Julie Charton
email: julie.charton@univ-lille2.fr

Rebecca Deprez-Poulain

Nathalie Hennuyer

Anne Tailleux

Bart Staels

Benoit Deprez

Novel non carboxylic acid retinoids: 1,2,4-oxadiazol-5-one derivatives

We have successfully obtained 1,2,4-oxadiazol-5-one bioisoteres of Am580 or Tazarotene-like retinoids. In particular compound 4 displays an EC 50 of 26 nM on RAR-β.

The biological activities of retinoids are mediated by two types of nuclear receptors, Retinoic Acid Receptors (RARs or NR1B : RARα, RARβ and RARγ), and Retinoid X Receptors (RXRs or NR2B : RXRα, RXRβ and RXRγ). At the molecular level RARs and RXRs form heterodimers. At the cellular level, these receptors are DNA binding proteins that regulate gene transcription in response to ligand binding. The success of RAR modulation in the treatment of acute promyelocyticleukaemia (APL) 5 has stimulated considerable interest in the development of RAR and RXR modulators. Most potent retinoids, such as Am580 or Tazarotenic acid have an aromatic carboxylic acid moiety instead of the polyenecarboxylic acid of retinoic acid (ATRA, Figure 1). In this family, the hydrophobic part and the linker (X) can be varied with retention of high activity. Tazarotene, an RAR-β,γ selective acetylenic retinoid, was chosen for development as a topical agent for psoriasis and acne. Am580 (RAR-α selective) has powerful and selective cyto-differentiating effects on APL. 6,7 Various acidic heterocycles are classically used by medicinal chemists as carboxylic acid bioisosteres. [START_REF] Wermuth | The Practice of Medicinal Chemistry[END_REF] Tetrazole is a common bioisostere of the carboxylic acid group but in the particular case of retinoids led to loss of activity. [START_REF] Dawson | [END_REF] On the contrary, thiazolidinedione moiety has been described by Tashima et al. to be an effective replacement of the carboxylic acid (Figure 2a). 10,11 More recently, bioisosteric replacement of carboxylic acid with a tropolone ring is reported (Figure 2b). 12,13 Another example of acidic heterocycle is the 1,2,4oxadiazol-5-one ring, found in AT1 antagonists 14 , COX inhibitors 15 , PLA2 inhibitors 16 and modulators of GluR [START_REF] Valgeirsson | 18 Measured with shake-flash method, UV detection[END_REF] . This heterocycle can replace carboxylic acids but displays geometrically different protomers and slightly different physico-chemical properties.

Compounds bearing this heterocycle have different lipophilic properties that may translate into improved bioavailability compared with their carboxylic analogues. Indeed, ΔlogD 7.4 between benzoic acid and 3-phenyl-1,2,4-oxadiazol-5-one = 0.7 log unit, the oxadiazolone being more hydrophobic than its carboxylic counterpart. 18 In our project aiming at synthesizing various analogues of pharmacologically active carboxylic acids, we explored the replacement of the carboxylic acid function by the 1,2,4-oxadiazol-5-one moiety in two series of retinoids: analogues of the tazarotene, a RAR-β,γ selective acetylenic retinoid, and Am580, an RAR-α selective retinoid (Figure 3). First, we investigated the synthesis of tazarotenic acid analogues. 19 Precursors 1 and 2 were synthesized as previously described from the corresponding nitrile in two steps (Scheme 1). 20

CN N H O N O R R i, ii R = p-I ; m-I 1 R = p-I 2 R = m-I Scheme 1.
Reaction conditions: i) NH 2 OH.HCl, DIEA, EtOH, reflux, 8 hrs, (1a: 75%, 2a: 95%). ii) CDI, DBU, dioxane, 110°C, 2hrs, (1: 80%, 2: 80%).

We have recently reported a strategy for solid phase synthesis of acetylenic derivatives of 3-aryl-1,2,4oxadiazol-5-one thanks to a Sonogashira reaction.

Compound 1 was thus grafted on resin and then reacted with 6-ethynyl-1,1,4,4-tetramethyl-1,2,3,4tetrahydro-naphthalene 3 to yield compounds 4 (Scheme 2).

H Br N O N O H O O O N N H O I iii i, ii 3 4
Scheme 2.

Reaction conditions: i) trimethylsilylacetylene (1.3 eq), PdCl 2 (PPh 3 ) 2 (0.05 eq), CuI (0.1 eq), NEt 3 , 70°C, 12 hrs. ii) KOH (0.1N), Isopropanol, 12hrs, room temp. iii) alkyne (10 eq), PdCl 2 (PPh 3 ) 2 (1.1 eq), CuI (2.2eq), DIEA (30 eq), DMF, rt, 3 hrs then 5% TFA/CH 2 Cl 2 , rt, 3 hrs

In the search of a more cost-effective synthesis, we also developed the first solution Sonogashira coupling with an unprotected 1,2,4-oxadiazol-5-one ring. Such strategy has been used for Suzuki-Miyaura reactions 21 and successfully applied for the synthesis of compounds 5-7 (Scheme 3). Some carboxylic acid analogues described in literature were also synthesized like compounds 8 and 9 in order to compare retinoidal activity of our oxadiazolone derivatives with known retinoic compounds. Reaction conditions: i) Alkyne (1.2 eq), PdCl 2 (PPh 3 ) 2 (0.05 eq), CuI (0.1 eq.), NEt 3 , DMF, 80°C, 5 hrs, 8a : 52 %, 9 : 20%, ii) NaOH (2 eq), EtOH, 8: 100%. Am580 and its 1,2,4-oxadiazol-5-one analog 10 were synthesized as described in Scheme 5. a The data are expressed as averages of % efficacy and EC 50 . Eff. : efficacy is relative to ATRA defined as 100%. b <10% at 10µM. c Efficacy is relative to Tazarotene defined as 100%.

Cl O N H O N H O N O i,ii iv,iv,v 10
Tazarotene and its analogue 8 are RAR-β,γ selective acetylenic retinoids, whereas analogue 9 is very active on the three subtypes. Interestingly, 1,2,4-oxadiazol-5one 4 and 5 activities differ from their carboxylic acids analogues. Indeed, compound 5 is slightly active on RAR-β. Compound 4, though nanomolar on RAR-β (EC 50 = 26 nM), is less active than 9 but displays a better selectivity on RAR-β,γ subtypes and an efficacy comparable with tazarotene. As expected, bended analogue 7 is not active since it is not linear enough to fit the RAR pharmacophore. In the second series, compound 10 is active on RAR-α with a similar efficacy to that of Am580 (40 versus 43 %). In the light of these results, both compounds 4 and 10 could be good starting points for further optimization. All these compounds were checked for cross-reactivity with the retinoid X receptor (RXR α, β, γ) and found to be inactive.

In conclusion, we have described the solid-phase or solution-phase syntheses of a new series of non carboxylic acid RAR ligands. We have shown that the 1,2,4-oxadiazol-5-one moiety can act as a bioisostere of the carboxylic acid function in retinoid structures. In particular the retinoidal activity of compound 4 (RAR-β,γ selective) is significant, considering that replacement of the carboxylic acid in retinoid structures with bioisosteric functional groups is generally ineffective. These non carboxylic acid type RAR ligands may exhibit different pharmacological behaviors from classical carboxylic acid compounds, as well as unique biological activity, and they may provide further scope for clinical applications.

Figure 1 :

 1 Figure 1: General structure of retinoids and examples. 2

Figure 2 :

 2 Figure 2 : Non-Benzoic Acid Retinoids.

Figure 3 :

 3 Figure 3: Generic structure of the potential retinoid 1,2,4-oxadiazol-5one derivatives.

Scheme 3 .

 3 Scheme 3. Reaction conditions: i) 1 or 2, PdCl 2 (PPh 3 ) 2 , CuI, NEt 3 , DMF, 80°C, 7 hrs, (5: 50%, 6: 83%, 7: 56%).

  Scheme 4. Reaction conditions: i) Alkyne (1.2 eq), PdCl 2 (PPh 3 ) 2 (0.05 eq), CuI (0.1 eq.), NEt 3 , DMF, 80°C, 5 hrs, 8a : 52 %, 9 : 20%, ii) NaOH (2 eq), EtOH, 8: 100%.

Table 1 :

 1 Transcriptional activation assay data. a

	Am580
	iii,
	The retinoidal activities of the synthesized compounds
	were examined in terms of potency and efficacy and

Scheme 5. Reaction conditions: i) H 2 N-C 6 H 4 -CO 2 Et, NEt 3 , THF, rt, 3 hrs, 85%. ii) NaOH, EtOH, H 2 O, 40°C, 12hrs, 90%. iii) H 2 N-C 6 H 4 -CN, NEt 3 , THF, rt, 12 hrs, 10a: 98%. iv) NH 2 OH.HCl, DIEA, EtOH, reflux, 6 hrs, 10b: 91%. v) CDI, DBU, dioxane, 110 °C, 2hrs, 10: 44%.

Acknowledgements

We are grateful to the Institutions that support our laboratory (Inserm, Université de Lille2 and Institut Pasteur de Lille). Data management was performed using Pipeline Pilot™ from Scitegic. We thank also the following Institutions or companies: CAMPLP and VARIAN.inc. This project was supported by the Fondation pour la Recherche Medicale (FRM) -Nord-Pas-de-Calais.

Supplementary data

Supplementary data associated with this article can be found for the online version.


Colloidal nanoparticles were prepared by aqueous self-assembly of amphiphilic β-cyclodextrins (βCDs) acylated on their secondary face with C14 chains to a total degree of substitution of 7.0, via a thermolysin-catalyzed transesterification process. The small-angle X-ray scattering pattern of the nanoparticles was consistent with a reverse hexagonal organization. Cryo-transmission electron microscopy images revealed particles with spectacular tortuous shapes and consisting of misoriented domains with a regular columnar hexagonal structure, separated by sharp interfaces. Edge dislocations as well as a variety of stepped tilt grain boundaries (GBs) composed of symmetrical and asymmetrical sections, together with one twist GB, were identified from axial views of the columnar organization. The tilt GB structure was analyzed using the concepts of coincidence site lattice and structural units developed to describe the atomic structure of interfaces in various types of polycrystals. The tilt GBs were described using sequences of βCD-C14 columns that differed by the number of neighboring columns (5, 6 or 7) and exhibiting distinctive contrasts. To our knowledge, this is the first time that these types of topological defects are described at the nanometric scale by direct observation of colloidal polycrystalline hexosomes of self-organized amphiphiles.

Introduction

One major challenge of nanomedicine is the elaboration of "nanovehicles" that deliver active compounds to a targeted site, at a sufficient concentration and without early degradation, in order to maximize the efficiency of the substance while limiting secondary effects. In the recent years, among a wide variety of colloidal vectors of bioactive molecules, liquid crystalline nanoparticles such as the so-called cubosomes or hexosomes [1][2][3][4][5] have attracted a significant attention due to their versatility as delivery systems of antifungal infection agents, 6 contrasting agents for theranostic applications [7][8][9][10] or in food additives, 11 to name but a few examples. For a better understanding of the encapsulating capacity and in order to optimize their formulation and lyoavailability, the knowledge of the morphology and molecular organization of these particles is crucial. Cryo-transmission electron microscopy (cryo-TEM) has been the technique of choice to directly visualize the shape, size distribution and ultrastructure of the colloidal nanosystems, 1,[12][13][14] in conjunction with dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) analyses.

