domain: chim sdv

text:
Cristiane Dos Santos

Cerqueira Alves

Yuri Kelvin

Silva Camacho Tavares

Guilherme Urias

Menezes Novaes

Victória Laysna

Anjos Santos

Ana Paula De Oliveira

Cintia Emi

Yanaguibashi Leal

Edilson Beserra De Alencar Filho

Christiane Adrielly Alves Ferraz

Laurent Picot

Jackson Roberto Guedes Da Silva Almeida

Raimundo Gonçalves De Oliveira Júnior
email: raimundo.goncalves-de-oliveira-junior@u-paris.fr

Anti-melanoma potential of inclusion complexes containing phyllacanthone in β-cyclodextrin and sulfobutyl-ether-β-cyclodextrin

Keywords: diterpene, Cnidoscolus, cyclodextrin, skin cancer, polymer Highlights

The bis-nor-diterpene phyllacanthone (PHY) was isolated from the chloroform fraction of the stem barks from Cnidoscolus quercifolius Pohl (Euphorbiaceae). PHY/β-CD and in PHY/SB-Eβ-CD inclusion complexes were obtained and characterized using NMR, SEM and FT-IR techniques. In silico approach show that most stable conformations presenting binding energies of -89.8068 Kcal/mol (PHY/β-CD) and -87.4032 Kcal/mol (PHY/SB-E-β-CD), corroborating FTIR and 1 H NMR results. The in vitro dissolution assay demonstrated a fast PHY release from both complexes, indicating that the complexation improved its solubility. PHY/β-CD and PHY/SB-E-β-CD inclusion complexes presented high entrapment efficiency rates (89.60% and 77.56%, respectively). PHY, PHY/β-CD and PHY/SB-E-β-CD inclusion complexes induced growth inhibition in A2058 melanoma cells when compared to the control group, suggesting that both (natural or chemically modified) CDs can be used as carriers of PHY in pharmaceutical formulations or drug delivery systems as a strategy to improve its stability and ensure better bioavailability.

Introduction

Phyllacanthone (PHY) is a bis-nor-diterpene previously isolated from the stem barks of Cnidoscolus quercifolius Pohl (Euphorbiaceae), a Brazilian medicinal plant popularly known as "favela" or "faveleira" [START_REF] Oliveira-Júnior | Antibacterial activity of terpenoids isolated from Cnidoscolus quercifolius Pohl (Euphorbiaceae), a Brazilian medicinal plant from Caatinga biome[END_REF]. It is a species with thick leaves, tuberous roots, and small white flowers, widely disseminated in the Caatinga biome and traditionally used for the treatment of infections, tumors, and inflammation (Santos;Silva;[START_REF] Santos | Supercritical CO 2 extraction of favela (Cnidoscolus quercifolius) seed oil: Yield, composition, antioxidant activity, and mathematical modeling[END_REF][START_REF] Sobrinho | Antiproliferative activity of species of the genus Cnidoscolus against HT-29, Hep-2 and NCI-H292 cells[END_REF]. Previous reports have demonstrated that stem barks of "favela" are rich in cytotoxic diterpenes. Known as favelines, these molecules bear a tricyclic benzylcycloheptene skeleton (C 6 -C 7 -C 6 ) and are of unique occurrence in the genus

Cnidoscolus. A recent study showed that PHY, majorly found in C. quercifolius, induces growth inhibition, cell cycle arrest, and tubulin depolymerization-mediated apoptosis in BRAF-mutated melanoma cells [START_REF] Oliveira-Júnior | Bis-nor-diterpene from Cnidoscolus quercifolius (Euphorbiaceae) induces tubulin depolymerization-mediated apoptosis in BRAF-mutated melanoma cells[END_REF].

As well as other aromatic diterpenes, PHY has high volatility and low solubility in water, which limits its pharmaceutical development. In an attempt to overcome these barriers, several carriers can be used to improve the physicochemical properties of this type of compound, and then contribute to better bioavailability (Abril-Sanchez et al., 2019). In the past decades, the use of cyclodextrins (CDs) has proven to be an effective strategy to enhance aqueous solubility and then better dissolution rates and bioavailability of non-polar anticancer drugs candidates [START_REF] Gandhi | The use of cyclodextrin inclusion complexes to improve anticancer drug profiles: a systematic review[END_REF][START_REF] Carneiro | Cyclodextrin-Drug Inclusion Complexes: In Vivo and In Vitro Approaches[END_REF].

CDs are macrocyclic and non-toxic oligosaccharides, usually formed by D-glucose units linked by α 1-4 glycosidic bonds. They are often represented as one cone truncated with a hydrophilic surface and a hydrophobic central cavity, capable to host hydrophobic molecules with low solubility in water. They confer chemical and physical stability to small molecules and their complexation in CDs' internal portion makes them ideal for solubilizing compounds in aqueous systems due to their surface polarity, forming inclusion complexes that can improve bioavailability, thermal stability (e.g. reducing evaporation losses) and also plasma stability, protecting the guest compound from enzymatic degradation after oral administration [START_REF] Marques | A review on cyclodextrin encapsulation of essential oils and volatiles[END_REF][START_REF] Oliveira-Filho | Biological activities and pharmacological applications of cyclodextrins complexed with essential oils and their volatile components: A systematic review[END_REF].

In addition to natural CDs (e.g. α-CD, β-CD and γ-CD), that differ in the number of D-glucose units and volume of the internal cavity, new chemically modified structures, such as hydroxypropyl-β-cyclodextrin (HP-β-CD), methyl-β-cyclodextrin (M-β-CD) and sulfobutyl-ether-β-cyclodextrin (SB-E-β-CD) were synthesized to improve the complexing properties. In fact, the solubility and complexing capacity of natural CDs are limited and could not be applicable to several compounds. Chemical modifications can be planned to overcome these limitations and to enhance CDs' ability to form inclusion complexes [START_REF] Brewster | Cyclodextrins as pharmaceutical solubilizers[END_REF][START_REF] Brewster | Cyclodextrins as pharmaceutical solubilizers[END_REF]. For example, electrostatic attraction between the positive charge of the guest and the negative charge of the SB-E-β-CD increases the effect of host-guest complexation and enhances solubility [START_REF] Fenyvesi | Methyl-beta-cyclodextrins: the role of number and types of substituents in solubilizing power[END_REF].

The present work sought to obtain and characterize the formation of inclusion complexes containing phyllacanthone isolated from stem barks of Cnidoscolus quercifolius in β-cyclodextrin and evaluate the effect of complexation on the cytotoxic activity of the bisnor-diterpene in human melanoma cells. In order to compare their complexation performance, inclusion complex using sulfobutyl-ether-β-cyclodextrin was also developed and tested.

Materials and methods

Drugs and chemicals

Phyllacanthone (PHY, >99%) was isolated from C. quercifolius stem-barks as described bellow. β-CD (>99%) was purchased from Sigma-Aldrich ® and SB-E-β-CD (Dexolve ® , >99%) was kindly provided by Cyclolab (Brazil). Organic solvents (ethanol, chloroform, hexane and methanol) were purchased from Synth ® lab (Brazil). Managing Genetic Heritage and Associated Traditional Knowledge (SisGen), under the record number AB179D2. The plant material was then dried in an oven with air circulation (40 °C, for 72 hours) and pulverized, yielding 900 g of stem barks powder.

Plant material

Stem barks of

Extraction and purification of phyllacanthone (PHY)

PHY was extracted and purified as previously described [START_REF] Oliveira-Júnior | Antibacterial activity of terpenoids isolated from Cnidoscolus quercifolius Pohl (Euphorbiaceae), a Brazilian medicinal plant from Caatinga biome[END_REF].

Dried and pulverized plant material was subjected to an exhaustive maceration using ethanol 95% as solvent. The extractive solution was filtered and concentrated in a rotary evaporator, yielding 61 g of ethanol extract. Part of the extract (30 g) was fractioned by vacuum liquid chromatography (VLC), using silica gel 60 as stationary phase. Hexane, chloroform, ethyl acetate, and methanol were used as mobile phase, in increasing order of polarity, resulting in the respective fractions.

Chloroform fraction (6 g, from which PHY was previously isolated) was applied on preparative TLC plates (silica gel 60, 2-25 µm, Merk ® ) and eluted with hexane/ethyl acetate (97:3). The entire process was monitored with a UV flashlight and a sub-fraction containing 700 mg of purified PHY was obtained and then analyzed by 1D and 2D NMR for structural characterization.

