Morfin I Combet

Chain conformation : a key parameter driving clustering or dispersion in polyelectrolytecolloid systems

Keywords: Small angle neutron scattering, Pluronic, hyaluronic acid, chain conformation, depletion, ionic strength

The work presents the characterization of Pluronic F127 micellar solutions in presence of hyaluronic acid in semi-dilute regime. The effects of the nature and salt concentration are investigated by differential scanning calorimetry and small angle neutron scattering. Hyaluronic acid reduces the critical micellar temperature to the same extend as an increase of the ionic strength. Within the investigated HA concentration range, the size and shape of the micelles are not modified by the addition of HA but their dispersion state depends on the salt concentration. By increasing the ionic strength we observe the formation of small micellar clusters which organize into a face-centered cubic liquid crystalline phase at high salt concentration. This behavior is reinforced by increasing the HA concentration. The nature of the salt play also a role and divalent cations such as Ca 2+ promote the clustering of micelles and their crystallization. The origin of the aggregative behavior is the change of the HA chain conformation -from stretched to coil-by addition of salt which in turn induces an excluded volume around the micelles and exerts a depletion interaction.

Introduction

In the medical field and especially for cancer treatment, nanocarrier systems with specific targeting are promising tools to reduce the dose and the side effects, to avoid burst release and prolong the circulation into the blood stream. In addition, they must be easily injectable, biocompatible and biodegradable. Among all the potential carriers, Pluronics copolymers are widely investigated. They are composed by two hydrophilic poly(ethylene oxide) (PEO) groups and a central hydrophobic poly(propylene oxide) (PPO) group. Their self-assembling properties (size and shape of the micelles, critical micellar concentration and temperature) can be varied by changing the EO/PO ratio and group lengths. F127 (EO 100 PO 70 EO 100 ) used in this work is involved in a considerable number of patents. The high capability of F127 micelles to load hydrophobic molecules, and more specifically drugs is widely recognized [START_REF] Kabanova | Pluronic block copolymers for overcoming drug resistance in cancer[END_REF].

Hyaluronic acid (HA) is a natural linear semirigid polyelectrolyte with a repeating negatively charged disaccharide unit composed of N-acetyl-glucosamine and glucoronic acid. HA is found in large quantities in animal and human body. It is a common component of synovial fluid and extracellular matrix. However it plays ambiguous roles. Whereas high molecular weight HA (> 500 kDa) is antiangiogenic, anti-inflammatory and immunosuppressive, low molecular weight (10 -500 kDa) is highly angiogenic and pro-inflammatory. HA is currently investigated as a target-specific material since many malignant cancer cells overexpress HA receptors and its incorporation in nanocarrier systems can be used as targeting ligand [START_REF] Rankin | Frankel Soft Matter[END_REF].

Considering the relevant properties of each of these macromolecules, several studies report on the potential applications of the mixed systems for tissue engineering and drug delivery. The presence of HA improves the mechanical and mucoadhesive properties and permits a sustained release of drugs [START_REF] Chen | [END_REF][START_REF] Mayol | [END_REF][START_REF] Mayol | [END_REF][START_REF] Jung | [END_REF][START_REF] Seol | [END_REF][8]. These studies are carried out mostly in the range of concentration where the Pluronic micelles form a gel i.e. at concentrations above 15 vol%, in the hexagonal phase. A detailed structural characterization of these complex systems is often lacking but dynamic light scattering characterization shows the formation of structures of few hundreds of nanometers [START_REF] Jung | [END_REF][START_REF] Gurav | [END_REF], i.e. more than ten times the micellar size. Before any therapeutic application, the knowledge of the structure appears mandatory, especially if aggregation occurs and might be an issue for injection and circulation into the blood vessels.

From a fundamental point of view, the phase behavior of small colloids and large polymers has attracted much interest in the past years due to its importance in many domains (from nanocomposites to care products) and more specifically for applications in protein crystallization giving to such polymer-colloid systems the name of "protein limit". The counterpart -large colloids and short polymers-is called "colloidal limit". In the latter case, the natural parameter to introduce is the Q parameter defined as R G /R, the ratio between the radius of gyration of the polymer and the radius of the colloid [START_REF] Asakura | [END_REF][START_REF] Asakura | Interaction between particles in solutions of macromolecules[END_REF]. The colloidal limit is simpler and can be understood by the fact that the polymers are excluded from the space between two adjacent colloids, and exert an unbalanced osmotic pressure which induces an attraction between the particles. In other words, the depletion of the polymers around particles causes entropy loss of polymer when the particles are homogeneously dispersed. The aggregation of the particles increases the accessible volume for the polymers and leads to an entropy gain. In this case, depletion interaction are pairwise additive. The radius of gyration fixes the range of depletion and the polymer concentration sets the strength. The model first proposed by Asakura-Oosawa [START_REF] Asakura | [END_REF][START_REF] Asakura | Interaction between particles in solutions of macromolecules[END_REF] and further developed by Vrij [START_REF] Vrij | [END_REF] gives accurate results for polymer in q solvent and Q < 1. In the protein limit and in a semi-dilute polymer solution, the correlations between the segments of the polymers induce correlations between the positions of the particles which behave as if they were attracted to each other [START_REF] De Gennes | Scaling concepts in polymer physics[END_REF]. The theoretical treatment and the phase diagram forecast are more difficult since many-body colloidal interactions are involved and there is in addition the necessity to take into account the polymer-polymer interactions and the chain conformation. The first scaling theories were developed the by Sear et al. [START_REF] Sear | [END_REF][15][START_REF] Sear | Entropy-driven phase separation in mixtures of small colloidal particles and semidilute polymers[END_REF]. The main hypotheses are the following: 1-the polymer is considered to be in good solvent conditions and does not adsorb on the particles; 2-all the interactions considered are excluded volume interactions, i.e two polymer segments or two particles or a polymer segment and a particle cannot occupy the same volume. The demixing occurs if the entropy of mixing decreases due to a loss of entropy of translation for the particles or a loss of entropy of conformation for the polymer. For a dilute polymer solution, the chains do not overlap and the system is miscible. When the polymer concentration reaches c* (semi-dilute regime), the chains start to overlap and form a network with a characteristic mesh size or correlation length x [START_REF] De Gennes | Scaling concepts in polymer physics[END_REF] in which the particles can enter. In consequence, in the protein limit and for polymer in semidilute regime, the particle-polymer interaction depends on the parameter Q defined here as the ratio D/ x (where D is the particle diameter) and scales with the polymer volume fraction. As the concentration of polymer increases, x decreases and the space available to the particle decreases until 𝜉~𝐷. In that limit, the free energy to insert a particle and push away the polymer becomes large and drives the phase separation [START_REF] Sear | Entropy-driven phase separation in mixtures of small colloidal particles and semidilute polymers[END_REF]. For 𝐷 < 𝜉 and low particle concentration, the position of the particles in the polymer solution are correlated over the polymer correlation length as if they were subject to an additional attractive potential. When the particle concentration increases, the correlation between the polymer segments are in turn modified by the particle inducing a drop of the correlation length [START_REF] Lim | Polymer crowding and shape distributions in polymer-nanoparticle mixtures[END_REF][START_REF] Kramer | [END_REF][START_REF] Le Coeur | Compression of random coils due to macromolecular crowding[END_REF]. Further theoretical developments were done within the PRISM (Polymer Reference Interaction Site Model) theory [START_REF] Fuchs | Structure and thermodynamics of colloid-polymer mixtures: A macromolecular approach[END_REF][START_REF] Fuchs | Structure of colloidpolymer suspensions[END_REF], which allowed direct determination of thermodynamic and structural properties of the colloid / polymer mixtures. More recently, full-monomer simulations were able to describe the phase boundary for Q ≥ 1 [START_REF] Bolhuis | Colloid-polymer mixtures in the protein limit[END_REF]. Based on the free-volume theory of Lekkerkerker [START_REF] Lekkerkerker | Phase-behavior of colloid plus polymer mixtures[END_REF] and Bolhuis [START_REF] Bolhuis | Colloid-polymer mixtures in the protein limit[END_REF], Fleer and Tuinier [START_REF] Fleer | Analytical phase diagram for colloid-polymer mixtures[END_REF][START_REF] Fleer | Analytical phase diagrams for colloids and non-adsorbing polymer[END_REF] have derived a general law that allows to predict the binodals and generate a universal scaling law, regardless the polymer and the colloid size.

Most of the experimental studies on depletion concerns nonionic polymers with charged colloids but few is known about the structure and interaction of polyelectrolytes with nonionic colloids. For potential applications as drug delivery systems, it is also mandatory to control the aggregation state and the stability, two factors that may affect the therapeutic efficiency. With this perspective, we are interested in the mixed system composed of Pluronic F127 micelles and HA. Since F127 and HA do not exhibit specific adsorption nor electrostatic interaction, a depletion phenomenon should be observed depending on the polyelectrolyte concentration or the ionic strength that screens the inter-and intrachain electrostatic repulsions. Two HA, with molecular masses of 12 and 300 kDa are used. We work in the Pluronic micellar phase (3 vol%) and different HA volume fractions above 1%, in the semi-dilute regime. The effects of the nature of the salt and its concentration on the formation, shape and organization of the micelles are probed by differential scanning calorimetry (DSC) and small angle neutron scattering (SANS).

Experimental

Material HA (sodium hyaluronate) with two molecular weights, HA12 (12.6 kDa) and HA300 (300 kDa) were kindly provided by Soliance. Pluronic F127, P123, NaCl, NaBr, KCl, KBr, CaCl 2 and D 2 O were purchased from Sigma-Aldrich and used without further purification. The physical parameters are recalled in Table 1 and2.

Sample preparation

The samples are all prepared in D 2 O is order to increase the scattering length density (SLD) contrast and to reduce the incoherent background for the SANS experiments. The sample composition is given by the volume fraction of F127 (F F127 ) and the volume fraction of HA (F HA ) defined as: Where V F127 , V D2O and V HA are the volumes of Pluronic, solvent (D 2 O or D 2 O + salt) and HA respectively. In the following, Φ '()* is kept constant at 3% (2.6 10 -3 mol/L).

Φ '()* = , -./0 , -./0 1, 2/3
A 3% F127 stock solution in D 2 O or D 2 O brine at the wanted salt concentration is prepared and gently stirred until the total dissolution of F127. The F127 stock solution is then added to the precisely weighted amount of HA to get the final desired concentration. The sample is regularly vortexed and left for equilibration at room temperature 2 days before use. The samples prepared with HA12 flow; with HA300 for concentrations above 2 vol%, the samples are sufficiently viscous so that they do not flow in an inverted test-tube. At room temperature, close to the F127 critical micellar temperature, all the samples are transparent.