Cyclodextrins (CDs), obtained by enzymatic conversion of starch by the CD glycosyl transferase, are macrocyclic oligosaccharides composed of α(1→4)-linked glucosyl units. The so-called α-, band gCDs, composed of 6, 7 and 8 glucosyl units, respectively, have been considered as building blocks in a variety of nanodevices used for drug delivery. [15][16][17] Since hydrophobic molecules can be entrapped in the apolar cavity of the CD, a higher solubility, stability or absorption of a large variety of drugs can be achieved. 18 Some inclusion compound products are commercial, which attests to the biocompatibility and safety of the CDs and their hydrophilic derivatives.

CDs can be chemically modified to prepare amphiphilic derivatives that have attracted a significant interest. 17,[19][20][21] They are prepared by grafting hydrophobic substituents on the primary and / or secondary hydroxy groups of the glucosyl units and can be designed to be incorporated in model bilayer membranes, [22][23][24] or to form aggregates that encapsulate and carry bioactive drugs to specific targets. [25][26] For instance, amphiphilic CDs have been prepared by grafting alkyl chains with different lengths on the secondary hydroxy groups of the CD, either chemically 19,27,28 or using thermolysin to catalyze the transesterification of vinyl fatty ester donors. 29,30 Upon nanoprecipitation, depending in the formulation and process parameters, these derivatives self-organize into nanoreservoirs or solid nanoparticles [31][32][33][34] whose drug encapsulation and release properties were evaluated in vitro and in vivo as potential long-circulating nanocarriers for vascular administration. [33][34][35][START_REF] Memisoğlu-Bilensoy | Microencapsulation[END_REF][START_REF] Mendez-Ardoy | [END_REF][38] In previous reports, we have described the morphology and structure of nanoparticles prepared by self-organization of a series of βCD-Cn (n = 8, 10, 12, 14) amphiphilic derivatives with various total degrees of substitution (TDS), after dissolution in acetone and nanoprecipitation in water. 30,32,39 The particles were characterized by cryo-TEM imaging of quench-frozen aqueous suspensions and SAXS. Two types of morphologies and ultrastructures were observed depending on the TDS of the parent derivative. The molecules with TDS < 5 formed nanospheres with an onion-like multilamellar organization whereas those with TDS > 5 self-assembled into barrel-like (n = 8, 10, 12) or more tortuous (n = 14) particles with a columnar inverse hexagonal structure. Ultrastructural models of both structures were proposed based on the analysis of the contrast distribution in cryo-TEM images showing different projections of the lattices. 40 Remarkably, the nanosystems formed from βCD-C14 with TDS = 7.0 exhibited unusual facetted shapes and were composed of several domains (or "grains") with different orientations with respect to one another, separated by sharp interfaces. 40 The present report focuses on the description of the morphology and structure of these βCD-C14 particles analyzed from high-resolution cryo-TEM images. In particular, we have described the columnar organization in a number of topological defects, namely dislocations and grain boundaries, at the nanoscale, using geometrical concepts (coincidence site lattices and structural units) previously developed to describe the structure of defects and interfaces in various types of polycrystals.

Experimental

Materials

βCDs (Kleptose ® ) were provided by Roquette Frères (Lestrem, France). Thermolysin (EC 3.4.24.27), a type X protease isolated from Bacillus thermoproteolyticus rokko, anhydrous DMSO (99%), and vinyl myristate (95%) were purchased from Sigma-Aldrich (l'Isle d'Abeau Chesnes, France). Acetone (Acros Organics, analytical grade) was from Fischer Scientific (Illkirch, France).

The nanoparticles were prepared in ultrapure water (Elga Purelab).

Synthesis and characterization of the βCD-C14 derivative

The βCD fatty ester was biosynthesized according to the procedure previously described by Choisnard et al. that involved C14 vinyl esters as acyl donors and thermolysin as biocatalyst. [30][31][32] The alkyl chains were grafted on the secondary face of the βCD, mostly at the C2 position of the glucosyl units. 29 The βCD fatty ester was analyzed by matrix-assisted laser desorption/ionization mass spectroscopy (MALDI-MS). According to the analytical method described by Choisnard et al., 39 the derivative was characterized by its average molar weight and its TDS, as defined by Challa et al. 41 The TDS was calculated using the formula given by Choisnard et al. 39 The βCD-C14 derivative used in the present study was characterized by = 2599.5 g mol -1 and TDS = 7.0. 40

Nanoparticle preparation

βCD-C14 nanoparticles were prepared using the solvent displacement method. 42 Ten milligrams of βCD-C14 derivative was dissolved in 10 mL acetone to reach a concentration of 1 mg mL -1 . The solutions were kept at 25 °C in a double envelope set-up and poured into an equal volume of distilled water under magnetic stirring (500 rpm). The nanoparticles spontaneously formed and the organic solvent was evaporated under reduced pressure. The aqueous suspension was concentrated to 1.5 mg mL -1 at 40 °C under vacuum, filtered through 0.8 µm filters and kept at room temperature.

The sample was further concentrated by centrifugation (9600 g) to about 5-10 mg mL -1 for cryo-TEM observation and SAXS analyses. The samples were prepared in triplicates.

Cryo-transmission electron microscopy (cryo-TEM)

Cryo-TEM specimens were prepared by quench-freezing thin liquid films of a 5-10 mg mL -1 nanoparticle suspension formed on Pelco NetMesh lacy carbon films into liquefied ethane, using a Leica EM-GP workstation. Once mounted in a Gatan 626 cryoholder precooled with liquid nitrogen, the specimens were observed at low temperature (-180 °C), under low dose illumination, using a Philips CM200 'Cryo' microscope operating at 80 kV. The images were recorded on Kodak SO163 films.

Small-angle X-ray scattering (SAXS)

SAXS analyses were performed on the BM02 beamline at the European Synchrotron Radiation Facility (Grenoble, France). The concentrated suspension was poured into a glass tube (3.0 mm outer diameter). The scattering pattern was recorded at room temperature, during 50 s, at an energy of 16.62

keV (λ = 0.0764 nm) with a CCD detector placed at a distance of 24 cm, and calibrated using a silver behenate standard. The SAXS profile was calculated as a radial average of the two-dimensional pattern and the profile of a glass tube containing only water was subtracted. The diffraction peaks were fitted with Gaussian functions and the unit cell parameters were calculated using a least-squares regression.

Scheme 1. Idealized structure of one molecule of βCD-C14 with TDS = 7. The alkyl chains, drawn in gray, are grafted at the C2 position on the secondary face of the βCD. 29 Hydrogen atoms were omitted for clarity.

Results

Morphology and ultrastructure of the βCD-C14 particles

The synthesis of the βCD-C14 derivative with TDS = 7.0 was described in the Experimental Section and a schematized 3D model of the molecule is shown in Scheme 1. The particles prepared by nanoprecipitation, briefly presented in our previous article, 40 exhibit a variety of shapes. In particular, a significant number of particles have a peculiar tortuous and facetted morphology (Fig. 1a). At higher magnification, lattice lines are visible whose patterns depend on the shape and orientation of the particles with respect to the electron beam. Most interestingly, a given tortuous particle can exhibit different patterns, suggesting that it consists of distinct domains with different mutual orientations. This is illustrated, for example, by the particle showed in Fig. 1c, as well as those in Supplementary Information Fig. S1. Some regions of the particles exhibit sets of parallel fringes whereas others contain highly contrasted hexagonal patterns (Fig. 1b and1d), the neighboring grains being separated by more or less clearly defined interfaces. In addition, as seen in Fig. 2, the SAXS profile recorded from a suspension of βCD-C14 particles contains three main peaks with a series of qi/q1 ratios (1, √3, 2) characteristic of a columnar reverse hexagonal structure. 40,43 From this profile, we calculated the hexagonal unit cell parameter ahex-SAXS = 4.90 nm. By analogy with the so-called HII phase proposed to describe the self-assemblies of various amphiphiles, [44][45][46][47] the βCD-C14 molecules are likely organized into columns with the alkyl substituents radiating outward and a central cavity hosting water molecules (Fig. 1e). Assuming that the hexagonal pattern of dark rings visible in some grains corresponds to an axial projection of the columns, the hexagonal lattice parameter is the distance between the centers of two neighboring columns. From the cryo-TEM images, we measured the parameter ahex-TEM = 4.8 nm (Fig. 1d), in good agreement with the value measured by SAXS.

The origin of the contrast in the cryo-TEM images of βCD-Cn nanoparticles has been explained in detail in our previous article, in particular for edge-on projections of the hexagonal columnar organization. 40 Here, our description of the structure focuses on the images showing end-on projections, i.e. when the electron beam is parallel to the column axis (Fig. 1e). Briefly, at an acceleration voltage of 80 kV and with a defocus of the objective lens ranging between -2.0 and -3.0 µm, the regions with the highest density and a repeat distance of around 4-5 nm in the lattice images are transferred with a dark contrast. 40 Assuming that the model proposed in Fig. 1e is correct and considering that the βCD macrocycle and ester bonds contain oxygen atoms (Scheme 1), the electron density of the cylinders formed by the superimposition of βCD moieties should be higher than that of the corona containing only alkyl chains. Therefore, the central part of the column, supposedly containing water molecules, should be clear, while the surrounding dark ring would correspond to the superimposition of βCD macrocycles, each ring separated by a continuous phase of alkyl chains with an intermediate gray level. The cryo-TEM images also reveal a dark outline that surrounds all the particles regardless of their size and shape (Fig. 1 and Fig. S1). Consistent with a mechanism previously described for the stabilization of various micellar structures, 44,45,48 a continuous layer of βCD-C14 should cover the particle surface, with the hydrophilic primary face of the βCD moiety oriented outward, in contact with water, thus ensuring a good colloidal stability (Fig. 1e). 40 TEM images of dry preparations of the βCD-C14 particles observed at low temperature are shown in Fig. S2. Since the images were recorded at a lower magnification to limit the radiation damage, the resolution is not as good as that in the cryo-TEM images. However, the presence of various sets of lattice fringes attests to the partial stability of the ultrastructure upon drying. In some regions, the particle orientation is such that an axial projection of the hexagonal lattice can be recognized (Fig. S1a).

Description of the topological defects

Two types of topological defects will be described in the following that could directly be visualized in cryo-TEM images of axial projections of the columnar domains: edge dislocations (Section 3.2.1) and tilt grain boundaries (Section 3.2.2). Many other types of linear and twodimensional defects likely exist but they could not be analyzed due to their misorientation with respect to the observation direction, with the exception of one twist grain boundary (Section 3.2.3).

The organization of βCD-C14 columns is such that, in axial projection, the two-dimensional hexagonal lattice can be described by means of (x,y) coordinates along the two [10] axes of the unit cell defined by parameter ahex = bhex and angle g = 120°.