Obtention of inclusion complexes

Inclusion complexes of PHY in β-CD (Sigma-Aldrich ® ) and in SB-E-β-CD (Dexolve ® , Cyclolab) were prepared in a 1:1 molar ratio (100 mg of PHY to 0.998 g of β-CD and 100 mg of PHY to 1.970 g of SB-E-β-CD). A solution of 75% water and 25% ethanol was added to the mixture, for a total of 250 mL of solution. The used amount of PHY was added to the used volume of ethanol, and the mixture was subjected to ultrasound (10 minutes at 25 °C). The water solutions and the respective CDs were added to the mixtures and placed in a magnetic stirrer for 48 h. PHY/β-CD solution was lyophilized, while PHY/SB-E-β-CD solution was left in an oven with circulating air (at 45 °C) in a glass container for 48 hours. Physical mixtures were prepared in the same molar ratio, ground, and stored in a freezer until future analyses [START_REF] Alves | Caracterização físico-química de complexos de inclusão contendo os monoterpenos cânfora e 1,8-cineol, constituintes majoritários dos óleos essenciais de Croton conduplicatus, em β-ciclodextrina[END_REF].

Scanning electron microscopy (SEM)

Surfaces of PHY, CDs, physical mixtures, and inclusion complexes were examined in a scanning electron microscope model TescanVEGA3. Samples were prepared by mounting the powders on carbon tape placed under aluminum foil. Subsequently, the powders were metalized with gold powder for 12 minutes and examined using SEM at 10 kV.

Fourier transform infrared (FTIR)

FTIR spectra were obtained using KBr pellets as solid support. To prepare the tablets, approximately 1 mg of the sample was used for 100 mg of KBr, which were macerated until a fine powder was obtained. The mixture was then subjected to a pressure of 78.5 KN, using a Shimadzu ® hydraulic press for a period of ten minutes. The samples were then analyzed in an IRTracer-100 spectrometer, Shimadzu ® , in the region between 4000 and 400 cm -1 , with 45 scans and a resolution of 8 cm -1 .

Nuclear magnetic resonance (NMR)

1D and 2D 1 H NMR experiments were acquired using D 2 O and conducted in a Bruker™ 400 MHz ASCEND III spectrometer, operating at 9.4 Tesla, equipped with a 5 mm direct detection probe and z-axis field gradient. 1 H NMR chemical shifts were expressed relative to the TMS signal at δ 0.00 (reference compound) and coupling constants (J) were expressed in Hz.

In silico procedures

Initially, a molecular docking analysis was performed using Autodock Vina [START_REF] Trott | Autodock vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading[END_REF] program and Autodock Tools [START_REF] Morris | Software news and updates AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility[END_REF] graphical interface, only to generate an initial pool of conformations for further semi-empirical refinement. The structure of βCD was taken from the RCSB-PDB crystallographic database (www.rcsb.org) PDB ID:

5MK9. The structure of SB-E-β-CD was drawn from the three-dimensional structure of 5MK9 in Chemsketch, following the sulfonation pattern of the inclusion complex used experimentally. Chimera software [START_REF] Pettersen | UCSF Chimera -A visualization system for exploratory research and analysis[END_REF] was used to add hydrogens. The 3D structure of PHY was obtained from PubChem (www.pubchem.ncbi.nlm.nih.gov), with hydrogens added also with Chimera software. For Vina calculations, the dimensions of gridbox were 14 x 13 x 13 Å and 21 x 21 x 21 Å with a default spacing of 1.0 Å between the grid points, centered on β-CD and SB-E-β-CD, respectively. Starting from the best poses found in Vina, semi-empirical calculations were performed using PM6-DH2 Hamiltonian with the MOPAC2016 package (Stewart, 2016). The final binding energies after semi-empirical calculations (ΔE), considering the heat of formation (ΔH° f) of each system, were evaluated as:

ΔE = ΔH° f (complex) -[ΔH° f (host) + ΔH° f (guest) ]

Entrapment efficiency

The real concentration of PHY complexed into CDs was determined using a protocol previously described [START_REF] Ferraz | Complexation with β-cyclodextrin enhances apoptosis-mediated cytotoxic effect of harman in chemoresistant BRAFmutated melanoma cells[END_REF]. Firstly, the superficially adsorbed PHY was removed from both complexes (PHY/β-CD and PHY/SB-E-βCD) by washing 10 mg of the sample with 10 mL of ethanol for 10 minutes with stirring. The resulting solution was centrifuged at 4500 g for 5 min (25 °C) in order to remove any remaining CD. Then, the supernatant containing the adsorbed PHY was collected and filtered through a membrane filter (0.22 μm filter, Millipore TM ). An aliquot (1000 μl) of the filtrate was added to quartz cuvettes for absorbance reading in a UV-Vis spectrophotometer at 260 nm. A standard curve (30-60 μg/ml, R 2 = 0.9911) was constructed under the same conditions to calculate the final PHY concentration (n=3). Finally, the entrapment efficiency (EE%) was calculated according to the equation: EE% = (mass of PHY recovered / mass of PHY in the complex) x 100

In vitro dissolution test

A dissolution profile of PHY and its inclusion complexes was performed as previously proposed [START_REF] Oliveira | β-Cyclodextrin complex improves the bioavailability and antitumor potential of cirsiliol, a flavone isolated from Leonotis nepetifolia (Lamiaceae)[END_REF]. Initially, the maximum wavelength of PHY was determined by scanning at wavelengths of 200-400 nm in saline solution (0.15 M) acidified to pH 1.5 with hydrochloric acid (37 ºC) using a UV-Vis spectrophotometer. A calibration curve was performed using concentrations between 2-30 µg.mL -1 (Y = 0.0033X + 0.0077, R 2 = 0.9911, where X is the PHY concentration and Y is the absorbance at 260 nm at 37 °C).

For the controlled release study, free PHY and its inclusion complexes were placed in 50 mL of saline solution, and the experiments were carried out in a magnetic stirrer with controlled temperature and agitation (37 ºC and 100 rpm, respectively) for a total time of 100 minutes. At periodic intervals, samples of the release medium were taken and analyzed by spectrophotometry at 260 nm to determine the amount of released PHY. Results were obtained from three independent experiments.

Evaluation of cytotoxic activity

The cytotoxic activity was determined by the MTT method, described by [START_REF] Mossman | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF] and adapted by [START_REF] Oliveira-Júnior | Bis-nor-diterpene from Cnidoscolus quercifolius (Euphorbiaceae) induces tubulin depolymerization-mediated apoptosis in BRAF-mutated melanoma cells[END_REF]. Samples were solubilized in DMSO (dimethylsulfoxide) and diluted in the culture medium (final DMSO concentration < 1%, used as control). A2058 melanoma cells (2 x 10 3 cells/well) were exposed to PHY and its inclusion complexes at the same concentration (50 µM) for 72 h. CDs and physical mixtures were also tested under the same conditions. After the incubation period, cell morphology was evaluated under an inverted phase contrast microscope (Nikon, Eclipse, France) and a MTT solution (5 g/L) was then added to each well. Formed formazan crystals were solubilized in 200 μL of DMSO and absorbance readings were performed at 550 nm, using the FLUOstar Omega microplate reader (BMG Labtech ® , France). The percentage of cell growth inhibition (GI%) was then calculated using the following equation:

GI% = 100 -[(Aa -Ab) / (Ac -Ab)] x 100
Where: Aa represents the absorbance of the wells containing cells treated with the samples;

Ab represents the absorbance of empty wells (blank) and Ac represents the absorbance of wells containing only cells grown in culture medium + final DMSO concentration. Results

were obtained from at least three independent experiments.

Statistical analysis

Statistical analysis was performed using one-way analysis of variance (one-way ANOVA) followed by Tukey's multiple comparison test. P values < 0.05 were considered statistically significant. All analyzes were performed using GraphPad software Prism ® 5.0 (GraphPad Prism Software, Inc., San Diego, CA, USA).

Results and discussion

SEM analysis

SEM analysis can visually demonstrate the morphological difference between isolated and complexed compounds, and morphological modifications can be assumed as proof of the formation of a solid inclusion complex with a CD's hydrophobic cavity [START_REF] Srinivasan | Study of inclusion complex between 2, 6-dinitrobenzoic acid and β-cyclodextrin by 1 H NMR, 2D 1 H NMR (ROESY), FT-IR, XRD, SEM and photophysical methods[END_REF][START_REF] Srinivasan | Study of inclusion complex between 2, 6-dinitrobenzoic acid and β-cyclodextrin by 1 H NMR, 2D 1 H NMR (ROESY), FT-IR, XRD, SEM and photophysical methods[END_REF]. The results of SEM analyses for PHY, β-CD, SB-E-β-CD, physical mixtures, and inclusion complexes are shown in Figure 1.

PHY exhibited plane-shaped crystals, while SB-E-β-CD has a spherical structure and β-CD has a very characteristic morphology, resembling large rectangles. Physical mixtures preserved the crystalline and amorphous structures of PHY and both CDs, respectively, presenting characteristics that can be attributed to the isolated carriers and, possibly, to the PHYthus, the compound is not inserted in the cavity of the host molecule. In fact, it is common to have preserved characteristics for both host and guest during SEM analysis of physical mixtures. In this case, CDs are often covered and/or surrounded by small crystals from the guest compound, maintaining the original morphology [START_REF] Domingues | Antinociceptive effects of bupivacaine and its sulfobutylether-β-cyclodextrin inclusion complex in orofacial pain[END_REF].

