, optimization, and perovskite solar cell device integration of ultrathin poly (3, 4-ethylene dioxythiophene) films via a single-step all-dry process[END_REF][START_REF] Kaviani | Electroactive and Conformal Coatings of oCVD Polymers for Oxygen Electroreduction[END_REF] and antimony(V) pentachloride (SbCl 5 ) [START_REF] Kaviani | Electroactive and Conformal Coatings of oCVD Polymers for Oxygen Electroreduction[END_REF][START_REF] Gharahcheshmeh | Optimizing the optoelectronic properties of face-on oriented poly(3,4-ethylenedioxythiophene) via water-assisted oxidative chemical vapor deposition[END_REF]. SbCl 5 appears to be cost effective and among the less toxic compounds of this list. To the best of our knowledge, the only works available on oCVD of PEDOT from SbCl 5 are that of Kaviani et al. [START_REF] Kaviani | Electroactive and Conformal Coatings of oCVD Polymers for Oxygen Electroreduction[END_REF] and of Gharahcheshmeh et al. [START_REF] Gharahcheshmeh | Optimizing the optoelectronic properties of face-on oriented poly(3,4-ethylenedioxythiophene) via water-assisted oxidative chemical vapor deposition[END_REF]. The latter recently studied the correlations existing between nanostructure, crystal state and electrical conductivity of PEDOT thin films, in order to optimize their optoelectronic properties. They succeeded in reaching significantly high electrical conductivities up to 7500 S/cm, by operating at 140 • C deposition temperature and in the presence of water vapor. Water vapor appears to facilitate the PEDOT linear polymerization forming the conductive quinoid structure, by exalting the oxidant SbCl 5 decomposition into Cl -dopant. Kaviani et al. [START_REF] Kaviani | Electroactive and Conformal Coatings of oCVD Polymers for Oxygen Electroreduction[END_REF] investigated the electrocatalytic activity in oxygen reduction reaction of PEDOT films deposited on carbon substrates without, however, correlating the films characteristics with the deposition conditions. This is the focus of the present work, with an ultimate objective to develop a robust one-step full dry oCVD process for the production of PEDOT thin films of controlled characteristics and properties.

We first study the stability over time of the vaporized SbCl 5 oxidant flow rate to guarantee a full control of the process, in particular of the film thickness. Then, we analyze the influence of the SbCl 5 /EDOT molar ratio, substrate temperature, total pressure and deposition duration on the mass, thickness, uniformity, morphology, chemical composition, covering p1ior deposition a 2 cm large cross-zone of the substrates with a Kapton tape (Fig. 1 b), allowing a 56 ± 3 cni2 deposition surface on the Si wafer. Three measurements were made at each position and then averaged. Scanning electron microscopy (SEM) (Helios NanoLab 600i) was used to check the film thickness on cross-sections of selected san1ples eut in liquid nitrogen and to investigate the smface morphology on top view micrographs.

Atomic Force Microscopy (AFM) maps were obtained using an Agi lent Technologies AFM/STM 5500 (PicoPlus) microscope. Film compo sition was analyzed using transmission Fourier transform infrared spectrometty (FTIR), pe1fom1ed in a PerkinElmer Spotlight 400 bench with 16 scans at 4 cm-1 resolution. Optical tt•ansn1ittance of films deposited on purpose on glass substrates (Deckglaser 0101062) was measured using an Agilent Caiy 5000 UV-Vis spectt•ometer wit11in the range 300-800 nm. A scan rate of 600 nm/n1in with 2 nm spectral band width (SBW) was applied. The film sheet resistance Rs (Q/0) was measured on the wafer substt•ates with a Signatone S-302-4 four-point probe station. A constant current of 200 nA was applied with a Keith ley 6220 instt• ument, and the corresponding voltage was read on a Keithley 2182A one. The electrical conductivity cr (S/cm) was deduced from Eq. ( 1) where t is the tl1ickness of the film (cm) [START_REF] Smits | Measurements of sheet resistivities with the four point probe[END_REF].

(1 -R,. t

Results and discussion

Prelimina,y srudy on che oxidanc fl ow race scabilicy

(1)

Continuous and constant presence of oxidant vapor is mandato1y to produce in a controlled way PEDOT films by oCVD, as explained in introduction. With the aim to probe the evaporation rate of SbCls, we monitored the weight Joss of the SbC1 5 bubbler for 3 evaporation du rations, 30, 60 and 120 n1in in the conditions of PEDOT deposition ex perin1ents, i.e. at a total pressure of 75 Pa under 10 sccm of N 2 . The results ai•e repo1ted in Fig. 2. The evaporated mass of SbC! 5 Jineai• ly increases from 134 to 551 mg when the evaporation time is increased from 30 n1in to 120 n1in, providing a constant flow rate of 4.5 mg/n1in (or 0.35 sccm) of gaseous SbCl 5 . lt is concluded that SbC1 5 can be vapo1ized in a continuous mode, thus offering the possibility to fully contt•ol the oCVD process.

lt is in1po1tant to note that due to the high saturation pressure of SbCls vapors (150 Pa at 20 ° C) in compai' ison to its pai t ial pressure in the reactor (close to 2.5 Pa), we did not observe any droplets of condensed SbCls forn1ing on the reactor walls or on a Si wafer intentionally placed for this purpose on the substrate holder, even at an1bient ten1perature. This is an advantage of the SbC1 5 involving oCVD process in compa1ison with FeC1 3 , since the cleaning procedure of the reactor between two deposition expe1iments is simplified.

U.o

,..-.., 0>500 -S • ,..-.., � 500 .l E (Il • 0 .4 8 E + .!!!., -u 400

Influence of che inlec SbClsfEDOT molar ratio

With the aim to investigate the influence of the composition of the input gas on the films chai• acteristics and on their electrical conductivity we pe1formed a selies of depositions at 25 °C fixed T su b and at different inlet SbCl 5 /EDOT molai• ratios in the range 0.18 to 3.00. For the ratios between 0.18 and 0.76, the EDOT flow rate was vai• ied between 2.1 and 0.85 sccm for a constant SbC1 5 flow rate of 0.26 sccm. For the SbCl 5 / EDOT ratios of 2.08 and 3.00, the EDOT flow rate was kept constant at 0.42 sccm and the SbC1 5 flow rate was vai•ied between 0.86 and 1.26 sccm. In ail nms, the total gas flow rate (EDOT + SbC1 5 + Nz) was kept constant at 10-11 sccm; i.e. close to the N 2 flow rate, except for the run with the lowest SbClsfEDOT ratio of 0.18 where, due to the high EDOT flow rate, the total gas flow slightly increased to 12.5 sccm.

Fig. 3a details the evolution of the mass and average thickness of the deposited films. lt appears that when the SbCls/EDOT ratio increases from 0.18 to 0.76 coITesponding to the decrease of EDOT flow rate, the mass of the film slightly increases whereas its thickness slightly de creases. This opposite valiation could be due to an evolution of the film porosity, since the FTIR results detailed later do not show any -OH peak indicating the absence of hydrophilic by-products related to possible antimony oxides. The weak mass evolution indicates that in this process window, the EDOT supply is not a mai•kedly lin1iting factor of the pro cess. For ratios higher than 0.76 coITesponding to increasing SbC! 5 flow rates, both the mass and t11ickness of the film shai•ply increase. This result clearly demonstt• ates that for these conditions, the deposition of background are present in other samples as well, but for the highest SbCl 5 /EDOT ratio analyzed (2.08), the large bright regions are replaced by larger particles of hundred nanometers in diameter. AFM maps of several samples (Fig. 5b) show that the bright particles or islands are significantly thicker than the underneath film. At a SbCl 5 / EDOT ratio 0.18, their height reaches 80 nm and increases to 180 nm and 400 nm for the ratios 0.42 and 0.76, respectively, all on a 10 × 10 µm 2 zone. These islands were not visible by profilometry, probably due to the pressure applied on the polymeric film by the stylus, which could crush them.

EDX and Raman analysis of these samples (Figures S1, S2 and S3 and Table S1 of Supplementary Materials) reveal all PEDOT signatures on the entire surface of the films, including the particles. According to localized Raman probing, the bright particles contain less quinoid (doped) PEDOT in comparison with the other zones and are consequently less conductive. This could explain the decrease of conductivity for the roughest films produced at the highest SbCl 5 flow rates tested.