Edge dislocations

Two examples of dislocations are shown in Fig. 3a and 3d. The complete particles from which these images were extracted are shown in Fig. S1d and S1e. The dislocations were indirectly detected by the small bending of the lattice induced by the displacement field around their core and the nature of the dislocations was determined by drawing Burgers circuits. [START_REF] Hull | Introduction to Dislocations[END_REF] Two possible but equivalent circuits were drawn (Fig. 3c and3f) from which a similar b(1,0) Burgers vector was deduced, that correspond to the distance between two columns along a (10) plane, equal to ahex. Since the contrast of the columns did not seem to be significantly modified around the defect, we assumed that the dislocations were of pure edge nature, i.e. their Burgers vectors were normal to the dislocation lines, without any additional twist component along the column direction.

Since the contrast in the core itself was not clearly defined, we marked the position of each visible column with their 6 neighboring columns in the hexagonal lattice. The core region was then described by a coordination defect that would be composed of the association of two different columns, one with 5 neighbors and the other with 7. Although putative in the present case, the geometry of this defect is likely since it is similar to those described for edge dislocations in materials with two-dimensional hexagonal lattices, such as block copolymer films, [START_REF] Hammond | [END_REF][51][52] charged polymer colloids, [START_REF] Irvine | Proc. Nat. Acad. Sci[END_REF] butterfly eyes, 54-

Tilt grain boundaries

A so-called tilt GB separates two grains misoriented around a rotation axis that is parallel to the boundary plane. [START_REF] Hull | Introduction to Dislocations[END_REF] In the present case, the tilt axis is also parallel to the axis of the columns formed by the βCD-C14 molecules. Since the hexagonal unit cell contains three sets of (10) planes, the rotation angle between two adjacent grains can be defined using several conventions. In the present article, the tilt angle q was defined as the smallest angle between a set of (10) planes chosen in each grain (Fig. S3).

The concept of coincidence site lattice (CSL) was developed to describe the structure of simple GBs in a variety of crystalline materials. [START_REF] Warrington | [END_REF][60][61] This description can also be applied to the present twodimensional hexagonal arrangement of βCD-C14 columns. A dichromatic pattern is defined as the two interpenetrating crystal lattices, one being rotated by an angle q with respect to the other around one site common to both lattices. For specific rotation angles, all sites in coincidence form a superlattice, the so-called CSL. [START_REF] Warrington | [END_REF]62 Examples of such dichromatic patterns and corresponding CSLs for a hexagonal unit cell are shown in Fig. S4. The unit cells in each grain are similar lozenges with a 120° angle and, due to the symmetry of the dichromatic pattern, the CSL unit cell is also a hexagonal unit cell described by a homothetic lozenge. The coincidence index Σ is defined by the ratio of the surface of the unit cell of the CSL to that of one elementary lattice. The CSL planes with lower indices thus contain the highest number of coincidence sites and can be used to define the position of possible low-energy symmetrical tilt GBs. [START_REF] Warrington | [END_REF] Examples of symmetrical tilt GBs between domains with a columnar hexagonal structure are shown in Fig. 4a-d, using circles and dots that would correspond to columns of self-organized βCD-C14 molecules. The GB with the lowest angle, namely S = 37 (q = 9.4°) can be described using the same 5/7 pair of coordination defects (or structural units -SUs) that was assumed to correspond to the core of the bulk b(1,0) edge dislocations (Fig. 3c and3f). The S = 37 GB, as well as those corresponding to S = 19 (q = 13.2°) and S = 7 (q = 21.8°) can be described by a specific sequence of 5/7 SUs separated by a number of 6-type columns, and their distance decreases with increasing tilt angle. For these three examples, the GB is a mirror plane. For the symmetrical S = 13 (q = 32.2°) GB, the 5/7 SUs are contiguous and organized in a zig-zag fashion to better minimize the local distortions. In this case, the GB is a mirror + glide plane. These SU sequences are very similar to those proposed to describe the structure of tilt GBs in a variety of two-or three-dimensional polycrystalline materials where atoms, molecules or particles are organized in hexagonal lattices through interactions of very different nature, exemplified in semiconductors, 61 graphene, 63-66 bubble rafts 67 magnetic bubble arrays, 56 block copolymer films, [START_REF] Hammond | [END_REF]51,68 or the cornea of some butterflies. The coincidence index S and corresponding tilt angle are given. The tilt angle was defined as in Fig. S2. For S = 37, 19 and 7, both grains are symmetrical and the GB is a mirror plane. For S = 13, the 5-and 7-type structural units adopt a zig-zag arrangement and the GB is a mirror + glide plane. Strictly, the GB described for q = 30° does not correspond to a coincidence. The (10) planes of grain 1 are parallel to the (11) planes of grain 2 but the distances between columns in both types of planes are incommensurable. Only a small portion of the GB is shown in order to introduce new tentative arrangements of SUs.

A special GB model corresponding to a tilt angle q = 30° is also shown in Fig. 4e. It is asymmetrical and is parallel to one set of (10) plane of grain 1 and one set of (11) planes of grain 2.

However, this GB does not correspond to a coincidence. Indeed, the repeat distance of the (11) planes is 2𝑎 cos 30°= 𝑎√3 and is incommensurate with the closest repeat distance along a (10) plane (i.e. 2a). In this type of GB, the mismatch is compensated by specific aperiodic groups of SUs. In the present case, the tentative column arrangement shown in Fig. 4e, and referred to as q30, was inspired by those proposed by Liu and Yakobson 57 and Yazyev 69 for a similar asymmetrical GB in polycrystalline graphene. It also involves 5/7-type SUs organized in two different motifs.

The structure of the various tilt GBs that were observed in the βCD-C14 particles was analyzed with respect to these reference structures, using recognizable sequences of 5/7-and 6-type SUs. In various materials, when a GB does not correspond to a perfect coincidence, the deviation from it can be accounted for by so-called secondary GB dislocations. [START_REF] Hull | Introduction to Dislocations[END_REF] However, we could not use this type of analysis since the GB sections in the hexosomes were rather short, preventing to define a clear reference coincidence over a long distance and draw proper Burgers circuits around defects.

While the images presented in Figs. 5 and 6 are close-up views of the tilt GBs in selected particles, lower magnification views of the whole corresponding facetted particles are shown in Fig. S1. While the contrast of the edge dislocation core was not well defined in the images of Fig. 3, the 5 and 7 SUs in GBs were directly recognizable. The core of the 7-type columns appeared to be slightly larger than that of the 6-type columns in the grains (Fig. 5a, 5d and 5g). On the contrary, the 5-type columns appeared as dark dots and not as rings like the 6-and 7-type columns. Considering the more constrained environment, it is likely that the βCD-C14 molecules could not organize to form a hollow column as described in Fig. 1e. All βCD macrocycles would rather superimpose, forming a single electron-dense column, with the alkyl moieties still pointing outward and facing the neighboring columns.

Four stepped tilts GBs were analyzed in Fig. 5 and described as composed of portions of reference symmetrical or asymmetrical GBs shown in Fig. 4. GB1 in Fig. 5b and 5c is composed of sections of symmetrical S = 7 (q = 23 ± 1°) in different planes, while GB2 in Fig. 5d and5e corresponds to S = 13 (q = 31 ± 1°). The GB in Fig. 5g A general observation was that the analyzed tilt GBs were not symmetrical and rather exhibited various stepped structures. In addition, the tilt angles were close but not equal to those of the main coincidences defined in Fig. 4. However, for the majority of analyzed GBs, S typically ranged between 23 and 31°, i.e. within the typical angular domain between S = 7 and 13, in line with the CSL model that claims that coincidences with the smallest indices are favored. Lee et al. made a similar observation on the statistics of tilt GBs occurring in the multidomain hexagonal arrays of nanonipples in the eye of the Mourning Cloak butterfly. 62 Energy calculations performed on grain boundaries in GaN semiconductors as a function of tilt angle also showed minima at S = 7 and 13. 61 The image in Fig. 6 shows a more complex configuration. A short section of symmetrical S = 13 can be recognized in the continuity of a lower angle section that contains SU sequences corresponding to symmetrical S = 19 and S = 31 (q = 17.9°) (Fig. 6c). Near the region where the GB changes directions, there is an edge dislocation that can still be considered isolated in grain 2 and not part of the GB. Complementary tilted views of this configuration are shown in Fig. S7.

Twist grain boundary

A twist GB separates two grains that are misoriented around a rotation axis that is perpendicular to the boundary plane. [START_REF] Hull | Introduction to Dislocations[END_REF] The image of the particle shown in Fig. 7 contains a small region where the axial projection of a few βCD-C14 columns can be recognized, around which the contrast pattern consists of more or less defined concentric fringes. In order to simulate this peculiar pattern, we have superimposed two identical images of a perfect hexagonal pattern of columns. The top image was made semi-transparent and incrementally rotated with respect to the image below. Qualitatively, a satisfactory fit with the experimental image was obtained with a rotation of 8°. This confirmed that the contrast could be explained by assuming that the two grains in the particle were rotated with respected to each other around an axis parallel to the column, thus separated by a twist GB. However, with this projection image, it was not possible to describe the GB itself and, in particular, determine whether it lay perfectly perpendicular to the rotation axis or with a more complex geometry like that of the stepped tilt GBs described in the previous section.

Discussion

To our knowledge, the formation of such complex morphologies has never been reported before for nanoparticles of self-assembled amphiphilic molecules and the question of the mechanism that drives the formation of such convoluted polycrystalline objects stays open. During nanoprecipitation, βCD-C14-rich droplets form upon dilution of the acetonic solution into water. When saturation is exceeded, the molecules self-assemble, forming nuclei that can grow into well-formed mesophases. [START_REF] Martínez-Muñoz | Nano-and Microencapsulation -Techniques and Applications[END_REF] However, the resulting particles are expected to adopt a shape that would minimize their surface-to-volume ratio to some extent, [START_REF] Lepeltier | [END_REF]72 although there might be a competition with a favorable growth along specific directions of the self-assembled mesophase. This tendency has previously been observed with βCD-C8 and βCD-C10 with TDS around 7, that self-assembled into individual barrel-like hexosomes elongated along the column axis. 40 However, a significant number of the βCD-C14 particles presented here were multidomain and exhibited shapes for which the surface-to-volume ratio was clearly not minimized. Among the series of tested βCD-Cn derivatives, βCD-C14 had the highest hydrophobicity due to the contribution of the longest alkyl moieties and the high degree of substitution (TDS = 7.0) and this characteristic likely promoted a kinematic freezing of the molecular arrangements at room temperature, as soon as a sufficient amount of acetone had been diluted in water upon nanoprecipitation. Indeed, the dislocations and GBs did not spontaneously migrate to the surface of the particles and they were immobilized like in a solid material.