Differential Scanning Calorimetry

DSC measurements were carried out with a Micro DSC III apparatus (Setaram, France) which allows high sensitivity with dilute solutions. The sample of about 0.4 g is filled in the Hastelloy vessel and the same amount of D 2 O is used as reference. A first fast heating / cooling cycle at 2 K/min between 4 and 60 °C is performed to ensure the homogeneity of the solution. Then 2 cycles at 0.5 K/min are repeated to check for the reproducibility and reversibility of the transition. The critical micellar temperature is determined from the temperature at the intersection of the tangent at the first inflection point of the peak with the baseline [START_REF] Hecht | Colloids Surface A: Physicochem[END_REF]. The enthalpy of micellization (ΔH m ) is calculated from the area of the micellization peak.

Small Angle Neutron Scattering

Most of the SANS measurements were carried out on the D33 instrument at the Institut Laue Langevin, Grenoble, France. Two wavelengths 6 and 13 Å with a wavelength spread Dl/l of 10% and two detector distances (d = 2 and 12 m) were used to cover a q-range from 1.5 10 -3 to 0.45 Å -1 where q is the wave vector transfer (q = 4p/l sinq) with 2q the scattering angle. Additional measurements were carried out on the PAXY instrument at the Laboratoire Léon Brillouin, Saclay, France. Four instrument configurations were used to cover a similar q-range (l = 5 Å, d = 1 and 3.5 m; l = 8 Å, d = 5 m; l = 15 Å, d = 7 m). Samples were placed in 1 mm thick Hellma cells and thermostatted through a circulation water bath system at 37°C. Data were corrected from electronic background and empty cell and normalized to the absolute scale (cm -1 ) using standard procedures implemented in the Lamp software at the ILL [29] and in PASINET at the LLB.

SANS data analysis

In this ternary system as it will be shown in the experimental part, the scattering is fully dominated by the scattering of the micelles for q < 0.15 Å -1 . Following the approach already used in [START_REF] Grillo | Structural Characterization of Pluronic Micelles Swollen with Perfume Molecules[END_REF], the general equation used to analyze the scattering intensity of the F127 micelles can be written:

𝐼 𝑞 = Φ : 𝑉𝑃 𝑞 𝑆 𝑞 + 𝐼 ?@ABC 𝑞 + 𝐼 D?E Eq. 1

with F m , the volume fraction of particle and V the particle volume, I bck the incoherent scattering contribution. The form factor P(q) describes the shape of the micelles and S(q) is the interparticle structure factor. I coils (q) is the scattering of a Gaussian coil. This contribution is added to better taking into account the PEO chains within the micellar shell and the HA macromolecules.

The scattering of the pure Pluronic micelles is fitted to a core-shell model combined with a hard-sphere structure factor. The core contains PPO and water, and the shell PEO and water. The resulting parameters are R c , the core radius, e shell , the thickness of the PEO shell, the core and shell SLD, r c , r sh and F m , the hard sphere volume fraction of micelles. A Gaussian coil with R G = 6 Å is added to improve the fitting at high q (> 0.2 Å -1 ), which mainly originates from the PEO segments. Polydispersity (with a Gaussian size distribution) is applied to the core and the shell independently. Finally, the instrument resolution is taken into account in the fitting procedure. The detailed equations of the form and structure factors used for the SANS data analysis are given in the Electronic Supporting Information.

In presence of HA the modification of the scattering intensity at low q values can be explained by a reorganisation of the micelle dispersion state. It is modelled with an attractive square-well structure factor, based on the Baxter's sticky hard-sphere model [31] for the micellar interactions. The potential V(r) has a fine width D and a depth U 0 (Figure SI 1). Considering r the distance between the particles, the potential is infinite for r < s = 2R; is equal to -U o for s < r < s+D and 0 for r > s+D. R is the radius of the micelles; s = 2R is the diameter of the micelles and D is the range of attraction of the order of the mesh size x of the polyelectrolyte network. This model has been successfully used to model the static structure of short-range attractive colloidal systems in a wide range of concentration and temperature [START_REF] Tuinier | Depletion interaction of casein micelles and an exocellular polysaccharide[END_REF]33]. At high q values a direct contribution of the HA macromolecule is also observed and taken into account in the Gaussian coil scattering.

Experimental Results

DSC Characterization

Typical DSC thermograms for a 3% F127 solution in D 2 O and in D 2 O/0.1 M NaCl during the heating phase are presented in Figure SI 2. The pronounced endothermic peak is the signature of the micellar formation induced by the dehydration of the PPO as the temperature increases and overcomes the critical micellar temperature. For the reference solution in D 2 O, the critical temperature is found at 22°C in good agreement with values reported in the literature [34,[START_REF] Alexandridis | Poly(ethylene oxide)-poly(propylene oxide )-poly (ethylene oxide) block copolymer surfactants in aqueous solutions and at interfaces: thermodynamics, structure, dynamics, and modeling[END_REF]. Figure 1 shows the effect of the salt concentration (NaCl) on the micellization temperatures without and with HA (F HA = 2%). Figure 2 shows the effect of HA concentration at a fixed NaCl concentration of 0.1 M. In Figure SI 3, the nature of the salt is investigated. It is well established that salt affects the water solvency. Water structuring salts, leading to an increase in self hydration through hydrogen bounding reduce the hydration of PPO and decrease its water solubility resulting in a lower critical micellar temperature. NaCl and KCl have a slight larger effect than NaBr and KBr. A linear relationship between the critical temperature and the salt concentration is observed as already reported [36,37]. Independently of its molecular mass, addition of the HA (polyanions and counterions) affects the transition in the same manner as a low molecular weight electrolyte, inducing a linear decrease of the critical temperature as its concentration increases. For all the samples, the enthalpy of micellization remains constant within the experimental errors with an average value of DH m = 355 kJ/mol. In the following of the paper, the experiments are performed at 37°C in a domain well above the critical micellar temperature for all the salt and HA concentrations investigated.

SANS Structural Characterization

Characterization of the individual components

In this paragraph, we briefly present the characterization of the binary systems (i.e., pure Pluronic or HA solutions) before to move to the ternary system.

Pluronic F127

The scattering of the 3% F127 solution in D 2 O is shown in Figure SI 4 together with the data analysis. At high angles, the nearly q -2 behavior comes from the Gaussian behavior of the PEO chains in the hydrophilic shell. A correlation peak is clearly visible at q = 0.018 Å -1 , corresponding in the real space to a distance of 350 Å between the micelles. The fit to the core-shell form factor described above, gives a core radius of 45 Å and a shell thickness of 60 Å. The experimental shell SLD found at 5.8 10 10 cm -2 corresponds to 87% of hydration: the shell is almost invisible by SANS. Due to the high hydration level, the micellar volume fraction F m within the core-shell description is equal to 11%, a value significantly higher than the volume of the dry Pluronic. The core SLD is equal to 0.8 10 10 cm -2 , corresponding to 10% of water. These values are in good agreement with previous studies on Pluronics [START_REF] Grillo | Structural Characterization of Pluronic Micelles Swollen with Perfume Molecules[END_REF][START_REF] Pedersen | [END_REF]39].

The effect of salt (NaCl) for concentrations up to 4 M is presented in Figure SI 5. Up to 0.5 M NaCl, the curves are all superimposed, the size, shape and organization of the micelles are not modified. The SLD of the shell remain at 5.8 10 10 cm -2 but that of the core decreases to 0.4 10 10 cm -2 , close to that of the PPO. Within the accuracy of the measurements and data analysis, the core does not contain water anymore. Between 1 and 2 M, one observes the shift of the oscillation toward lower q together with the increase of the intensity at small q which indicates a growth of the micelles. From 2 to 4 M a transition from spherical to cylindrical micelles occurs induced by the dehydration of the PEO segments in agreement with the literature [START_REF] Kadam | [END_REF][START_REF] Fan | [END_REF].

Finally, Figure SI 6 shows that the 4 monovalent salts investigated (NaCl, NaBr, KCl and KBr) at 0.1M as well as the divalent salt CaCl 2 at 0.03, 0.05 and 0.1M do not modify the scattering of the micelles.

Hyaluronic Acid

HA is a semirigid polyelectrolyte. The total persistence length L T is the sum of two contributions, the intrinsic persistence length, L 0 and the electrostatic persistence length L e . L e depends on the ionic strengthfixed by the dissociated counterions and the added salt concentrations. It can be calculated with the model developed by Odjik, Skolnick and Fixman [42][START_REF] Skolnick | Electrostatic Persistence Length of a Wormlike Polyelectrolyte[END_REF][START_REF] Fixman | The flexibility of polyelectrolyte molecules[END_REF]. Assuming a fully stretched polyion (rod limit) with a contour length L c , the radius of gyration is equal to 𝑅 G = 𝐿 ? 12 and the overlap concentration is given by c*=𝑀w

M N 𝜋𝑅 G N 𝑁 Q .
One obtains c* values of 6 and 1.2 10 -2 g/L for HA12 and HA300 respectively. In a more realistic case, by considering a "wormlike" chain of persistence length L T (with L T = 90 Å), the Benoit and Doty equation [START_REF] Benoit | [END_REF][START_REF] Buhler | Chain persistence length and structure in hyaluronan solutions : ionic strength dependence for a model semirigid polyelectrolyte[END_REF] gives 𝑅 G of 69 Å and 470 Å leading to c* values of 15 and 1.1 g/L for HA12 and HA300. These values are only indicative. However in the following, at HA concentrations higher than 1 vol% (16.9 g/L) we can admit that we are always above c*.

In the case of a depletion mechanism as recalled in the introduction, the mesh size of the polymer network x is used to quantify the width of the attractive potential. In order to estimate thes correlation length, scattering experiments have been carried out on HA12 and HA300 solutions (2%) in D 2 O and in 0.1M NaCl. The results are presented in Figure SI 7 and SI 8. Below 0.01 Å -1 one observes an upturn whose origin has been largely debated, and is related to the formation of large aggregates [START_REF] Buhler | Chain persistence length and structure in hyaluronan solutions : ionic strength dependence for a model semirigid polyelectrolyte[END_REF][START_REF] Buhler | Chain persistence length and structure in hyaluronan solutions : ionic strength dependence for a model semirigid polyelectrolyte[END_REF][START_REF] Salamon | Probing the Mesh Formed by the Semirigid Polyelectrolytes[END_REF].

In absence of salt and more clearly for the SAXS data, the so-called polyelectrolyte peak is visible. Its position q m provides a direct measurement of the correlation length x (x = 2p/q m ). It is close to 80 Å independently of the molecular mass of the polyelectrolyte as we are in the semi-dilute regime.