However, both complexes showed alterations in CDs morphology and a reduction in the crystallinity of PHY. Inclusion complexes exhibited quite singular morphological changes, linked to a reduction in particle size and to a transformation to an amorphous structure (when compared with isolated CDs and physical mixtures).

FTIR analysis

FTIR is a useful tool for the characterization of vibrational patterns because it is able to show changes in characteristic bands of CDs, such as broadening, narrowing, disappearance, or change in intensity, which may be indicative of the formation of inclusion complexes [START_REF] Mura | Analytical techniques for characterization of cyclodextrin complexes in the solid state: A review[END_REF]. FTIR spectra of PHY, CDs, inclusion complexes, and physical mixtures are shown in Figure 2.

Analyses of PHY/β-CD and PHY/SB-E-β-CD inclusion complexes spectra show a reduction in the intensity of the characteristic bands of PHY. Absorption bands are clearly noted in the region of 3000 cm -1 (O-H stretching), 2900 cm -1 (C-H stretching), at 1154 cm -1 (C-O stretching) and 1026 cm -1 (C-O-C stretching), all characteristic of β-CD and SB-E-β-CD, with a slight displacement of the O-H and C-H stretching bands. This finding suggests that PHY, in both complexes, is interacting more strongly with the CDs' cavity, resulting in greater conformational restriction, reducing the free movement of the included molecules and then decreasing the intensity of the signal [START_REF] Eid | Characterization of the inclusion complex of zerumbone with hydroxypropyl-βcyclodextrin[END_REF]. This absorption pattern is not observed in the physical mixtures spectra, which is not as deformed as in the absorption pattern of inclusion complexes.

NMR analysis

The interaction of CDs with an encapsulated compound during formation of an inclusion complex can be demonstrated by spectral variations through changes in chemical shifts, mainly through variations of internal hydrogens (Bernini et al., 2004). Thus, the formation of inclusion complexes was observed based on the chemical shifts of the CDs protons that remain inside the cavity, hydrogen 3 (H-3) and hydrogen 5 (H-5), which will be altered once the guest molecule is inside of it [START_REF] Jahed | NMR ( 1 H, ROESY) spectroscopic and molecular modelling investigations of supramolecular complex of β-cyclodextrin and curcumin[END_REF]. Chemical shifts are shown in Table 1 and 1 H NMR spectra are presented in Figure 3.

The experiment for PHY/β-CD and PHY/SB-E-β-CD inclusion complexes showed that internal hydrogens (H-3, H-5, and H-6) were shifted in a lower frequency region, indicating that they were shielded in the presence of the guest molecule. To confirm this mode of complexation, 1 H-1 H ROESY NMR experiment was carried out, since it is considered one of the most effective techniques to study the internal interactions of host-guest in inclusion complexes [START_REF] Ali | Validating strategy of quantitative ROESY analysis for structure determination of cyclodextrin inclusion complexes[END_REF]Muzzafar, 2019). However, no correlations between the hydrogens of the molecules were observed, probably due to overlapping signals. To determine the most stable poses of the inclusion complexes, considering a gap of 1 H-1 H ROESY information, an in silico study can be assigned [START_REF] Oliveira | β-Cyclodextrin complex improves the bioavailability and antitumor potential of cirsiliol, a flavone isolated from Leonotis nepetifolia (Lamiaceae)[END_REF].

In silico procedures

To confirm and better understand the complexation mode of PHY into the CDs' internal cavity, molecular docking studies were applied to characterize the three-dimensional structure of inclusion complexes, based on the binding energies. During PH6-DH2 calculations, it is established that lower energy values indicate better guest/host interaction (Sapte; Pore, 2016).

The conducted molecular docking studies of PHY/β-CD PHY/SB-E-β-CD inclusion complexes help understand the binding affinity and stability of the interactions. Data predicted that β-CD and SB-E-β-CD can encapsulate PHY within their hydrophobic cavity, and PM6-DH2 calculations suggested that the most stable conformations (Figure 4) show binding energies of -89.8068 Kcal/mol (PHY/β-CD inclusion complex) and -87.4032

Kcal/mol (PHY/SB-E-β-CD inclusion complex).

Properties such as size, charge and polarity of the molecule can influence the ability and stability of inclusion complex formation [START_REF] Faucci | Computer-aided molecular modeling techniques for predicting the stability of drug-cyclodextrin inclusion complexes in aqueous solutions[END_REF][START_REF] Faucci | Computer-aided molecular modeling techniques for predicting the stability of drug-cyclodextrin inclusion complexes in aqueous solutions[END_REF][START_REF] Faucci | Computer-aided molecular modeling techniques for predicting the stability of drug-cyclodextrin inclusion complexes in aqueous solutions[END_REF]. The results indicated that PHY was preferably inserted through the substituted benzene, corroborating FTIR and 1 H NMR results (absence of peaks from axial deformation attributed to a methoxyl group and more significant interaction between H-3, H-5 and H-6 with a possible guest molecule). Based on this findings, the possible inclusion mode of PHY in both β-CD and SB-E-β-CD cavities is presented in Figure 5.

In vitro dissolution test

CDs are widely used by pharmaceutical industries for various purposes, including improvement of thermal and plasma stability, as well as enhancement of aqueous solubility, since most drug candidates are hydrophobic. Considering the low solubility of PHY as a limiting factor for its pharmaceutical development, we performed the dissolution test. In order to mimic stomach conditions, the dissolution test was carried out in pH-adjusted and temperature-controlled saline solution (pH 1.5 at 37 °C) [START_REF] Oliveira | β-Cyclodextrin complex improves the bioavailability and antitumor potential of cirsiliol, a flavone isolated from Leonotis nepetifolia (Lamiaceae)[END_REF].

As shown in Figure 6, there was no dissolution of PHY into the medium during the entire experiment time (100 minutes). However, when complexed with both CDs, PHY exhibited a high release rate in the first five minutes, especially for PHY/SB-E-βCD (>90%).

After 30 min, both complexes presented a dissolution rate >95%, indicating that complexation of PHY in βCD or SB-E-βCD may significantly enhance its solubility under the tested conditions. This observation is often well associated to a greater in vivo bioavailability of organic compounds. An improved solubility combined with the permeation capacity intrinsic to hydrophobic molecules usually contribute to a better bioavailability [START_REF] Zhou | β-Cyclodextrin inclusion complex: preparation, characterization, and its aspirin release in vitro[END_REF].

Entrapment efficiency

The entrapment efficiency (EE%) is a quantitative parameter used to determine the real amount of the guest molecule present in the inner cavity of the CDs. A high EE% indicates efficiency of the chosen complexation method and ensures better dissolution rate and consequently better bioavailability [START_REF] Kumar | Ultrasound processed nanoemulsion: A comparative approach between resveratrol and resveratrol cyclodextrin inclusion complex to study its binding interactions, antioxidant activity and UV light stability[END_REF]. In the present study, the EE% obtained for PHY/β-CD and PHY/SB-E-β-CD were of 89.60% and 77.56%, respectively, representing a satisfactory complexation yield. Many factors can influence the interactions between guest molecules and CDs, including the type of cyclodextrin (which differ in the size of the cavity), the complexation method and the level of hydrophobicity of the guest molecule [START_REF] Fenyvesi | Cyclodextrins in food technology and human nutrition: benefits and limitations[END_REF]. The high encapsulation efficiency values observed for both inclusion complexes indicate that a large amount of PHY was actually inserted into the CDs internal cavity.

Evaluation of cytotoxic activity

Phyllacanthone is the major diterpene found in C. quercifolius. In a recent study, we have shown that this compound inhibits cell proliferation and migration, and induces apoptosis in chemoresistant melanoma cells by disrupting the cell cytoskeleton.

Immunoassays and molecular docking experiments revealed PHY induces tubulin depolymerization through a strong interaction with the colchicine-specific binding site (Oliveira-Junior et al., 2022). Despite its anti-melanoma potential, PHY has poor solubility, which limits its efficacy and hinders its pharmaceutical development. Its complexation in two different CDs could improve its physicochemical properties without compromising its antimelanoma activity. This hypothesis was confirmed by testing PHY alone and complexed with both β-CD and SB-E-β-CD. As shown in Figure 7, PHY 50 µM exhibited a percentage of cell growth inhibition of 35.50 ± 4.81%. PHY/β-CD and PHY/SB-E-β-CD inclusion complexes showed a percentage of inhibition of 42.98 ± 3.70% and 39.95 ± 2.5%, respectively, both with a statistically significant difference when compared to control group (p<0.05).