Sb was equally detected by EDX and XPS all over the surface of the films (Figures S1 andS4 and Table S2 of Supplementary Materials), at an average concentration not exceeding 5 at.%, in agreement with recently published results [START_REF] Gharahcheshmeh | Optimizing the optoelectronic properties of face-on oriented poly(3,4-ethylenedioxythiophene) via water-assisted oxidative chemical vapor deposition[END_REF]. The presence of Sb may be due to the formation of Sb-complexes on the PEDOT film through polymerization and reduction of adsorbed SbCl 5 after losing two chlorine atoms. SbCl 3 is indeed the most common oxidant by-product of SbCl 5 decomposition [START_REF] Gharahcheshmeh | Optimizing the optoelectronic properties of face-on oriented poly(3,4-ethylenedioxythiophene) via water-assisted oxidative chemical vapor deposition[END_REF]. Based on this assumption, the formation of bonds between SbCl 3 and the oxygen in monomer ethylenedioxy rings results in trapping Sb during the film growth, as proposed by Kaviani et al. [START_REF] Kaviani | Electroactive and Conformal Coatings of oCVD Polymers for Oxygen Electroreduction[END_REF] for PEDOT films containing comparable amounts of Sb. The concentration of Sb in the films is lower than that of Fe in PEDOT films deposited from FeCl 3 [START_REF] Mirabedin | An out of the box vision over oxidative chemical vapor deposition of PEDOT involving sublimed iron trichloride[END_REF]. This difference can be attributed to the fact that, in contrast to FeCl 3 , gaseous SbCl 5 cannot condensate on the film surface in the adopted operating conditions. Fig. 6a presents FTIR spectra of the films processed with SbCl 5 /EDOT ratios between 0.18, 0.42, 0.76 and 2.08. Their intensity is normalized to that of the C-O-C stretching bond at 1091 cm -1 , since the corresponding peak is not modified by polymerization. Table S3 in Supplementary Materials details the attribution to the different bond vibrations of all peaks in the 2000-400 cm -1 range. All PEDOT peaks are observed in all samples and no peak is observed in this range corresponding to SbCl 5 and its byproducts. No OH peak is observed at 3400 cm -1 meaning that PEDOT-SbCl 5 films are not contaminated by humidity. This is differentiating advantage compared to PEDOT-FeCl 3 films [START_REF] Mirabedin | An out of the box vision over oxidative chemical vapor deposition of PEDOT involving sublimed iron trichloride[END_REF].

The normalization of the spectra with regard to the C-O-C stretching peak at 1091 cm -1 allows comparing the C C antisymmetric stretching peak at 1527 cm -1 . It is known that the intensity of the C C peak increases with the conjugation length of PEDOT [START_REF] Im | Systematic control of the electrical conductivity of poly (3, 4-ethylenedioxythiophene) via oxidative chemical vapor deposition[END_REF] and that such an increase enhances the electrical conductivity of conductive polymers [START_REF] Cheng | Reactive vapor deposition of conjugated polymer films on arbitrary substrates[END_REF]. The histogram of Fig. 5b shows that the under peak area of the C C peak decreases with increasing the SbCl 5 /EDOT ratio, which is in agreement with the conductivity results of Fig. 3b.

The results presented in this section allow concluding that the most conductive PEDOT films formed at 25 • C are the most uniform and smoothest ones, deposited with the lowest SbCl 5 (0.35 sccm) and the highest EDOT (2 sccm) flow rates tested, corresponding to an inlet SbCl 5 /EDOT molar ratio of 0.18.

Influence of the substrate temperature

Convergent literature information [START_REF] Lock | Oxidative chemical vapor deposition of electrically conducting poly (3, 4-ethylenedioxythiophene) films[END_REF][START_REF] Drewelow | Factors controlling conductivity of PEDOT deposited using oxidative chemical vapor deposition[END_REF][START_REF] Gleason | Fabrication of Organic Surfaces and Devices[END_REF] concludes on the improvement of the conductivity when T sub is increased, due to the reorganization of the conjugation in the backbone of the polymer. In the present section, we investigate the influence of T sub in the range 25-70 • C, for the lowest SbCl 5 /EDOT ratio tested in the previous section (0.18). Fig. 7a details the evolution of the film thickness and mass as a function of the inverse T sub . It is recalled that all runs were performed PEDOT is controlled by the SbCl 5 flow rate, as observed with other oxidants in particular FeCl 3 [START_REF] Mirabedin | An out of the box vision over oxidative chemical vapor deposition of PEDOT involving sublimed iron trichloride[END_REF]. The deposition rate varies from 0.059 mg/min (1.8 nm/min) to 0.268 mg/min (15 nm/min), for SbCl 5 /EDOT ratios 0.18 and 3.00, respectively. These values are higher than the only deposition rates values available in the literature for PEDOT oCVD from SbCl 5 [START_REF] Gharahcheshmeh | Optimizing the optoelectronic properties of face-on oriented poly(3,4-ethylenedioxythiophene) via water-assisted oxidative chemical vapor deposition[END_REF], which are between 0.45 and 3.75 nm/min, probably because of the lower total pressure (20 Pa) and higher substrate temperature these authors used. The electrical conductivity of the films is presented in Fig. 3b as a function of the SbCl 5 /EDOT ratio. It remains in the same range of values (470-570 S/cm) when decreasing the EDOT flow rate, for SbCl 5 /EDOT ratios between 0.18 and 0.76. The highest conductivity obtained at ambient temperature in the present series of experiments without any post deposition treatment is 571 S/cm, which is higher than the conductivities obtained for PEDOT-SbCl 5 films at 100 • C after post deposition acid rinsing [START_REF] Kaviani | Electroactive and Conformal Coatings of oCVD Polymers for Oxygen Electroreduction[END_REF], but much lower than the values obtained at 140 • C using water vapor [START_REF] Gharahcheshmeh | Optimizing the optoelectronic properties of face-on oriented poly(3,4-ethylenedioxythiophene) via water-assisted oxidative chemical vapor deposition[END_REF]. The PEDOT-SbCl 5 conductivities are generally higher than those of the PEDOT-FeCl 3 films deposited at ambient temperature [START_REF] Mirabedin | An out of the box vision over oxidative chemical vapor deposition of PEDOT involving sublimed iron trichloride[END_REF]. The conductivity is much lower, below 100 S/cm, for higher SbCl 5 /EDOT ratios of 2.08 and 3, corresponding to the higher SbCl 5 flow rates tested. Let us note that for the conditions tested, the film thickness has no noticeable impact on the electrical conductivity, as will be detailed later. The overall decrease of the electrical conductivity with increasing the oxidant/EDOT ratio is in agreement with previous observations on oCVD PEDOT films from SbCl 5 [START_REF] Gharahcheshmeh | Optimizing the optoelectronic properties of face-on oriented poly(3,4-ethylenedioxythiophene) via water-assisted oxidative chemical vapor deposition[END_REF], FeCl 3 [START_REF] Mirabedin | An out of the box vision over oxidative chemical vapor deposition of PEDOT involving sublimed iron trichloride[END_REF] and VOCl 3 [START_REF] Gharahcheshmeh | Tuning, optimization, and perovskite solar cell device integration of ultrathin poly (3, 4-ethylene dioxythiophene) films via a single-step all-dry process[END_REF]. Since the conductivity of the film deposited at a SbCl 5 /EDOT ratio of 3 is close to 0, we did not consider this sample in the following sections.

The profiles of the film thickness over the 10 cm Si wafer are shown in Fig. 4 along the x1 axis (described in Fig. 1b) for the samples deposited with SbCl 5 /EDOT ratios 0.18 and 2.08. The average thickness of the film processed at a 0.18 ratio is constant along the wafer diameter, at 50 ± 5 nm. A similar behavior was observed for films processed with SbCl 5 /EDOT ratios as high as 0.76. The average thickness of the film processed with a ratio of 2.08 is significantly higher with a mean value of 480 nm and a strong deviation of ±60 nm, probably due to the higher roughness of the film, as detailed below.

