Gaëlle Le Fer

Clémence Le Coeur

Jean-Michel Guigner

Catherine Amiel

Gisèle Volet

Amphiphilic diblock and triblock copolymers based on poly(2-methyl-2-oxazoline) and poly(D,L-lactide): Synthesis, physicochemical characterizations and self-assembly properties

Keywords: poly(2-methyl-2-oxazoline), block copolymers, micelles, Small-Angle Neutron Scattering

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

Introduction

Amphiphilic block copolymers capable of self-assembling into micellar structures drain a large interest over the last decades to improve hydrophobic drug encapsulation and delivery because of the mesoscopic size range and the high drug-loading capacity of the inner core. [START_REF] Adams | Amphiphilic block copolymers for drug delivery[END_REF][START_REF] Kwon | Block copolymer micelles as long-circulating drug vehicles[END_REF][START_REF] Kataoka | Block copolymer micelles for drug delivery: design, characterization and biological significance[END_REF][START_REF] Biswas | Recent advances in polymeric micelles for anti-cancer drug delivery[END_REF] In this aim, several ABA poloxamer poly(ethylene glycol)-b-poly(propylene oxide)-bpoly(ethylene glycol) (PEG-b-PPO-b-PEG), commercially available under the name pluronic received attention because of their self-assembly in core-shell micelles capable of passing physiological barriers such as the blood-brain barrier. [START_REF] Kabanov | A new class of drug carriers: micelles of poly (oxyethylene)-poly (oxypropylene) block copolymers as microcontainers for drug targeting from blood in brain[END_REF][START_REF] Meng | Pluronic F127 and D-α-Tocopheryl Polyethylene Glycol Succinate (TPGS) Mixed Micelles for Targeting Drug Delivery across The Blood Brain Barrier[END_REF] The PPO block ensures the hydrophobic cohesion and subsequently the encapsulation of hydrophobic active compounds while the PEG block is the shell-stabilizing segment. Due to its high water solubility, remarkable stealth properties and apparent low toxicity, many efforts have been carried out to elaborate a large library of amphiphilic copolymers based on PEG. In this way, Kataoka and co-workers [START_REF] Scholz | A novel reactive polymeric micelle with aldehyde groups on its surface[END_REF][START_REF] Nagasaki | The reactive polymeric micelle based on an aldehyde-ended poly (ethylene glycol)/poly (lactide) block copolymer[END_REF] performed a one-pot synthesis of α-acetal-poly(ethylene glycol)-b-poly(D,Llactide) (α-acetal-PEG-PLA). Then, the dialysis against water was employed to prepare polymeric micelles of 31 nm, after the solubilization of block copolymer in dimethylacetamide (DMAc), a good solvent for both segments. More recently, amphiphilic copolymers based on PEG and poly(amino acid) block able to form nanometric structures showed a large interest for drug and gene delivery, [START_REF] Osada | Polymeric micelles from poly (ethylene glycol)poly (amino acid) block copolymer for drug and gene delivery[END_REF] in part because polypeptide conformation can influence their aggregation behavior in aqueous medium. [START_REF] Lin | Aggregate structure change induced by intramolecular helix-coil transition[END_REF][START_REF] Priftis | Self-Assembly of α-Helical Polypeptides Driven by Complex Coacervation[END_REF] However, several studies revealed important drawbacks of PEG. Due to the intensive use of PEG, PEGylated systems have lost their stealth function when applied in vivo, because of the existence of non-specific and specific recognition by the immune system. Indeed, specific antibodies were detected in the serum of patients treated with PEG-asparaginase [START_REF] Armstrong | Antibody against poly (ethylene glycol) adversely affects PEG-asparaginase therapy in acute lymphoblastic leukemia patients[END_REF] and PEGuricase. [START_REF] Sherman | PEG-uricase in the management of treatment-resistant gout and hyperuricemia[END_REF] In addition, PEG-specific antibodies were identified in 25 % of patients who had never received treatment based on PEG, due to the presence of PEG in many food and cosmetic products. [START_REF] Leger | AMER ASSOC BLOOD BANKS 8101[END_REF][START_REF] Armstrong | AMER SOC HEMATOLOGY 1900 M STREET. NW SUITE 200[END_REF] These antibodies have a neutralizing effect and cause a significant loss of the expected therapeutic effect.