In presence of 0.1M of salt, the peak disappears due to the screening of the electrostatic repulsions between the chain segments [START_REF] Nierlich | [END_REF]. HA chains adopt a random coil configuration with a persistence length close to the intrinsic one [START_REF] Buhler | Chain persistence length and structure in hyaluronan solutions : ionic strength dependence for a model semirigid polyelectrolyte[END_REF]. This persistence length has been estimated by light scattering and SANS in dilute conditions (below the overlap concentration c*) and at high ionic strength (0.1M NaCl) when all the electrostatic contributions are screened so that the electrostatic persistence length L e is negligible and the total persistence length L T is equal to the intrinsic persistence length L 0 . The different studies report L 0 values between 50 and 90 Å [START_REF] Bonnet | Suppression of aggregation in natural-semiflexible/flexible polyanion mixtures, and direct check of the OSF model using SANS[END_REF][START_REF] Shi | Role of the ratio of biopolyelectrolyte persistence length to nanoparticle size in the structural tuning of electrostatic complexes[END_REF]. This remains comparable to the radius of the F127 micelles and for this intrinsic rigidity we can infer that the chain cannot wrap around a single micelle. For semi-dilute solution of neutral polymer, the scattering length function can be described by the Ornstein-Zernike equation leading to the extraction of the correlation length x. We admit that this behavior is still valid for HA in presence of 0.1 M NaCl. In good solvent conditions, x scales with the concentration c according [START_REF] Buhler | Chain persistence length and structure in hyaluronan solutions : ionic strength dependence for a model semirigid polyelectrolyte[END_REF]:

𝜉 ∼ 𝑐 TU.** Eq. 2 It should be noticed that for hyaluronan, a larger exponent of 0.9 has been measured in [START_REF] Buhler | Chain persistence length and structure in hyaluronan solutions : ionic strength dependence for a model semirigid polyelectrolyte[END_REF] and it is explained by the fact that the behavior of the chains is close to Gaussian chains without excluded volume effects.

The weakness of the signal in the SANS data with the upturn at low q, does not allow any accurate determination of the correlation length. In the following, it will be estimated at 𝜉 U ≈ 60 Å from the SAXS data for the 2% HA concentration, a value slightly smaller than the correlation length in absence of salt. The mesh size for a HA concentration c (in vol%) is therefore obtained following:

𝜉 = 𝜉 U (𝑐 2) TU.**
Eq. 3 The mesh size value is taken as an estimation of the width D of the square-well potential.

Characterization of the ternary system

Effect of salt, HA concentration and molar mass SANS data of F127 at F F127 = 3% in pure D 2 O as a function of the volume fraction of HA are shown in Figure 3 (left) for HA12 and Figure 4 (left) for HA300. The signal is fully dominated by the scattering of the micelles and is almost not modified by the presence of HA. We observe a small increase of the intensity at low q for F HA300 > 4%. Also, at high q above 0.15 Å -1 , slight modifications are observed due to the scattering of polyelectrolyte. In the middle q domain, the overlap between the curves is the evidence that the size and the shape of the micelles are not modified by the presence of HA.

The situation considerably differs in presence of 0.1 M of NaCl (figure 3 and4, right). As in pure D 2 O, the small increase of the intensity at high q is the signature of the HA chains. In the middle q range, the scattering of the micelles dominates and all the curves are superimposed. The oscillation of the form factor remains at the same q-position. At low q, one observes a significant increase of the scattering intensity that reaches a plateau. This is the signature of small clusters of micelles in the solution. The effect of HA differs thus from a salt effect (Figure SI 4) inducing the transition from spherical to cylindrical micelles. Above volume fractions of F HA = 9% and 5% for HA12 and HA300 respectively, the samples become turbid and the scattering curves present the characteristic diffraction peaks of a face centered cubic phase. It should be noted that due to the viscosity of the samples, there is no macroscopic phase separation. From the first peak position (corresponding to the 111 reflection), we calculate a center-to-center distance between the micelles of 213 Å and 208 Å for HA300 at 5 and 6% respectively and 198 Å with HA12 at 9%. The micelles are closely packed. This crystalline structure is usually obtained in pure F127 in salt free water solutions at volume fractions higher than 20% [START_REF] Wanka | [END_REF].

Below the HA concentration leading to the crystallization of micelles, the scattering data are fitted with a core-shell model for the form factor and a square-well structure factor, enabling to reproduce the micelle aggregation and the cluster formation. These different contributions are shown in Figure SI 9. The potential width D is fixed to the mesh size of the HA network according to Eq.3. The results are shown with full lines in Figure 3 and Figure 4 for HA12 and HA300 respectively and in Figure SI 10 with a shift in intensity to improve the visual clarity. The summaries of the fit parameters are given in Table 3 and Table 4. The data analysis confirms that the size of the micelles is not modified by the presence of HA. From the position of the maximum of the structure factor, an average distance between the center of mass of the micelles in the cluster is calculated (D m ). It decreases from 350 Å without HA to 168 Å and 198 Å at the highest HA12 and HA300 concentrations just before their crystallization. The PEO shells are slightly overlapping and this explain the small decrease down to 5.6 10 -6 Å -2 of the average SLD. Finally, the increase of the scattering at low q is consistent with an increase of the depth of the potential and therefore to more attractive interactions between the micelles. At a given HA volume fraction, the potential is deeper for HA300 than HA12, and the crystallization of the micelles occurs at lower volume fraction for HA300 (~ 5%) than for HA12 (~ 9%). The number of micelles per clusters N mic is estimated from the radius of gyration of the clusters calculated from the Guinier regime at low q (Eq SI 8). It varies between 1 and 25 as the HA concentration increases. For F HA12 ≤ 2%, N mic is found at 1 with however a distance between adjacent micelles of 225 Å (against 350 Å in absence of HA). This shows that the micelles are no longer homogeneously distributed in the solution. This number is also an average, and it cannot be excluded that single micelles and clusters are already coexisting. The size of the micellar clusters grows when the HA concentration increases. At a fixed HA concentration, the size of the clusters is larger with the largest polyelectrolyte molecular mass.

A complementary measurement shows that by increasing the Pluronic concentration to 5%, for a same HA300 volume fraction, the attractive interaction between the micelles is larger and the micellar cristallisation occurs at a smaller volume fraction of HA (between 2 and 4%) (Figure SI 11 and table SI 1).

Effect of the salt (NaCl) concentration

In this part, the effect of the salt (NaCl) concentration is explored. The volume fraction of HA is fixed at 2% and the salt concentration kept below 1 M, to avoid the growth of the micelles and their shape transition (Figure SI 5). The scattering results are presented in Figure 5 and Figure SI 12. The fitting parameters are summarized in Tables 5 and6. For HA chains, above salt concentration of 0.1M, the electrostatic persistence length L e is reduced to 1.5 Å [START_REF] Buhler | Chain persistence length and structure in hyaluronan solutions : ionic strength dependence for a model semirigid polyelectrolyte[END_REF] and the total chain stiffness is only due to the intrinsic stiffness of the chain. The folding of the HA network is mostly complete [52] and the value of the potential width is kept constant at 60 Å. The size of the cluster increases with the salt concentration. At a fixed salt concentration, before the crystallization, the clusters are larger with HA300 than with HA12. Consequently, the attractive potential determined from the data analysis is deeper for HA300. It should be pointed that the 0.5 M concentration with HA12 corresponds certainly to a concentration very close to the 2-phases domain as shown with a drastic increase of the forward scattered intensity. Inside the clusters, by increasing the salt concentration from 0.05 M to 0.5 M, the center-to-center distance of the micelles decreases from 224 to 200 Å. The micelles are then in contact.

The micelle crystallization occurs at lower salt concentration for HA300 (> 0.1 M) than HA12 (> 0.5 M). For HA12, as the sample remains relatively fluid, a phase separation occurs, in which the supernatant is enriched in micelles almost in contact (Figure SI 13). With HA300, the sample is viscous, there is no macroscopic phase separation. From the first Bragg peak position, considering a face centered cubic lattice, we calculate a center-to-center distance of 235 Å between the micelles, thus slightly larger than their diameter. ) q (Å -1 )

HA300 NaCl

Table 6: F127 F F127 = 3%, HA300, F HA = 2%, T = 37°C. Effect of the NaCl concentration. Fit parameters to a coreshell form factor model and a square-well potential for the structure factor. Same parameter definition as in Table 3. Effect of the nature of the salt Does the nature of salt impact the micellar stability and organization? We work at volume fractions of 3% for F127 and 2% for HA. Four monovalent salts, NaCl, KCl, NaBr, KBr at 0.1 M are used and the divalent salt CaCl 2 is chosen for its relevance in biological systems. For further comparison with the monovalent salts, three concentrations, 0.033, 0.05 and 0.1 M are investigated, corresponding to ionic strengths of 0.1, 0.15 and 0.3 M. The SANS data are presented in Figure 6 In presence of the monovalent salts the scattering curves are almost identical with HA12. The data analysis show that the attractive potential is slightly higher for NaBr and KBr (0.7 k B T) compared to NaCl and KCl (0.6 k B T). With HA300, the scattering curves are almost superimposed for NaCl and KCl, and superimposed for NaBr and KBr with an attractive potentials equal to 1.2-1.3 k B T for the former salts and 1.8 k B T for the latter ones. The clusters are significantly larger with HA300 than with HA12. The center-to-center distance between the micelles is also a few Angströms smaller with NaBr and KBr than with NaCl and KCl. For HA12 with NaCl and KCl, the micelles are separated by a tenth of Angströms, in the other cases, the micelles are in contact.

C
In presence of the divalent salt CaCl 2 , the ionic strength is the relevant parameter. The 0.1 and 0.15 M concentration with monovalent salts can be compared with the 0.03 and 0.05 M concentrations with CaCl 2 . For both HA12 and HA300, the divalent salt induces a larger attractive potential than the monovalent salts, and thus larger cluster size. In the cluster, the micelles are in contact. For HA300, the crystallization of the micelles occurs between 0.1 and 0.15 M in ionic strength as previously observed with NaCl (Figure 5). Their center-to-center distances are 243 Å in 0.05 M CaCl 2 solution and 236 Å in 0.1 M CaCl 2 solution. Table 7: F127 F F127 = 3%, HA12 F HA = 2%, T = 37°C. Effect of the nature of the salt. Fit parameters to a core-shell form factor model with a square-well potential for the structure factor. Same parameter definition as in Table 3.

Influence of the size of the Pluronic micelles

Pluronics form a wide group of PEO-PPO-PEO triblock copolymers and offer the possibility to tune easily the size of the micelle by keeping the same chemical characteristics. P123 was then chosen to keep the same number of PO units for the core but has a smaller shell with 30 EO units. The scattering of P123 with HA12 and HA300 at different volume fractions is presented in Figure SI 15 and fit parameters are given in Table SI 2. The SANS data analysis shows that the pure P123 micelles in brine solution have a total radius of 73 Å. As for F127, the radius of the core is 45 Å but the hydrophilic shell thickness is smaller (28 Å) and the average of SLD of 2.10 -6 Å -2 indicates that the shell contains only 23% of water. We can note that at the Pluronic concentration of 3%, the structure factor is close to the unity for P123 and the scattering curve reaches a plateau at 100 cm -1 whereas an interaction peak was clearly visible for F127.