Fig. 5a presents FEG SEM surface micrographs of the films deposited with SbCl 5 /EDOT ratios 0.18, 0.42, 0.76 and 2.08. Low magnification micrographs reveal two different zones, namely a dark continuous surface covered by bright particles. The high magnification micrograph of the film deposited at a SbCl 5 /EDOT ratio 0.18 presents three zones: large bright regions over the surface, which are encountered with small bright particles all over a rough dark background. The small particles and dark during 30 min. The increase of T sub from 25 to 50 • C results in the decrease of both the mass and the thickness, from 1.78 to 0.08 mg and from 54 nm to 12 nm, respectively. The deposition rate decreases from 0.059 mg/min to 0.003 mg/min. Operating at 70 • C did not result in film formation, verified by sample weighting, profilometry and FTIR. This influence of T sub on the mass and thickness of the film is in agreement with recent results obtained at 90 and 140 • C [START_REF] Gharahcheshmeh | Optimizing the optoelectronic properties of face-on oriented poly(3,4-ethylenedioxythiophene) via water-assisted oxidative chemical vapor deposition[END_REF]. It is however opposite to what we observed during a previous study with FeCl 3 [START_REF] Mirabedin | An out of the box vision over oxidative chemical vapor deposition of PEDOT involving sublimed iron trichloride[END_REF]. Considering that the SbCl 5 flow rate is the limiting factor of the oCVD process in the conditions tested as previously demonstrated, this behavior may be due to the low and decreasing SbCl 5 concentration existing near the heated substrate with increasing T sub . This is in agreement with the results of Li et al. [START_REF] Li | Deposition behavior of polyaniline on carbon nanofibers by oxidative chemical vapor deposition[END_REF] who found for the oCVD of PANI from aniline and SbCl 5 that the process is most likely limited by reactant adsorption on the substrate surface because higher temperatures generally reduce vapor-to-surface adsorption (or sticking coefficient) and, therefore, reduce the amount of reactant at a surface available for reaction. T sub has no clear influence on the film uniformity as shown in Figure S5 of Supplementary Materials.

The results presented in Fig. 7b reveal that the electrical conductivity slightly increases from 571 to 770 S/cm with increasing T sub . The most conductive film is obtained at the highest T sub tested allowing the formation of the film (50 • C) [START_REF] Mirabedin | An out of the box vision over oxidative chemical vapor deposition of PEDOT involving sublimed iron trichloride[END_REF]. We confirm the positive impact of T sub on the electrical conductivity of oCVD PEDOT, as found elsewhere [START_REF] Drewelow | Factors controlling conductivity of PEDOT deposited using oxidative chemical vapor deposition[END_REF][START_REF] Gharahcheshmeh | Optimizing the optoelectronic properties of face-on oriented poly(3,4-ethylenedioxythiophene) via water-assisted oxidative chemical vapor deposition[END_REF][START_REF] Gleason | Fabrication of Organic Surfaces and Devices[END_REF][START_REF] Andrew | Melding vapor-phase organic chemistry and textile manufacturing to produce wearable electronics[END_REF][START_REF] Bilger | A critical review of reactive vapor deposition for conjugated polymer synthesis[END_REF]. The conductivity of 770 S/cm at T sub at 50 • C is slightly higher than that of Kaviani et al. [START_REF] Kaviani | Electroactive and Conformal Coatings of oCVD Polymers for Oxygen Electroreduction[END_REF] at 120 • C (701 S/cm) for oCVD of PEDOT from SbCl 5 . Even if this article does not detail all the deposition parameters, this may be due to the use of smaller SbCl 5 / EDOT ratios in the present work (0.18 vs. 3 in [START_REF] Kaviani | Electroactive and Conformal Coatings of oCVD Polymers for Oxygen Electroreduction[END_REF]). This behavior could be attributed to the lower deposition rate, allowing a possible organization of the polymer chain in a way that could increase the conjugation length and improve the conductivity. The maximum conductivity we obtained is however much lower than the recent one of Gharahcheshmeh et al. [START_REF] Gharahcheshmeh | Optimizing the optoelectronic properties of face-on oriented poly(3,4-ethylenedioxythiophene) via water-assisted oxidative chemical vapor deposition[END_REF], confirming their statement that process parameters profoundly affect the texture and nanostructure of oCVD PEDOT thin films. A lower total pressure (20 Pa), a higher T sub (140 • C) and the addition of water vapor probably explain this improvement.

Fig. 8 presents FEG SEM surface micrographs of films deposited at 25 and 50 • C. Increasing T sub leads to a size reduction of the large bright islands. The highest magnification views show that the surface morphology is modified by increasing T sub through the formation of nodules whose size is ca. 50 nm. Raman analysis (Figure S6) revealed that both the bright islands and the zones with smaller particles in SEM images for films deposited at 50 • C contain quinoid PEDOT, which is coherent with the fact that the film morphology evolution is not detrimental to the conductivity.

The normalized FTIR spectra are presented in Fig. 9a for the samples with T sub equal to 25, 35 and 50 • C. All the peaks are associated to those of PEDOT. Again, no OH peak was observed at 3400 cm -1 . The intensity of the C C stretching peak at 1527 cm -1 slightly increases with T sub for these normalized transmittance spectra, as presented in Fig. 9b. As can be seen, the under curve area slightly increases with T sub , in agreement with the electrical conductivity evolution (Fig. 7b).

XRD analysis revealed that the films do not diffract X-rays, even for the highest T sub tested (Figure S7 in Supplementary Material), in agreement with the results of Kaviani et al. [START_REF] Kaviani | Electroactive and Conformal Coatings of oCVD Polymers for Oxygen Electroreduction[END_REF].

4. Influence of che cocal pressure

The concentration of the gas species increases with the total pressure, which may result in higher deposition rates. In order to probe this trend, two deposition experin1ents were perfom1ed at T sub 50 °C, SbCls/EI)OT ratio 0.18 and two total pressures, 75 and 135 Pa. The obtained results are resumed in Table 1. As can be seen, the deposited mass increases from 0.08 to 3.46 mg and the average thickness increases from 12 to 287 nm when the pressure is increased. A Jess than twofold increase of the pressure then leads to a 43 fold increase of the mass of the films while at the sanie time, the electiical conductivity decreases by 30% from 770 ± 56 S/cm to 537 ± 116 S/cm. lt is worth noting that deposition at 135 Pa results in a st:rong decrease of films uniformity (Figure S8 of Supplen1entaiy Materials). The film thickness at 75 Pa is 12 ± 4 nm, while a thickness profile vaiying between 120 nm and 600 nm is observed at 135 Pa. The higher deposition rate is atmbuted to the increased reactants concentration at higher total pressure, while the sinmltaneously observed thickness non uniformity could be due to a higher roughness of the film, as obse1ved in Section 3.2 for the highest flow rates of SbCls tested.

The normalized FTIR spectra ai• e presented in Fig. 10a for the two san1ples. Ali the peaks are associated to those of PEDOT. In addition, an OH peak at 3350 cm-1 is present for the film at 135 Pa (not shown), probably due to an excess of hydrophilic by-products related to the higher SbCl 5 concentration, since the chai• acterization was performed under an1bient air. Fig. 1 Ob focuses on the intensity of the peak associ ated to the C C conjugation bond. The ai• ea corresponding to the film processed at 135 Pa (estimated for two zones due to the film heteroge neity) is lower than that relative to the sample at 75 Pa, which is coherent with the conductivity results.

As a condusion of this pait, depositing at higher pressure for the conditions tested is unfavorable for obtaining unifom1 films with higher elecmcal conductivity.

5. Influence of che deposicion duracion

The demonstrated stable evaporation of SbCls and the resulting continuous PEDOT deposition allow investigating the effect of deposi tion tin1e on the chai• acteristics of the films. Four deposition tin1es, 30, 60, 90 and 150 min, were studied for identical deposition conditions, nan1ely total pressure 75 Pa, SbClsfEDOT ratio 0.18 and T sub 50 •c.

First, the influence of films thickness on their optical transmittance was investigated. PEDOT films were deposited on purpose on 24 x 24 nuu 2 microscope cover glass substrates (Deckglaser 0101062). A small Si coupon (2 x 2 cni2), paitially covered with Kapton was also positioned on the substrate holder in each expe1iment, in order to measure the thickness of the deposited film. The small size of the san1ples and the precision linut of the balance did not allow the detem1ination of the mass of the films.