To solve this problem, different polymer families are considered as possible alternatives to PEG for biomedical uses: poly(vinylpyrrolidone) (PVP), poly(N-(2-hydroxypropyl) methacrylamide) (PHPMA), synthetic hydrophilic polypeptides and poly(oxazoline)s. [START_REF] Barz | Overcoming the PEG-addiction: well-defined alternatives to PEG, from structure-property relationships to better defined therapeutics[END_REF] Particularly poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx) are often compared to PEG because they present similar chain flexibility in physiological media, [START_REF] Gubarev | Conformational properties of biocompatible poly (2-ethyl-2oxazoline) s in phosphate buffered saline[END_REF] same stealth behavior towards plasmatic protein [START_REF] Hoogenboom | Poly (2-oxazoline) s: a polymer class with numerous potential applications[END_REF][START_REF] Adams | Poly (2-oxazolines) in biological and biomedical application contexts[END_REF][START_REF] Luxenhofer | Poly (2oxazoline) s as Polymer Therapeutics[END_REF] and a better biocompatibility. [START_REF] Luxenhofer | Structure-property relationship in cytotoxicity and cell uptake of poly (2-oxazoline) amphiphiles[END_REF][START_REF] Bauer | Poly (2-ethyl-2-oxazoline) as Alternative for the Stealth Polymer Poly (ethylene glycol): Comparison of in vitro Cytotoxicity and Hemocompatibility[END_REF] Amphiphilic diblock and triblock copolymers based on PMeOx have been reported in the literature. [START_REF] Nardin | Polymerized ABA triblock copolymer vesicles[END_REF][START_REF] Rasolonjatovo | Poly (2-methyl-2-oxazoline)-b-poly (tetrahydrofuran)-b-poly (2-methyl-2-oxazoline) Amphiphilic Triblock Copolymers: Synthesis, Physicochemical Characterizations, and Hydrosolubilizing Properties[END_REF][START_REF] Kempe | Discovering new block terpolymer micellar morphologies[END_REF][START_REF] Bonné | Aggregation behavior of amphiphilic poly (2-alkyl-2-oxazoline) diblock copolymers in aqueous solution studied by fluorescence correlation spectroscopy[END_REF][START_REF] Krumm | Well-Defined Amphiphilic Poly (2-oxazoline) ABA-Triblock Copolymers and Their Aggregation Behavior in Aqueous Solution[END_REF] In these works, hydrophobic blocks were of different natures, going from fluid blocks made of low glass transition temperature (Tg) polymers such as polydimethylsiloxane, [START_REF] Nardin | Polymerized ABA triblock copolymer vesicles[END_REF] poly(tetrahydrofuran) [START_REF] Rasolonjatovo | Poly (2-methyl-2-oxazoline)-b-poly (tetrahydrofuran)-b-poly (2-methyl-2-oxazoline) Amphiphilic Triblock Copolymers: Synthesis, Physicochemical Characterizations, and Hydrosolubilizing Properties[END_REF] or poly(2-(1-ethylheptyl)-1,3-oxazoline) [START_REF] Kempe | Discovering new block terpolymer micellar morphologies[END_REF] or poly(2-nonyl-2-oxazoline) [START_REF] Bonné | Aggregation behavior of amphiphilic poly (2-alkyl-2-oxazoline) diblock copolymers in aqueous solution studied by fluorescence correlation spectroscopy[END_REF] to frozen blocks made of high Tg polymers such as poly(2phenyl-2-oxazoline). [START_REF] Krumm | Well-Defined Amphiphilic Poly (2-oxazoline) ABA-Triblock Copolymers and Their Aggregation Behavior in Aqueous Solution[END_REF] Despite the well-known interesting and often reported properties of poly(lactide) (PLA), such as biocompatibility and biodegradability, [START_REF] Anderson | Biodegradation and biocompatibility of PLA and PLGA microspheres[END_REF][START_REF] Ramot | Biocompatibility and safety of PLA and its copolymers[END_REF] and extensive description within PEG/PLAbased systems both in term of synthesis strategies, biofunctionalization, colloidal stability, physico-chemical characterization and drug encapsulation, [START_REF] Scholz | A novel reactive polymeric micelle with aldehyde groups on its surface[END_REF][START_REF] Nagasaki | The reactive polymeric micelle based on an aldehyde-ended poly (ethylene glycol)/poly (lactide) block copolymer[END_REF][START_REF] Riley | Colloidal stability and drug incorporation aspects of micellar-like PLA-PEG nanoparticles[END_REF][START_REF] Riley | Physicochemical evaluation of nanoparticles assembled from Poly (lactic acid)-Poly (ethylene glycol)(PLA-PEG) block copolymers as drug delivery vehicles[END_REF][START_REF] Salem | Synthesis and characterisation of a degradable poly (lactic acid)-poly (ethylene glycol) copolymer with biotinylated end groups[END_REF][START_REF] Riley | Core-shell structure of PLA-PEG nanoparticles used for drug delivery[END_REF] only a few works were related to self-assembly study of amphiphilic block copolymer combining poly (2-oxazoline) with PLA, which behave as frozen block at ambient temperature. [START_REF] Wang | Synthesis and characterization of temperature-and pHsensitive hydrogels based on poly (2-ethyl-2-oxazoline) and poly (D, L-lactide)[END_REF] Moreover, the control of the characteristics of polymeric micelles, such as size, shape and surface properties characterization, have crucial importance, to optimize drug encapsulation and then delivery as well as to maintain colloidal staibilty. [START_REF] Decuzzi | Size and shape effects in the biodistribution of intravascularly injected particles[END_REF][START_REF] Yang | Advances in particle shape engineering for improved drug delivery[END_REF] In addition, the distribution of drug-loaded polymeric micelles in the body may be determined mainly by their size and morphology and is less affected by the properties of loaded drugs if these are embedded in the inner core of the micelles. Indeed, a nanometric size range from 10 to 200 nm is a crucial factor in determining their body disposition, especially when an enhanced permeation retention effect (EPR effect) is involved. [START_REF] Yokoyama | Characterization and anticancer activity of the micelle-forming polymeric anticancer drug adriamycin-conjugated poly (ethylene glycol)-poly (aspartic acid) block copolymer[END_REF][START_REF] Maeda | Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review[END_REF][START_REF] Kobayashi | Improving conventional enhanced permeability and retention (EPR) effects; what is the appropriate target?[END_REF][START_REF] Matsumura | A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs[END_REF] Consequently, a thorough characterization of their size and structure at nanometer resolution is a prerequisite. It should be emphasized that contrary to the literature works where the middle block was hydrophobic, the hydrophilic block was set in the middle of the triblock copolymers synthesized, leading to another variety of supramolecular structures. Two processes were used, the nanoprecipitation and the dialysis to rationalize the relationship between copolymer structure, method of self-assembly and the morphology of the micelles by using dynamic light scattering (DLS), small angle neutron scattering (SANS) and cryo-TEM.

Materials and methods

Materials

The high-molecular-mass poly(D,L-lactide) (PLA) was purchased from Cargill Laboratories (Cedar Grove, NJ, USA). The monomer 2-methyl-2-oxazoline (Sigma-Aldrich, Saint-Louis, MO, USA, purity 99 %) were dried overnight over calcium hydride and purified by distillation under an argon atmosphere. Sodium azide (NaN3) (BioUltra, purity ≥ 99.5 %), anhydrous acetonitrile (ACN) (purity 99,8%), 1-iodobutane (purity 99%), 1,3-diiodopropane (purity 99%) were purchased from Sigma Aldrich and used as received. Dimethylsulfoxide (DMSO), diethyl ether and dichloromethane were purchased from Carlo Erba (Val de Reuil, France) and used without extra purification. N,N-Dimethylformamide (DMF) was purchased from VWR (Radnor, Pennsylvania, USA). Copper commercially available nanopowder (Cu NPs) with nominal size from 20 nm to 40 nm was purchased from Alfa Aesar (Haverhill, MA, USA).