Microphotographs (Figure 7C) for the cytotoxic activity of PHY and inclusion complexes showed a reduction of the cell population promoted by the treatments. These findings are in accordance with previous reports by [START_REF] Oliveira-Júnior | Bis-nor-diterpene from Cnidoscolus quercifolius (Euphorbiaceae) induces tubulin depolymerization-mediated apoptosis in BRAF-mutated melanoma cells[END_REF], that described the moderate concentration dependent cytotoxic potential and cell inhibition promoted by PHY against A2058 melanoma cells. SB-E-β-CD, has been extensively described for improving physical-chemical properties and consequently the bioactivity of terpenes. The addition of the hydroxypropyl or sulfobutyl groups to the outer surface of β-CD considerably improves its solubility. In this work, we chose to use SB-E-β-CD to assess whether its polyanionic character could have any additional influence on the complexation of phyllacanthone, since HP-β-CD is neutral just like its natural precursor cyclodextrin (β-CD). Moreover, native and modified CDs, particularly SB-E-βCD, are widely used as excipients in pre-clinical safety studies because of their excellent physicochemical and pharmacological properties and limited/reversible toxicity, particularly when dosed orally [START_REF] Lima | Inclusion of terpenes in cyclodextrins: Preparation, characterization and pharmacological approaches[END_REF]. There is still a significant lack of clinical studies validating their use in humans. We hope that these preliminary results will encourage the use of SB-E-β-CD to enable clinical trials with terpenes such as PHY.

Conclusions

In this report, we have demonstrated that the complexation of PHY with β-CD and SB-E-β-CD may be a promising alternative for solving the physicochemical and bioavailability problems of this diterpene. Both inclusion complexes were characterized by FTIR, NMR and microscopic studies. In addition, we determined the mechanism of complexation using an in silico approach. Both inclusion complexes preserved the cytotoxic activity of PHY against melanoma cells and exhibited increased dissolution rates, suggesting that the complexation improved PHY aqueous solubility. Altogether, these findings encourage the use of both (natural or chemically modified) CDs as carriers of PHY in pharmaceutical formulations or drug delivery systems as a strategy to improve its stability and ensure better bioavailability in vivo.

Authors contributions

Figure 1 -

 1 Figure 1 -Scanning Electron Microscopy of PHY (A1, A2, A3); β-CD (B1, B2, B3); physical mixture of PHY/β-CD (C1, C2, C3); PHY/β-CD inclusion complex (D1, D2, D3); SB-E-β-CD (E1, E2,E3); physical mixture of PHY/SB-E-β-CD (F1, F2, F3); PHY/SB-E-β-CD inclusion complex (G1, G2, G3). Magnifications of 1000x, 3000x, and 5000x, respectively.

Figure 2 -

 2 Figure 2 -FTIR characterization for (A) -PHY (red), β-CD (blue), PHY/β-CD physical mixture (PM, green), PHY/β-CD inclusion complex (IC, pink); and (B) -PHY (red), SB-E-β-CD (blue), PHY/SB-E-β-CD physical mixture (PM, green), PHY/SB-E-β-CD inclusion complex (IC, pink).

Figure 3 -

 3 Figure 3 -1 H NMR spectra obtained for β-CD (A), PHY/β-CD (B), SB-E-β-CD (C) and PHY/SB-E-β-CD (D) in D 2 O.

Figure 4 -

 4 Figure 4 -Most stable docking conformations for PHY/β-CD (A1/A2) and PHY/SB-E-β-CD (B1/B2) in two visualization modes.

Figure 5 -

 5 Figure 5 -Possible inclusion mode of PHY in the internal cavity of β-CD and SB-E-β-CD.

Figure 6 -

 6 Figure 6 -Graph for the in vitro release rate, demonstrating the variation in the concentration of PHY in saline solution when in an inclusion complex with β-CD and SB-E-β-CD (for a total time of 100 minutes).

Figure 7 .

 7 Figure 7. Cytotoxic activity of PHY (50 µM), PHY/β-CD and PHY/SB-E-β-CD inclusion complexes and physical mixtures (A and B). Data are expressed as mean of %GI (Growth Inhibition) ± SEM, *p < 0.05 (vs. control group) according to one-way ANOVA followed by Tukey's test. Microphotographs of cells after 72h of exposure to PHY, PHY/β-CD and PHY/SB-E-β-CD inclusion complexes and physical mixtures show the reduction of the cell population after treatments. The scale (red bar) corresponds to 100 µm (C).

  The plant material was previously identified by a botanist, and a comparison was made with a specimen already deposited(voucher #19202) in the HerbariumVale do São Francisco (HVASF) of UNIVASF, being registered in the National System for

Cnidoscolus quercifolius Pohl were collected in Petrolina-PE, in a Caatinga zone from the Federal University of Vale do São Francisco (UNIVASF) (09°19'43'' S / 40°33'09'' W).

Table 1 -

 1

	Proton	β-CD	PHY/β-CD	Δδ
	H-2	3.6104	3.6099	0.0005
	H-3	3.9918	3.9907	0.0011
	H-4	3.6760	3.6754	0.0006
	H-5	3.8806	3.8782	0.0024
	H-6	3.9045	3.9033	0.0012
	Proton	SB-E-β-C	PHY/SB-E-β-CD	Δδ
	H-2	3.6348	3.6340	0.0008
	H-3	4.0650	4.0625	0.0022
	H-4	3.6499	3.6495	0.0004

1 H NMR, chemical shifts (δ, ppm) in D 2 O of β-CD/SB-E-β-CD and changes observed for the inclusion complexes (PHY/β-CD and PHY/SB-E-β-CD).

Δδ = δ CD -δ inclusion complex (positive or negative values). Data obtained from Bruker Avance III 400 MHz. The most significant Δδ were observed for H-3, H-5 and H-6.

Acknowledgments

The authors thank CAPES-FACEPE [88887.615975/2021-00] for financial support, the Franco-Brazilian Network on Natural Products (FB2NP) and CycloLab ® for providing Dexolve ® (SB-E-β-CD).

CSCA, CAAF and RGOJ contributed to PHY isolation; CSCA, YKSCT, GUMN, VLAS and APO performed complex inclusion preparation and characterization (NMR, FTIR and SEM analyses); CSCA and GUMN performed entrapment efficiency and in vitro dissolution studies; LP and RGOJ performed all cytotoxic activity evaluation; CEYL and EBAF performed molecular docking analysis; CSCA, RGOJ and JRGSA participated in the design of manuscript, data analysis and interpretation; JRGSA and RGOJ supervised the entire study.

Conflict of interest

The authors declare no conflict of interest.


Michal Ronovsky

Lujin Pan

Malte Klingenhof

Isaac Martens

Raphael Chattot

Lukas Fusek

Peter Kus

Marta Mirolo

Fabio Dionigi

Harriet Burdett

Jonathan Sharman

Peter Strasser

Alex Martinez Bonastre

Jakub Drnec

Assessing Utilization Boundaries for Pt-Based Catalysts in an Operating Proton-Exchange Membrane Fuel Cell

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

Introduction

As we try to reduce our carbon footprint, a hydrogen economy is becoming integral to transitioning to a carbon-neutral future. Given the critical role of noble metals for Oxygen Reduction Reaction (ORR) catalysis, much research has been dedicated to improving the catalytic activity of Pt by modifying the electronic and structural properties via Pt alloying with transition metals (Co, Ni, Fe, Ir) [START_REF] Marković | Oxygen Reduction Reaction on Pt and Pt Bimetallic Surfaces: A Selective Review[END_REF], [START_REF] Asset | A Review on Recent Developments and Prospects for the Oxygen Reduction Reaction on Hollow Pt-Alloy Nanoparticles[END_REF], [START_REF] Kim | Alloy Nanocatalysts for the Electrochemical Oxygen Reduction (ORR) and the Direct Electrochemical Carbon Dioxide Reduction Reaction (CO RR)[END_REF], [START_REF] Gasteiger | Activity Benchmarks and Requirements for Pt, Pt-Alloy, and Non-Pt Oxygen Reduction Catalysts for PEMFCs[END_REF]), (C. [START_REF] Zhang | A Review of Pt-Based Electrocatalysts for Oxygen Reduction Reaction[END_REF], [START_REF] Sharma | Zoom in Catalyst/Ionomer Interface in Polymer Electrolyte Membrane Fuel Cell Electrodes: Impact of Catalyst/Ionomer Dispersion Media/Solvent[END_REF], [START_REF] Lin | High Durability of Pt-Ni-Ir/C Ternary Catalyst of PEMFC by Stepwise Reduction Synthesis[END_REF]. In 2007, Stamenkovic et al. identified the most active surface: Pt3Ni(111), which led to the development of PtNi octahedra NPs with 10-fold activity gain due to maximizing the surface of (111) facets [START_REF] Stamenkovic | Trends in Electrocatalysis on Extended and Nanoscale Pt-Bimetallic Alloy Surfaces[END_REF], [START_REF] Cui | Octahedral PtNi Nanoparticle Catalysts: Exceptional Oxygen Reduction Activity by Tuning the Alloy Particle Surface Composition[END_REF]. However, transferring the activity measured in rotating disc electrode (RDE) experiments to PEMFC and reaching stability targets remains a significant challenge [START_REF] Pan | Current Challenges Related to the Deployment of Shape-Controlled Pt Alloy Oxygen Reduction Reaction Nanocatalysts into Low Pt-Loaded Cathode Layers of Proton Exchange Membrane Fuel Cells[END_REF].