Fig. 1 la presents the evolution of the film thickness and conductivity as a function of the deposition tin1e. The thickness increases lineai• ly from 13 nm at 30 n1Îll to 58 nm at 150 nun, co1Tesponding to a 0.4 nm/ nun constant deposition rate, showing the stability of the polymeriza tion process.

Ali samples present sinillai• electrical conductivity close to 700 S/cm, indicating that for the conditions tested, conductivity is not influenced by the film thickness between 13 and 58 nm. In addition, the evaporated masses of EDOT and SbCl 5 during each run (not shown) also lineai• ly correlate with the total evaporation time for each compound, confim1ing the stability and reproducibility of the EDOT and oxidant evapora tion rates.

Fig. 11 b shows the optical tra11Sn1Îttance (T0/4,) of the films and their evolution as a function of the deposit duration, for the wavenumber range 300-800 nm, close to the visible one. Using an as received glass coupon as the reference, the 13 nm thick film deposited during 30 nun presents a 96% transpai•ency at 550 nm. This wavenumber is typically chosen as it represents the maximal intensity of sunlight in the region of green light [START_REF] Badday | Application of heteropolyacid-based heterogeneous catalysts for conversion of oleochemicals into renewable fuels and other value-added products[END_REF] and the highest sensitive wavenumber to human eyes [START_REF] Qiu | Persistent Phosphors: From Fundamentals to Applications[END_REF].

This value decreases slightly to 95% and 93% with increasing thickness. These results indicate that the optical characteristics of such oCVD PEDOT films are compatible with optoelectronic applications like OLEDs and solar cells which require transparent anodes [START_REF] Gao | Advanced nanomaterials for solar cells and light emitting diodes[END_REF]. The transmittance values are comparable with those of Gharahcheshmeh et al. for PEDOT-SbCl 5 (97% for 10 nm) [START_REF] Gharahcheshmeh | Optimizing the optoelectronic properties of face-on oriented poly(3,4-ethylenedioxythiophene) via water-assisted oxidative chemical vapor deposition[END_REF], PEDOT-VOCl 3 (T 93% for 70 nm and 97% for 10 nm) [START_REF] Gharahcheshmeh | Tuning, optimization, and perovskite solar cell device integration of ultrathin poly (3, 4-ethylene dioxythiophene) films via a single-step all-dry process[END_REF] and slightly higher than PEDOT-FeCl 3 by Kovacik et al. (T 90% for 27 nm) [START_REF] Kovacik | Scale-up of oCVD: large-area conductive polymer thin films for next-generation electronics[END_REF] and PEDOT-FeCl 3 by Lock et al. [START_REF] Lock | Oxidative chemical vapor deposition of electrically conducting poly (3, 4-ethylenedioxythiophene) films[END_REF] (T 84% for 35 nm).

Fig. 12 presents AFM maps of two films deposited on Si for 30 min and 90 min, on two areas, of 10 × 10 µm 2 and a 1 × 1 µm 2 . Maps of the film surface after 30 min of deposition reveal the presence of a dense film with isolated, 40-50 nm high peaks. A similar morphology prevails on surfaces after 90 min deposition, however with an increasing peaks height, which reaches 90-100 nm. These islands probably do not correspond to the rich benzoid form particles in the bright zones of Fig. 5, since the film conductivity is unchanged, which could be due to the positive combination of the low EDOT/oxidant ratio, low total pressure and high T sub used. Observations of smaller areas on the smoothest zones (Fig. 12c and12d) indicate that the surface of the film deposited for 30 min is composed of a puzzle of beads whose lateral dimension is in the order of 50 nm. These beads are getting more faceted, larger and rougher with increasing the deposition time. The RMS roughness over the smooth surface parts increases from 4 nm at 30 min to 8 nm at 90 min. It is worth noting that this evolution of morphology and roughness does not modify the electrical conductivity of the films.

Study of the uniformity at large scale

In a similar way as in our previous work on PEDOT-FeCl 3 , an experiment was conducted involving ten 1 × 1 cm 2 Si coupons fixed vertically on a scaffold structure into the reactor, with the aim to get insight into the PEDOT deposition mechanisms [START_REF] Mirabedin | An out of the box vision over oxidative chemical vapor deposition of PEDOT involving sublimed iron trichloride[END_REF]. The coupons were placed over four levels of the scaffold at a 5 cm distance from each other, as schematized in Fig. 13. In addition, a Si wafer was also placed on the substrate holder. One third of each coupon was covered by Kapton for thickness measurements. The inlet molar SbCl 5 /EDOT ratio was 0.18, T sub was fixed at 25 • C and the run duration was 30 min. Two positions on each substrate were simultaneously probed for film thickness and conductivity.

The thickness values of all samples are reported in Fig. 13. It is shown that the deposition occurred uniformly on all ten positions within the reactor volume with a 52 nm average film thickness. A similar thickness was measured on the sample positioned at the top of the reactor, which is close to the one reported for the same deposition conditions in Section 3.2. The standard deviation is of 8% over the ten coupons. Similarly, the average electrical conductivity is close to 600 S/cm for all samples, as for the same deposition conditions of Section 3.2.

No solid residues were found in the reactor. This information combined with the result about the film thickness uniformity allows concluding that there are no gas phase reactions taking place during the deposition. As the diffusivity of the gas phase is high due to the prevailing low operating pressure, this result indicates that the oxidant and EDOT concentrations are identical at every point into the reactor. In the opposite case, the presence of gas phase reactions resulting in various gaseous intermediate species would have considerably reduced the precursors concentration along the scaffold height, leading to a decreasing deposition rate profile from the gas inlet to the reactor outlet. As a conclusion, over and above film uniformity, there are only surface reactions involved in the Step Growth Polymerization of PEDOT by SbCl 5 , similarly to the oCVD process with FeCl 3 [START_REF] Mirabedin | An out of the box vision over oxidative chemical vapor deposition of PEDOT involving sublimed iron trichloride[END_REF].

Finally, this scaffold experiment demonstrates that the developed oCVD process is fully reproducible and that PEDOT can be uniformly deposited on several tens of cm 2 substrates. The obtained results pave the way towards the scaling up of the PEDOT-SbCl 5 oCVD process over large surfaces.

Conclusions

SbCl 5 was investigated as oxidant for the production of PEDOT films, with the aim to develop a robust, full dry oCVD process. The stability over time of SbCl 5 evaporation was demonstrated by dedicated evaporation experiments, thus offering the possibility to control the oCVD process. Correlations between key process parameters (including inlet molar oxidant/EDOT ratio, substrate temperature, total pressure and deposition duration), film characteristics and properties were for the first time thoroughly studied.

The electrical conductivity of the films increased by decreasing the oxidant/EDOT ratio and by increasing the substrate temperature, as for the classical FeCl 3 oCVD oxidant. An opposite influence of the substrate temperature was observed on the deposited mass and thickness, which can be attributed to the significantly lower oxidant/EDOT ratios used with SbCl 5 compared to values reported in studies with FeCl 3 , in addition to the higher volatility of the former. An increase of total pressure was detrimental to the film uniformity and conductivity, but increased considerably the deposition rate. The morphology of the film is clearly sensitive to the deposition conditions. In particular, the presence of poorly conductive benzoid PEDOT islands on the film can be highly reduced by optimizing the deposition parameters. The absence of post deposition liquid rinsing allowed developing a full dry process, opening the way to the treatment of fragile substrates, like paper or fabrics. Finally, the thickness uniformity of deposited films all over the oCVD reactor was proved for depositions at 75 Pa using a scaffold structure. This uniformity associated with the whole results obtained indicate that for the conditions tested, the process operates in the kinetically limited regime, with an overall polymerization kinetics limited by the SbCl 5 local concentration near the substrate. This study demonstrates the reproducibility and the capability of this fully dry SbCl 5 oCVD process to be scaled up over larger surfaces. This opens the way to its industrial development in the fabrication of optoelectronic or sensing devices. It is worth noting that the absence of water in this oCVD process portends improved durability over time of the devices which could use PEDOT-SbCl 5 oCVD in substitution of the classical PEDOT:PSS route.