Polymers synthesis

Synthesis of functionalized polymers: alkynyl-terminated poly(D,L-lactide) and azideterminated poly(2-methyl-2-oxazoline)

Alkynyl-terminated poly(D,L-lactide) (PLA≡) oligomers were prepared via transesterification of high-molecular-mass PLA ( = 8.72×10 4 g.mol -1 , Đ = = 2) as previously reported. in dry acetonitrile (ACN) (12.0 mL) was stirred at 80 °C, under dry argon. After 5 hours, the necessary time for total monomer consumption, determined by the kinetic study previously conducted, [START_REF] Fer | Copolymers based on azidopentyl-2-oxazoline: synthesis, characterization and LCST behavior[END_REF] polymer was functionalized by adding an excess of NaN3, and the reaction mixture was stirred for 24 hours still at 80 °C. The polymer obtained was purified by precipitation in diethyl ether, filtered and dried under vacuum at 60 °C to afford a white powder (yield = 95 %). Two polymers were synthesized with different number-average molecular masses : 2.2×10 3 and 5.5×10 3 g.mol -1 . A similar procedure was used to synthesize telechelic polymers N3-PMeOx-N3 using a bifunctional initiator, the 1,3diiodopropane and knowing the polymerization constant. [START_REF] Volet | Click-Chemistry: An Alternative Way to Functionalize Poly (2-methyl-2-oxazoline)[END_REF] The telechelic polymers obtained had of 2.2×10 3 and 8.2×10 3 g.mol -1 .

Synthesis of amphiphilic copolymers

Nanoprecipitation and dialysis methods

Self-assembly of diblock copolymers were prepared using the nanoprecipitation method: 1 ml of solution of copolymer in acetonitrile (ACN) with a concentration of 2 g.L -1 was stirred and added dropwise in 2 mL of ultrapure water at 25 °C stirred at 220 rpm. The organic solvent was removed from the dispersion by evaporation under reduced pressure at room temperature and the volume was adjusted by addition of ultrapure water to obtain a concentration of 1 g.L - 1 .

Self-assembly of triblock copolymers were prepared with the same nanoprecipitation procedure and by the dialysis method. Concretely, a solution of copolymer in ACN at a concentration of 1 g.L -1 was dialyzed against water for 2 days (Spectra/Por dialysis tubing, molecular weight cut off 6-8000 Da). The volume of water was 500 mL and was renewed 5 times per day. Finally, the volume of the suspension was adjusted by evaporation under vacuum to obtain a concentration of 1 g.L -1 .

Instrumentation and Measurements

Fourier-transform infrared spectroscopy (FTIR) spectra were recorded on a Bruker Tensor 27 instrument equipped with a Digi Tect DLaTGS detector, 32 scans were collected at a resolution of 1 cm -1 using an ATR accessory (single-reflection horizontal ATR, Miracle, Pike Technologies). Chemical structures of the polymers were investigated using a Raman apparatus Xplora from Horiba Jobin Yvon (Longjumeau, France) equipped with a laser emitting at 638 nm. The acquisition time was fixed at 1 min. Santa Barbara, CA, USA). In the case of DMF eluent, SEC was performed on a Waters equipment with refractive index detector and a Shodex KD-806M column that was calibrated with poly(ethylene oxide) standards. The chromatographic analysis of polymers was done with all polymer solutions at a concentration of 10 mg mL -1 .

Thermogravimetric analyses (TGA) were performed on a Setaram Setsys Evolution 16 apparatus by heating the samples at a rate of 10 °C.min -1 from 20 °C to 800 °C under argon atmosphere.

To determine the hydrodynamic radius (Rh) and the polydispersity index (PDI, a dimensionless measure of the broadness of the size distribution), dynamic light scattering (DLS) measurements were performed on a Malvern Instruments Zetasizer Nano ZS operating with a He-Ne laser source at the wavelength of 633 nm. All measurements were performed at 25 °C and at a scattering angle of 173°. The concentration of the solutions was 1 mg.mL -1 .

The autocorrelation functions (g1(t)) were analyzed in terms of relaxation time distribution (τ) (equation 1).

g1(t) = ( ) exp ( ) (1)
Hydrodynamic radius (Rh) was determined from the Stokes-Einstein relation (equation 2).

Rh = (2)

Where D0 is diffusion coefficient, ηs ids the viscosity of the solvent, T is absolute temperature and kB the Boltzmann constant. The correlation functions were also analyzed using the cumulants method to derive the mean radii and polydispersity index (PDI) of the nanoparticles. In case of PDI lower than 0.3, the distribution width, σDLS, can be approximated as (PDI) 1/2 .

For the cryo-transmission electron microscopy (Cryo-TEM), a drop of NPs suspension was deposited on "quantifoil"® (Quantifoil Micro Tools GmbH, Germany) carbon membrane.

The excess of liquid on the membrane was blotted out with a filter paper and, before evaporation, the membrane was quench-frozen in liquid ethane to form a thin vitreous ice film in which NPs were captive. Once mounted in a Gatan 626 cryo-holder cooled with liquid nitrogen, the samples were transferred in the microscope and observed at low temperature (-180 °C). Cryo-TEM images were recorded on an ultrascan 2k ccd camera (Gatan, USA), using a LaB6 JEOL 2100 (JEOL, Japan) cryo-microscope operating at 200kV with a JEOL low dose system (Minimum Dose System, MDS) to protect the thin ice film from any irradiation before imaging and reduce the irradiation during the image capture.

Small-angle neutron scattering (SANS) measurements were performed at the Laboratoire Le ón Brillouin ("ORPHEE" reactor, CEA Saclay) on the "PACE" spectrometer. The experimental scattering vector q (q = (4π / λ) sin(θ/2)) range was 0.0032 < q (Å -1 ) < 0. Scattering intensities from solutions were corrected for empty cell scattering and sample transmission. I(q) is in absolute scale (cm -1 ). For all samples, the solvent scattering intensity was subtracted, but we have not succeeded in completely removing the sample background (which is inferior to 1×10 -3 cm -1 ). All colloidal suspensions were prepared as previously explained and then dialyzed against D2O, and the concentration was 1 g.L -1 unless specified.

Fits: 2 types of model fitting provided by SasView software (http://www.sasview.org) were used. Polydisperse sphere model describes a sphere with a lognormal distribution on the radius rs (Equation 3).

!(", $) = % & '3)(* + -* +-./ )0 +1 (", $)2² (3)
Where Psp(q,r) is the form factor for a monodisperse spherical particle with uniform scattering length density ρs, ρs value has been fixed as the scattering length density of PLA.