As with F127, we observe for the lowest polyelectrolyte concentrations an increase of the scattering intensity at low q. The depth of the attractive potential is 1.2 k B T for HA12 at F HA = 2% and 0.4 k B T for HA300 at F HA = 1%. With F127 at the same HA concentrations, we found 0.66 k B T (HA12) and 1 k B T (HA300). The appearance of the crystalline phase occurs at significantly lower HA volume fractions for P123 than for F127 i.e. 4% vs 7% with HA12 and 2% vs 4% with HA300.

Discussion

Colloids -polyelectrolytes systems exhibit a large variety of phase diagrams and structures due to a complex interplay between the different attractive (van der Waals, depletion, electrostatic) and repulsive forces (hydrophobic, steric, and electrostatic). Addition of salt modifies not only the ionic strength and the electrostatic interaction but also the water solvency and the hydrogen bonding.

For oppositely charged systems, the electrostatic interaction (and the release of counterions) is the motor of the complexation. The length of the polyelectrolyte chain, its concentration and the salinity play a role on the structure of the complexes. The phase diagram and the structure of the complexes formed by lysozyme and PSSNa have been investigated in [START_REF] Gummel | [END_REF]. It is shown that for a given [PSS] / [lysozyme] ratio, gels are formed with the long chains and dense packed globules are observed with short chains. However, the crossover between these two regimes can be tuned with the chain concentration or the ionic strength. SANS combined with contrast variation experiments evidenced the reduction of the persistence length of the polyelectrolyte chain in presence of protein in the gel phase and a more collapsed state of the polyions in the globules. When PSS is replaced by HA, the semirigid nature of HA compared to that of PSS brings an additional constrain, hindering the chain wrapping around the colloids. Effectively, in the HA / lysozyme system, the formation of rod-like shape structures which extend on larger scales than the persistence length of HA is evidenced due to the electrostatic repulsion between neighboring proteins attached to the HA backbone [START_REF] Morfin | [END_REF]. Such behavior has been further generalized as a function of the ratio between the total persistence length of the polyelectrolyte and the colloids [START_REF] Shi | Role of the ratio of biopolyelectrolyte persistence length to nanoparticle size in the structural tuning of electrostatic complexes[END_REF]. In addition to the electrostatic interaction, hydrogen bonds can play an important role in the structure and dynamics of hyaluronic gels [START_REF] Bełdowski | Physical crosslinking of hyaluronic acid in the presence of phospholipids in an aqueous nanoenvironment[END_REF] and in general in polyelectrolytes complexes. It has been shown that at low pH, F127 monomers can interact with the carboxylic group of polyacids. The more hydrophobic chains bind with the PPO blocks of F127 inducing drastic change in the critical micellar temperature, and the more hydrophilic wrap around the individual F127 micelles forming nanosized dispersed complexes [56].

Unlike the previously described studies, Pluronic are nonionic and none of the structural organizations described above fully corresponds to those found in the HA / F127 system. When the attractive electrostatic interaction is reduced new competing interactions are revealed. For non-adsorbing polymers a depletion effect can be observed leading to a macroscopic phase separation into colloidsrich and colloids-poor phases. This depletion interaction depends on the excluded volume that is controlled by the nature of the different constituents, their concentration and the solution conditions. Despite the importance of the depletion interaction for the phase behavior in colloidal dispersions and therefore, its role in industrial formulation and biology, only limited experimental structural studies on model systems have been carried out. In this context, scattering experiments provide a unique way to reveal the nature of the interactions prior to the phase separation. In particular, SANS combined with contrast matching can be used to reveal the colloids partial structure factor that is directly linked to the interaction potential. Depletion effects in neutral particle / polymer model systems have been investigated by SANS [START_REF] Tuinier | Depletion interaction of casein micelles and an exocellular polysaccharide[END_REF][START_REF] Ye | Depletion interactions in colloid-polymer mixtures[END_REF][58][59]. First measurements were carried out on calcium carbonate particles [START_REF] Ye | Depletion interactions in colloid-polymer mixtures[END_REF]58] stabilized with by an adsorbed surfactant layer in presence of polyethylene polypropylene copolymers in decane (R G /R » 2). No macroscopic phase separation was observed but an increase of the scattered intensity was detected at low q. The colloids partial structure factor could be fitted using an attractive interaction potential developed by Vrij and valid for small polymer chains [START_REF] Asakura | Interaction between particles in solutions of macromolecules[END_REF]. An effective attraction between colloids could be evidenced in presence of polymers. This depletion induced attraction was found to increase with polymer concentration and level off slightly above c*.

Charged colloidal dispersion with neutral polymers shows a more complex phase diagram due to the repulsive interactions between particles [60-62]. The phase behavior now depends on the balance between the repulsive (colloids-colloids, polymer-polymer) and attractive (depletion) interactions. This subtle interplay can be varied by changing the electrolyte or the polymer concentration. SANS can link the phase diagram with the colloidal organization and the particle interaction under salt and polymer addition. Charged silica particles with poly(ethylene glycol) in water have been recently reported (0

.3 < R G /R < 0.7, c < c*) [61-62].
In salt-free solutions, electrostatic repulsion between charged colloids stabilizes the dispersion: a single homogeneous phase is observed even at high polymer concentration. Addition of electrolyte screens the electrostatic and a depletion induced microscopic phase separation appears when increasing the polymer concentration (turbid solutions due to particle association). Further addition of polymers modifies the strength of the depletion attraction and a transition to a single homogenous phase is observed (reentrant phase). When poly(ethylene glycol) is replaced by block copolymers (Pluronic P85) [60], the depletion effect is enhanced when the temperature is increased due to the formation of spherical Pluronic micelles and the induced variation of the excluded volume.

As in previous studies, experimental work on depletion usually concerns nonionic polymers and neutral or charged colloids in dilute regime. Semi-dilute polymer solutions in which the mesh size fixes the interaction range are rarely considered. Furthermore, there is a lack of studies on polyelectrolytes although they form a very important class of water-soluble macromolecules and have many implications in biology and technology. The present study gives new experimental structural findings on depletion effects in charged polymers / neutral colloids using a simple model system based on linear polysaccharides and Pluronic micelles. HA / F127 mixtures in aqueous solution have been investigated in semidilute regime by DSC and SANS. We explored the role of different parameters such as the HA concentration (above c*), the chain length, the nature and salt concentration as well as the size of the micelles (F127, P123) on the critical micellar temperature, the structural organization and the stability of the solutions.

The size ratio Q characterizing our system is determined from the estimated radius of gyration of the polyions R G and the radius of the micelles R (R » 105 Å). Q is close to 1 for HA12 (69 < R G < 92 Å) and in between 5 to 21 for H300 (470 Å < R G < 2200 Å). We are in the protein limit for HA300 and in an intermediate situation for HA12. Mixtures are in semi-dilute regime for all investigated polymer concentrations (from 1 to 7%). Only the lowest volume fraction (1%) with HA12 is close to c*. In this entangled regime the range of attraction of the depletion equals the correlation length x of the polymer network. The organization of the solution should no longer be sensitive to the molar mass of the chains. x varies with the polymer concentration and the ionic strength of the solutions. For salt concentration around 0.1 mol/L, the Debye screening length is of the order of 10 Å i.e. much less that the intrinsic persistence length L 0 of polymer. Electrostatic interaction between charged monomers is screened and polyions can be treated as neutral chains in good solvent conditions. For salt-free and brine solutions (c salt > 0.1 mol/L), x only depends on the polymer concentration as given by eq. 3. In this system, the radius of the particles is larger than the correlation length (from 75 to 40 Å), the size ratio Q now defined as D/x lies in between 3 and 5.

In this new polymer / colloid system, SANS intensity is dominated by the signal of the micelles. Scattering experiment gives a direct insight into the F127 partial structure factor, and therefore, to the form factor P(q) and the dispersion state of the Pluronic micelles (S(q)). SANS measurements are very sensitive to the salt addition and show substantial modifications for certain compositions. Within the investigated HA and salt concentrations, the size and shape of the micelles as well as the SLD of the PEO shell are not modified by the presence of polymers. These observations are proofs that there are neither specific interaction nor adsorption between HA and the micelles. This is a very important point since the absence of adsorption is a prerequisite for depletion effects. On the other hand, clear modifications are observed at low q. The increase of the intensity is the signature of the formation of small clusters of micelles in the solution. Structural modifications are therefore mainly related to the dispersion state of the colloids as expected for an attractive depletion interaction [59,61,62]. In order to reveal precisely the nature of structural arrangements, we introduced a structure factor S(q) based on a sticky hard-sphere model [31] in which the range of attraction is directly linked to the depletion layer thickness i.e. the correlation length x of the polymer network.

The main results of the study can be summarized as follow:

(1) the presence of HA modifies the critical micellar temperature of the Pluronic copolymers to the same extend as an increase of the ionic strength. (2) In salt free solutions, micelles are homogeneously distributed in the system and not perturbed by the presence of HA. (3) By increasing the ionic strength the micellar organization is clearly changed: we observe the formation of clusters that evolve to a face-centered cubic liquid crystalline phase at high salt concentration. This behavior is reinforced by increasing the HA concentration and the molecular weight of the polyelectrolyte chains. It announces a macroscopic phase separation for non-viscous systems (HA12). ( 4) The nature of the salt plays also a role and divalent cations such as Ca 2+ promote the clustering of the micelles and their crystallization.

Without salt, we observe a single homogeneous phase whatever the HA molar mass or concentration. This situation is very similar to the symmetrical system where charged colloids are mixed with neutral polymers [61,62] and stabilize the solutions. There are strong electrostatic repulsions between the semirigid polyions. A single phase in which the micelles are homogeneously distributed in the HA network is energetically more favorable than a phase separation that would decrease the available volume for the polyelectrolyte, reducing the distances between the charged chains.

In presence of salt (0.1 M NaCl solution), the repulsive inter and intra chain repulsions are screened, the chains can get closer without a high energy cost. In addition, salt induces a change of the conformation of the polyelectrolyte chains and a reduction of the correlation length which in turn enhances the excluded volume for the micelles and induces an attractive depletion force. The micelles form first clusters which get bigger as the HA concentration increases. The intermicellar distance D m is reduced until the crystallization occurs. Due to the high viscosity of the solutions, mixtures are turbid, but no macroscopic phase separation is observed. This general scenario is depicted in Figure 8. The structural organization and the formation of finite size micellar clusters is similar that found in neutral calcium carbonate particles / polyethylene polypropylene copolymers systems [START_REF] Ye | Depletion interactions in colloid-polymer mixtures[END_REF]58] or in charged silica particles / poly(ethylene glycol) [61,62] except that no real crystallization (into a well ordered lattice) was observed in these systems.