Fig. 1 .

 1 Fig. 1. Schematic view of (a) the oCVD reactor, (b) the substrate with the cross in Kapton tape.

Fig. 2 .

 2 Fig. 2. Evolution of the SbC1 5 evaporated mass versus evaporation time.

  Fig. 3. (a) Average film thickness and mass and (b): Electrical conductivicy as a function of the SbClsfEDOT ratio (T oub of 25 •c, total pressure of 75 Pa and deposition duration of 30 min).

Fig. 4 .

 4 Fig. 4. Thickness profiles along the x1 axis for films deposited with inlet molar SbCl 5 /EDOT ratios of 0.18 and 2.08 (T sub of 25 • C, total pressure of 75 Pa and deposition duration of 30 min). Note the same scale for the two ordinates.

Fig. 5 .

 5 Fig. 5. (a) Surface FEG SEM micrographs and (b) AFM views of films deposited with different SbCl 5 /EDOT ratios (T sub of 25 • C, total pressure of 75 Pa and deposition duration of 30 min).

3 Fig. 6 .Fig. 7 .

 367 Fig. 6. (a): FTIR spectra obtained for the films with different SbCls/EDOT ra tios, (b): Under curve areas of the corresponding C = C peak (1527 cm 1 ).

Fig. 8 .Fig. 9 .

 89 Fig. 8. FEG SEM views at two magnifications of films prepared at T sub of 25 • C and 50 • C (total pressure of 75 Pa, SbCl 5 /EDOT ratio of 0.18 and deposition duration of 30 min).

Fig. 10 .

 10 Fig. 10. (a) FTIR spectra of samples produced at 75 and 135 Pa, (b) Evolution of the C = C stretching peak (T sub of 50 • C, deposition duration of 30 min, SbCl 5 /EDOT ratio of 0.18).

Fig. 11 .

 11 Fig. 11. Evolution of (a): film thickness and conductivity and (b): film UV-Vis transmittance as a function of deposition duration (T sub of 50 • C, total pressure of 75 Pa, SbCl 5 /EDOT ratio of 0.18).

Fig. 12 .

 12 Fig. 12. AFM maps of two films deposited during 30 min (left) and 90 min (right). The upper and lower maps correspond to 10 × 10 µm 2 and 1 × 1 µm 2 areas, respectively (T sub of 50 • C, total pressure of 75 Pa, SbCl 5 /EDOT ratio of 0.18).

Fig. 13 .

 13 Fig. 13. Thickness values (in nm) over ten 1 × 1 cm 2 Si coupons on a scaffold structure.

Table 1

 1 Mass, deposition rate, thickness and electrical conductivity of PEDOT films deposited at two different total pressures of 75 and 135 Pa (T sub of 50 • C, deposition duration of 30 min, SbCl 5 /EDOT ratio of 0.18).

	Pressure	Film mass	Deposition rate	Thickness	Conductivity (S/
	(Pa)	(mg)	(mg/min)	(nm)	cm)
	75	0.08 ±	0.003 ± 0.001	12 ± 4	770 ± 56
		0.04
	135	3.46 ±	0.115 ± 0.002	287 ± 267
		0.05

Acknowledgments

We are indebted to C. Tendero and O. Marsan (CIRIMAT), C. Josse and T. Hungria (UMS Castaing), and M.L. de Solan Bethmale (LGC SAP) for their contribution concerning sample characterizations. We would like to thank J. Compain (LGC), D. Samelor, and D. Sadowski (CIRIMAT) for their help in preparing the experimental setup.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi. org/10.1016/j.apsusc.2021.149501.

Supplementary Materials

Liquid antimony pentachloride as oxidant for robust oxidative chemical vapor deposition of Poly(3,4ethylenedioxythiophene) films

Milad Mirabedin a, b , Hugues Vergnes a , Nicolas Caussé b , Constantin Vahlas b , Brigitte Caussat a* a Laboratoire de Génie Chimique, Université de Toulouse, 4 allée Emile Monso, 31030 Toulouse cedex 4, France.

b CIRIMAT, Université de Toulouse, 4 allée Emile Monso, 31030 Toulouse cedex 4, France.

EDX analyses of PEDOT simple with SbCl5/EDOT=0.42

As shown in Figure S1, EDX analysis of these samples revealed presence of all PEDOT elements (C, S, …) on the surface. Additionally, the film with SbCl5/EDOT=0.42 was analyzed on a ≈ 5.5 µm line by EDX, across the dark background and the bright particles. We observe that all detected elements except Si show an increase in intensity when probing the particles.

Fluctuations in Si intensity are associated with variable presence of other elements on the surface, attributed to the local increase in film thickness due to the presence of small particles. This evolution is confirmed when probing the large particle in the middle of the micrograph, with a significant increase in all components and a sharp decrease in Si intensity.

It is concluded that the PEDOT elements (C, S, O, Sb and Cl) are present everywhere on the film, as was the case for those deposited from FeCl3. Presence of all elements are higher on the big particle probably because of the smaller distance between the surface and the probe due to the height of these particles. The relative intensity of the probed particles remains qualitatively similar to the one of the background, in contrast to films deposited from FeCl3, which were locally richer in O and Fe. Such difference in composition of films deposited from the two chemistries can be attributed to the fact that, in contrast to FeCl3, gaseous SbCl5 cannot condensate in the adopted operating conditions. It can also be due to the lower oxidant/EDOT ratios used for the SbCl5 experiments and to the fact that SbCl5 is less moisture and air sensitive. The homogenous presence of Sb in EDX and independent XPS analyses suggest that some SbCl5 might create Sb-complexes on the PEDOT film after losing two chlorine atoms through polymerization and reduction. With this hypothesis, Sb remains trapped in the film forming links between SbCl3 and the oxygen in monomer ethylenedioxy rings, as proposed by Kaviani et al. [1] for PEDOT films containing 2% mole Sb.

Raman analyses of the sample with SbCl5/EDOT=0.42

The surface of the film with SbCl5/EDOT ratio 0.42 was locally analyzed by Raman spectrometry with the aim of identifying the possible differences between the large particles and the two other zones on the surface. Raman laser beam can analyze a smaller zone of the surface (probe size 700 nm) in comparison with the 1 cm probed area by FTIR. Considering the 700 nm diameter limit, we carefully chose these 3 areas to investigate their chemical composition. These zones correspond to large particles (Zone 1), small particles (Zone 2) and the dark base (Zone 3) in Figure S2. Raman intensity of the small particles (zone 2) and of the background (zone 3) in the SEM micrograph indicates similar positions for all peaks, confirming the presence of PEDOT in the small particles (Figure S3). The intensity of all PEDOT peaks is systematically stronger on large particles (zone 1), attributed to the higher thickness of the film on this zone. This is why the oxyethylene ring deformation peak at 990 cm -1 , which is seen on the large particles in zone 1 is covered by the silicon peak on other zones. A significant difference to note is the shift of the Cα=Cβ symmetric peak in Table S1, from 1434 cm -1 to 1447 cm -1 on all large particles of zone 1, in comparison with the other zones. We attribute this shift to the non-uniform presence of quinoid (doped) and benzoid (undoped) PEDOT in the film, as is reported in [1,2]. It appears that zone 1 is poorer in quinoid (doped) PEDOT in comparison with the other zones and consequently less conductive. Let us note that this shift does not appear in the most conductive film with SbCl5/EDOT=0.18.

The Raman spectra of the sample deposited with the higher SbCl5/EDOT ratio of 0.76, show that the Cα=Cβ symmetric peak is present at 1447 cm -1 . No peak shift is observed between big and small particles on this sample. All peaks are similar to the peak at zone 1 on sample with ratio =0.42 with less quinoid PEDOT. In samples with less SbCl5/EDOT ratio, we see a difference in the position of Cα=Cβ symmetric peak for different zones. Zone 1 with large particles is shifted to right so has less doped PEDOT. As the ratio increases, although again these three zones are observed, there is less difference between quinoid and benzoid Cα=Cβ symmetric peak positions as they both tend to shift to the benzoid form (shift to right). This means that these films with higher ratios have more undoped PEDOT. We see then a correlation between the position of Cα=Cβ symmetric peak and the electrical conductivity as a function of the SbCl5/EDOT ratio.