Two mechanisms can be proposed to explain the polycrystallinity of the particles: 1) individual small hexosomes randomly collided in the aqueous suspension and formed stable aggregates; 2) at an early stage of nanoprecipitation, several nuclei simultaneously formed and grew inside βCD-C14-rich droplets. The confinement of these small proto-hexosomes in the droplets would promote their clustering into larger particles. The diversity of shape, size and orientation of the constituting domains is in favor of a clustering mechanism. However, the random flocculation of the formed hexosomes in aqueous suspension seems unlikely considering the continuity of the facetted surfaces and the regular layer of βCD-C14 molecules covering the particles to preserve the colloidal stability. In addition, the significant number of tilt GBs would require a reorientation of the grains or a reorganization of the columns to yield neighboring grains with a common tilt axis. The early clustering of neighboring proto-hexosomes inside droplets is more likely as long as a residual amount of acetone is available to maintain some molecular mobility. Then, the overall structure is frozen.

To shed additional light on the mechanism at work during particle formation, it would thus be interesting to compare the morphology adopted by the self-assembled βCD-C14 molecules when i) the nanoprecipitation is carried at temperatures higher than 25 °C and ii) the particles shown in the present article are submitted to a hydrothermal annealing treatment that may induce structural transition / reordering. 44 If the molecular mobility is increased at a higher temperature, one can expect the topological defects to migrate to the particle surface, and the various domains to coalesce and reorganize into unique hexosomes with a bulkier shape that would minimize their surface-to-volume ratio. Optimizing such annealing treatments may allow for a better control of the particle morphology and ultrastructure for nanomedicine applications.

The columnar inverse hexagonal structure can simply be described as a parallel packing of cylinders. The formation of edge dislocation and tilt boundaries would thus be energetically favorable since all cylinders remain parallel in these configurations. However, we restricted our description to the topological defects that were conveniently oriented in the ice film, i.e. with the columns aligned parallel to the electron beam direction. Many different grain boundaries existed in many other particles, some of which are visible in the images shown in Supplementary Information, but they were not properly oriented and could not be analyzed. Moreover, in many cases, the GBs could not be analyzed because small twist components likely induced local blurring of the image contrasts.

Therefore, since our geometrical description was only successful for a relatively small number of particles of the population, we could not provide any meaningful statistics on the occurrence of such and such type of defects.

Visualizing topological defects in radiation sensitive materials is still a substantial challenge for electron microscopists, which explains why such images are extremely rare in the literature. This study was carried out by recording cryo-TEM images of the nanosystems on photographic films that require a sufficient electron dose in order to achieve a good contrast. There is no doubt that higher resolution information on the particle ultrastructure and the topological defects would be collected using new-generation cryomicroscopes equipped with highly sensitive digital camera and software allowing automated low-dose image recording. 78 Tomographic approaches such as that used to visualize the three-dimensional molecular organization in monoglyceride-based cubosomes 79 would also be helpful to analyze a wider variety of topological defects, making a direct comparison with image simulations from numerical molecular models possible.

Conclusions

Recent works have reported on topological defects -namely, disclinations -in facetted liquid crystalline vesicles [START_REF] Xing | Proc. Nat. Acad. Sci[END_REF] and striped block polymersomes, [START_REF] Wong | [END_REF]75 but little is known about the highresolution structure of topological defects in soft colloids. Grain boundaries and dislocations have been observed by Hudson and coll. in RuO4-stained thin films of columnar hexagonal and cubic assemblies of a thermotropic amphiphilic dendrimer. 76,77 More recently, Liu et al. have characterized the complex topology of twins and five-fold twin boundaries in double-diamond cubosomes from heteroclusters. 78 However, to our knowledge, this is the first time that topological defects are described in solid hexosome nanoparticles of a self-assembled amphiphile, with a resolution of a few nanometers. Based on cryo-TEM images of projections of the hexagonal structure along the column axis, the organization of βCD-C14 assemblies, and the structure of edge dislocations and stepped tilt grain boundaries, were analyzed in particles whose convoluted morphology and polycrystalline structure are likely due to the combination of a clustering taking place in the early stages of nanoprecipitation and a kinematic freezing in a water-rich environment due to the high hydrophobicity of the seven C14 moieties grafted on the secondary face of the βCD macrocycle. The topological defects in this supposedly "soft" assemblies were surprisingly well described using the concepts of coincidence site lattice and structural units initially defined for solid polycrystalline materials like semiconductors and metals and, due to the hexagonal symmetry of the unit cell, the coordination defects were geometrically similar. All analyzed tilt GBs were described as sequences of 5/7 and 6-type structural units defined with respect to symmetrical boundaries with a high degree of coincidence.

Figure 1 .

 1 Figure 1. Cryo-TEM images of particles prepared by nanoprecipitation of the bCD-C14 derivative with TDS = 7: a) general view: note the presence of elongated and tortuous particles; b,c) examples of facetted particles showing the hexagonal organization of bCD-C14 columns. In b, the particle is made of only one grain and the hollow columns appear as dark circles seen in axial view. The arrow points to one structural defect near the edge of the particle. In c, the particle is composed of several domains misoriented with respect to one another, separated by grain boundaries. Various projections can be observed depending on the orientation of the grains. d) Higher magnification image of the hexagonal network of bCD-C14 columns seen along the column axis. e) Scheme describing the organization of bCD-C14 molecules in the projection of the columns and in the outer layer in contact with water revealed by the continuous dark fringe in image d. ahex is the parameter of the twodimensional hexagonal unit cell.

Figure 2 .

 2 Figure 2. SAXS profile recorded from a suspension of bCD-C14 particles that confirms the hexagonal organization. The qi/q1 ratios are indicated above each peak, with q1 = 1.49 nm -1 .

Figure 3 .

 3 Figure 3. Examples of b(1,0) edge dislocations in bCD-C14 hexosomes: a,d) cryo-TEM images; b,e) enlargement of images a and b onto which the traces of (10) planes have been superimposed. The location of the edge dislocation is indicated with a ^ symbol in front of the extra half-plane. c,f) Corresponding models of column organization. The black circles are projections of bCD-C14 hollow columns organized in a hexagonal structure. The blue and yellow disks are columns with 5 and 7 neighboring columns, respectively. Two possible Burgers circuits are drawn around the dislocation cores, both resulting in the same b(1,0) Burgers vector (red arrow). Larger views of the particles are given in Fig. S1d,e.

54

 54

Figure 4 .

 4 Figure 4. Geometrical models describing tilt GBs, using specific arrangements of SUs. The black circles are axial projections of bCD-C14 hollow columns organized in hexagonal lattices. The blue and yellow disks correspond to columns surrounded by 5 and 7 neighboring columns, respectively.The coincidence index S and corresponding tilt angle are given. The tilt angle was defined as in Fig.S2. For S = 37, 19 and 7, both grains are symmetrical and the GB is a mirror plane. For S = 13, the 5-and 7-type structural units adopt a zig-zag arrangement and the GB is a mirror + glide plane. Strictly, the GB described for q = 30° does not correspond to a coincidence. The (10) planes of grain 1 are parallel to the (11) planes of grain 2 but the distances between columns in both types of planes are incommensurable. Only a small portion of the GB is shown in order to introduce new tentative arrangements of SUs.

  and 5h contains two sections of symmetrical S = 7 in different planes and one portion of S = 13, resulting in a global tilt angle of 23 ± 1°. The GB in Fig. 5j and 5k is composed of two sections of q30 separated by a step and one section of symmetrical S = 7 GB, resulting in a global tilt angle of 27 ± 1°. Tilted views of this GB highlighting the local distortion of the lattice planes in the vicinity of the boundary are shown in Fig. S5. The SU sequence in the stepped GB shown in Fig. S6 is more complex although short sections from S = 7 and q30 GBs can be recognized (q = 25 ± 1°).

Figure 5 .

 5 Figure 5. Cryo-TEM images of various stepped tilt grain boundaries (GBs) observed in bCD-C14 hexosomes with tentative descriptive models of column organization. The black circles in the schemes are projections of bCD-C14 hollow columns organized in a hexagonal lattice. The blue and yellow disks are columns with 5 and 7 neighboring columns, respectively. The tilt angles measured between the adjacent grains are 23 ± 1° (GB1) and 31 ± 1° (GB2) in image a, 23 ± 1° in image f, and 27 ± in image i. In c, e, h and k, local GB planes with corresponding coincidence indices are proposed based on the sequences of SUs defined in the models of perfect GBs (Fig.4). Larger views of the whole particles are available in Fig.S1a-c. Additional tilted views of the GB in i are provided in Fig.S5.

Figure 6 .

 6 Figure 6. Cryo-TEM images of a stepped tilt grain boundary (GB) and an edge dislocation (D) observed in a bCD-C14 hexosome (a,b) with descriptive models of column organization (b,c). The black circles are projections of bCD-C14 columns organized in a hexagonal structure. The blue and yellow disks are columns with 5 and 7 neighboring columns, respectively. In c, the Burgers circuit around the dislocation core is drawn in gray and the red arrow indicates the b(1,0) Burgers vector. The location of the edge dislocation is indicated with a ^ symbol in front of the extra half-plane. A larger view of the particle is shown in Fig. S1f. Complementary tilted views are provided in Fig. S7.

Figure 7 .

 7 Figure 7. Twist boundary in a bCD-C14 hexosome around an axis that is parallel to the bCD-C14 column (cryo-TEM image). The projection of the grains rotated with respect to one another is blurred except in the vicinity of the rotation axis. Inset: simulation of the contrast pattern observed in the experimental image: a partially transparent image of a perfect hexagonal network was superimposed on the same image and rotated in plane by 8°.

Figure S2 .

 S2 Figure S2. TEM images of polycrystalline bCD-C14 particles. After deposition of the suspension and air-drying, the grid was mounted in a Gatan 626 specimen holder and flash-frozen in liquid nitrogen before being transferred in the microscope. The preparation was observed under low dose conditions, at low temperature. The arrows point at regions corresponding to axial projections of the hexagonal columnar organization, exemplified by the inset in image a that corresponds to the framed region.

Figure S3 .

 S3 Figure S3. Definition of the tilt angle between two adjacent grains separated by a symmetrical tilt grain boundary. The rotation angle q between two adjacent grains is defined as the smallest angle between (10) planes from each grain. Example of a S = 7 boundary with a tilt angle q = 21.8°. The yellow and blue disks are columns with 7 and 5 neighboring columns, respectively.

Figure S4 .

 S4 Figure S4. Two-dimensional dichromatic patterns formed by rotating one elementary hexagonal lattice with respect to the other by the angle indicated in parentheses around one site common to both crystals. The elementary lattices are drawn with blue and red dots, respectively, and the unit cells of both lattices are indicated by blue and red rhombi. The coincidence site lattice (CSL), drawn with gray lines, is characterized by the coincidence index Σ defined by the ratio of the surface of the unit cell of the CSL (indicated by a gray rhombus) to that of one elementary lattice. All CSLs have been oriented so that one plane of maximum coincidence is vertical.

Figure S5 .

 S5 Figure S5. Two views of the cryo-TEM image of the stepped grain boundary analyzed in Figure 5i-k. The original image has been rotated to help visualize the tilt angle between the two grains and the local distortion of the lattice planes in the vicinity of the boundary (indicated by arrows). The images have been prepared using the free Blender software (www.blender.org).

Figure S6 .Figure S7 . 6 .