How can we understand the enhanced clustering as the ionic strength increases above 0.1 M? For HA chains, at a salt concentration of 0.1 M, the electrostatic persistence length L e is reduced to 1.5 Å [START_REF] Buhler | Chain persistence length and structure in hyaluronan solutions : ionic strength dependence for a model semirigid polyelectrolyte[END_REF] and the total chain stiffness is only mainly due to the intrinsic stiffness of the chain L 0 . The folding of the coil is mostly complete [52]. The enhanced clustering of the micelles observed above 0.1 M is not due to the increase of excluded volume but to the weakening of the steric repulsion between the micelles. If salt affects the nonionic species in a lesser extent than charged colloids, it has nevertheless an influence on the Pluronic, both on the PO and EO groups, through solvation competition. On one side, the decrease of the critical micellar temperature in presence of salt is due to a reduced hydration of the PO groups and that all the more that the salt is cosmotropic. One the other side, the salt affects the hydrogen bonds between PEO and water and modify the PEO solubility. The differences observed between the different salts can be related to the Hofmeister classification [63][START_REF] Ataman | Properties of aqueous salt-solutions of poly(ethylene oxide)[END_REF][START_REF] Zhang | Interactions between macromolecules and ions: The Hofmeister series[END_REF]. At low salt concentration, it has been shown that subtle changes of the PEO layer coating silica particles, drive attraction [START_REF] Zackrisson | [END_REF]67]. Similarly, in our system, the weakening of the PEO layer and thus of the steric repulsion, is at the origin of the enhanced formation of micellar clusters when the ionic strength increases. Nevertheless, at a given ionic, the differences between the different salts cannot been fully rationalized. Larger clusters are formed in presence of NaBr, KBr and CaCl 2 . Compare to Na + , Br + is more chaotropic but Ca 2+ is more cosmotropic. More systematic investigations would be then necessary to clarify the effect of the nature of the ions.

An important point concerns the role of the HA molar weight on the organization and the stability of the solutions. At a given HA volume fraction, larger clusters are formed with the largest HA chain which also induces the crystallization of the micelles at a lower HA concentration. Similar effects are found when increasing the ionic strength. However, since we are in semi-dilute regime, attractive depletion interaction should no longer be dependent on the molar mass of the polymer chains as long as the solvent quality is preserved. For a given salt and HA concentration, the attractive interaction between the micelles is apparently larger for HA 300 than for HA12 while x is identical. We believe that this unexpected effect is related to the low molar weight of HA12 and its high polydispersity (PDI = 1.6). In this system, we are in an intermediate situation in between the colloidal and protein limits (0.7 < R G /R <0.9). The average extended length of the chains only corresponds to 3 or 4 intrinsic persistence lengths L 0 Due to the large polydispersity, very short chains (extended length close to 1 or 2 L 0 ) cohabit with longer ones. In this inhomogeneous polymer solution, shorter chains are no longer flexible and do not show any conformational variation under salt addition. Their contribution to the excluded volume is negligible and the phase boundary is displaced to higher polymer concentration.

Finally, in a depletion process, the attractive strength is related to the size ratio between the species. The P123 micelles being smaller than the F127 micelles, it was expected that their crystallization occurs at larger volume fraction [START_REF] Sear | Entropy-driven phase separation in mixtures of small colloidal particles and semidilute polymers[END_REF], but we observed the inverse trend. P123 micelles have a smaller PEO shell than F127 micelles. As for the salt effect, the steric repulsion between them is reduced and the crystallization occur at a lower depletion attraction. It is anticipated that a temperature increase reducing the water solvation and thus the size of the PEO shell, will also promote the clustering.

A rigorous and quantitative data analysis of the HA / Pluronic scattering would require the calculation of a dedicated potential of interaction between the two species which is above the scope of the paper. Asakura-Oosawa potential [START_REF] Asakura | [END_REF][START_REF] Asakura | Interaction between particles in solutions of macromolecules[END_REF] and models derived from it [START_REF] Vrij | [END_REF][START_REF] Edwards | [END_REF] would certainly better reproduce the physics of the system. We are aware that the square-well potential chosen here to model the attractive depletion interaction is very raw. However, it gives a good agreement with the experimental neutron data and has already been used in several studies to model the depletion attraction [START_REF] Muratov | [END_REF]. One principal limitation is the impossibility to decouple numerically the width from the depth of the potential. To fix the width, the mesh size is needed but it is unknown in presence of micelles. Colloids and polymer exert a mutual influence. The impact of polymer on the colloids is easy to proof but the reverse is more subtle. With synthetic deuterated polymer, SANS in contrast matching conditions has allowed to extract the chain conformation close to the liquid-liquid phase boundary showing that colloids induce a significant shrinking (up to 50%) of the polymer chains [START_REF] Kramer | [END_REF][START_REF] Le Coeur | Compression of random coils due to macromolecular crowding[END_REF][START_REF] Gummel | [END_REF]. The chain behavior of natural polyelectrolytes in presence of colloids is still unknown and certainly deserve to be explored.

Conclusion and perspective

The model system composed of F127 Pluronic micelles and HA illustrates the well-known phenomenon that long range correlations in polymer solution induce an effective attraction between colloids that are immersed in the solution [START_REF] De Gennes | Scaling concepts in polymer physics[END_REF][START_REF] Sear | [END_REF]. Because the polymer correlations depend not only on intrinsic polymer characteristics (mass, charge, rigidity,...) but also on environmental parameters that affect the chain solvency (temperature, ionic strength, nature of the solvent,…), the spatial organization and interaction between the colloids can be modified accordingly as evidenced by SANS. Addition of salt screens the electrostatic repulsion between the chains and induces a conformational change of the HA chain that increases the excluded volume. The micelles form first clusters with a finite size and then crystallize when the HA concentration increases. Although nonionic, Pluronic and their PEO moieties are also affected by the salt. The dehydration of the hydrophilic layer weakens the steric repulsion and also promotes the formation of clusters. Due to the possibility to easily vary either the ionic strength, the mass of the HA chain or the size of the Pluronic micelles, this system could be largely investigated to confront the existing theories with experiments to improve the understanding of the depletion attraction in the so-called "protein limit". In the medical field, macromolecular crowding and related effects due to excluded volume and increase of the viscosity, are at the origin of protein aggregation and fibrillation observed for example in Parkinson or Alzheimer diseases [START_REF] Munishkina | The effect of macromolecular crowding on protein aggregation and amyloid fibril formation[END_REF]. In consequence, disequilibrium of the blood saline composition could play a role in the formation of protein aggregates. Finally, the F127 / HA system appears as a good candidate to encapsulate active molecules to obtained a biocompatible solution for a local application. The incorporation of the linalool molecule in this system will be the topic of a forthcoming paper.

Acknowledgement:

The authors thank the ILL for the beam time on the D33 instrument and the Partnership for Soft Condensed Matter (PSCM) for the sample preparation and complementary measurements (DSC). The neutron data set are available at doi:10.5291/ILL-DATA.9-12-417 and doi:10.5291/ILL-DATA.9-13-564 [START_REF] Grillo | Formation of pluronic micellar clusters induced by the change of chain conformation of hyaluronic acid after addition of salt[END_REF]. IG is very grateful to A. Brûlet and to the Laboratoire Léon Brillouin (LLB, Saclay, France) for the sabbatical period during when the first SANS test measurements were done. We are also grateful to the LLB for the beam time on PAXY and A. Brûlet, A. Chennevière and F. Cousin are thanked for their help on the instrument and data reduction. Xray measurements were performed on the beam line D2AM at the ESRF. Prof. Th. Zemb is gratefully acknowledged for his comments to clarify the presentation and helpful suggestions for the future research routes. Isabelle Morfin and Jérôme Combet express their fond acknowledgement to their colleague and friend Isabelle Grillo, recently deceased, who initiated this work.

Figure 1 :

 1 Figure 1: Evolution of the F127 critical micellar temperature (determined by DSC) as a function of the NaCl concentration (c NaCl ). (¨ black) F F127 = 3%; (r red ) F F127 = 3%, F HA12 = 2%; (¡ blue) F F127 = 3%, F HA300 = 2% . The dotted lines are guides for the eyes.

Figure 2 :

 2 Figure 2: Evolution of the F127 critical micellar temperature (determined by DSC) as a function of the HA concentration (c HA ) or volume fraction (F HA ). ( red) F F127 = 3% and HA12 in D 2 O; ( blue) F F127 = 3% and HA300 in D 2 O; (r red) F F127 = 3% and HA12 in D 2 O/0.1M NaCl; (¡ blue) F F127 = 3% and HA300 in D 2 O/0.1 M NaCl.

Figure 3 :

 3 Figure 3: Evolution of the scattered intensity as a function of the volume fraction of HA12. F F127 = 3%. T = 37°C. Left: in D 2 O. (¡ grey) F HA = 0%, (¨ dark blue) F HA = 2%. Right: in NaCl 0.1 M. (¡ grey) F HA = 0%, (¨ dark blue) F HA = 1%, (¯ blue) F HA = 2%, (¡ green) F HA = 4%, (¨ red ) F HA = 5%, (r orange) F HA = 6%, (¯ purple) F HA = 7%, (+ black) F= 9%. Full lines correspond to the best fits of the scattering data before crystallization threshold.

Figure 4 :

 4 Figure 4: Evolution of the scattered intensity as a function of the volume fraction of HA300. F 127 = 3%. T = 37°C. Left: in D 2 O. (¡ grey) F HA = 0%, (¨ dark blue) F HA = 1%, (¯ blue) F HA = 2%, (¡ green) F HA = 4%, (¨ orange ) F HA = 5.5%. Right: in NaCl 0.1 M. (¡ black) F HA = 0%, (¨ dark blue) F HA = 1%, (¯ blue) F HA = 2%, (¡ green) F HA = 4%, (+ orange) F HA = 5%, (¨ red ) F HA = 6%. Full lines correspond to the best fits of the scattering data before crystallization threshold.

Figure 5 :

 5 Figure 5: Evolution of the scattered intensity for a solution of F F127 = 3%, as a function of the NaCl concentration. T = 37°C. Left: HA12 F HA12 = 2%. (¡ black) NaCl 0 M, (¨ dark blue) NaCl 0.05 M, (¯blue) NaCl 0.1 M, (¡ green) NaCl 0.15 M, (¨ orange) NaCl 0.2 M, (r red) NaCl 0.5 M. Right: F HA300 = 2%. (black) NaCl 0M, (¨ dark blue) NaCl 0.05 M, (¯ blue) NaCl 0.1 M, (¡ green) NaCl 0.15 M, (¨ orange) NaCl 0.2 M. Full lines correspond to the best fits of the scattering data before crystallization threshold.

  and 7 and Figure SI 14.

Figure 6 :

 6 Figure 6: SANS data showing the effect of the nature of the salt. F F127 = 3%. T = 37°C. Left: HA12 F HA12 = 2%. (x black) NaCl 0 M, (¡ grey) NaCl 0.01 M, (¯ blue) KCl 0.01 M, (¡ red) NaBr 0.01 M, (¯ orange) KBr 0.01 M. Right: HA300 F HA300 = 2%. (x black) NaCl 0 M, (¡ grey) NaCl 0.01 M, (¯ blue) KCl 0.01 M, (¡ red) NaBr 0.01 M, (¯ orange) KBr 0.01 M. Full lines correspond to the best fits of the scattering data.