Polydisperse core-multishell model (Equation 4) consists in a core of radius rc, spherical shells with thicknesses δ1 (δ1 = rs1 -rc) and δ2 (δ2 = rs2 -rs1), rs1 and rs1 being respectively the radius of the first and the second shell. We applied a lognormal distribution for rc, rs1 and rs2.

!4", $ 5, $ +6 , $ +7, 8 = % & 93) 5 (* 5 -* +6 )0 +1 (", $ 5 ) + 3) +6 (* +6 -* +7 )') +6 0 +1 (", $ +6 ) - ) 5 0 +1 (", $ 5 )2 + 3) +7 (* +7 -* +-./ )') +7 0 +1 (", $ +7 ) -) +6 0 +1 (", $ +6 )2; ² (4)
Where 0 +1 (", $) has been previously defined. ρc, ρs1, ρs2, ρsol are respectively the neutron scattering length density of the core, the shell 1, the shell 2 and the solvent.

Results and discussion

Synthesis and characterization of di-and triblock copolymers.

First, the functionalized polymers were prepared by cationic ring opening polymerization (CROP) for poly(2-methyl-2-oxazoline) (PMeOx) and by transesterification for poly(D,Llactide) (PLA). In a second step, the block copolymers were built by "click" chemistry. The CROP of 2-methyl-2-oxazoline was performed, as described previously, [START_REF] Krumm | Well-Defined Amphiphilic Poly (2-oxazoline) ABA-Triblock Copolymers and Their Aggregation Behavior in Aqueous Solution[END_REF][START_REF] Kempe | Discovering new block terpolymer micellar morphologies[END_REF] in anhydrous ACN at 80 °C, using 1-iodobutane or 1,3-diiodopropane as initiator and NaN3 as termination agent to obtain α-azide-terminated poly(2-methyl-2-oxazoline) (PMeOx-N3) and α-, ω-azidefunctionalized poly(2-methyl-2-oxazoline) (N3-PMeOx-N3) respectively. The polymers were fully characterized by [START_REF] Adams | Amphiphilic block copolymers for drug delivery[END_REF] H NMR and SEC. The 1 H NMR spectra of the PMeOx-N3 and N3-PMeOx-N3 (Figure S1 and S2) were performed in D2O and CDCl3 respectively. The degrees of polymerization and consequently the number-average molecular masses ( < = ) of the polymers were calculated using the integration of the peaks 1 at 2.08 ppm, corresponding to the repetitive unit and the integration of the peak a at 0.92 ppm corresponding to the protons of methyl end group in the case of PMeOx-N3 or the integration of the peaks a at 1.18 ppm corresponding to the methylene protons of the bifunctional initiator in the case of N3-PMeOx-N3. The polymers obtained were characterized by SEC in aqueous solution with LS detector to obtain absolute number-average molecular masses >?@ reported in Table S1. A good agreement was observed between the < = , >?@ and the expected molecular masses calculated from the feed ratio AB1A5 AC . Chromatograms highlighted monomodal distributions (Figure S3) with narrow dispersities indicating the controlled character of the polymerizations. The presence of the azide end groups, for all polymers, was confirmed by FTIR with the band at 2097 cm -1 (Figure S4). Alkynyl-terminated PLA (PLA≡) was previously prepared by direct transesterification reaction as reported elsewhere. [START_REF] Kempe | Discovering new block terpolymer micellar morphologies[END_REF] This method has the major advantage to produce oligomers with an alkynyl end group in a onestep reaction. The average-number molecular mass and the chemical structure of the alkynylterminated PLA were previously confirmed by SEC, [START_REF] Adams | Amphiphilic block copolymers for drug delivery[END_REF] H NMR and Raman spectroscopy. [START_REF] Fer | Biocompatible Soft Nanoparticles with Multiple Morphologies Obtained from Nanoprecipitation of Amphiphilic Graft Copolymers in a Backbone-Selective Solvent[END_REF] A series of five oligomers were prepared with number-average molecular masses ranging from 1.2×10 3 to 5.2×10 3 g.mol -1 with dispersity values from 1.3 to 1.5.

The di-and triblock copolymers were prepared using the copper-catalyzed Huisgen The copolymers were characterized by SEC in DMF using a poly(ethylene oxide) (PEO) calibration curve. The chromatograms have shown monomodal distributions, as reported in Figure S5 for a diblock and a triblock copolymer. This is a first indication of the coupling efficiency between the different blocks. The quantitative results are reported in Table S2. For comparison, the homopolymers PLA≡ , PMeOx-N3 and N3-PMeOx-N3 were also analyzed in DMF. For PLA≡ , there is a good agreement between the >?@ and AB1A5 AC but the molecular weights determined for PMeOx are lower than the expected values and are also lower than the molecular weights determined by light scattering detector in aqueous eluent (Table S1). This discrepancy could be attributed to a more compact conformation of PMeOX chains than PEO standards. The molecular weights determined for the copolymers indicated also a similar discrepancy between the molecular weights determined from the PEO calibration curve and AB1A5 AC and this can be attributed to the more compact conformations of the copolymers in DMF than PEO chains. Nevertheless, the molecular weights increased, as expected, with the molar masses of each block.

The efficiency of the coupling reaction between the azido and alkynyl end groups was then demonstrated by FTIR and Raman spectroscopy, two perfectly complementary techniques.

The success of the cycloaddition reaction, for the diblock and triblock copolymers, was confirmed by FTIR with the total disappearance of the azide signal at 2097 cm -1 (Figure 1A)

and by Raman spectroscopy (Figure 1B) with the disappearance of the signal corresponding to the alkyne end function of the PLA at 2131 cm -1 . f: hydrophilic weight ratio (ratio of the mass of the hydrophilic part to the total mass)

Self-assembly study of the diblock copolymers

Self-assemblies of the diblock copolymers were prepared by nanoprecipitation, that is commonly known for resulting in kinetically trapped structures due to rapid solvent exchange. [START_REF] Lepeltier | Nanoprecipitation and the "Ouzo effect": Application to drug delivery devices[END_REF] First, the PMeOx-b-PLA diblock copolymers were dissolved in acetonitrile (ACN) (2 g L -1 ), which is a solvent for both PMeOx and PLA polymers and has a good miscibility with water. The solution was added dropwise to deionized water, under moderate stirring, inducing the self-assembly process. The organic solvent was then removed under vacuum and water added to reach the desired concentration. Herein, the different diblock copolymers used were, in increasing order of hydrophilic weight ratio f: PMeOx2200-b-PLA5200 (f = 30 %), PMeOx2200-b-PLA2800 (f = 44 %), PMeOx5500-b-PLA5200 (f = 52 %) and PMeOx5500-b-PLA2800 (f = 66 %).