 S6S76 Figure S6. Example of a stepped tilt grain boundary (GB) in a bCD-C14 multidomain hexosome with a descriptive model of column organization. The black circles are projections of bCD-C14 columns organized in a hexagonal structure. The yellow and blue disks are columns with 7 and 5 neighboring columns, respectively. The tilt angle measured between the adjacent grains is 25 ± 1°. A larger view of the particle is shown in Fig. S1g.

Acknowledgements

The experiments were conducted with the financial support of Agence Nationale de la Recherche (grant #ANR-11-BS10-0007) and Institut de Chimie Moléculaire de Grenoble (ICMG, UAR 2607).

The authors thank the NanoBio-ICMG platform for granting access to the Electron Microscopy facility, ESRF for beamtime provision (proposal #02-01-775), Cyrille Rochas (CERMAV) for his excellent technical assistance during the data collection, Delphine Levilly (DPM) for the synthesis of the cyclodextrin ester, Yu Ogawa (CERMAV) for building the molecular model of βCD-C14, Pierre Sailler (CERMAV) for assistance in using the Blender software, and Henri Chanzy for his critical reading of the manuscript. We acknowledge support from LabEx ARCANE and CBH-EUR-GS (grant #ANR-17-EURE-0003) and the Glyco@Alps program (grant #ANR-15-IDEX-02). CERMAV and DPM are part of Institut Carnot PolyNat (grant #ANR-11-CARN-030-01). J.-L.P. would like to express a heartfelt thank you to Jean-Michel Penisson and the late Jany Thibault for introducing him to the concept of coincidence site lattice many years ago.

Supplementary Information

Tilt grain boundaries and dislocations in these particles have been described in the main manuscript (Figures 567).


Théodore Magne

Richard Paridaens

Florent Ravelet

Sofiane Khelladi

Farid Bakir

Petar Tomov

Loïc Pora

Effect of degassing phenomenon of the aviation fuel cavitation dynamics in a case of a three-bladed inducer

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

Cavitation phenomenon is vastly present in different technical devices such as hydrofoils, pumps and convergent-divergent nozzles observed in various fields of engineering. One particular field of interest is the cavitation in the low pressure stage of a fuel pump, located at the interface between the aircraft and its engine. Cavitation phenomenon can generate vibrations, noise, and instabilities in the flow, which can reduce the main fuel pump hydraulic performance. Moreover, a degassing phenomenon from the aircraft fuel can take place during aircraft climbing. As a result, both phenomena can be coupled, which can lead to the emergence of a complex multiphase flow, which needs a thorough research and exploration.

An experimental work is conducted on two 3-bladed axial inducers of a low pressure stage fuel pump at partial flow rates in cavitating and non-cavitating regimes for two different working fluid. The two different fluid are: water with added dissolved CO 2 and a jet fuel. Futhermore, two different procedures will be tested, for the first procedure, the depressure is achieved with a vacuum pump, for the second procedure it is done by closing an upstream valve. Typical Net Positive Suction Head (NPSH) curves and characteristic curves are obtained for numerous rotational rate and flow coefficient. The resulting cavitation inception and dynamics are different for each case study. The cavitation dynamics is observed by means of a high-speed camera.

The experiments were conducted in a closed water loop at Dynfluid's facility. The latter consists of a 1000 L fuel tank and a test section, where the three-bladed inducer is located in a 80 mm diameter circular duct. The test section is transparent in order to perform visualization with the high-speed camera at a sampling rate of 1 kHz. Additionally, the outer casing of the test section has a rectangular shape to minimize the effect of a light refraction. The high-speed camera is not fixed on the loop, so that any vibrations from the rotating machinery can be omitted. The water temperature is equal to 18 • C ± 1 • C and it is constantly monitored during the tests. The inducer rotating speed ranges from 0 rpm to 8000 rpm ± 10 rpm. One of the water tank is partially filled with liquid so that a free surface is created inside. A vacuum pump is connected to the free surface in order to decrease the pressure in the system and to simulate the aircraft climbing in altitude. Moreover, a specific device is placed in one of the tanks in order to inject a controlled quantity of CO 2 and to dissolve it in water. The valve needed for the ARP procedure is mounted at almost 26 D H upstream the test section. The fluid motion can be achieved by the inducer only or by a coupling with a centrifugal pump present in the loop. The flow rate ranges from 0 m 3 • h -1 to 40 m 3 • h -1 . In the present study, the centrifugal pump is used for fluid flows higher than 10 m 3 • h -1 . It has been verified that there is no impact on the inducer hydraulic performances when the centrifugal pump is activated. The fluid flow is controlled by multiple manual valves. The pressure measurements are taken on multiple locations: 20 D H in upstream the inducer and 11.25 D H downstream. There are also two average pressure tap locations, which are located 1 D H upstream the tip leading edge of the inducer, the one at the outlet is at 0.75 D H downstream.

The NPSH curves and ARP curves are plotted for various inducer operating points. The similarity of these curves is dependent on the properties of the working fluid. In fact, for the case with gassed water the procedure does not change the performance of the inducer at a given running condition. While for the jet fuel case, differences in the cavitation inception can be found .

Figure 1 :

 1 Figure 1: Schematic view of the Dynfluid test facility. The flow direction is indicated with the flesh. The injection of CO 2 takes place on the bottom of tank A. The circuit pressure is driven by the vacuum pump. The test section is fully transparent between tank A and tank B.

Figure 2 :

 2 Figure 2: Different views of the inducer U1

Effect of degassing phenomenon of the aviation fuel cavitation dynamics in a case of a three-bladed inducer

  AFM/SHF Conference on Hydraulic Machines and Cavitation November 6-7, 2019, Sion, Switzerland Cavitation and Multiphase flow Key words: cavitation dynamics, degassing, jet fuel, inducer, NPSH Corresponding author: theodore.magne@safrangroup.com

	Théodore MAGNE, Richard PARIDAENS, Florent Ravelet,
	Sofiane KHELLADI, Farid BAKIR
	DynFluid Laboratory
	ENSAM ParisTech
	151 Blvd de l'Hopital
	75013 Paris, FRANCE
	Petar TOMOV, Loïc PORA
	Fuel Equipment Design Office
	SAFRAN Aircraft Engines
	77550 Rond-Point René Ravaud, FRANCE
	Proposed session:


C Duroux

A Hagège

CE-ICP-MS to probe Aβ 1-42 / copper (II) interactions, a complementary tool to study amyloid aggregation in Alzheimer's Disease

Keywords: amyloid-, oligomerization, copper speciation, CE-ICP-MS hyphenation, labile complexes, voltage induced dissociation

CE-ICP-MS to probe Aβ1-42/copper (II) interactions, a complementary tool to study amyloid aggregation in Alzheimer's disease

Coraline Duroux, Agnès Hagège

Introduction

Alzheimer's disease (AD) is one of the most common forms of dementia in the world, characterized by progressive cognitive and memory impairments. However, the treatments are further hampered by the lack of understanding of the highly complex mechanisms at the origin of AD. While it now seems accepted that amyloid-β peptides (A), mainly Aβ1-40 and Aβ1-42, play a central role, their mode of action is not yet understood. Although the aggregation of the peptide appears to be a crucial step, many questions remain unsolved about the toxicity of oligomerized species of A. Toxicity, initially attributed to the presence of senile plaques, [START_REF] Hardy | Alzheimer's Disease: The Amyloid Cascade Hypothesis[END_REF] seems to be now caused by small soluble oligomers of these peptides. [START_REF] Cizas | Size-dependent neurotoxicity of β-amyloid oligomers[END_REF] In addition, the involvement of metals such as copper (Cu), zinc (Zn), and iron (Fe) seem to be accepted and is supported by various observations. As far as Cu is concerned, micro-PIXE analyses performed by Lovell et al. indicated a significant increase in Cu in amyloid plaques rims. [START_REF] Lovell | Copper, iron and zinc in Alzheimer's disease senile plaques[END_REF] Cu and Zn observed in brain by SXRF microprobe were also reported to be localized in high -sheet content regions. [START_REF] Miller | Synchrotron-based infrared and Xray imaging shows focalized accumulation of Cu and Zn co-localized with β-amyloid deposits in Alzheimer's disease[END_REF] However, even if a co-localization of Cu with these amyloid plaques was ascertained [START_REF] Lovell | Copper, iron and zinc in Alzheimer's disease senile plaques[END_REF][START_REF] Miller | Synchrotron-based infrared and Xray imaging shows focalized accumulation of Cu and Zn co-localized with β-amyloid deposits in Alzheimer's disease[END_REF] , some authors reported a non-significant increase of Cu amounts associated with Aβ plaques in brain regions of Alzheimer patients, compared to the basal Cu levels. [START_REF] James | Iron, Copper, and Zinc Concentration in Aβ Plaques in the APP/PS1 Mouse Model of Alzheimer's Disease Correlates with Metal Levels in the Surrounding Neuropil[END_REF] Conversely, ICP measurements of 7 brain regions from 9 cases with AD revealed a copper deficiency in all AD brain regions. [START_REF] Xu | Evidence for widespread, severe brain copper deficiency in Alzheimer's dementia[END_REF] Apparently contradictory findings were also reviewed about the influence of Cu(II) on aggregation. [START_REF] Rana | Cu and Zn interactions with Aβ peptides:consequence of coordination on aggregation and formation of neurotoxic soluble Aβ oligomers[END_REF] Both promoting [START_REF] Sarell | Substoichiometric Levels of Cu 2+ Ions Accelerate the Kinetics of Fiber Formation and Promote Cell Toxicity of Amyloid-β from Alzheimer Disease[END_REF][START_REF] Atwood | Dramatic Aggregation of Alzheimer Aβ by Cu(II) Is Induced by Conditions Representing Physiological Acidosis[END_REF] and inhibiting effects [START_REF] House | Copper Abolishes the β-Sheet Secondary Structure of Preformed Amyloid Fibrils of Amyloid-β42[END_REF][START_REF] Zou | Cu 2+ Inhibits the Aggregation of Amyloidβ-Peptide(1-42) in vitro[END_REF][START_REF] Pedersen | Cu(II) Mediates Kinetically Distinct, Non-amyloidogenic Aggregation of Amyloid-β Peptides[END_REF] of copper binding on aggregation were reported. Aβ1-42 aggregation is an intricate problem since many parameters, such as pH, temperature, and concentrations affect this phenomenon. However, Cu(II) appears to be one of the driving forces in the conformational changes of the Aβ1-42 in different oligomerized forms with various structures and sizes, such as pre-fibrillar oligomers, fibrillar oligomers and annular protofibrils. [START_REF] Viles | Metal ions and amyloid fiber formation in neurodegenerative diseases.Copper, Zinc and Iron in Alzheimer's, Parkinson's and Prion disease[END_REF] Pedersen et al. proposed the existence of copper-dependent aggregation pathways. [START_REF] Pedersen | Cu(II) Mediates Kinetically Distinct, Non-amyloidogenic Aggregation of Amyloid-β Peptides[END_REF] It was suggested that the presence of Cu (II) in high concentration leads to the formation of amorphous deposits prone to precipitation. However, under stoichiometric ratios, Cu(II) might accelerate -sheet formation, promoting the appearance of thioflavin-T reactive fibrils species. [START_REF] Smith | Concentration Dependent Cu 2+ Induced Aggregation and Dityrosine Formation of the Alzheimer's Disease Amyloid-β Peptide[END_REF] Little data have been published on the aggregation kinetics of Aβ1-42 upon addition of sub-equimolar amounts of Cu(II). In these conditions, Cu was found to lead to the formation of oligomers and even to the disassembly of fibres. [START_REF] Matheou | Cu 2+ accentuates distinct misfolding of Aβ (1-40) and Aβ (1-42) peptides, and potentiates membrane disruption[END_REF] Although many studies have been devoted to this problem, most of them were focused on amyloid-β peptide's conformation, structure and properties variations upon copper addition. Developments of speciation approaches allowing the study of the association of metals with the oligomers of these Aβ are scarce. No changes in Cu(II) coordination environment were detected in EPR when bound to A1-40 in various oligomeric states [START_REF] Sarell | Substoichiometric Levels of Cu 2+ Ions Accelerate the Kinetics of Fiber Formation and Promote Cell Toxicity of Amyloid-β from Alzheimer Disease[END_REF] which seems to preclude its use in speciation analysis. The coupling of ion mobility with mass spectrometry was used to demonstrate the stabilization of metal-induced compact forms of Aβ1-40 oligomers, from trimers to pentamers, in the presence of metal ions such as Cu 2+ and Zn 2+ . Although recorded mass spectra correspond to metal-free oligomers, the slow interconversion between compact species and extended species allowed to retain the metalbound drift time profile in their metal-free form. [START_REF] Sitkiewicz | Factors Influencing Compact-Extended Structure Equilibrium in Oligomers of Aβ1-40 Peptide-An Ion Mobility Mass Spectrometry Study[END_REF] The aim of the presented research was to propose an analytical approach to assess the distribution of Cu (II) among the different A1-42-species. Directly assessing metal-Aβ complexes requires an analytical method allowing a minimal disruption of the complexes. Capillary zone electrophoresis coupled to ICP-MS was successfully used for the analysis of several metal-protein complexes. [START_REF] Michalke | Manganese speciation in paired serum and CSF samples using SEC-DRC-ICP-MS and CE-ICP-DRC-MS[END_REF][START_REF] Aleksenko | Metallomics for drug development: an integrated CE-ICP-MS and ICP-MS approach reveals the speciation changes for an investigational ruthenium(III) drug bound to holo-transferrin in simulated cancer cytosol[END_REF][START_REF] Huynh | Investigation of uranium interactions with calcium phosphate-binding proteins using ICP/MS and CE-ICP/MS[END_REF][START_REF] Poléc | Investigation of metal complexes with metallothionein in rat tissues by hyphenated techniques[END_REF] However, owing to the small size of the peptides and oligomers and the lability of copper complexes, analysis of Cu(II) complexes of A1-42 remains a difficult challenge. Capillary zone electrophoresis was demonstrated to be able to separate A monomer and oligomers, [START_REF] Sabella | Capillary electrophoresis studies on the aggregation process of β-amyloid 1-42 and 1-40 peptides[END_REF][START_REF] Colombo | CE can identify small molecules that selectively target soluble oligomers of amyloid β protein and display antifibrillogenic activity[END_REF][START_REF] Picou | Analysis of Aβ (1-40) and Aβ (1-42) monomer and fibrils by capillary electrophoresis[END_REF][START_REF] Brinet | An improved capillary electrophoresis method for in vitro monitoring of the challenging early steps of Aβ1-42 peptide oligomerization: Application to anti-Alzheimer's drug discovery[END_REF][START_REF] Bisceglia | An integrated strategy to correlate aggregation state, structure and toxicity of Aβ 1-42 oligomers[END_REF] but to our knowledge, only one study looked at the fate of both amyloid-β peptides and Cu. However, only total Cu concentrations were measured by ICP-MS after centrifugation and no speciation data were provided. [START_REF] Pedersen | Cu(II) Mediates Kinetically Distinct, Non-amyloidogenic Aggregation of Amyloid-β Peptides[END_REF] Here the use of coupling of capillary electrophoresis to ICP-MS has been proposed to study the influence of Cu(II) on A1-42 aggregation and evaluate the Cu content of oligomers during the aggregation process.