Several factors play the role. Octahedra (oh) NPs (5-8 nm) have a smaller electrochemical surface area (ECSA) than commercial 2-3 nm Pt catalysts. Thus, octahedra must accommodate and withstand a larger current per nanoparticle. Accommodating increased current density (per nanoparticle) requires a well-structured boundary that allows sufficient oxygen diffusion as well as proton and electron conductivity [START_REF] Ly | Integrating Nanostructured Pt-Based Electrocatalysts in Proton Exchange Membrane Fuel Cells[END_REF], (O'Hayre and Prinz 2004), [START_REF] Kongkanand | The Priority and Challenge of High-Power Performance of Low-Platinum Proton-Exchange Membrane Fuel Cells[END_REF]. Further, withstanding potential changes during PEMFC operation is the biggest challenge because the dissolution of nickel or platinum degrades the finely-tuned structure and thus lowers the catalytic activity. One way to stabilize the structure is to dope oh-PtNi with a small amount of additional metal such as Ir, Mo, or Rh [START_REF] Dionigi | Controlling Near-Surface Ni Composition in Octahedral PtNi(Mo) Nanoparticles by Mo Doping for a Highly Active Oxygen Reduction Reaction Catalyst[END_REF], [START_REF] Li | One-Nanometer-Thick PtNiRh Trimetallic Nanowires with Enhanced Oxygen Reduction Electrocatalysis in Acid Media: Integrating Multiple Advantages into One Catalyst[END_REF]. While this is a promising improvement, and the stability objectives are reached in the RDE configuration, the stability still does not satisfy the target when tested in the MEA configuration following the DOE testing protocol. For another class of ORR catalysts, PtCo intermetallics and benchmark Pt catalysts, it has been observed that a stress test's potential limits and potential cycling protocol greatly impact the overall stability [START_REF] Gatalo | Importance of Chemical Activation and the Effect of Low Operation Voltage on the Performance of Pt-Alloy Fuel Cell Electrocatalysts[END_REF], [START_REF] Ahluwalia | Potential Dependence of Pt and Co Dissolution from Platinum-Cobalt Alloy PEFC Catalysts Using Time-Resolved Measurements[END_REF], [START_REF] Myers | Potentiostatic and Potential Cycling Dissolution of Polycrystalline Platinum and Platinum Nano-Particle Fuel Cell Catalysts[END_REF]). Faster scan rates generally lead to less dissolution; thus, square-wave test profiles mimic more realistic catalyst usage [START_REF] Kneer | Effect of Dwell Time and Scan Rate during Voltage Cycling on Catalyst Degradation in PEM Fuel Cells[END_REF]. It has been hypothesized that this is linked to the Pt redox dynamics, where the oxidation extent of surface Pt atoms leads to various degrees of dissolution and degradation. To this extent, much research has focused on the Pt oxidation-dissolution link. Many publications highlighted the influence of upper potential limit (UPL) in an accelerated stress test, making clear that increasing the UPL increases the extent of oxidation and, in turn, the extent of dissolution [START_REF] Cherevko | Dissolution of Platinum in the Operational Range of Fuel Cells[END_REF], [START_REF] Topalov | Towards a Comprehensive Understanding of Platinum Dissolution in Acidic Media[END_REF], [START_REF] Huang | Transient Platinum Oxide Formation and Oxygen Reduction on Carbon-Supported Platinum and Platinum-Cobalt Alloy Electrocatalysts[END_REF], [START_REF] Imai | In Situ and Real-Time Monitoring of Oxide Growth in a Few Monolayers at Surfaces of Platinum Nanoparticles in Aqueous Media[END_REF], [START_REF] Myers | Potentiostatic and Potential Cycling Dissolution of Polycrystalline Platinum and Platinum Nano-Particle Fuel Cell Catalysts[END_REF], [START_REF] Drnec | Initial Stages of Pt(111) Electrooxidation: Dynamic and Structural Studies by Surface X-Ray Diffraction[END_REF], (Ruge et al. 2017b), [START_REF] Wang | Effect of Voltage on Platinum Dissolution[END_REF], [START_REF] Fuchs | Structure Dependency of the Atomic-Scale Mechanisms of Platinum Electro-Oxidation and Dissolution[END_REF]. However, only a limited amount of research focused on the influence of lower potential limit (LPL) (H. [START_REF] Zhang | The Impact of Potential Cycling on PEMFC Durability[END_REF], [START_REF] Uchimura | Electrocatalyst Durability under Simulated Automotive Drive Cycles[END_REF]). In the most recent work, Đukić et al. pointed out the importance of controlling LPL to improve the PEMFC catalyst's lifetime, which is somewhat linked to the reduction process (Đukić et al. 2022). Nevertheless, the actual mechanism between LPL and the stability of the catalyst in the MEA remains an open question.

Here we look at the dissolution mechanics of a PtNiIr/C octahedra catalyst (doped with <1 at. % of Ir) and compare it with a reference Pt/C catalyst. With a focus on LPL, we investigate the linkage between the catalyst degradation and operational boundaries in 5 cm 2 PEMFC optimized for high-energy X-ray diffraction (XRD) measurement. The fast acquisition rate enabled by the EBS-ESRF allows tracking oxidation/reduction processes during the PEMFC operation with a high temporal resolution. Moreover, the determination of potential onsets of oxidation and reduction and their relation to the observed degradation is presented. We conclude that Pt and Ni dissolution is proportional not only to the extent of Pt oxidation but, interestingly, also to the extent of Pt reduction. Limiting LPL is possible if the longevity of the catalyst layer is a concern. However, such an approach greatly limits the maximum power density obtainable from the PEMFC.

Results and discussion

For the experiments shown in this work, we use the catalyst-coated membranes (CCMs) of two types: (i) "Pt(JM)" benchmark CCM with Pt (volume weighted diameter is 2.9 nm as determined using XRD) on both cathode (0.187 mg Pt /cm 2 ) and anode (0.079 mg Pt /cm 2 ) that serves as reference material; (ii) "oh-PtNiIr" CCM (volume weighted diameter is 7.1 nm) with PtNiIr (0.100 mg Pt /cm 2 and 0.017 mg Ni /cm 2 ) at the cathode and Pt at the anode (0.079 mg Pt /cm 2 ). See the experimental section for more details. Interestingly, we find that the oh-PtNiIr catalyst incorporated in the CCM already has a different composition from the assynthesized oh-PtNiIr powder: EDX analysis shows that the oh-PtNiIr powder has an atomic Pt:Ni ratio of 2.20, whereas the as-prepared oh-PtNiIr CCM has the ratio of 6.35. This suggests that the catalyst lost 57% of its Ni content during the ink-making and printing. Such a high loss of transition metal alloying element has been observed and discussed previously in the work of [START_REF] Gatalo | Importance of Chemical Activation and the Effect of Low Operation Voltage on the Performance of Pt-Alloy Fuel Cell Electrocatalysts[END_REF], [START_REF] Dubau | Tuning the Performance and the Stability of Porous Hollow PtNi/C Nanostructures for the Oxygen Reduction Reaction[END_REF]. It has been pointed out that the CCM-making process, together with the activation potential cycling, alters the catalyst morphology and composition to such an extent that the catalyst is no more the same material as synthesized, questioning the common approaches to catalyst activity testing using the RDE technique [START_REF] Chattot | Break-In Bad: On the Conditioning of Fuel Cell Nanoalloy Catalysts[END_REF]. Our results provide further evidence that this is also the case for multimetallic oh-PtNiIr. In contrast to compositional changes, the structural changes are less noticeable in the ink-making/printing step (Figure 1a,b). Qualitatively, the edges of octahedral nanoparticles are less pronounced in the catalyst coated on the membrane than in the catalyst powder. Figure 1c shows the catalyst after 10k cycles of a standard (0.6 to 0.95) V accelerated stress test (AST). At the end of life (EOL), the atomic Pt:Ni ratio is 8.33, meaning an additional 21% of Ni content leaches out. Thus, almost three times more Ni content dissolved during the ink-making/printing procedure compared to the AST. It is clear that to retain the composition of state-of-the-art multimetallic catalysts during the MEA preparation, serious attention must be focused on adapting the inkmaking/printing procedure. From Figure 1c, we can see that Ni dissolution itself is not the only degradation mechanism. Nanoparticles also aggregate, coalesce, ripen, and carbon support corrodes [START_REF] Cherevko | Stability and Dissolution of Electrocatalysts: Building the Bridge between Model and 'real World' Systems[END_REF], [START_REF] Prokop | Review of the Experimental Study and Prediction of Pt-Based Catalyst Degradation during PEM Fuel Cell Operation[END_REF], (J. [START_REF] Zhang | Stabilizing Pt-Based Electrocatalysts for Oxygen Reduction Reaction: Fundamental Understanding and Design Strategies[END_REF], [START_REF] Okonkwo | Platinum Degradation Mechanisms in Proton Exchange Membrane Fuel Cell (PEMFC) System: A Review[END_REF]). In the following section, we perform a series of ASTs discussing the effects of lower potential boundaries on catalyst degradation.