The resulting suspensions were characterized by dynamic light scattering (DLS) at 173°, the results showed the presence of nano-objects with hydrodynamic radius (Rh) between 14 nm and 24 nm according to the composition of the amphiphilic copolymer, as well as objects of several hundred nanometers (as shown in the Figure 5.A for the distribution based on PMeOx2200-b-PLA2800). The distribution widths (σDLS) are high, of the order of 0.5, because of the bimodal size distribution (only in intensity, volume size distributions showed a unimodal distribution). The Rh (from volume size distribution) and σDLS values are reported in Table 3.

The cryo-TEM micrographs of the suspension based on PMeOx2200-b-PLA2800 (Figure 5.B) allowed observing spherical objects with an average radius of 15 nm (average radius was calculated on the basis of at least 100 nano-objects using imageJ software) which is extremely close to the Rh obtained by DLS. It should be noted the absence of large aggregates, around about 200 nm, in cryo-TEM, suggesting that they are few, which is consistent with their presence in DLS only in intensity, not in volume. Small-angle neutron scattering (SANS) was used to probe more precisely and quantitatively the structure of the nano-objects and to measure better averaged nanoscopic dimensions of the objects through an appropriate model. As said before, the suspensions were analyzed in D2O at a concentration of 1 g.L -1 at room temperature. Figure 6 shows the scattered intensity as a function of scattering vector (q) for nano-objects based on PMeOx2200-b-PLA5200 (f = 30%) (pink circle), PMeOx2200-b-PLA2800 (f = 44%) (red square) and PMeOx5500-b-PLA52000

(f = 52 %) (orange triangle) (for better visibility, scattered intensities of red and orange curves were multiplied by a power of 10 on the graph). The general appearance of the three curves was similar for large and medium q values. Indeed, the intermediate q value regime (2.0×10 -2 < q (Å -1 ) < 8.0×10 -2 ) is characterized by a variation in q -4 and might correspond to a compact structure. While for larger values of q (8.0×10 -2 < q (Å -1 ) < 4.4×10 -1 ), the decrease of log I was less abrupt. This variation might be characteristic of an interface of dangling polymer chains. However, the absence of a variation in q -2 was due to the impossibility to subtract more incoherent from the solvent. Nevertheless, for small values of scattering vector q, it is possible to observe, for samples based on PMeOx2200-b-PLA2800 and PMeOx5500-b-PLA5200, a plateau (Guinier regime) characteristic of a size approachable by SANS, while for samples based on PMeOx2200-b-PLA5200 this plateau 1 was not reached which might correspond to a greater polydispersity and / or larger radius of 2 gyration.

3

The SANS data were fitted using a polydisperse sphere model. The dispersities due to the 4 detector, on the I and q values, have been considered. The traces of the fits obtained are in 5 black on the Figure 6. The parameters of the fits (RSANS and σSANS) are reported in Table 3. One can notice that RSANS were always lower than Rh for all the samples analyzed by the two 13 methods. For instance, for PMeOx2200-b-PLA2800, RSANS = 9.9 nm which was lower than Rh = 3. In all cases, RSANS was lower than LC(PLA). Moreover, the thickness of the corona layer, roughly estimated from the difference between Rh and RSANS were also always lower than LC(PMeOx). The aggregates had thus likely a micellar core-shell structure with a core made of PLA blocks' self-assembly and a corona of non-extended PMeOx.

The results showed that with the same size of the hydrophilic PMeOx block, the core radius increased with an increase of the hydrophobic PLA block polymer length, from 9.9 nm for PMeOx2200-b-PLA2800 to 16.7 nm for PMeOx2200-b-PLA5200. Moreover, the thickness of the PMeOx shell increased with the PMeOx length, as roughly represented by the difference Rh -RSANS which was about 5.0 -7.0 nm for PMeOx2200 and 16.0 nm for PMeOx5200. These observations are consistent with previous studies on other linear amphiphilic diblock copolymers. [START_REF] Szymusiak | Core-Shell Structure and Aggregation Number of Micelles Composed of Amphiphilic Block Copolymers and Amphiphilic Heterografted Polymer Brushes Determined by Small-Angle X-ray Scattering[END_REF] In order to demonstrate the coherence of this analysis, aggregation numbers Nagg were estimated assuming that the core was made of only PLA blocks and contained no solvent (equation 6):

^_``= ^ abc ( 4e$ Xf$g 3 3 ) (6)
Where hij was the PLA number-average molecular mass obtained by 1 H NMR, NA the Avogadro number, d the density of the PLA block equal to 1.25 g.cm -3 and rcore estimated as RSANS.

The data are presented in Table 3. Nagg are ranging from 500 to 2800. These are relatively high values compared to the ones reported for micellar surfactants (less than few hundred). [START_REF] Israelachvili | Intermolecular and surface forces[END_REF] The unusually high Nagg values were mainly due to the formation of "irregular" micelles made of a frozen PLA core. However, these values were always lower than the maximum Nagg values calculated for a given PLA block and obtained by replacing rcore by Lc(PLA) in equation 6. (Nagg) max were equal to 1700 and 10600 for PLA blocks of 2800 and 5200 g.mol -1 respectively. More quantitatively, Nagg decreased when the hydrophilic weight fraction f increased.