Materials and methods

Products

Aβ1-42 and Aβ1-16 (purity ≥ 95%) were purchased as lyophilized powder from GeneCust (Boynes, France) and used without further purification. Tris (Trizma® base), sodium chloride, hydrochloric acid 37%, tributyl phosphate (TBP) 99% and EDTA were obtained from Sigma-Aldrich. Nitric acid 1% was obtained by dilution in water of HNO3 65% (Suprapur® quality) purchased from Merck. Stock solution of Cu(II) at 500 µM in 10 mM Tris, 15 mM NaCl at pH 7.4 was obtained via dissolution of CuCl2.2H2O purchased from Merck. Acetone (0.1% in water) and tributylphosphate (TBP 0.03% in DMSO 2%) purchased from Sigma-Aldrich were used as neutral species markers for CE-UV and CE-ICP-MS respectively. Since Cu is one of the most difficult element to analyze at trace levels, extreme caution was taken to avoid external contamination (see SI). All products were analysed regarding to their Cu content by ICP-MS and compared with a reference value obtained for copper at 5 ppb. Products with contamination greater than the one measured for HNO3 1% were discarded (a few tens of ppt). Before use, labware was cleaned three times with nitric acid 1%. These precautions drastically improve the background level which can however not fall below 600 cps. Fused-silica capillaries were purchased from Polymicro Technologies (Phoenix, AZ). A hydroxypropyl cellulose (HPC) thermal coating of the capillaries (I.D of 75 µm, O.D of 375 µm, 64 cm total length) [START_REF] Shen | High-Efficiency Capillary Isoelectric Focusing of Peptides[END_REF] was performed using a HPC solution, 0.05 g/ mL in water in order to limit the interactions between the silanols of the capillary surface and the copper ions.

Peptide samples preparation

Stock solutions of Aβ1-42 at 250 µM were prepared by direct dissolution of lyophilized Aβ1-42 in 0.1% (m/v) ammonium hydroxide aqueous solution. Stock solutions of Aβ1-16 at 500 µM were prepared by direct dissolution of lyophilized Aβ1-16 in 10 mM Tris, 15 mM NaCl at pH 7.4. Aβ peptides concentration was determined by UV measurements (Thermo Scientific™ NanoDrop™ UV-Vis Spectrophotometer) at 280 nm (ε = 1490 M -1 cm -1 for both peptides, from UniProtKB/Swiss-Prot Database). These solutions were checked to be stable over one week, when stored at 4°C. Before each study, Aβ peptides were diluted in 10 mM Tris, 15 mM NaCl at pH 7.4 and varying volumes of the stock solution of Cu(II) were added in order to obtain a final peptide concentration of 100 μM in 8 mM Tris, 10 mM NaCl at pH 7.4, in the presence of different concentrations of Cu(II).

Taylor dispersion analysis (TDA) for Aβ1-42 size measurements TDA experiments were conducted using a Beckman P/ACE MDQ instrument in HPC capillaries. After injection (0.5 psi, 5 s, i.e. 23.5 nL), samples were mobilized with an applied pressure of 0.7 psi. The mobilization medium used was a Tris 10 mM, NaCl 15 mM buffer at pH 7.4 and detection was performed by UV at 200 nm with a data acquisition rate of 16 Hz (detection length: 54 cm). Between runs, the capillary was flushed at 10 psi for 5 min with the mobilization medium. The taylorgrams obtained by TDA can be assimilated to the sum of several Gaussians signals:

𝑆(𝑡) = ∑ √ exp - ( ) (1)
where t0 is the peak residence time and Ai and σi are the area under the curve and temporal variance associated to the different species I, respectively. Under these experimental conditions, the molecular diffusion coefficient D is given by equation 2 and can be related to the hydrodynamic radius of the species.

𝐷 = = (2)
where rc is the capillary radius, kB is the Boltzmann constant, T is the temperature, η is the viscosity, and Rh is the hydrodynamic radius of the solute. For a Tris 10 mM, NaCl 15 mM buffer, the viscosity was previously measured and found to be 0.908. Deconvolution and integration of the peak was performed using the Origin 8.5 software, assuming ε200nm to be equal for all Aβ1-42 species.

Aggregation monitoring using CE-UV CE experiments were carried out on a Beckman P/ACE MDQ instrument, using HPC capillaries. The background electrolyte (BGE) was 10 mM Tris, 15 mM NaCl at pH 7.4. Prior to the first use, the capillary was flushed with water and BGE for 5 min under 10 psi. Samples were introduced into the capillary by hydrodynamic injection under 0.5 psi for 5 sec (i.e. 23.5 nl). Separations were performed at 25°C at -20 kV with an assisted pressure of 0.1 psi. Since amyloid- compounds have relatively low electrophoretic mobility, this additional pressure was necessary to compensate the absence of electroosmotic mobility in a neutral capillary such as HPC. The assisted pressure leads to a reduced residence time in the capillary but also to the formation of a Poiseuille flow affecting the peak width, and consequently the resolution. Detection was performed by UV at 200 nm at 54 cm from the inlet with an acquisition rate of 16 Hz. Data were collected using the 32 Karat Software. Between runs, the capillary was rinsed with the BGE for 5 min under 10 psi. Calculation of Aβ1-42 species content was performed using the classical correction of peak areas in CE (measured area divided by migration time), assuming ε200nm to be equal for all Aβ1-42 species.

Determination of copper distribution using CE-ICP-MS

For CE-ICP-MS hyphenation, the ICP-MS instrument used as detector was an Agilent 7700 equipped with a MicroMist nebulizer. The spectrometer was controlled by the MassHunter software. Hyphenation was achieved via a home-made sheath-flow interface. The capillary goes first through a microcross piece toward the tip of the nebulizer. The electrical connection was achieved by the ground electrode positioned through the third inlet of the microcross. The sheath liquid (Tris 5 mM, pH 7.4 containing 10 µM EDTA to warrant the absence of copper sorption on ICP-MS glass parts) was introduced by self-aspiration to the fourth inlet of the interface. The sheath liquid solution height was adjusted by means of an elevator to both eliminate the suction effect due to the nebulizer and avoid any flow reversal into the capillary following the procedure described elsewhere. [START_REF] Chamoun | Sensitivity enhancement in capillary electrophoresis-inductively coupled plasmamass spectrometry for metal/protein interactions analysis by using large volume stacking with polarity switching[END_REF] The optimal flow was estimated to be around 450 µL.min -1 by measuring the sheath flow consumption over a 30 min period. This interface was checked not to introduce significant external dispersion (see supplementary information). Samples were introduced into the HPC capillary by hydrodynamic injection under 1 psi for 10 sec (i.e. around 90 nL). Electrophoretic separations were performed at 25°C at -7 kV assisted by a pressure of 0.3 psi. Cu was detected by ICP-MS at m/z = 65 (dwell time 1 s) at the end of the capillary (64 cm). ICP operating conditions were: carrier gas flow rate: 1 L.min -1 , makeup gas flow rate: 0.1 L.min -1 , plasma gas flow rate: 15 L.min -1 , auxiliary gas flow rate: 0.9 L.min -1 , radiofrequency power 1550 W for the plasma, other parameters were tuned in order to maximize the signal. Between runs, the capillary was rinsed with nitric acid 1% for 10 min under 5 psi and with the BGE for 5 min under 5 psi. Integration of the Cu(II) containing peaks was performed using Origin 8.5 by minimizing an exponentially modified Gauss function, as proposed by Sauge-Merle et al. [START_REF] Sauge-Merle | Revisiting binding of plutonium to transferrin by CE-ICP-MS[END_REF] , when using low resolution conditions (i.e. 5kV + 0.5 psi in their case).