XPS results of a PEDOT-SbCl5 film prepared under SbCl5/EDOT=0.18

Figure S4 presents the high resolution XPS spectra of a PEDOT-SbCl5 film prepared under SbCl5/EDOT=0.18 and Tsub=25°C. Table S2 presents the corresponding XPS elemental analysis. Sb was probed at an average concentration not exceeding 5 at.%, in agreement with the EDX results. Effect of substrate temperature on film thickness uniformity

Raman analysis of the sample with SbCl5/EDOT=0.18 and Tsub=50°C

The Raman spectra are shown in Figure S6 for bright particles and small particles seen on the surface in Figure 8. These results are significantly different from those in Figure S3 as the bright particles on the surface show the Cα=Cβ symmetric peak in the same position as the small particles, both at 1435 cm -1 . Again, observation of the peak at this wavenumber shows more quinoid (doped) PEDOT in the film, by increasing the substrate temperature [1,2].


Josette Linda

Diderot Toussi Matchi

Diderot Tchamo Noungoue

Isabelle Kuhn

Jérôme Boissier

Jean Claude Tchouankeu

Marc Nothisen

Guilhem Chaubet

Delphine Garnier

Sylvain Ursuegui

Silvère Augustin Ngouela

Alain Wagner

Manniindole, an indole derivative from the roots of Anonidium mannii

Keywords: Anonidium mannii, Annonaceae, phytochemical and biological investigation, indole alkaloid, antischistosomal activity, enzymatic activity (SmNACE), cytotoxicity

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

The genus Anonidium, belonging to the Annonaceae family, comprises 7 tropical African tree and shrub species [START_REF] Pellegrin | Les Annonacées du Gabon[END_REF], but this genus is not well studied. Anonidium mannii (Oliv.) Engl. & Diels, is known to be located to Central Africa [Cameroon (Bankomo, Makenene, Ndikinemeki, Kribi), Gabon and Congo], and has been used in the respective countries of origin for the treatment of various ailments including female infertility [START_REF] Abondo | Traditional medicinal plants[END_REF], treatment of abscess [START_REF] Taffou | Anti-yeast potential of some annonaceae species from Cameroonian biodiversity[END_REF], open wounds, hemorrhoids, intestinal spasms, diarrhea [START_REF] Muganza | In vitro antiprotozoal and cytotoxicactivity of 33 ethonopharmacologically selected medicinal plants from Democratic Republic of Congo[END_REF], arthritis, rheumatism, dysentery, paralysis, epilepsy, convulsion, stomach trouble [START_REF] Erhenhi | Known medicinal and aphrodisiac plants of Urhonigbe forest reserve, Edo State, Nigeria[END_REF], cancer [START_REF] Kuete | Cytotoxicity and modes of action of the methanol extracts of six Cameroonian medicinal plants against multidrug-resistant tumor cells[END_REF]) malaria [START_REF] Betti | An ethnobotanical study of medicinal plants among the Baka Pygmies in the Dja Biosphere Reserve, Cameroon[END_REF]) and Schistosomiasis [START_REF] Messi | Contribution à l'étude des plantes médicinales du Cameroun: le cas des plantes utilisées en médecine traditionnelle pour le traitement des maladies parasitaires[END_REF]. Otherwise, Schistosomiasis, also known as bilharzia, is a disease caused by parasitic worms infecting people worldwide. This neglected tropical disease (OMS 2010;[START_REF] Zhang | Control of neglected tropical diseases needs a long-term commitment[END_REF]) comes second after malaria as the most devastating parasitic disease in terms of impact. It is estimated that 600 million people are at risk of infection, 200 million people are infected, and at least 200 thousand deaths per year are associated with the disease (OMS 2014). The parasites that cause schistosomiasis live in certain types of freshwater snails, from which their infectious form, known as cercariae, can emerge into the water and infect humans through skin contact. In most human infections, acute schistosomiasis is caused by Schistosoma mansoni, S. haematobium, or S. japonicum, and is characterized by fever, headache, myalgia, and respiratory symptoms, and occasionally by eosinophilia and painful hepato-and/or splenomegaly. No vaccine is currently available and the antiparasitic drug mostly used to treat schistosomiasis is Biltricide, which active ingredient (Praziquantel) belongs to the alkaloid family (OMS 2020;[START_REF] Doenhoff | Praziquantel: its use in control of schistosomiasis in sub-Saharan Africa and current research needs[END_REF]). However, it is only effective against adult worms and it has showed many side effects. More worrying, because the question of the reduction of the efficacy of praziquantel molecule was raised recently, it is thus urgent to find new, safe antischistosomal drugs and new therapeutic targets [START_REF] Fallon | Drug-resistant schistosomiasis: resistance to praziquantel and oxamniquine induced in Schistosoma mansoni in mice is drug specific[END_REF]. SmNACE is an ecto-enzyme recently discovered which has a favorable topology because it is one of the rare identified and characterized target on the tegument of adult schistosomes responsible for serious clinic troubles. In fact, SmNACE is extremely interesting due to its accessibility of drugs and thus a potential target for conception of future therapeutic agents [START_REF] Goodrich | Production of calcium-mobilizing metabolites by a novel member of the ADP-ribosyl cyclase family expressed in Schistosoma mansoni[END_REF][START_REF] Kuhn | Identification by high-throughput screening of inhibitors of Schistosoma mansoni NAD(+) catabolizing enzyme[END_REF][START_REF] Jacques | Discovery of potent inhibitors of Schistosoma mansoni NAD catabolizing enzyme[END_REF].

The Annonaceae family is known to contain acetogenins, alkaloids, essential oils, terpenoids, and some flavonoids [START_REF] Leboeuf | The phytochemistry of the annonaceae[END_REF]. Previous phytochemical investigation on the specie Anonidium mannii revealed the presence of alkaloids, phenols, polyphenols, saponins, tannins and steroids [START_REF] Djeussi | Antibacterial activities of selected edible plants extracts against multidrug-resistant Gram-negative bacteria[END_REF][START_REF] Ngangoue | A phenanthridin-6(5H)one derivative and a lanostane-type triterpene with antibacterial properties from Anonidium mannii (Oliv). Engl. & Diels (Annonaceae)[END_REF]. Some prenylated indoles and bisindole alkaloids have been identified from the stem bark of A. mannii [START_REF] Achenbach | Constituents of West African medicinal plants. XVIII: The Annonidines -A new class of prenylated bisindole alkaloids from Anonidium mannii[END_REF] and some have been reported to have the following biological activities: antibacterial, immunosuppressive and radical scavenging [START_REF] Southon | Dictionary of alkaloids[END_REF]. As a part of our ongoing project on the structurally and biologically interesting secondary metabolites from Anonidium mannii, and based on previous reports on the antiparasitic activity of this species, the aim of this work was to carry out a phytochemical and biological investigation on the roots of the plant collected from Cameroon.

Results and discussion

Herein report the isolation and characterization by the spectroscopies means, of the novel indole derivative 1, together with four known compounds identified as, aristolactam A II 2 [START_REF] Sun | Aristolactam A II[END_REF], aristolactam B II 3 [START_REF] Akasu | Aristolactam BII)[END_REF]), piperolactam D 4 (Desai et al. 1990) and polycarpol 5 (Hammonière et al. 1976) respectively (Figure 1); and also their cytotoxicity, enzymatic and antischistosomal activities evaluation Compound 1 was isolated as a yellow-orange amorphous powder, which gave a positive Dragendorff test characteristic of alkaloid. Its molecular formula C18H23N3O, could be deduced from its positive high resolution electrospray ionization mass spectrum (HRESI-MS) which showed protonated molecule [M + H] + ion peak at m/z 298. 1924 (calc. 298. 1919), indicating nine degrees of unsaturation. Its infrared spectrum displayed maxima at around 3 480 cm -1 which could be due by a vibration of the N-H group for a indole skeleton [START_REF] Benesova | Isolation and structure of two new indole alkaloids from Riccardia sinuata (HOOK.) Trev[END_REF], and we can also observe absorptions attributable to the N-carbamoylpyrrolidine at (cm - 1 ) 3270 and 3100 (NH2); 1651 (amid carbonyl, N2C=O); 1519, 1494 (N-CO-N) [START_REF] D'ambrosio | Carbamoylpyrrolidine and 7-Chlorocavernicolenone, Two new metabolites of the Mediterranean sponge Aplysina (=Verongia) cavernicola[END_REF]).