Accelerated stress tests

In laboratory conditions, catalyst degradation is studied by square-wave accelerated stress tests. U.S. DRIVE Fuel Cell Technical Team adopted the standard of cycling from (0.6 to 0.95) V with a 3 s dwell time at both lower potential limit and upper potential limit. This test mimics the dynamics of the typical FC operation with Pt dissolution and Ostwald ripening as preferential degradation pathway [START_REF] Sasaki | Dissolution and Stabilization of Platinum in Oxygen Cathodes[END_REF], [START_REF] Topalov | Towards a Comprehensive Understanding of Platinum Dissolution in Acidic Media[END_REF], [START_REF] Cherevko | Dissolution of Platinum in the Operational Range of Fuel Cells[END_REF], [START_REF] Sandbeck | Particle Size Effect on Platinum Dissolution: Considerations for Accelerated Stability Testing of Fuel Cell Catalysts[END_REF], [START_REF] Ehelebe | Platinum Dissolution in Realistic Fuel Cell Catalyst Layers[END_REF]. Even though the mechanism of Pt degradation and dissolution as a function of upper potential limit is well known [START_REF] Lopes | Dynamics of Electrochemical Pt Dissolution at Atomic and Molecular Levels[END_REF], [START_REF] Khedekar | Probing Heterogeneous Degradation of Catalyst in PEM Fuel Cells under Realistic Automotive Conditions with Multi-modal Techniques[END_REF], [START_REF] Bi | PEM Fuel Cell Pt ∕ C Dissolution and Deposition in Nafion Electrolyte[END_REF], (H. [START_REF] Zhang | The Impact of Potential Cycling on PEMFC Durability[END_REF], [START_REF] Martens | Decoupling Catalyst Aggregation, Ripening, and Coalescence Processes inside Operating Fuel Cells[END_REF])(Martens2019), the exact nature of the rather surprising linkage between LPL and Pt degradation is still unclear [START_REF] Gatalo | Importance of Chemical Activation and the Effect of Low Operation Voltage on the Performance of Pt-Alloy Fuel Cell Electrocatalysts[END_REF])(H. [START_REF] Zhang | The Impact of Potential Cycling on PEMFC Durability[END_REF])), (Đukić et al. 2022), [START_REF] Gatalo | Importance of Chemical Activation and the Effect of Low Operation Voltage on the Performance of Pt-Alloy Fuel Cell Electrocatalysts[END_REF], [START_REF] Uchimura | Electrocatalyst Durability under Simulated Automotive Drive Cycles[END_REF].

In order to study the effect of lower potentials on the aging of oh-PtNiIr and Pt(JM) catalysts, we perform two ASTs with different LPLs (0.6 to 0.95 V and 0.7 to 0.95 V). We use a custom-built 5 cm 2 fuel cell operating at 80 °C, at 80% relative humidity, flowing H2/N2 at 200 sccm to mimic the conditions of commercial devices [START_REF] Martens | X-Ray Transparent Proton-Exchange Membrane Fuel Cell Design for in Situ Wide and Small Angle Scattering Tomography[END_REF], [START_REF] Martens | Towards Comprehensive Understanding of Proton-Exchange Membrane Fuel Cells Using High Energy X-Rays[END_REF]. Both Pt(JM) and oh-PtNiIr MEAs made from as-prepared CCMs with Sigracet 22BB GDLs were conditioned at 500 mA/cm 2 for approximately two hours. Throughout the 10k cycles of each AST, we follow structural changes of the catalyst with XRD [START_REF] Martens | X-Ray Transparent Proton-Exchange Membrane Fuel Cell Design for in Situ Wide and Small Angle Scattering Tomography[END_REF]. In general, we track four XRD parameters: scale factor, microstrain, lattice parameter, and crystallite size.

1. Scale factor, i.e. scattering intensity, corresponds to the total amount of scatterers. Martens et al. proposed that scale factor decreases at oxidation potentials due to the formation of an amorphous oxide layer on Pt NPs (Martens, Chattot, et al. 2019). The scale factor is thus a direct indicator of the amorphous oxide growth/reduction onset.

2. Microstrain originates from local defects in the lattice [START_REF] Leineweber | Understanding Anisotropic Microstrain Broadening in Rietveld Refinement[END_REF][START_REF] Chattot | Surface Distortion as a Unifying Concept and Descriptor in Oxygen Reduction Reaction Electrocatalysis[END_REF]. Such defects can be generated by various surface processes and through diffusion distributed in the bulk of the NP, giving rise to the broadening of the diffraction peaks [START_REF] Chattot | Surface Distortion as a Unifying Concept and Descriptor in Oxygen Reduction Reaction Electrocatalysis[END_REF]) [START_REF] Kawaguchi | Electrochemically Induced Strain Evolution in Pt-Ni Alloy Nanoparticles Observed by Bragg Coherent Diffraction Imaging[END_REF]. One mechanism causing the microstrain increase is a place exchange, where the structural defects are formed during the extraction of Pt atom [START_REF] Drnec | Initial Stages of Pt(111) Electrooxidation: Dynamic and Structural Studies by Surface X-Ray Diffraction[END_REF]) [START_REF] Fuchs | Structure Dependency of the Atomic-Scale Mechanisms of Platinum Electro-Oxidation and Dissolution[END_REF]) (Ruge et al. 2017a) [START_REF] Jacobse | Atomic-Scale Identification of the Electrochemical Roughening of Platinum[END_REF], (You et al. 1994), [START_REF] Nagy | Applications of Surface X-Ray Scattering to Electrochemistry Problems[END_REF], (You and Nagy 1994). Another possibility is the dissolution of less noble metal from alloyed NPs, where the process leaves the defects throughout the bulk of nanoparticles. [START_REF] Kongkanand | Surface Platinum Electrooxidation in the Presence of Oxygen[END_REF]. 3. Lattice parameter is a global measure of strain. In bimetallic systems, the changes can be caused by dissolution, following Vegard's law [START_REF] Vegard | Die Konstitution der Mischkristalle und die Raumf�llung der Atome[END_REF]) [START_REF] Denton | Vegard's Law[END_REF], or by the evolution of surface tension due to the field effect and adsorption phenomena [START_REF] Weissmüller | Charge-Induced Reversible Strain in a Metal[END_REF]) [START_REF] Chattot | Electrochemical Strain Dynamics in Noble Metal Nanocatalysts[END_REF]). 4. Crystallite size is the size of the coherently scattering domain. The size of the scattering domain does not change during aggregation, but it is the degradation process of coalescence and ripening when the size of the scattering domain changes. Thus, depending on the degradation process, one nanoparticle can be composed of a few such domains [START_REF] Martens | Decoupling Catalyst Aggregation, Ripening, and Coalescence Processes inside Operating Fuel Cells[END_REF]. Harsher degradation leads in general to a more pronounced crystallite size increase.

Figure 2 shows the evolution of crystallite sizes of Pt(JM) and oh-PtNiIr and lattice parameter of oh-PtNiIr throughout 10k cycles. As expected, the 0.6 V LPL AST is harsher as the crystallite size of the NPs increases throughout 10k cycles by 42% for Pt(JM) and 30% for oh-PtNiIr, whereas in the case of the 0.7 V LPL AST by only 17% for Pt(JM), and 10% for oh-PtNiIr. The degradation and ripening of NPs are the fastest at the beginning of the AST for both catalysts. In Figure 2b, we show the evolution of the lattice parameter of the oh-PtNiIr catalyst for 0.6 V LPL AST (stars) and 0.7 V LPL AST (diamonds). Nickel dissolution is more substantial in the case of 0.6 V LPL AST as the lattice parameter increases by 0.4%, corresponding to an 18% loss of Ni at atomic percent, which is in line with the 21% loss of Ni at atomic percent measured by EDX. In contrast, in the case of the 0.7 V LPL AST, Ni loss was only 4% at atomic percent. Similar to the size increase, Ni dissolution is also the fastest at the beginning of the AST. These observations agree with the previously reported dependency of dissolution on LPL for Pt (H. [START_REF] Zhang | The Impact of Potential Cycling on PEMFC Durability[END_REF]) and Pt-alloyed nanocatalysts [START_REF] Gatalo | Importance of Chemical Activation and the Effect of Low Operation Voltage on the Performance of Pt-Alloy Fuel Cell Electrocatalysts[END_REF].