Results and discussion

Identification of Aβ1-42 species

In order to follow Aβ1-42 aggregation, attempts to identify the different of Aβ1-42 species within electrophoregrams were performed. For that purpose, two different solutions containing 100 µM Aβ1-42 were prepared: one solution prepared in self-aggregating conditions, i.e. incubated for 48h at room temperature in the electrolyte at pH 7.4, another one in conditions limiting aggregation, i.e. freshly prepared in NH4OH 0.1%. Analyses of both samples were realized in capillary electrophoresis at -20 kV + 0.1 psi in a HPC-modified capillary using Tris 10 mM, NaCl 15 mM, pH 7.4 as background electrolyte. The resulting electropherograms are presented in Figure 1.

The electrophoretic mobilities (µep) were determined by subtracting the Poiseuille flow contribution, using acetone as a neutral marker (NM) and thanks to the equation 3:

µ = * ( - ) (3)
where Ld is the length of detection, Lt is the total length of the capillary, V is the applied voltage, tm is the migration time of the analyte peak and tNM is the migration time of the neutral marker peak.

Apart from the first peak attributed to the presence of NH4OH by direct injection of a 0.05% NH4OH, both mobilograms exhibit two main peaks (1 and 2), with electrophoretic mobilities of -2x10 -8 m²V -1 s -1 and -1x10 -8 m²V - 1 s -1 respectively (Fig. 1a). Peak areas were measured for both peaks and the respective percentages of both species were calculated from the corrected peak areas (peak area/migration time). Results are presented in Table 1.

In parallel, TDA experiments were performed using the same capillary and the same electrolyte as mobilization buffer. The taylorgrams were shown to be successfully fitted by a trimodal fitting, indicating the presence of three different species in the two samples (Fig. 1b). The sharpest deconvoluted peak was attributed to NH4OH since the diffusion coefficient experimentally determined ((1.3 ± 0.3) 10 -9 m 2 .s -1 ) was similar to the one tabulated for NH4OH at 20°C (1.5.10 -9 m 2 .s -1 ). Both hydrodynamic radii and percentages of the other two species were calculated and compared in Table 1.

Based on these results, the presence of two different populations containing Aβ1-42 was assumed: one fast migrating population (µep ~ -2 x10 -8 m 2 .s -1 .V -1 ) with a large hydrodynamic radius, attributed to large oligomers, and one population migrating slower (µep ~ -1 x10 -8 m 2 .s -1 .V -1 ) with a smaller hydrodynamic radius, attributed to monomers and small oligomers. This assumption is reinforced by the data obtained by Deleanu et al. [START_REF] Deleanu | Unraveling the Speciation of β-Amyloid Peptides during the Aggregation Process by Taylor Dispersion Analysis[END_REF] , who proposed possible conformations of Aβ1-42 in relation with their size determined by TDA. They found a population of monomers and small oligomers, with a mean hydrodynamic radius around 1.9 nm, and a population of larger oligomers, with a mean radius around 5.1 nm. Moreover, several groups reported the µep of Aβ1-42 oligomers and monomer at pH 7.4 [START_REF] Sabella | Capillary electrophoresis studies on the aggregation process of β-amyloid 1-42 and 1-40 peptides[END_REF][START_REF] Colombo | CE can identify small molecules that selectively target soluble oligomers of amyloid β protein and display antifibrillogenic activity[END_REF][START_REF] Picou | Analysis of Aβ (1-40) and Aβ (1-42) monomer and fibrils by capillary electrophoresis[END_REF] and showed that oligomers present a mobility which is twice the one of monomers.

Separation conditions preserving copper complexes

The separation of Aβ1-42 species is well-described in literature [START_REF] Sabella | Capillary electrophoresis studies on the aggregation process of β-amyloid 1-42 and 1-40 peptides[END_REF][START_REF] Colombo | CE can identify small molecules that selectively target soluble oligomers of amyloid β protein and display antifibrillogenic activity[END_REF][START_REF] Picou | Analysis of Aβ (1-40) and Aβ (1-42) monomer and fibrils by capillary electrophoresis[END_REF][START_REF] Brinet | An improved capillary electrophoresis method for in vitro monitoring of the challenging early steps of Aβ1-42 peptide oligomerization: Application to anti-Alzheimer's drug discovery[END_REF][START_REF] Bisceglia | An integrated strategy to correlate aggregation state, structure and toxicity of Aβ 1-42 oligomers[END_REF] and was performed at -20kV + 0.1 psi on a neutral HPC capillary without further improvement.The repeatability of mobilities was determined by performing three CE separations of a partially aggregated peptide Aβ1-42. Results indicated a relative standard deviation not exceeding 5% for mobilities allowing the use of this method for the monitoring of the different species over time. However, the separation of the Cu(II)-complexes monitored by CE-ICP-MS in such analytical conditions is severely marred by the observation of strongly tailing peaks, due to the partial dissociation of the complexes during separation. Optimization of separation conditions was carried out, to face the disequilibrium effects linked to the analysis of copper complexes. Indeed, kinetically fast metals like Cu(II) may form labile complexes, which are likely to dissociate during the separation. [START_REF] Sonke | Disequilibrium effects in metal speciation by capillary electrophoresis inductively coupled plasma mass spectrometry (CE-ICP-MS); theory, simulations and experiments[END_REF] As this dissociation may be increased by the application of voltage, experiments were carried out with different voltages. For that purpose, Aβ1-16 was chosen as reference compound since it contains the same complexation site than Aβ1-42 and is not subject to aggregation. Figure 2 presents the results obtained for the CE-ICP-MS analysis of solutions containing 100 µM Aβ1-16 and either 10 µM or 50 µM Cu(II). The electropherograms show distorted peaks, composed of a nearly Gaussian part corresponding to the Aβ1-16-Cu(II) complex, and a tailing part, corresponding to the copper dissociated from this complex (Fig. 2a). For the different voltages, Gaussian peak, tailing part and Cu(II) peak released by HNO3 rinses areas were calculated (Fig. 2b). These results show that an increase of voltage leads to an increase of the tailing part of the peak (Fig. 2c and2d). Moreover, the washing step using HNO3 1% reveals a small but increasing amount of Cu adsorbed on the HPC capillary. Applying a high voltage such as -20kV leads to the dissociation of 2/3 of Cu(II) initially complexed, for both Cu(II) concentrations. By decreasing the voltage to -7 kV, more than 80% of the complex remains intact. However, these conditions dramatically affect the resolution and any additional decrease of the voltage would prohibit the separation of the Aβ1-42 species.

As a consequence, -20kV + 0.1 psi was maintained in association with the UV detection to separate Aβ1-42 species with a good resolution and -7kV + 0.3 psi was applied when using the ICP-MS detection to perform the separation of Cu(II) complexes while limiting the dissociation. As Aβ1-42 undergoes self-aggregation over the time, repetability on peak areas, linearity and limit of quantification cannot be evaluated using such a sample. Consequently, Aβ1-16 was used instead of Aβ1-42, to overcome variations due to speciation changes during aggregation (see supplementary information). Although all the analytical figures of merit (Table 3) cannot be directly transposed to the analysis of Aβ1-42, they may be representative of the method.

Does copper accelerate the aggregation of the Aβ1-42 peptide?

This CE-UV method was used in order to study the influence of the presence of Cu on the aggregation kinetics of the Aβ1-42 peptide. For that purpose, kinetic monitoring was carried out, for several Aβ1-42 : copper initial ratios (1: 0, 1: 0.1 and 1: 0.5). Incubations of 100 µM of Aβ1-42 in the presence of different Cu(II) concentrations (10 and 50 µM) were performed at room temperature. The solutions were analysed at different incubation times and compared with the results obtained for a 100 µM of Aβ1-42 solution in the absence of Cu. All the results are averages from two different kinetic experiments for each Aβ1-42 : Cu(II) ratio and are presented in Fig. 3. All the electropherograms (Fig. 3a) exhibit two main peaks, with electrophoretic mobilities -2x10 -8 m²V -1 s - 1 and -1x10 -8 m²V -1 s -1 . Previous affinity capillary electrophoresis studies performed in the presence of Cu(II) in the electrolyte have shown that A and A-Cu complexes exhibit only slight differences in electrophoretic mobilities (see supplementary information). Peaks 1 and 2 were therefore attributed to large oligomers and small oligomers/monomer respectively. Peak 1, observed in many studies in the absence of copper, was not observed by Pedersen et al. in the presence of this metal with A1-40. [START_REF] Pedersen | Cu(II) Mediates Kinetically Distinct, Non-amyloidogenic Aggregation of Amyloid-β Peptides[END_REF] Indeed, it was postulated that A1-40 displays a different aggregation pathway, which does not lead to the formation of transient soluble oligomers. [START_REF] Deleanu | Unraveling the Speciation of β-Amyloid Peptides during the Aggregation Process by Taylor Dispersion Analysis[END_REF] Fig. 3b shows the percentage of Aβ1-42 involved in large oligomers in the soluble fraction. This was determined by calculating the ratio of large oligomeric forms area to the total area in UV, using corrected areas to correct the influence of the different velocities on the signal measurement and assuming ε A in monomer to be equal to ε A in oligomer species at 200 nm. The distribution of Aβ1-42 between the different soluble species was calculated as follows: %Aβ1-42, species I = ∑ (4)

Such calculations allow to compensate for the absence of calibration curve (impossible to obtain for Aβ1-42). Surprisingly, no immediate precipitation of A1-42 was observed whatever the copper concentration in the sample. This was verified by measuring both UV absorbance at 280 nm and copper concentration by ICP-MS at the beginning and the end of the experiment. Moreover,  corrected peak area was found to be constant over the time with a relative standard deviation of 10%. In our case, percentages can be directly converted into A1-42 concentrations.