Discher [START_REF] Discher | Polymer vesicles[END_REF]49 proposed an unified empirical rule based on the ratio f of the molecular mass of the hydrophilic part to the total molecular mass: polymersomes are generally observed in the f range 25 % -40 %, worm like micelles in the f range 40 % -50 %, and spherical micelles for higher f values. Several authors verified this rule and reported the formation of core-shell micellar structures for PMeOx-based diblock copolymers in which the hydrophilic weight ratio f was larger than 50 % 26,50 but also for triblock copolymers with a hydrophobic middle block. For instance the self-assembly of poly(2-methyl-2-oxazoline)-b-poly(tetrahydrofuran)b-poly(2-methyl-2-oxazoline) (PMeOx-b-PTHF-b-PMeOx) amphiphilic triblock copolymers was previously reported by Rasolonjatovo et al., [START_REF] Rasolonjatovo | Poly (2-methyl-2-oxazoline)-b-poly (tetrahydrofuran)-b-poly (2-methyl-2-oxazoline) Amphiphilic Triblock Copolymers: Synthesis, Physicochemical Characterizations, and Hydrosolubilizing Properties[END_REF] they showed the increase of the hydrodynamic radius of micelles when the PMeOx length increased and the PTHF length remained constant. On the other hand, well defined polymersome morphologies have been obtained with PMeOx-b-PDMS diblock copolymers as well as PMeOx-b-PDMS-b-PMeOx triblock copolymers within the expected hydrophilic weight ratio range, between 20 and 30 %. [START_REF] Nardin | Polymerized ABA triblock copolymer vesicles[END_REF][START_REF] Dieu | Polymersomes conjugated to 83-14 monoclonal antibodies: in vitro targeting of brain capillary endothelial cells[END_REF] When the hydrophobic middle block (poly(2-phenyl-2-oxazoline)) was glassy, polymersomes were also observed, for a hydrophilic weight ratio of 50 % but in coexistence with micelles. [START_REF] Krumm | Well-Defined Amphiphilic Poly (2-oxazoline) ABA-Triblock Copolymers and Their Aggregation Behavior in Aqueous Solution[END_REF] Le Meins et al. [START_REF] Meins | Recent trends in the tuning of polymersomes' membrane properties[END_REF] recalled that for polymers, the obtained morphologies not only results from geometrical parameters but an entropy and enthalpy loss must be taken into account during the self-assembling process. In our case, multiple morphologies were expected as f was varied between 30 and 66 %. However, only the spherical micelles morphologies were clearly identified in this work. The other morphologies (vesicles, wormlike micelles) were not reached. This could be due to the kinetically trapped system led by the kinetic of solvent diffusion faster than the kinetics of self-assembly (by nanoprecipitation), leading to frozen morphologies. Similar findings have also been reported of PEG-co-PLA copolymers with f ratio close to 0.2 using a flash nanoprecipitation [START_REF] Szymusiak | Core-Shell Structure and Aggregation Number of Micelles Composed of Amphiphilic Block Copolymers and Amphiphilic Heterografted Polymer Brushes Determined by Small-Angle X-ray Scattering[END_REF] method whereas micrometric sized vesicles where observed using a slow water dissolution process with f in the range 0.2 -0.42. 54 This phenomenon confirms that even if it is reported that the morphologies are in part predetermined by the macromolecular composition, the material morphology can also be controlled by the self-assembly process used. [START_REF] Huang | Biologically active polymersomes from amphiphilic glycopeptides[END_REF][START_REF] Fer | Design and self-assembly of PBLG-b-ELP hybrid diblock copolymers based on synthetic and elastinlike polypeptides[END_REF] Self-assembly study of the triblock copolymers

Next we studied the self-assembly behavior of the ABA triblock copolymers, in a B-selective solvent. For this purpose, six copolymers with different compositions were synthesized: three copolymers having a hydrophilic middle block of 8.2×10 3 g.mol The distribution widths σDLS are high, of the order of 0.6 and in the same range as for the diblock copolymers, because of the bimodal size distribution (only in intensity, volume size distributions showed a unimodal distribution). Rh and σDLS values are reported in Table 4.

It should be outlined that the contribution of the large size aggregates on the intensity signal is somewhat larger in the case of the samples obtained by the dialysis method. Moreover, a discreet signal corresponding to these large aggregates is persistent on the volume signal (Figure 8, PLA2200-b-PMeOx2200-b-PLA2200). The intensity representation gives a strong importance to the aggregates, so we chose to retain the Rh value in volume distribution in order to characterize the sizes of the majority of objects.

The cryo-TEM micrographs of the suspension based on PLA2200-b-PMeOx8200-b-PLA2200 copolymer (Figure 7.B) showed spherical structures with an average radius of 9.5 nm which was smaller than the hydrodynamic radius obtained by DLS (17.0 nm). This can be explained by the influence of the largest structures on the hydrodynamic diameter obtained by DLS, but also one should consider that hydrodynamic diameters take into account the solvent displaced by the nano-objects and this could result in an overestimate of the size.

The self-assemblies from triblock copolymers were characterized by SANS in D2O at 1 g.L -1 and at room temperature. Figure 9 and Figure S7 show the plots of the scattered intensity as a function of q from the different samples obtained by nanoprecipitation and dialysis respectively. For better visibility, intensity was multiplied by a power of 10 on the graph. Similar to the SANS results obtained for the diblocks, the six curves were characterized by a variation in q -4 in the intermediate q value regime (2.0×10 -2 < q (Å -1 ) < 8.0×10 -2 ) that may correspond to a compact structure. While for larger values of q (8.0×10 -2 < q (Å -1 ) < 4.4×10 - 1 ), the decrease of log I was less abrupt. This variation might be characteristic of an interface coated with hydrated polymer chains. However, the absence of a variation in q -2 was due to the impossibility to subtract more incoherent from the solvent.

The low q behavior (3.0×10 -3 < q (Å -1 ) < 2.0×10 -2 ) was quite different from the one observed for the diblock copolymers. In all cases, there was an intensity increase as q decreased which should be characteristic of an aggregation phenomenon. The self-assemblies were thus made of finite size aggregates, as can be extrapolated from the combination of DLS, cryo-TEM and SANS results. The SANS data were fitted and the traces of the fits obtained are in black in Figure 9. A polydisperse sphere model was used in most cases. As was highlighted in the diblock selfassembly study, SANS probes mainly the dense PLA core because the hydrated PMeOx corona has a contrast length density close to the one of D2O. Consequently, the fits parameters, RSANS and σSANS, are thus characteristic of the core radius of the micelles. RSANS were in a close range 6.0 -8.2 nm for the nanoprecipitation method and 6.9 -11.5 nm for the dialysis method, without a marked dependence upon the PLA chain length. Interestingly, RSANS for the triblocks were smaller than the ones determined for the diblocks (9.5-16.7 nm)

whereas Rh of the triblocks had comparable values to the ones of the diblocks. One can notice that RSANS were always lower than Rh as expected, the latter being sensitive to the hydrated micellar corona and to the aggregation phenomenon. Interestingly, for PLA1200-b-PMeOx2200b-PLA1200, a polydisperse core-multishell model was found to be the most appropriate model to fit the SANS data. It consists in a dense PLA core (ρPLA, R c SANS and σ c SANS), a hydrated shell (ρD2O, R 1 SANS and σ 1 SANS) and an external PLA shell (ρPLA, R 2 SANS and σ 2 SANS). Again, in this case, the total radius (R 2 SANS= 7.1 nm) was in the same range as for the other triblocks and much lower than Rh.