Figure 7 :

 7 Figure 7: SANS data showing the effect of CaCl 2 . F F127 = 3%. T = 37°C. Left: HA12 F HA12 = 2%. (r green) CaCl 2 0.033 M, (¡ grey) CaCl 2 0.05 M, (¯ blue) CaCl 2 0.1 M. Right: HA300 F HA300 = 2%. (r green) CaCl 2 0.033 M, (¡ grey) CaCl 2 0.05 M, (¯ blue) CaCl 2 0.1 M. Full lines correspond to the best fits of the scattering data before crystallization.

Figure 8 :

 8 Figure 8: Schematic representation of the Pluronic micellar organization in presence of HA. The relative size between the micelles and the polymer network tries to respect the real organization in HA300 / F127 mixtures.

Table 1 :

 1 Selected physical parameters of the different chemicals used: chemical formula, molecular weight, density, molecular volume, neutron scattering length density r. The PO and EO density values are taken as determined in[START_REF] Sommer | [END_REF].

		Formula	Mw (g/mol)	Density	Molecular volume (Å 3 )	r (neutron) (cm -2 )
	F127	(EO)100(PO)65(EO)100	12570	1.14	19000	-
	P123	(EO)20(PO)65(EO)20	5800	1.018	8700	-
	(PO)	(OCH(CH 3 )CH 2 )	58	1.01	96	0.34 10
	(EO)	(C 2 H 4 O)	44	1.14	62	0.67 10
	HA (repeat unit) C 28 H 43 N 2 O 23 Na	401	1.695	770	2.31 10
	solvent	D 2 O	20	1.104	30.1	6.36 10

Table 2 :

 2 Characteristics of HA: molecular weight Mw, degree of polymerization DP, polydispersity index PDI, contour length of the fully extended chain Lc calculated with a monomer size of 10.2 Å f[START_REF] Buhler | Chain persistence length and structure in hyaluronan solutions : ionic strength dependence for a model semirigid polyelectrolyte[END_REF].

	Name	Mw (kDa)	DP	PDI	L c (nm)
	HA12	12.6	31	1.6	32
	HA300	300	750	2.0	760

Table 3 :

 3 F127 F F127 = 3%, NaCl 0.1 M, HA12, T = 37°C. Effect of HA concentration. Fit parameters from the SANS data analysis to a core-shell model for the form factor and a square-well potential for the structure factor. Distance between micelles calculated from the position of the first maximum of the structure factor. F HA : HA volume fraction; F m : volume fraction of micelles; Rc: core radius; e sh : shell thickness; r c , r sh , r solv : scattering length densities of the core, shell and solvent; D m : average distance between micelles; N mic : number of micelles in cluster; x: square-well potential width; U 0 : depth of the square-well potential.

	F HA							0	1%	2%	4%	5%	6%	7%
	F m							0.11	0.11	0.1	0.1	0.092	0.085	0.09
	Rc (Å)							45	45	45	46	47	47	47
	e sh (Å)						60	60	60	60	60	60	60
	r c (10	-6 Å	-2 )	0.8	0.4	0.4	0.4	0.4	0.4	0.4
	r sh (10	-6 Å	-2 )	5.8	5.8	5.8	5.8	5.8	5.6	5.6
	r solv (10	-6 Å	-2 )	6.4	6.4	6.4	6.4	6.4	6.4	6.4
	D m (Å)						350	224	215	208	198	178	168
	N mic							1	1	1	2	5	9	25
	x (Å)							-	75	60	47	44	41	40
	U 0 (k B T)					-	0.5	0.66	1.34	2.1	2.5	3.7

Table 4 :

 4 F127 F F127 = 3%, NaCl 0.1 M, HA300, T = 37°C. Effect of HA concentration. Fit parameters from the SANS data analysis to a core-shell model for the form factor and a square-well potential for the structure factor. Same parameter definition as in Table3.

	F HA %					1%	2%	4%
	F m						0.10	0.11	0.11
	R c (Å)						45	45	45
	e sh (Å)					60	60	60
	r c (10	-6 Å	-2 )	0.4	0.4	0.4
	r sh (10	-6 Å	-2 )	5.8	5.8	5.8
	r solv (10	-6 Å	-2 )	6.4	6.4	6.4
	D m (Å)					214	207	198
	N mic						5	6	20
	x (Å)						75	60	47
	U 0 (k B T)				1	1.45	2.8

Table 5 :

 5

	100							HA12 NaCl	100
	I(cm -1 )	1 10							I(cm -1	10 1
	0.1							0.1
	0.01							0.01
		0.001				0.01	0.1	q (Å -1 )	0.001	0.01	0.1
	C NaCl (mol/L)	0.05	0.1	0.15	0.20	0.50
	F m							0.1	0.1	0.1	0.1	0.1
	Rc (Å)							45	45	45	45	45
	e sh (Å)						60	60	60	60	60
	r c (10	-6 Å	-2 )	0.4	0.4	0.4	0.4	0.4
	r sh (10	-6 Å	-2 )	5.8	5.8	5.8	5.8	5.8
	r solv (10	-6 Å	-2 )	6.4	6.4	6.4	6.4	6.4
	D m (Å)						224	217	209	207	201
	N mic							1	1	3	5	80
	x (Å)							60	60	60	60	60
	U 0 (k B T)					0.038	0.66	1.05	1.4	2.33

F127 F F127 = 3%, HA12, F HA = 2%, T = 37°C. Effect of the NaCl concentration. Fit parameters from analysis the SANS data to a core-shell model for the form factor model and a square-well potential for the structure factor. Same parameter definition as in Table

3

.

Table 8 :

 8 F127 F F127 = 3%, HA300 F HA = 2%, T = 37°C. Effect of the nature of the salt. Fit parameters to a coreshell form factor model and a square-well potential for the structure factor. Same parameter definition as in Table3.

	salt	NaCl	KCl	NaBr	KBr	CaCl 2	CaCl 2	CaCl 2
	F HA	0.02	0.02	0.02	0.02	0.02	0.02	0.02
	C salt (mol/L)	0.1	0.1	0.1	0.1	0.033	0.05	0.1
	F m	0.10	0.10	0.10	0.10	0.10	0.10	0.10
	Rc (Å)	45	45	45	45	45	45	45
	e sh (Å)	60	60	60	60	60	60	60
	r c (10 -6 Å -2 )	0.4	0.4	0.4	0.4	0.4	0.4	0.4
	r sh (10 -6 Å -2 )	5.8	5.8	5.8	5.8	5.8	5.8	5.8
	r solv (10 -6 Å -2 )	6.4	6.4	6.4	6.4	6.4	6.4	6.3
	D m (Å)	217	222	210	207	214	202	202
	N mic	1	1	3	2	4	15	45
	x (Å)	60	60	60	60	60	60	60
	U 0 (k B T)	0.6	0.55	0.7	0.7	0.9	1.85	2.29


T Morota
email: morota@eps.s.u-tokyo.ac.jp

S Sugita

Y Cho

M Kanamaru

E Tatsumi

N Sakatani

R Honda

N Hirata

H Kikuchi

M Yamada

Y Yokota

S Kameda

M Matsuoka

H Sawada

C Honda

T Kouyama

K Ogawa

H Suzuki

K Yoshioka

M Hayakawa

M Hirabayashi

H Miyamoto

T Michikami

T Hiroi

R Hemmi

O S Barnouin

C M Ernst

K Kitazato

T Nakamura

L Riu

H Senshu

H Kobayashi

S Sasaki

G Komatsu

N Tanabe

Y Fujii

T Irie

M Suemitsu

N Takaki

C Sugimoto

K Yumoto

M Ishida

H Kato

K Moroi

D Domingue

P Michel

C Pilorget

T Iwata

M Abe

M Ohtake

Y Nakauchi

K Tsumura

H Yabuta

Y Ishihara

R Noguchi

K Matsumoto

A Miura

N Namiki

S Tachibana

M Arakawa

H Ikeda

K Wada

T Mizuno

C Hirose

S Hosoda

O Mori

T Shimada

S Soldini

R Tsukizaki

H Yano

M Ozaki

H Takeuchi

Y Yamamoto

T Okada

Y Shimaki

K Shirai

Y Iijima

H Noda

S Kikuchi

T Yamaguchi

N Ogawa

G Ono

Y Mimasu

K Yoshikawa

T Takahashi

Y Takei

A Fujii

S Nakazawa

F Terui

S Tanaka

M Yoshikawa

T Saiki

S Watanabe

Y Tsuda

Sample collection from asteroid 162173 Ryugu by Hayabusa2: implications for surface evolution

The near-Earth asteroid 162173 Ryugu is thought to be a primitive carbonaceous object and to contain hydrated minerals and organic molecules. We report sample collection from Ryugu's surface by the Hayabusa2 spacecraft on 21 February 2019. Touchdown images and global observations of surface colors are used to investigate the stratigraphy of the surface around the sample location and across Ryugu. Latitudinal color variations suggest reddening of

exposed surface material by solar heating and/or space weathering. Immediately after touchdown, Hayabusa2's thrusters disturbed dark fine grains that originate from the redder materials. The stratigraphic relationship between identified craters and the redder material suggests that surface reddening occurred over a short period of time. We suggest that Ryugu has previously experienced an orbital excursion near the Sun.

Main Text:

Hayabusa2 is a sample-return mission to 162173 Ryugu, a carbonaceous near-Earth asteroid (NEA) [START_REF] Watanabe | Hayabusa2 mission overview[END_REF]. The spacecraft launched on 3 December 2014 and arrived at Ryugu on 27 June 2018. Initial global observations showed the asteroid has a spinning top-shape and rubblepile structure [START_REF] Watanabe | Hayabusa2 arrives at the carbonaceous asteroid 162173 Ryugu-A spinning top-shaped rubble pile[END_REF][START_REF] Hirabayashi | The western bulge of 162173 Ryugu formed as a result of a rotationally driven deformation Process[END_REF]. The surface has generally uniform spectra [START_REF] Kitazato | Surface composition of asteroid 162173 Ryugu as observed by the Hayabusa2 NIRS3 instrument[END_REF][START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF]. Small variations in surface color are quantified using the spectral slope from b-band (0.48 µm) to x-band (0.86 µm) (hereafter, b-x slope) [START_REF] Watanabe | Hayabusa2 arrives at the carbonaceous asteroid 162173 Ryugu-A spinning top-shaped rubble pile[END_REF][START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF]6) observed with the telescopic optical navigation camera (ONC-T) (Fig. 1A) [START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF]. Two compositional types of material are present on the surface with different colors: bluer material distributed at the equatorial ridge and in the polar regions, and redder material in the mid-latitude regions [START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF]. However, the cause of these spectral variations is not understood.

On 21 February 2019, Hayabusa2 conducted its first sample collection from Ryugu, performing a touchdown on the surface. During the touchdown operation, Hayabusa2 took images of Ryugu's surface with resolutions as high as ~1 mm pixel -1 . Those images allow us to observe the surface response to the physical disturbances generated by the touchdown, including the sampling projectile collision and firing of the thruster gas jets.