Figure 2: a) crystallite size evolution of Pt(JM) (black) and oh-PtNiIr (red/green) catalysts during 10k cycles of two ASTs. In the case of (0.6 to 0.95) V AST, Pt(JM) size increase is 42% and for oh-PtNiIr 30%, whereas in the case of (0.7 to 0.95) V AST, Pt(JM) size increase is 17% and for oh-PtNiIr 10%. b) lattice parameter evolution of oh-PtNiIr for (0.6 to 0.95) V and (0.7 to 0.95) V AST.

X-ray diffraction coupled with cyclic voltammetry

To build the connection between the LPL and dissolution mechanism, we follow the cyclic voltammetry with XRD in the course of several cycles (XRD-CV). CV measures current density which originates from overlaying non-faradaic and faradaic processes. Coupling with XRD, we further distinguish contributions from adsorption and place-exchange-induced oxidation as the first is measured by changes in lattice parameter and the latter by a decrease in scale factor. The top part of Figure 3 shows the evolution of scale factor (brown) and microstrain (green) during CV (blue) (50 mV/s) for Pt(JM). The bottom part shows the same for oh-PtNiIr. As the first CV cycle is clearly different from the subsequent ones due to impurity removal and reconstruction of the surface, only the second cycle is shown [START_REF] Chattot | Break-In Bad: On the Conditioning of Fuel Cell Nanoalloy Catalysts[END_REF]). (For more information about the measurement protocol, see the experimental section in supporting information.)

To ambiguously follow the growth of surface oxide, we start the anodic sweep at 0.05 V where the catalyst is fully reduced. In general, the XRD scale factor follows the oxidation hysteresis apparent in CV. It is constant up to the onset of the oxidation potential, after which it decreases until the reverse potential of 1.23 V. At the beginning of the cathodic sweep, the scale factor rests at its lowest value and starts increasing when the potential crosses the onset of the reduction potential. After passing a reduction region (highlighted in red), the scale factor returns close to its original value. There is a slight slope between the end of the reduction region and 0.05 V in the cathodic sweep, pointing to the fact that the catalyst is not yet entirely reduced. A small number of catalytic sites, presumably highly oxophilic defects, are much harder to reduce giving rise to this slope [START_REF] Janik | On the Presence of Surface Bound Hydroxyl Species on Polycrystalline Pt Electrodes in the 'hydrogen Potential Region' (0-0.4 V-RHE)[END_REF], [START_REF] Chattot | Building Practical Descriptors for Defect Engineering of Electrocatalytic Materials[END_REF]). Nevertheless, the reduction is ~97% complete at the end of the reduction region. Further, there is a slight difference between both oxidation and reduction onsets for Pt(JM), but no difference is observed for oh-PtNiIr within our 0.02V resolution limit.

In agreement with the CV profiles, the reduction region is shifted towards higher potentials for the oh-PtNiIr compared to the Pt(JM) catalyst. This is a consequence of OH bond weakening, which is related to a d-band shift induced by alloying with less noble metal [START_REF] Hammer | Theoretical Surface Science and Catalysiscalculations and Concepts[END_REF], (J. K. [START_REF] Nørskov | Towards the Computational Design of Solid Catalysts[END_REF]), (Jens K. [START_REF] Nørskov | Density Functional Theory in Surface Chemistry and Catalysis[END_REF], [START_REF] Kitchin | Role of Strain and Ligand Effects in the Modification of the Electronic and Chemical Properties of Bimetallic Surfaces[END_REF]), [START_REF] Xin | Effects of D-Band Shape on the Surface Reactivity of Transition-Metal Alloys[END_REF]. Clearly, a d-band shift not only enhances ORR activity but also shifts the oxidation onset to higher potentials, defining the stability window of the catalyst [START_REF] Drnec | Initial Stages of Pt(111) Electrooxidation: Dynamic and Structural Studies by Surface X-Ray Diffraction[END_REF]. This is then expressed by lesser oxidation during AST and, therefore, slower degradation and higher stability of PtNi catalysts overall (Figure 2a).

Next, we turn to the general trend of microstrain, distinguishing between the formation of amorphous surface oxide and other processes causing the increase of general disorder of atoms within the NPs. Such processes can be triggered by a place exchange during the oxidation onset, in which the localized surface strain caused by Pt-O swap, or dealloying where defects formed due to the Ni dissolution, inhomogeneously propagates to the bulk of the particle [START_REF] Kawaguchi | Electrochemically Induced Strain Evolution in Pt-Ni Alloy Nanoparticles Observed by Bragg Coherent Diffraction Imaging[END_REF]. Microstrain follows a similar trend as the scale factor: it is constant in the (0.3 to 0.9) V range and then increases until sweep reversal. Upon reversal, it holds its value, and after the 0.9 V is crossed, it starts to decrease again until it reaches a plateau at 0.6 V. No difference between anodic and cathodic microstrain onsets is observed. An additional feature is present in the range of (0.05 to 0.3) V, where microstrain drops to its lowest value at 0.05 V. This part is associated with the HUPD region, in which adsorbed hydrogen likely further anneals the surface and, subsequently, the bulk of NPs, leaving behind a fully reduced, well-ordered catalyst without structural impurities in the lattice.

For Pt(JM), microstrain onset is at higher potentials than scattering intensity onset. This is due to the fact that a sufficient amount of amorphous oxide must be present on the surface before the surface microstrain propagates to the bulk and is therefore detectable by X-ray measurement. For oh-PtNiIr, microstrain increases in the anodic scan even before the formation of the amorphous oxide. This might be due to the preferential oxidation of Ni Finally, we connect the degradation data from ASTs with the oxidation/reduction dynamics deduced from XRD-CV. First, oh-PtNiIr does not oxidize to such a large extent at UPL as Pt(JM), giving the of-PtNiIr catalyst better overall stability. Second, the 0.6 V LPL results in a fully reduced oh-PtNiIr catalyst, while at 0.7 V LPL, the reduction is only partial for both Pt(JM) and oh-PtNiIr. Given that the reduction region is where the most dissolution happens, the dissolution amount must be proportional to both the degree of oxidation and the degree of reduction, and this is clearly shown by the XRD-CV measurement. This result also implies that the most oxophilic sites which are more difficult to reduce, likely the least coordinated sites, are also prone the most to dissolution. Therefore, one of the strategies to limit the dissolution is to keep those vulnerable sites permanently oxidized.

Such knowledge has a direct implication for the operation of a fuel cell, especially involving high-active shape-controlled platinum alloys. Their activity is strongly bound to a specific crystal structure, and the extent of surface restructuring during the reduction will affect this activity to a larger extent than other catalysts. In general, if we want to preserve high performance, we must carefully select the operational boundaries and limit both the lower and higher potential boundaries regardless of the catalyst type. Moreover, in the automotive industry, where multimetallic catalysts will be used to lower the stack's cost, the loss in power density at higher LPL can be compensated by an increased exchange current density of a more active catalyst and its higher electric efficiency.

Conclusion

We have shown the practicality of the XRD-CV technique to explain the degradation behavior and provided further important insight into catalyst degradation mechanisms in the operational PEMFC. We have found specific onsets of key crystallographic parameters (microstrain and scale) associated with the oxidation, reduction, and degradation for two types of catalysts. For oh-PtNiIr, platinum oxidation and reduction are shifted towards higher potentials compared to the Pt(JM) catalyst due to the d-band shift, and subsequent weakening of the OH bond, induced by alloying with a less noble metal. By employing two AST with different lower potential limits (0.6 to 0.95) V and (0.7 to 0.95) V, we confirm the dependence of catalyst degradation on the lower potential limit. By combining these two results, we conclude that the extent of degradation is proportional not only to the degree of oxidation but also to the degree of reduction within a cycle, independently of the catalyst's nature. In order to preserve the crystalline structure of oh-PtNiIr and thus its high catalytic activity, an operational boundary has to be set on both lower and higher potential limits. This approach will work best for automotive applications where a multimetallic catalyst reduces the stack's price and its increased exchange current density with higher electric efficiency do not reduce the power density. We think that limiting LPL is a crucial step for implementing alloyed catalysts into PEMFCs and FC automotive as such. Moreover, the presented simple and fast methodology can be further developed to serve as a benchmark measurement to explore the operation boundaries for all kinds of Pt-based catalysts.