In all the cases, the aggregation number has been deduced from a similar calculation as for the diblock copolymers (equation 6). In the case of the analysis by a core-multishell model, the term 4/3π(rc) [START_REF] Kataoka | Block copolymer micelles for drug delivery: design, characterization and biological significance[END_REF] has been replaced in the same equation by the total volume of the PLA domains (in the core and in the shell). As was expected from the values of RSANS, Nagg were smaller than the ones estimated for the diblock copolymers. Values depended on the preparation method. Nagg were in the range 100-400 for the nanoprecipitated samples and were generally larger for the dialyzed samples 240-830.

aggregate radii were in the range 15.0 -30.0 nm although DLS highlighted the presence of a small proportion of larger objects of several hundred nanometers. The SANS and cryo-TEM measurements revealed the compact structure of the micellar cores and SANS showed their aggregation into connected structures. Several authors have reported upon the self-assemblies of ABA triblock copolymers in a B-selective solvent. [START_REF] Liu | Regular and irregular micelles formed by A LEL triblock copolymer in aqueous solution[END_REF][START_REF] Agrawal | Structural Characterization of PLA-PEO-PLA SoluWons and Hydrogels: Crystalline vs Amorphous PLA Domains[END_REF] Two types of association mechanisms were described: (i) a closed association mechanism leading to flower-like micelles where the hydrophilic middle block is looped at the outer rim of the micelle, the core being composed of the hydrophobic block; and (ii) an open association mechanism with the formation of loose aggregates. Formation of micelles was reported using a crystalline PLLA block in PLLA-b-PEtOx-b-PLLA by Hsiue and co-workers [START_REF] Wang | New amphiphilic poly (2-ethyl-2-oxazoline)/poly (L-lactide) triblock copolymers[END_REF] . In the case of the same amorphous PLA blocks as in the present study, a closed association mechanism was also reported as micelles were detected and analyzed in PLA-b-PEO-b-PLA copolymers of f ratio larger than 0.5. [START_REF] Liu | Regular and irregular micelles formed by A LEL triblock copolymer in aqueous solution[END_REF][START_REF] Agrawal | Structural Characterization of PLA-PEO-PLA SoluWons and Hydrogels: Crystalline vs Amorphous PLA Domains[END_REF] The results of Bhatia and co-workers were also in good agreement with the formation of flower-like micelles where the core sizes and aggregation numbers increased with the PLA molecular mass. It should be noted that the core sizes were on the same order as for the micelles of this study (for comparable PLA number-average molecular mass).

Moreover, networks of bridged micelles were reported as the concentration was increased above 100 g.L -1 but there was no report of the formation aggregates of bridged micelles at concentration as low as 1 g.L -1 as in the present work.

As for the diblocks, there is the question of the micelles' morphology as a function of the f parameter . Additionnaly to the geometrical and energetic parameters already mentioned for the diblocks, one must take into account the entropy loss associated to the looping of the middle block. This last constraint might be less strong when the middle block has a larger molecular mass. In the experiments, there were two series of triblock copolymers, three copolymer samples with a short PMeOx block (2200 g.mol -1 ) and three other samples with a PMeOx block more than three times longer (8200 g.mol -1 ). As f is larger than 50 % for these last three copolymers, spherical micelles were expected, and the results were in good agreement whatever the preparation method chosen. The lower core radius, in comparison to the ones of the diblocks, could be attributed to the looping penalty of the middle block. The series with shorter PMeOx block had f values in a 21-48 % range. For PLA4200-b-PMeOx2200b-PLA4200, f = 21 %, spherical micellar morphologies were obtained using nanoprecipitation and dialysis method. This is in good agreement with the Discher morphology rules [START_REF] Discher | Polymer vesicles[END_REF]49 . For the two other triblock copolymers with f values of 34 and 48 %, vesicular morphologies could be expected following the same rule. A core-multishell morphology have been detected for PLA1200-b-PMeOx2200-b-PLA1200 using the nanoprecipitation preparation method and appearing as a shoulder in the intermediate q-range of the plot. This morphology might exist for other samples (such as samples with f = 34%) but could not be always detected by SANS due to smoothing polydispersity effects. The core-multishell morphologies were not observed for the diblocks, neither from the SANS plots nor from the cryo-TEM pictures. In the case of the triblocks, the formation of core-multishell structures should be favoured because of the looping penalty of the short PMeOx block.

The following association behavior is thus proposed as a function of the parameter f in Scheme 2. Whatever the value of f, bridged micelles of finite size were formed but those might coexist with isolated micelles which have not been represented in Scheme 2 for the sake of simplicity. The micellar core may have an inner structure depending on f as well as the middle block molecular mass. Indeed, for very close f values, 48 and 50 %, coremultishell and core-shell structures were respectively observed due to the PMeOx block length increase.

Scheme 2: Association scheme of the triblock copolymers as a function of the hydrophilic ratio f. Isolated micelles may coexist with bridged micelles in each case but for simplicity it has not been drawn.

However, the technique of self-assembly used has a great influence on the self-assembly process. Systematic experiments provided information about the location of the "Ouzo domain", in which only nanoparticles are obtained, and identified the parameters controlling the size, polydispersity and the crystallinity of nanoparticles. [START_REF] Pucci | Impact of the formulation pathway on the colloidal state and crystallinity of poly-ε-caprolactone particles prepared by solvent displacement[END_REF] The copolymer composition has a significant influence but the mixing time of the organic solution containing the amphiphilic copolymer with the aqueous phase is a parameter of first importance. When the kinetics of self-assembly is slown down by a rapid solvent mixing, here by nanoprecipitation or dialysis, copolymers do not have enough mobility during the self-assembly to reach the most stable morphology, the system is trapped under a metastable structure, often of reduced mean size, as the polydisperse core-multishell and micellar structures obtained in this study.