The touchdown site was selected based on established engineering safety criteria and scientific merits (2, 6, 7). Regional variations in the mixing ratio between redder and bluer materials are measured using the spectral slope (Fig. 1A). Spectral variations between candidate touchdown sites are much smaller than those within each site [START_REF] Watanabe | Hayabusa2 arrives at the carbonaceous asteroid 162173 Ryugu-A spinning top-shaped rubble pile[END_REF], implying that the surface materials are locally well-mixed. A sampling operation at any of the candidate sites should therefore collect both redder and bluer components. Safe landing locations were limited by the high boulder abundances [START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF][START_REF] Michikami | Boulder size and shape distributions on asteroid Ryugu[END_REF]. We initially intended the site designated L08-B, one of the lowest boulder density areas on Ryugu, as the primary landing site [START_REF] Watanabe | Hayabusa2 arrives at the carbonaceous asteroid 162173 Ryugu-A spinning top-shaped rubble pile[END_REF]6,[START_REF] Kikuchi | Design and reconstruction of the Hayabusa2 precision landing on Ryugu[END_REF] and deployed a target marker (TM) on October 23, 2018 to facilitate navigation. Based on the location where the TM settled and the detailed search for areas without boulders taller than 65 cm, which could reach Hayabusa2's reaction control system (RCS) during a touchdown, we finally chose L08-E1 (Fig. 2A andS5).

Hayabusa2 performed multiple low-altitude (~40 m) descent maneuvers near the L08 region, during which we conducted high-resolution (>0.01 m pixel -1 ) spectral and morphological observations. The touchdown site is generally slightly bluer than the global average, but reddish areas are found within L08-E1 (Fig. 1E and2C). These reddish areas are on average slightly darker than bluer areas (Fig. S6), a trend that is observed globally [START_REF] Watanabe | Hayabusa2 arrives at the carbonaceous asteroid 162173 Ryugu-A spinning top-shaped rubble pile[END_REF][START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF]. The reddish areas are limited to parts of individual boulders, with most boulder surfaces in this location being blue (Fig. 2C). These observations are compatible with the interpretation that Ryugu's boulders are originally bluer, with redder materials produced by surface metamorphic processes such as space weathering [START_REF] Lantz | Ion irradiation of carbonaceous chondrites: A new view of space weathering on primitive asteroids[END_REF], thermal metamorphism by solar heating [START_REF] Hiroi | Thermal metamorphism of the C, G, B, and F asteroids seen from the 0.7 μm, 3 μm, and UV absorption strengths in comparison with carbonaceous chondrites[END_REF], and/or pulverization by small impacts [START_REF] Cloutis | Spectral reflectance properties of carbonaceous chondrites 4: Aqueously altered and thermally metamorphosed meteorites[END_REF]. Redder materials may have been shed from boulder surfaces by impact disruption and/or thermal fatigue. The bluer surfaces of boulders remain un-reddened, implying that the timescale for surface reddening is longer than boulder resurfacing by impact disruption and/or thermal fatigue.

There are two morphological types of sub-meter-sized boulders around the touchdown site: dark ragged boulders and bright boulders with smooth surfaces (Fig. 2D andS7A). These types of boulders are observed in the ten-meter size range in remote images [START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF], and on smaller scales in images taken on the surface by the Mobile Asteroid Surface Scout (MASCOT) lander [START_REF] Jaumann | Images from the surface of asteroid Ryugu show rocks similar to carbonaceous chondrite meteorites[END_REF]. Images taken during the descent operation show that there is also a submeter-scale heterogeneity in surface reflectance of the bright boulders: the edges of many boulders are brighter than the planar surfaces of the same boulders (yellow arrows in Fig. 2E andS7B). This may be due to a higher abrasion rate at the boulder edges. One boulder in Fig. 2E appears to have broken in two, with the possible broken surface ~1.5 times brighter than surfaces of the surrounding boulders. Because the interior/less-processed part of the boulder is exposed on the boulder edges and broken surface, these observations suggest that boulder surfaces are generally darkened by exposure to space.

The Hayabusa2 spacecraft has a sampling mechanism [START_REF] Tachibana | Hayabusa2: Scientific importance of samples returned from C-type near-Earth asteroid (162173) 1999 JU3[END_REF] similar to that of the original Hayabusa spacecraft [START_REF] Yano | Touchdown of the Hayabusa Spacecraft at the Muses Sea on Itokawa[END_REF]. Ejecta are generated by the impact of a 1-cm-diameter tantalum projectile fired at a speed of ~300 m s -1 during the first contact with the surface, which are caught with a sampler horn extended from the bottom of the spacecraft [START_REF] Tachibana | Hayabusa2: Scientific importance of samples returned from C-type near-Earth asteroid (162173) 1999 JU3[END_REF][START_REF] Yano | Touchdown of the Hayabusa Spacecraft at the Muses Sea on Itokawa[END_REF][START_REF] Sawada | Hayabusa2 sampler: Collection of asteroidal surface material[END_REF].

The combination of the impact of the projectile fired from the sampler system and the operation of the RCS thrusters during the touchdown produced a large amount of debris from Ryugu's surface (Fig. 2G and Movie S1) (6). The video obtained by the nadir-viewing wideangle optical navigation camera (ONC-W1) indicates that large boulders (up to 1 m in the longest dimension) were moved horizontally by >5 m during the touchdown (Fig. S9). However, most of the debris disturbed during the touchdown was small pebbles and fine grains whose diameters are less than the pixel size (which varied from 1 to a few mm) of the W1 images (Fig. S8) (6). This high mobility of fragmental debris deposits (i.e., regolith) during the touchdown indicates that the cohesive forces between boulders/pebbles may be very weak, or that they depend on the particle sizes. This interpretation is consistent with the observed deficit of small craters [START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF][START_REF] Hirata | The spatial distribution of impact craters on Ryugu[END_REF], mass movement along slopes (i.e., mass wasting) on crater walls [START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF], and the crater formation by Hayabusa2's artificial impact experiment [START_REF] Arakawa | An artificial impact on the asteroid 162173 Ryugu formed a crater in the gravity-dominated regime[END_REF].

Immediately after the RCS thrust upon touchdown, the entire field-of-view of ONC-W1 was darkened uniformly. A dark ragged boulder, which we nickname "Turtle Rock" simultaneously became as bright as the surrounding brighter boulders (Fig. 2F, and 2H) (6). Turtle Rock was also moved by the RCS exhaust. These observations suggest that dark fine grains were originally present on the surfaces (or inside pores) of darker and redder boulders, before being lifted by the RCS thrusting. Such fine grains were not visible in the surface images taken by MASCOT [START_REF] Jaumann | Images from the surface of asteroid Ryugu show rocks similar to carbonaceous chondrite meteorites[END_REF]. The sampling process produced a cloud of dark fine grains that expanded from the touchdown site and extended to a zone ~10-m in diameter, centered at the touchdown site (Fig. 2G and2H). We estimate the total mass of the fine-grained cloud to be ~12 kg (6). The pre-touchdown color of this region was slightly bluer than the surrounding region, but it became redder after the deposition of the lofted dark fine grains (Fig. 2G and S11) (6). Observations with the Near Infrared Spectrometer (NIRS3) of the region before and after the touchdown showed little change in the OH band depth, although the NIRS3 spectra after the touchdown is slightly bluer and brighter (Fig. S12). This is consistent with the global lack of correlation between the spatial distribution of the OH band depth and the red-blue color variations observed by ONC [START_REF] Kitazato | Surface composition of asteroid 162173 Ryugu as observed by the Hayabusa2 NIRS3 instrument[END_REF][START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF]. We conclude that dark fine red grains originally concentrated on the boulder surfaces or in voids on the boulders, are not compositionally distinct from the subsurface, and the disruption by the touchdown event resulted in more even distribution of the grains across the affected area.

Globally, the b-x slope varies with latitude (Fig. 1A). We found that the b-x slope also correlates with the crater distribution. Fresh and stratigraphically younger craters >20 m in diameter have interiors that are spectrally bluer than their surroundings (Fig. 1C). This implies that the redder materials were covering the bluer materials, with the latter exposed by the crater formation. This is consistent with the stratigraphic relationship between the redder and bluer materials inferred from the global b-x slope distribution [START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF]. On the other hand, stratigraphically older craters tend to have redder interiors, and the color of the crater interiors is similar to that of surrounding materials (Fig. 1C). We investigated the contrasts in spectral slopes between crater interior surfaces and surrounding areas, which we define as the area within a distance of one crater radius outward from the crater rim (6). The histogram of the contrast in b-x spectral slope shows a bimodal distribution (Fig. 3), indicating that craters on Ryugu can be divided into two groups: red craters whose interior has b-x slope similar to that of their surroundings, and blue craters whose interior is bluer than their surroundings.

A potential explanation of the latitudinal variation in b-x slope is that the exposure of Ryugu's materials to space has led to their reddening, and mass wasting from the equator and polar regions (topographic highs) to the mid-latitude regions (topographic lows) has exposed fresh bluer subsurface materials [START_REF] Watanabe | Hayabusa2 arrives at the carbonaceous asteroid 162173 Ryugu-A spinning top-shaped rubble pile[END_REF][START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF]. The polar regions exhibit bluer spectra than the equatorial ridge (Fig. S3), suggesting that the reddening process seems to depend on illumination by the Sun (Ryugu's obliquity is 171.6˚ (2)). The color variation of crater interiors can be explained by their stratigraphic relations; the craters with redder interiors were formed before surface reddening occurred, while the bluer craters were formed after the surface reddening and the underlying bluer materials were exposed by the impacts. The bimodal distribution of the contrast in b-x spectral slope (Fig. 3) suggests that the surface reddening has not been active throughout the whole Ryugu's history and occurred mainly within a period after the formation of redder craters and before the formation of bluer craters. The surface reddening might not be currently active, or is active but slow compared with the resurfacing processes of boulder surfaces as discussed above (Fig. 2C).