Providing Potential Reviewer Names

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Figure 1 :

 1 Figure 1: Transmission electron microscopy images of oh-PtNiIr catalyst at three stages: a) as-prepared powder, b) as-prepared CCM, and c) end-of-life catalyst after 10k cycles of (0.6 to 0.95) V AST. The ink-making/printing procedure leached out 58% of Ni content and made the edges of octahedra less pronounced. The AST leached out only 23% of Ni content but caused severe structural degradation.

  atoms (A.[START_REF] Politano | Segregation and Selective Oxidation of Ni Atoms in Pt3Ni(111) in a Low-Pressure Oxygen Environment[END_REF],[START_REF] Politano | Unveiling the Oxidation Processes of Pt3Ni(1 1 1) by Real-Time Surface Core-Level Spectroscopy[END_REF], with the suggested formation of subsurface Ni-O species[START_REF] Lee | Surface Segregation and Oxidation of Pt3Ni(111) Alloys under Oxygen Environment[END_REF], leaving behind structural defects even before the Pt-O place exchange kicks in. Additionally, it is likely that only a small amount of Ni-O species is necessary to trigger defect propagation into the bulk as the oh-PtNiIr catalyst has intrinsically higher microstrain due to alloying. The potential window of microstrain decrease in the cathodic scan is shorter, suggesting that the relaxation processes are faster for oh-PtNiIr. Considering that the Ni-O bond is weaker than Pt-O (A.[START_REF] Politano | Segregation and Selective Oxidation of Ni Atoms in Pt3Ni(111) in a Low-Pressure Oxygen Environment[END_REF], faster overall kinetics likely translates to faster Ni-O reduction (and dissolution) compared to Pt-O. Consequently, oh-PtNiIr alloy pays the price for better kinetics by higher sensitivity to secondary metal leaching while cycling.

Figure 3 .

 3 Figure 3. X-ray diffraction coupled with cyclic voltammetry. Scale factor (brown) and microstrain (green) were obtained with XRD during (0.05 to 1.23) V CV with a 50 mV/s scan rate. The upper half corresponds to the Pt(JM) and the lower part to the oh-PtNiIr. An increase in defect concentration during oxidation increases the microstrain value. The catalyst's oxidation is accompanied by a reversible decrease in scattering intensity. In the raw data, the cathodic dissolution region is highlighted by a red curve that extends into a simplified diagram where the dissolution region is highlighted by a red box. Black vertical dash-dot lines mark the potential boundaries of two types of ASTs to connect XRD-CV with long-term stability.

Acknowledgments

This project has received funding from the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No 847439.

Supporting Information

Experimental Catalyst powders

Johnson Matthey supplied carbon-supported Pt nanoparticles with average sizes of 2.9 nm as determined by XRD. The oh-PtNiIr catalyst powder was prepared on Vulcan XC72R by a seed-mediated solvothermal method (manuscript in preparation). Two batches of ~500 mg each were prepared. The atomic composition of each batched analyzed with ICP-OS yielded Pt 68.2 Ni 31.2 Ir 0.5 and Pt 68.3 Ni 31.3 Ir 0.4 with Pt wt% of 30.1 and 29.0, respectively. For the first batch, TEM measurements showed a number-weighted nanoparticle size of (3.8 +-1.6) nm. For the second batch, two particle sizes were identified. Bigger nanoparticles had the number-weighted NP size (4.5 +-1.7) nm, and smaller nanoparticles were (2.7 +-1.7) nm in size. The volume-weighted nanoparticle size determined by XRD was 5.9 nm. A slightly larger volume-weighted NP size is a sign of non-symmetrical size distribution with the tail towards larger NPs, which is in line with TEM of the second batch. Batches were mixed to make the ink for CCMs.

Membrane electrode assemblies

CCMs printed from the catalyst powders were supplied by Johnson Matthey. For both types of CCMs, the anode is carbon-supported Pt with 0.079 mg Pt /cm 2 loading. MEAs were prepared by cutting the CCM into 5 cm 2 pieces and aligning SGL 22BB GDL on both sides. For Pt(JM), the cathode loading is 0.187 mg Pt /cm 2 with the average nanoparticle size determined by XRD of 2.9 nm. For oh-PtNiIr, the cathode loading was 0.100 mg Pt /cm 2 , 0.017 mg Ni /cm 2 , with the average nanoparticle size determined by XRD of 7.1 nm. In this case, quantitative single-nanoparticle size distribution analysis with TEM would not give a reasonable value as NPs formed chains of several crystallites, as seen in Figure 1b.

Fuel cell and operation protocol

As-prepared MEAs were used in a custom fuel cell described in previous literature [START_REF] Martens | X-Ray Transparent Proton-Exchange Membrane Fuel Cell Design for in Situ Wide and Small Angle Scattering Tomography[END_REF]). The cell was operated at 80 °C, with 80% relative humidity on the anode and cathode. MEAs for the AST experiments were conditioned at constant current density for (1 to 3) h: oh-PtNiIr 0.6 V LPL @ 140 mA/cm 2 ; 0.7 V LPL @ 400 mA/cm 2 ; Pt(JM) 0.6 V LPL @ 500 mA/cm 2 ; 0.7 V LPL @ 500 mA/cm 2 . MEAs for the XRD-CV experiments were conditioned at 0.6 V for approximately 2 h (Figure S1). In this case, constant potential conditioning was chosen to limit dissolution by oxidation and reduction. For both types of conditioning and the ASTs, the gas flow was 200/100 sccm O 2 /H 2 with an additional 0.5 bar backpressure. For XRD-CV, the gas flow was 100/100 sccm N 2 /H 2 with an additional 0.01 bar backpressure. Electrochemistry SP240 Biologic potentiostat was connected in the 4 electrodes setup. Voltage cables were connected directly to graphite flow fields, and the current was collected from the adjacent copper blocks [START_REF] Martens | X-Ray Transparent Proton-Exchange Membrane Fuel Cell Design for in Situ Wide and Small Angle Scattering Tomography[END_REF]. Cyclic voltammetry was performed from 0.05 V to 1.23 V at 50 mV/s, flowing N 2 /H 2 300/200 sccm at 80 °C, with 80% relative humidity. The protocol starts at 0.47 V, after compensation for ohmic drop at 85% amplitude. The first scan from 0.47 V to 1.23 V and back to 0.05 V is referred to as the 1st cycle. The second cycle shown in the Results and Discussion chapter starts at 0.05 V. Figure S2 shows the first two cycles for Pt(JM). We see that during the first cycle (dashed line) the catalyst oxidizes at higher potentials than in the subsequent cycle. This is due to impurity desorption from an overall restructuring of the surface [START_REF] Chattot | Break-In Bad: On the Conditioning of Fuel Cell Nanoalloy Catalysts[END_REF]). These impurities have to be removed by oxidation and reduction during the first cycle to get a clean surface that represents the actual catalytic properties of the catalyst. It shows the importance of reporting at least the second cycle to obtain relevant electrochemical information. In our experiments, subsequent cycles had the same characteristics as the second cycle. X-ray diffraction XRD data were collected at the ID31 beamline at the European Synchrotron Radiation Facility. A Pilatus 2M CdTe detector was used at a distance of 0.95 m. The photon wavelength was 0.161 A (77.00 keV), just below the Pt K edge. The beam size was approximately 5 × 20 µm (vertical x horizontal) with a flux of 10 13 photons/s. An exposure time was 1 s per point for AST data and 0.1 s per point for XRD-CV. No significant beam damage was detected. Raw diffraction patterns (Figure S3a) were radially integrated using PyFAI [START_REF] Kieffer | PyFAI: A Python Library for High Performance Azimuthal Integration on GPU[END_REF]. Integrated 1D powder diffractograms were loaded into GSAS-II, and sequential Rietveld analysis was performed [START_REF] Toby | GSAS-II: The Genesis of a Modern Open-Source All Purpose Crystallography Software Package[END_REF].

Transmission electron microscopy

The measurements were conducted using a Tecnai G2 20 s-Twin microscope, equipped with a LaB6-cathode and a GATAN MS794 P CCD-detector at ZELMI Centrum, Technical University of Berlin. TEM samples were ultrasonicated in i-PrOH and drop-dried on copper grids.

Inductively Coupled Plasma Optical Emission Spectrometry ICP-OES was used to determine the elemental composition of the various catalysts. Samples were prepared by dissolving 5 mg of the catalyst powder with 20 mg of NaClO 3 in a mixture of HCl and HNO 3 (3:1). The solutions were kept for 20 min at 180 °C using a Microwave Discover SP-D (CEM Corporation). The heating ramp was 15 °C/min. Subsequently, the solutions were diluted with Milli-Q® water, filtered, and brought to a known volume. To calculate the concentration of the different solutions, five standards of Pt, Ni, and Ir were prepared with concentrations of (1, 2, 7, 12, 20) mg/L for each element.

Energy-dispersive X-ray EDX, used for additional determination of elemental composition within samples, was carried out using XFlash 6|10 spectrometer (Bruker) installed within the MIRA III scanning electron microscope (Tescan). The primary electron beam energy was 20 keV. The area for spectra acquisition was 150um x 150um. Additionally, an area of 1000um x 1000um was used to rule out local inhomogeneities. PB-ZAF approach was used for peak deconvolution.

Author Information and Notes

Notes

The authors declare no competing financial interest.