The 13 C NMR spectrum (Table S1) displayed 18 carbon signals which were sorted by DEPT into two methyls, four sp 3 methylenes, six methines (three aromatics, two olefinics and one sp 3 ) five sp 2 quaternary carbons and one carbonyl at c157.2 characteristic of N-carbamoyl group.

The 1 H NMR spectrum of 1 (Table S1) showed in the low field region, two broad singlets at H 10.80 and 7.03 which might confirm the existence in 1 of an indoyl N-H and olefinic (H-2) protons respectively [START_REF] Benesova | Isolation and structure of two new indole alkaloids from Riccardia sinuata (HOOK.) Trev[END_REF], and furthermore it exhibited also three coupled aromatic protons each other at H 7.34 (1H, bd, J=7.8 Hz, H-4), H 6.90 (1H, (1H, t, J=7.5 Hz, H-5) and H 6.85 (1H, bd, J=7.0 Hz, H-6), this assume that the single aromatic cycle of 1 was trisubstituted. In addition, this spectrum showed the characteristic resonances of a 3,3dimethylallyl moiety [H 1.71 (6H, s, H-4''and H-5''); 3.50 (2H, d, J=7.4 Hz, H-1'') and 5.43 (1H, m, H-2'']. After all, we can also observe aliphatic protons at H [1.86 (2H, m, H-3'); 1.89 (1H, m, H-4a), 2.17 (1H, m, H-4b); 3.43 (1H, m, H-2a); 3.51 (1H, m, H-2b) and 5.11 (1H, bd, J=7.2 Hz; H-5')], together with amide protons (CONH2) at H 5.45 (2H, s,); According to its molecular formula, all these data corroborate the presence in the compound 1 of a Ncarbamoylpyrrolidine group. Combined analysis of these data above indicated that compound 1 had a framework of an indole type alkaloid which was substituted by prenyl and Ncarbamoylpyrrolidine groups respectively. In its 1 H-1 H COSY spectrum, coupling was observed between olefinic proton (H-2) and N-H proton of the pyrrole ring of the indole moiety and this assume that C-3 was substituted, and associate with the HMQC, that spectrum revealed connectivity of pyrrolidine ring (C-2', C-3' , C-4' and C-5' resp.) and prenyl group (C-1'', C-2''and C-4''/5'' resp.). Key HMBC correlations between aliphatic protons (H-2" and H-1") of the 3,3-dimethylallyl moiety and aromatic carbons (C-7 and C-6; C-7a resp.) of the indole skeleton allowed us to fix the prenyl group on C-7, and interaction between H-5' and C-2 enabled us to attach N-carbamoylpyrrolidine on carbon C-3. This was confirmed by its NOESY correlations observed between H-5' and H-2 and (CONH2) protons respectively (Figure S2). The compound 1 was showed optical activity, due to its chiral carbon (C-5) which deflected the polarized light to the left (levorotatory) based on the polarimetry performed. Hence, its structure was identified as (-)-3-(5'-N-carbamoylpyrrolidine)-7-(3''-methyl-2''-butenyl) indole and has been given the trivial name manniindole.

In the meantime, new compound 1, and compounds 2, 3 and 4 were subjected to antischistosomal, enzymatic and/or cytotoxicity activities, the results were quite encouraging.

The human lung cancer cells (A549) and the human hepatocarcinoma cells (Huh7) were chosen for evaluating the cytotoxicity of compound 1 due to the fact that the lung and liver are the focal points of pathogenic insult and subsequent pathological damage in schistosomiasis (De [START_REF] Oliveira | Schistosoma mansoni infection causes oxidative stress and alters receptor for advanced glycation end product (RAGE) and tau levels in multiple organs in mice[END_REF]. At the concentration C=100 M, the percentage of cell viability is about 15% (Huh7 cells) and 80% (A549 cells). This showed that the compound 1 was toxic at this concentration for Huh7 cells line and no toxic for A549 cells line (Figure S3).

The new compound 1 and the compounds 2 and 3 were evaluated in vitro for their activity against adult Schistosoma mansoni. Only compound 2 displayed quite good worm killing capability after 6 h at the concentrations of 100 M, Praziquantel was taken as positive control (Table S2).

The compounds 1, 2, 3 and 4 were also tested for their ability to inhibit the catalytic activity of recombinant SmNACE in vitro using 1,N 6 -etheno NAD + as substrate. This enzyme was chosen due to its expression on the outer surface of the Schistosoma mansoni adult worm tegument. In these sense, it could be an interesting pharmacological target for antischistosomal therapy [START_REF] Goodrich | Production of calcium-mobilizing metabolites by a novel member of the ADP-ribosyl cyclase family expressed in Schistosoma mansoni[END_REF]. The IC50 values were determined fluorometrically using 1,N 6etheno NAD + as substrate. Only the compound 4 showed a significant inhibition with a IC50 value about 10-20 M compared to the reference SmNACE inhibitor (Table S3).

Experimental

General methods

IR spectrum was obtained using a Thermo Electron (Nicolet 380) FT-IR spectrometer. Column chromatography was carried out using silica gel (Merck 60-120, 70-230 and 230-400 mesh). Rotating power was recorded on a P-2000 Jasco polarimeter. Thin layer chromatography was performed on percolated 0.5mm thickMerck Si gel 60 F254 aluminium sheets. Separated compounds were visualized under UV light and by spraying with H2SO4-EtOH (1:9, v/v) followed by mild heating for about 2-3 min. The mass spectra were recorded on an Agilent MS instrument (Agilent Technologies 6520, Accurate mass Q-Tof). NMR spectra were recorded on a Bruker Avance DRX-500 instrument operating at 500 MHz ( 1 H) and 125 MHz ( 13 C), using a deuterated dimethylsulfoxyde (DMSO-d6) as solvent. Chemical shifts () were quoted in parts per million (ppm) from internal standard tetramethylsilane (TMS) and the coupling constants (J) are given in Hz. Different mixtures of n-hexane, EtOAc, CH2Cl2 and MeOH were used as eluting solvents. They were distilled prior to use.

Plant material

Roots of Anonidium mannii were collected in 2016 at Mount Kalla (Latitude 3° 30'. North, Longitude 11° 13'. East), from Bankomo locality, Mefou et Akono Sub-division, Center Region of Cameroon and identified by M. Victor NANA, a botanist of National Herbarium, Yaounde, Cameroon; where a voucher specimen is deposited under the voucher number 45582HNC.

Extraction and isolation

The air-dried and powdered roots (3.4 kg) of A. mannii was macerated in 25 L of a mixture of EtOH-H2O (8:2) for 72 h at room temperature. After filtration and solvent evaporation, a residue of 130.41 g was obtained. A part of the crude extract (100 g) was subjected to flash column chromatography (FCC) on silica gel employing a step gradient of hexane-ethyl acetate and ethyl acetate-methanol to afford ten fractions Fr1-Fr10 based on TLC monitoring.

Fraction Fr6 (2.51 g) was purified employing a step gradient of hexane (1:0)-ethyl acetate (7:3) to yield polycarpol 5 (15.0mg). A part of the fraction Fr9 (3.24 g), eluted with a gradient dichloromethane (1:0)-methanol (9.5:0.5) lead to aristolactam AII 2 (8mg). Fraction Fr10 (9.1 g) was purified by flash chromatography (MPLC) over silica gel with a gradient ethyl acetate (1:0)-methanol (8.5:1.5) and subfractions [30-55] (250mg) were purified by semi-preparative HPLC using a gradient (5-95% CH3CN, in 60min) to give piperolactam D 4 (11.2mg), aristolactam BII 3 (12mg) and manniindole (1) (6mg). For 1 H and 13 C NMR data, see Table S2.