We interpret the crater size-frequency distributions (SFDs) using collision frequency models derived for the asteroid main belt (6, [START_REF] Bottke | The fossilized size distribution of the main asteroid belt[END_REF][START_REF] O'brien | The collisional and dynamical evolution of the main-belt and NEA size distributions[END_REF]. From the SFD of red craters larger than 100 m in diameter (Fig. 3D), we estimate the time between the formation of Ryugu to the surface reddening event to be 8.5 Ma. This is much younger than the breakup time of candidate parent asteroids, Eulalia and Polana (several hundred Ma to ~1 Ga) [START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF][START_REF] Bottke | In search of the source of asteroid (101955) Bennu: Applications of the stochastic YORP model[END_REF], suggesting that Ryugu is the product of more than one generation of parent body disruption and/or global resurfacing processes such as the spinning top-shape formation had occurred until 8.5 Ma ago. The observed number density of blue craters is ~30 times lower than that of red craters (Fig. 3D). We estimate a model age for the reddening event of about 0.3 Ma, from the observed SFD of blue craters larger than 30 m diameter based on the main-belt collision frequency model (Fig. S14A). Using an alternative NEA collision frequency model, the age of the reddening event is estimated to be about 8 Ma, because there is a much lower collision frequency for bodies in NEA orbits (6, [START_REF] Bottke | The fossilized size distribution of the main asteroid belt[END_REF][START_REF] O'brien | The collisional and dynamical evolution of the main-belt and NEA size distributions[END_REF]. We interpret these ages as upper and lower limits of age estimates of the surface reddening. The NEA model age is younger than the typical dynamical lifetime of NEAs (~10 Ma) [START_REF] Gladman | The near-Earth object population[END_REF] and the median orbital lifetime of Ryugu (~40 Ma) (22). We therefore suggest that the reddening of Ryugu's surface occurred after its orbit shifted from the main belt to its current near-Earth orbit [START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF].

The deficit of craters smaller than 100 m in diameter on Ryugu's surface suggests that crater erasure processes have occurred [START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF]. Existing small craters must therefore have formed geologically recently. However, smaller craters (<10 m in diameter) do not always exhibit bluer interiors, which would be expected if they were all young. Some small craters exhibit a redder, not bluer, interior than the surroundings materials (Fig. 1E). Streaked patterns of redder materials such as ejecta deposits are visible across the whole surface (Fig. S4B). In addition, a streaked pattern of redder materials elongated from a boulder suggests that a mass of redder material collided with the boulder and dispersed (Fig. S4F). Redder materials may have been disrupted and redistributed by impacts, thermal fatigue, and mass wasting, which may have resulted in the formation of a mixed layer of redder and bluer materials after the surface reddening event (Fig. 4). This interpretation is supported by the distribution of redder materials on boulder surfaces and the existence of dark reddish fine grains observed in the touchdown operation. The thickness of the mixed layer of redder and bluer materials is estimated to be a few meters, derived from the minimum crater size (~10 m in diameter) that penetrates to the underlying blue materials. The presence of ejecta rays with a length of a few tens of meters that consist primarily of redder materials (Fig. S4F) implies that the redder material layer originally had a minimum thickness of a few tens of centimeters (6). Solar heating is more likely than space weathering to be the source of the reddening of Ryugu's surface, because space weathering typically affects only a thin layer of about 100 nm, while the diurnal and annual thermal skin depths (depth at which temperature decays to 1/e the surface temperature) are ≲10 cm and ~1.5 m, respectively [START_REF] Delbo | Temperature history and dynamical evolution of (101955) 1999RQ36: a potential target for sample return from a primitive asteroid[END_REF]24).

We suggest that a surface reddening event within a short period of time could be explained if Ryugu underwent a temporary orbital excursion near the Sun, causing higher surface heating. Such solar heating is consistent with the apparent deficiency in C-type asteroids bearing aqueous alteration features at 0.7-µm band in the NEA population [START_REF] Marchi | Heating of near-Earth objects and meteoroids due to close approaches to the Sun[END_REF]. However, solar heating on Ryugu during an orbital excursion cannot account for the low abundance of hydrous minerals revealed by global observations [START_REF] Kitazato | Surface composition of asteroid 162173 Ryugu as observed by the Hayabusa2 NIRS3 instrument[END_REF][START_REF] Sugita | The geomorphology, color, and thermal properties of Ryugu: Implications for parent-body processes[END_REF], because the bluish/brighter areas on Ryugu, which did not experience the intense solar heating, also have low abundance of hydrous minerals (4).

The large local variations in the spectral slope and albedo within the sampling site suggest that both bluer and redder components were likely collected during the touchdown. We predict that the returned sample will contain a mix of altered and unaltered materials, with the former recording a solar heating event. occurred within a short period after the emplacement of red craters and before the formation of blue craters. We interpret the cause of the surface reddening as solar heating while Ryugu came temporally closer to the Sun than at present. Between the formation of Ryugu's spinning topshape and the surface reddening we estimate a time of 9 Ma, based on the CSFDs of red craters. From the CSFDs of blue craters, the age of the surface reddening is estimated to be 0.3 Ma using the main-belt collision frequency model and 8 Ma using the NEA collision frequency model. We interpret these as upper and lower limits of age estimates of the surface reddening. After the surface reddening, the redder materials were disrupted and redistributed by impacts, thermal fatigue and mass wasting from the equator to mid latitude regions. A layer of mixed red and blue material subsequently formed on Ryugu's surface.

Figure 1 .

 1 Figure 1. Spectral slope of Ryugu's surface. (A) Global map of b-x slope (µm -1 ), indicated by the color bar, superimposed on a v-band image map. The white arrow indicates the location of the first touchdown (TD) point (4.30˚N, 206.47˚E). Craters larger than 20 m in diameter are shown by black circles. Dashed lines indicate areas shown in other panels. (B) v-band image (hyb2_onc_20180801_183933_tvf) obtained from 5.1 km altitude. Yellow arrows indicate craters with bluer interior than the surrounding materials. (C) b-x slope of the same region as panel B. Craters larger than 20 m in diameter are shown by white dashed circles. Blue craters B1 and B2 are higher in the stratigraphy than red craters R1, R2 and R3. There is no blue crater stratigraphically superposed by red craters, suggesting that the blue craters are younger than the red craters. (D) v-band image and (E) b-x slope image showing a 9-meter-sized crater with redder interior than the surrounding materials, close to the touchdown point marked with a white cross. Panels C and E are on the same color scale as A.

Figure 2 .

 2 Figure 2. Touchdown site before, during, and after the touchdown operation. (A) Boulder and crater map around the touchdown site L08-E1. The light blue arrows indicate the location of the target marker (TM) (4.04˚N, 206.01˚E) and the touchdown point of the sampler horn (TD) (4.30˚N, 206.47˚E) (7). The light blue circle indicates the L08-E1 area. The white dashed circles indicate craters. Boulder heights (H) were estimated from their shadow lengths; those with H > 1.8m are outlined in red, and H > 0.65 in pink. The boulder nicknamed "Turtle Rock" is indicated by the yellow arrow. The white box indicates the region shown in later panels. (B) and (C) p-b ratio images (6) calculated from b-and p-band (0.95 µm) images obtained during the touchdown rehearsal operation, from two different altitudes (hyb2_onc_20181015_134707_tbf and hyb2_onc_20181015_134655_tpf). The dashed boxes in B indicate regions shown in the other panels. (D) ONC-W1 image during the spacecraft descent before the touchdown (hyb2_onc_20190221_222859_w1f). The dark ragged Turtle Rock and an example of bright boulders with smooth surfaces (BB) are outlined in yellow and cyan dashed lines, respectively. The white dashed box indicates the area shown in panel E. (E) Close-up of the same image, with yellow arrows indicating fresh bright spots at corners and a possible broken plane of boulders. (F) and (G) ONC-W1 images obtained about 7 and 47 seconds after the touchdown, showing debris lifted from the surface (hyb2_onc_20190221_222917_w1f and hyb2_onc_20190221_222957_w1f). (H) ONC-T ul-band (0.39 µm) image obtained at 76 m altitude after the touchdown (hyb2_onc_20190221_223156_tuf). Turtle Rock was lifted clear of the surface by the exhaust from Hayabusa2's RCS thrusters, indicated by the yellow arrows in panels F-H.

Figure 3 .

 3 Figure 3. Crater statistics on Ryugu. Histograms of the differences in b-x slopes between the interior and the surroundings of craters larger than 10 m in diameter, in the latitude ranges (A) ±50˚, (B) ±10˚, and (C) -50˚ to -10˚ and 10˚ to 50˚. Bar colors are illustrative only. (D) Crater size-frequency distribution (CSFD) in the latitude range of ±50˚. Black, red and blue squares indicate crater frequencies of all craters, red craters (defined as having a difference of b-x slope of <0.025) and blue craters (difference of b-x slope of >0.025), respectively. Gray curves indicate cratering chronology models fitted to the data for red and blue craters. The dashed line indicates the empirical saturation level. Error bars are calculated by ±N 1/2 /A, where N is the cumulative number of craters and A is the area of the latitude range of ±50˚. The resulting model

Figure 4 .

 4 Figure 4. A schematic illustration of our suggested evolution of Ryugu. Surface reddening

Acknowledgments: The Hayabusa2 spacecraft was developed and built under the leadership of Japan Aerospace Exploration Agency (JAXA), with contributions from the German Aerospace Center (DLR) and the Centre National d'Études Spatiales (CNES), and in collaboration with NASA, Nagoya Univ., Univ. of Tokyo, National Astronomical Observatory of Japan (NAOJ), Univ. of Aizu, Kobe Univ., and other universities, institutes, and companies in Japan. We thank the many engineers, including Noriyasu Inaba at JAXA and Tetsuya Masuda, Seiji Yasuda, Kouta Matsushima, and Takeshi Ohshima at NEC Corp. for their dedicated work on the Hayabusa2 mission, Koshi Sato at NEC Corp. for ONC development, and Shingo Kashima at NAOJ for optical calculations. We are grateful to B.E. Clark and two anonymous reviewers for thoughtful and constructive comments in their review of this paper. We also thank W. F. Bottke for useful comments on the orbital evolution of NEAs. Funding: T.Mo., S.Su., N.S., S.Ka., M.M., S.Sa., M.Ab., R.N., A.M., K.W., and S.W. was supported by KAKENHI from the Japan Society for the Promotion of Science (JSPS) (Grant Nos. 17H06459, 19H01951, 17H01175, 17KK0097, 19H00727, 18H01267, 18K11610, 19K03955, 16H04044, 19K03958). This study was supported by the JSPS Core-to-Core program "International Network of Planetary Sciences." T.H. acknowledges funding support from NASA EW and PDART programs. M.Hi. acknowledges support from NASA/Solar System Workings (NNH17ZDA001N/80NSSC19K0548) and Auburn University's intramural research grant. O.S.B. acknowledges support from OSIRIS-REx under Contract NNM10AA11C issued through the NASA New Frontiers Program..D.D. and C.M.E. acknowledge funding through the NASA Hayabusa2 Participating Scientist Program. P.M. acknowledge funding from the French space agency CNES and from Academies of Excellence: Complex systems and Space, environment, risk, and resilience, part of the IDEX JEDI of the Université Côte d'Azur, and from the European Union's Horizon 2020 research and innovation programme under grant agreement No 870377

website (http://www.darts.isas.jaxa.jp/pub/hayabusa2/paper/Morota_2020/). Additional data from the mission are delivered to the DARTS archive at https://www.darts.isas.jaxa.jp/planet/project/hayabusa2/, and higher-level data products will be available in the Small Bodies Node of the Planetary Data System https://pdssmallbodies.astro.umd.edu/ one year after departure from the asteroid."

Supplementary Materials:

Materials and Methods Supplementary Text