Data obtained at t=0 for the copper-free sample illustrate the strong ability of Aβ1-42 peptide to instantaneously self-aggregate upon dissolution in the buffer despite the presence of NH4OH. This could be due to the high concentration of A1-42 (100 µM) used in this study, similar to concentrations usually used by others in in vitro kinetics studies. Aβ1-42 involved in large oligomerized forms was found to be about 20%. By comparing the initial repartition for each Cu(II): Aβ1-42 ratio, it could be noticed that the initial percentage of oligomerization increases in the presence of Cu(II) in the sample, to reach up to 35% for both initial ratios. This instantaneous aggregation observed in the presence of Cu(II) was already observed in studies on A1-40. [START_REF] Atwood | Dramatic Aggregation of Alzheimer Aβ by Cu(II) Is Induced by Conditions Representing Physiological Acidosis[END_REF][START_REF] Pedersen | Cu(II) Mediates Kinetically Distinct, Non-amyloidogenic Aggregation of Amyloid-β Peptides[END_REF] The examination of the oligomerization kinetics curves (Fig. 3b) shows a similar profile with a propensity of the peptide to aggregate either with or without Cu(II). Whatever the Cu(II) concentration is, the percentage of peptide involved in oligomeric forms increases almost linearly with the incubation time to reach a plateau. Therefore, the slope of the linear part reflects the apparent oligomerization velocity (in %.h -1 ). Results are summarized in Table 2. The apparent velocities either in the absence of copper or for 10 μM Cu(II) were similar, which does not reflect any significant influence of Cu for such a sub-stoichiometric ratio. However, in the presence of 50 µM Cu (II) in the sample, velocity was found ca. 10 times higher, indicating an increase in the kinetic of aggregation. The percentage at the plateau does not seem to depend on Cu(II) concentration and discrepancies observed with and without Cu seem to be due to the differences observed à t=0.

How is copper distributed among the different oligomeric forms?

Monitoring of the distribution of Cu among the different forms of the peptide was performed by CE-ICP-MS analyses of 100 µM Aβ1-42 samples containing either 10 or 50 µM Cu(II). Experiments were performed in duplicates and results are presented in Fig. 4. For both samples (Fig. 4a andb), electropherograms show the presence of two negative copper complexes (peaks 1 and 2). The negative mobilities, calculated using TBP as a neutral marker (m/z = 31), are in agreement with the formation of negatively charged Aβ-Cu complexes. As a consequence of the combination of the application of low voltage and high pressure, the resolution was strongly affected. This peak overlapping hinders any accurate quantitative measurements but the separation remained sufficient to be able to distinguish both monomeric and oligomeric states of Aβ1-42. Moreover, a significant peak broadening was observed for the so called-monomeric species, compared to the results obtained in UV (Fig. 3a), and might suggest the co-migration of both the monomer and the smallest oligomers, already observed in UV. In the presence of 50 µM of Cu(II) (Fig. 4b), an additional unknown peak was revealed, which might result from the presence of ammonium hydroxide in the solution, since a copper species with similar mobility was also observed in blank samples.

Calculation of copper percentages were performed as previously for the different peaks in the soluble fraction: %Cu, species I = ∑

The absence of copper precipitation was checked by copper ICP-MS analyses at the beginning and at the end of each experiment. Figure 4 shows a decrease of the "monomer and small oligomers" fraction for both stoichiometries as the aggregation progresses (Fig. 4c and4d). In the 1: 0.1 A: Cu sample, an increase over time of the percentage of Cu present in the larger oligomers fraction was observed, until nearly all the soluble copper was incorporated in this fraction (Fig. 4c). This increase is closely correlated to the increase of the A1-42 peptide involved in the larger oligomers forms (Fig. 3b). Such a trend might indicate a similar complexation of Cu in the different forms. Mold et al. assumed that it could reveal a kinetically favoured aggregation, to the detriment of complexation. As a result, this could explain the similarity of the conversion of monomer into larger oligomers in the absence and in the presence of 10 µM Cu. [START_REF] Mold | Copper prevents amyloid-β1-42 from forming amyloid fibrils under near-physiological conditions in vitro[END_REF] Increasing Cu(II) concentration to 50 µM led to a quite different behaviour. Cu involved in the larger oligomers fraction slightly increased, until a plateau representing 40-45% of the soluble copper (Fig. 4d). Comparison of this distribution with the one of A1-42 peptide in the soluble forms (Fig. 3b) suggests clearly that Cu is mainly present in the "monomer and small oligomers" fraction. Based on the researches so far, the aggregation process could be enhanced via the formation of an intermolecular copper complex.

Conclusion

CE-ICP-MS was used to monitor for the first time the distribution of Cu between soluble species of Aβ1-42 and confirmed that Cu exhibits an affinity for both monomeric and oligomeric Aβ1-42 species and lead to the formation of anionic species. The investigation of Aβ1-42 oligomerization over time showed that Cu influences the rate of oligomerization and tends to distribute in both small and large oligomers. A clear difference in copper distribution was observed depending on the Cu(II) concentration used, which seems to be in agreement with the formation of intramolecular at low Cu(II) concentrations and intermolecular complexes for higher concentrations. Although the A1-42 concentrations used may not be representative of in vivo conditions and could favour selfaggregation, we showed that this method can provide valuable information in A / copper complexation studies.

Moreover, such a study was impaired by the poor resolution of the separation, resulting from mild separation conditions optimized to avoid complexes dissociation. Since, a total separation of the species was not possible, an accurate quantitation of copper in the different species is precluded. Non-separative methods, such as Taylor dispersion analysis, allowing the discrimination of species according to their diffusion coefficient, could also be envisaged. The ability of TDA to be coupled with ICP-MS should provide useful information concerning the size of the copper species formed during the in vitro aggregation process. It is also of importance to be able to separate monomer from small oligomers and further investigations are still ongoing to achieve this goal. Samples: 100 µM Aβ1-42 at t=0 after dilution in 0.1% NH4OH (A,B) and at t=48h after dilution in 10 mM Tris buffer containing 0.04% NH4OH at pH 7.4 (c,d). CE experimental conditions: HPC coated -capillary (75 µm i.d. x 64 cm), buffer: 10 mM Tris, 15 mM NaCl at pH 7.4, -20 kV with an assisted pressure of 0.1 psi, 25°C. Detection: UV at 200 nm at 54 cm from the inlet. TDA experimental conditions: HPC coated -capillary (75 µm i.d. x 64 cm), buffer: 10 mM Tris, 15 mM NaCl at pH 7.4, mobilisation pressure: 0.7 psi, 25°C. Detection: UV at 200 nm at 54 cm from the inlet.

Figures

  Figures

Figure 1 :

 1 Figure 1: Electrophoretic profiles of obtained by CE-UV (a,c) and taylorgrams obtained by TDA-UV (b,d).Samples: 100 µM Aβ1-42 at t=0 after dilution in 0.1% NH4OH (A,B) and at t=48h after dilution in 10 mM Tris buffer containing 0.04% NH4OH at pH 7.4 (c,d). CE experimental conditions: HPC coated -capillary (75 µm i.d. x 64 cm), buffer: 10 mM Tris, 15 mM NaCl at pH 7.4, -20 kV with an assisted pressure of 0.1 psi, 25°C. Detection: UV at 200 nm at 54 cm from the inlet. TDA experimental conditions: HPC coated -capillary (75 µm i.d. x 64 cm), buffer: 10 mM Tris, 15 mM NaCl at pH 7.4, mobilisation pressure: 0.7 psi, 25°C. Detection: UV at 200 nm at 54 cm from the inlet.

Figure 2 :

 2 Figure 2: Influence of the voltage applied in CE on the Aβ1-16 -Cu(II) dissociation Electropherograms obtained in CE-ICP-MS for a 100 µM Aβ1-16 and 10 µM Cu(II) sample (a) and a 100 µM Aβ1-16 and 50 µM Cu(II) sample (c) at -20kV (in purple), -15kV(green), -10kV (red) and -7kV (blue). Electropherograms are shifted in cps for sake of clarity. Areas attributed to the gaussian peak (white bars), the tailing part (blue bars) and the Cu(II) released by the HNO3 rinse at the end of the separation (dotted bars) for a 100 µM Aβ1-16 and 10 µM Cu(II) sample (b) and a 100 µM Aβ1-16 and 50 µM Cu(II) sample (d).

Figure 3 :

 3 Figure 3: Monitoring in CE-UV of 100 µM of Aβ1-42 aggregation over time in 10 mM Tris 15 mM NaCl (pH 7.4). (a) Electropherograms for different incubation times in the absence or the presence of either 10 or 50 µM of Cu(II). (b) Percentage of Aβ1-42 involved in large oligomeric forms in the soluble fraction over incubation time in the absence () or of 10 (), and 50 µM () of Cu(II). Electropherograms are shifted in absorbance for sake of clarity.

Figure 4 :

 4 Figure 4: Repartition of copper between the different oligomerization forms of 100 µM Aβ1-42 over time followed by CE-ICP-MS in the presence of 10 µM (a, c) or 50 µM (b, d) of Cu(II). (a, b) Electropherograms (shifted in 65 Cu signal intensity for sake of clarity, background signal is around 800 cps for all the electropherograms) Peak 1 represents copper present in the large oligomeric forms, peak 2 represents both small oligomers and monomer Aβ / Cu(II) complexes, and peak 3 is likely to be the Cu(II) released from the monomer. (c, d) Percentages of soluble copper in small () and large () oligomeric forms of Aβ1-42 peptide over time in the presence of 10 or 50 µM of Cu(II). Experimental conditions: HPC coated -capillary (75 µm i.d. x 64 cm), Buffer: 10 mM Tris, 15 mM NaCl at pH 7.4, -7 kV with an assisted pressure of 0.3 psi, 25°C. Detection: ICP-MS at m/z = 65 at the end of the capillary.

Figure 4

 4 Figure 4

Table 1 :

 1 Electrophoretic mobilities and hydrodynamic radii of the different species present in A1-42 samples

		A1-42 100 µM in NH4OH 0.1%		A1-42 100 µM in Tris, pH 7.4
	CE experiments	µep,1 (x 10 8 m 2 .V -1 .s -1 ) -0.98 ± 0.02	%species 1 74 ± 4	µep,2 (x 10 8 m 2 .V -1 .s -1 ) -2.10 ± 0.05	%species 2 26 ± 2	µep,1 (x 10 8 m 2 .V -1 .s -1 ) -0.94 ± 0.03	%species 1 53 ± 4	µep,2 (x 10 8 m 2 .V -1 .s -1 ) -2.13 ± 0.07	%species 2 47 ± 4
	TDA	RH,1 (nm)	%species,1 RH,2 (nm)	%species 2 RH,1 (nm)	%species,1 RH,2 (nm)	%species 2
	experiments	1.3	72 ± 7	6.6 ± 0.9	28 ± 3	1.3 ± 0.2	54 ± 6	4.2 ± 0.7	46 ± 6

Table 2 :

 2 Influence of amyloid-: Cu(II) stoichiometry on the apparent aggregation velocity

	Amyloid-: Cu(II) initial ratio	vmax, app (%.s -1 )	% monomers involved in oligomeric
			forms at the plateau
	1: 0	1.9x10 -6	65
	1: 0.1	2.0x10 -6	77
	1: 0.5	1.4x10 -5	74

Table 3 :

 3 Repetability, linearity and limits of detection determined in both CE-UV and CE-ICP-MS using A1-16 and

	A1-42
		CE-UV	CE-ICP-MS
	Migration times RSD a	3%
	Peak area RSD b	< 5 %	< 15 %
	Linearity b	r 2 = 0.9997 (range : 5 -100µM)	r 2 = 0.991 (range : 1.25 -25µM)
	Limit of quantification b	6.5 µM Aβ1-16	0.6 µM Cu
	a measured using A1-42
	b measured using A1-16

Acknowledgements

This work was supported by the Doctoral School of Chemistry, University of Lyon, France (grant to CD). The authors have declared no conflict of interest.