Bioassay

Cytotoxicity activity

Cytotoxicity was tested against the Huh7 and A549 cells using a MTS assay. Huh7 cells (Human Hepato carcinoma cells) are cultured in DMEM medium 1 g/L glucose (SIGMA), 2M Glutamine and 10% FCS (fetal calf serum, SIGMA). The A549 cells (Human Lung Tumor cells) are cultured in HamF12 medium, 2 M Glutamine and 10% FCS. 24 hours before the toxicity experiment, the cells are seeded in a 96-well plate (Nunc Edge 2.0, ThermoScientific) at the rate of 4000 cells/well (Huh7); 6000 cells/well (A 549) in 100 L and cultured in an incubator at 37°C. with 5% CO2. The extracts to be tested are diluted in medium with serum and added at a rate of 20 L/well to obtain the desired final concentration. All tests were done in triplicate. 48 hours after the addition of the extracts, 20 L of MTS solution (Cell Titer 96 R Aqueous One Cell Proliferation Assay Solution, Promega) are added to each well. After 1 hour of incubation at 37°C., the ODs are read at 490nm and 700nm (Safas, Monaco).

The OD or absorbance of the extracts to be assayed (490 nm) are analyzed after deducting the OD at 700 nm, the OD of the plastic, the OD of the cells alone.

Praziquantel and DMEM medium were used as positive and negative controls, respectively.

Determination of activity against adult Schistosoma mansoni (in vitro)

The host-parasite system used was an albino variety of Biomphalaria glabrata from Brazil and a strain of Schistosoma mansoni from Puerto Rico (NMRI strain). Female hamsters (Mesocricetus auratus; Janvier Labs; Le genest-Saint-Isle, France) were percutaneously exposed to 400 cercariae following standard procedures previously published [START_REF] Dumont | Influence of pattern of exposure, parasite genetic diversity and sex on the degree of protection against reinfection with Schistosoma mansoni[END_REF]. Forty days' post-exposition adult S. mansoni were recovered from the hepatic portal system and mesenteric veins by hepatic perfusion technique [START_REF] Boissier | Preliminary study on sex related inflammatory reactions in mice infected with Schistosoma mansoni[END_REF]. Living worms were immediately deposited in RPMI 1640 culture medium (supplemented with 5% inactivated foetal calf serum (iFCS) and 100 U/mL penicillin and 100 g/mL streptomycin (Invitrogen, Carlsbad, CA) at 37°C in an atmosphere of 5% CO2. Ten to twelve worms with equilibrated sex ratio were deposited in each well of 24 well plates. For the determination of activity against adult flukes all compounds were initially tested at a concentration of 100 lg/mL, using DMSO stock solutions (conc 10 g/mL; final concentration of DMSO: 0.2) diluted in supplemented RPMI 1640 medium with a final volume of 2mL per well. Wells with RPMI and DMSO in medium served as negative controls. PZQ served as positive control, and concentrations of 10, 50, and 100 g/mL were used to evaluate the shistosome's mortality. Each test was performed in duplicate or triplicate. Parasites were subsequently observed for body contractility and movement each hour for 6 hours. Parasites showing no body contractions during a 30-s observation were considered dead [no worm started to move again after 30 s without motor activity [START_REF] Boissier | In vitro activities of trioxaquines against Schistosoma mansoni[END_REF]].

Enzymatic activity (in vitro)

SmNACE activity was determined by a fluorometric assay using 1, N 6 -etheno NAD + (-NAD + , Sigma) as substrate. This assay consists in measuring the appearance of the reaction product -ADP-ribose by the increase of fluorescence at em = 410nm (exc = 310 nm) at 37°C in 10mM potassium phosphate buffer, pH 7.4, containing 0.05% (w/v) emulphogen (1 mL final volume) in a quartz tank.

In the spectrofluorimeter (Shimadzu RP-5301 PC), the buffer was added to the tank at 37°C and with stirring. The substrate was then added; its final concentration being 20 M. After that, inhibitors were introduced. Before introducing the enzyme, the tracing was started in order to see the basic fluorescence F0. Once the enzyme has been added, the kinetics were exploited at the end of the measurement time (2 minutes) using a non-linear regression program (GraphPad, Prism) for the determination of catalytic activity. All the inhibitors were initially tested at a concentration of 100mM diluted in DMSO (less than 2% added). In case that we observed fluorescence or quenching, the concentrations were decreased. So, concentrations of 10 M, 100 M, 1 mM, 10mM and 100mM were used. Each test was performed twice.

The approximate IC50 values were determined based on concentrations giving approximately 50% inhibition.

Conclusion

In conclusion, phytochemical and biological investigation which was carried out on the roots of Anonidium mannii led to the isolation of five major compounds among which one, 1, was newly described. Though there are well known in the Annonaceae family, this is the first time that lactams were found in A. mannii. Those isolated compounds were submitted for the first time to the combined biologic tests on the schistosomes (parasitical and enzymatical tests). Two compounds, respectively 2 and 4, were potentially having good activity on adult Schistosoma mansoni in vitro and good inhibition of a new member of the ADP-ribosyl cyclase family of enzymes (SmNACE) in the Platyhelminthes trematodes, Schistosoma mansoni and Schistosoma japonicum. *Experiments were performed twice. The others were performed three times. For each experiment, 10-12 worms were used with equilibrated sex-ratio.

Table S3. Approximate IC 50 values for the inhibition of SmNACE by compounds.

The roots extract was active on SmNACE with inhibition rate of 76.13%. at C=100 µg/mL being a mixture of compounds, its IC 50 could not be determined.

Compound

Figure S2 .

 S2 Figure S2. Key HMBC, COSY and NOESY Correlations between indole moiety and the two substituents of 1.

Figure S3 .

 S3 Figure S3. The cell viability of Compound 1 and Praziquantel assayed by MTT.

(*

  Cyanidin and Delphinidin are the natural products reference SmNACE inhibitors[START_REF] Kuhn | Identification by high-throughput screening of inhibitors of Schistosoma mansoni NAD(+) catabolizing enzyme[END_REF]).

Table S1 .

 S1 13 C (125 MHz) and 1 H NMR (DMSO-d 6 , 500 MHz) [J (Hz), δ (ppm)] data for compound (1). were based on HSQC, HMBC, COSY and NOESY experiments.

	δ 13 C -121.8 117.8 125.1 116.4 118.7 119.7 124.5 135.5 N-CO-NH 2 157.2 1 2 3 3a 4 5 6 7 7a 2' 46.1 3' 23.4 4' 33.7 5' 53.6 1″ 29.1 2″ 122.2 3″ 131.9 4″ 25.6 Assignments Attributions 5″ 17.7	1 δ 1 H (nH, mult., J (Hz)) 10.79 (1H, s) 7.02 (1H, s) --7.34 (1H, d, J = 7.5) 6.90 (1H, t, J = 7.5) 6.85 (1H, d, J = 7.5) --5.45 (2H, s) 3.43 (1H, m) 3.51 (1H, m) 1.86 (2H, m) 1.89 (1H, m) 2.17 (1H, dd, J = 13.4 ; 6) 5.11 (1H, bd, J = 7.2) 3.50 (2H, d, J = 7.4) 5.43 (1H, dd, J = 16.1 ; 8.7) -1.71 (3H, s) 1.71 (3H, s)

Table S2 .

 S2 Effects of the extract and the isolated compounds on adult Schistosoma mansoni in vitro.

	Extract/Compound	Concentration	Mobile worm
	(No.)	(μM)	after 6 hours of
			incubation
			(%)
	Roots extract	100	44.4
		50	100
		10	Not Done
	1 Manniindole	100*	70
		50*	95
		10*	100
	2 Aristolactam AII 100	9.1
		50	66.7
		10	71
	3 Aristolactam BII 100	62.5
		50	77.3
		10	94.4
	Praziquantel	100	0
		50	7
		10	Not Done
	Control (RPMI)	-	100

Acknowledgments

The authors are thankful to AUF for providing a fellowship to J L T M.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethic statement

The laboratory where the experiments were done has permission A66040 from «Ministère de l'Enseignement supérieur de la Recherche et de l'Innovation (France)» for animal experimentation. Experimenters possess the certificate for animal experimentation (authorization 007083, decree 87-848 and 2012201-0008). Housing, breeding and animal care followed the national and European ethical requirements.