Consequently, these structures were obtained by the combination of parameters, among them, the copolymer characteristics and the used self-assembly routes. reported for this kind of triblock copolymers. This process could be favored to reduce the entropy loss coming from the looping of the PMeOx middle blocks when forming simple flower-like micelles. The amphiphilic copolymers of this study displayed a rich variety of controlled self-assembled morphologies which could be promising candidates for drug delivery applications.

  In this context, we propose the elaboration of amphiphilic diblock and triblock copolymers poly(2-methyl-2-oxazoline)-b-poly(D,L lactide) (PMeOx-b-PLA) and poly(D,L lactide)-bpoly(2-methyl-2-oxazoline)-b-poly(D,L lactide) ((PLA-b-PMeOx-b-PLA) and their selfassembly in water.

  : PMeOx-b-PLA and PLA-b-PMeOx-b-PLA The amphiphilic diblock and triblock copolymers, poly[(2-methyl-2-oxazoline)-b-poly(D,Llactide)] (PMeOx-b-PLA) and poly[(D,L-lactide)-b-(2-methyl-2-oxazoline)-b-(D,L-lactide)] (PLA-b-PMeOx-b-PLA), were obtained by coupling the alkynyl-PLA with the azide end group of the functionalized PMeOx-N3 or N3-PMeOx-N3. Typically, to obtain the diblock copolymer (PMeOx2200-b-PLA2800), PLA≡ (2.8×10 3 g.mol -1 , 100 mg, 3.57×10 -2 mmol) and 1.1 equivalent of PMeOx-N3 (2.2×10 3 g.mol -1 , 87.0 mg, 3.95×10 -2 mmol), were dissolved in 10 mL of dry DMSO and 10 mg of commercially available copper nanoparticles (Cu NPs) were added. The reaction mixture was then subjected to microwave irradiation under stirring at 80 °C for 25 min. Cu NPs were removed by centrifugation at 4000 rpm for 10 min. The supernatant was centrifuged a second time and purified by dialysis against water (molecular weight cut off: 3.5 KDa or 6-8 KDa depending on the PMeOx molecular mass). The diblock copolymer was recovered by freeze-drying (yield = 79 -85 %). The amphiphilic triblock copolymers PLA-b-PMeOx-b-PLA were obtained from a similar procedure using the telechelic polymers N3-PMeOx-N3 (yield = 73 -92 %).

  44 and was covered by three sample-to-detector distances and two different wavelengths (1 or 3 m at the neutron wavelength of 5 Å and 4.5 m at 13 Å). The samples were loaded into Hellma quartz cells with a 2 mm optical path length. The cells were placed in a sample changer, and the scattering for each sample was measured for about 1.5 hour at room temperature.

  Scheme 1. Synthesis of PMeOx b-PLA and PLA-b-PMeOx b-PLA by Huisgen 1,3-dipolar cycloaddition with copper-nanoparticles (Cu NPs) as catalyst.

Figure 1 .

 1 Figure 1. (A) FTIR of spectra of PMeOx2200-N3 and PMeOx2200-b-PLA2800 and (B) RAMAN spectra of PLA≡ and PMeOx2200-b-PLA2800

Figure 2 . 1 H

 21 Figure 2. 1 H NMR spectrum of a diblock copolymer PMeOx2200-b-PLA2800 in DMSO-d6

Figure 3 . 1 H

 31 Figure 3. 1 H NMR spectrum of a triblock copolymer PLA2200-b-PMeOx2200-b-PLA2200 in CDCl3.

10 11 12 Table 2 . 1 fractions 3 4

 12213 Triblock copolymers PLA-b-PMeOx-b-PLA: molecular characteristics and weight hydrophilic weight ratio (ratio of the mass of the hydrophilic part to the total mass) Thermal stability of the diblock and triblock copolymers was studied by thermogravimetric 5 analyses (TGA) (Figure 4) and compared with the degradation profiles of the PMeOx-N3 or 6 N3-PMeOx-N3 and the PLA≡.

7 19Figure 4 .

 74 Figure 4. TGA curves for PLA≡, PMeOx-N3 and PMeOx b-PLA (A) and for PLA≡ and N3-PMeOx-N3 and PLA-b-PMeOx b-PLA (B).

Figure 5 .

 5 Figure 5. (A) DLS size distribution and (B) cryo-TEM micrograph of nano-objects of PMeOx2200-b-PLA2800 (f = 44 %) obtained by nanoprecipitation.

Figure 6 .

 6 Figure 6. Scattered intensity as a function of scattering vector for nano-objects of PMeOx2200b-PLA5200 (f = 30 %), PMeOx2200-b-PLA2800 (f = 44 %), and PMeOx5500-b-PLA5200 (f = 52 %) (the black line is the fitting curve by a polydisperse sphere model).

6 7 3 .

 63 Table Characteristics of PMeOx-b-PLA self-assemblies obtained by nanoprecipitation. Hydrodynamic radii and σDLS values determined by DLS9 (b) Determined by fitting the SANS curves using a polydisperse sphere model.10 (c) Aggregation number.

14 14. 5

 145 nm whereas the distribution widths were comparable from both techniques. This is in 15 agreement with a core-shell structure of the nano-assemblies, i.e. a denser polymer core and a corona made of hydrated PMeOx chains. Indeed, the hydrodynamic radius includes the solvent displaced by the nano-object whereas SANS should probe only the dense core, the scattering length density of the hydrated corona layer being very close to the one of D2O. It is thus reasonable to assume that RSANS corresponds to the core radius.It was possible to calculate the contour length of each block LC(PLA) and LC(PMeOx), to verify the possibility of a micellar core-shell structure by determining the maximum radius that could have a core only made of PLA and the maximum extension of a PMeOx external layer (equation 5):W X (PLA) = aPLA × DPPLA and W X (PMeOx) = aPMeOx × DPPMeOx(5)In which DPPLA and DPPMeOx are the degrees of polymerization of PLA and PMeOx respectively, and aPLA and aPMeOx are the lengths of a repeating unit in PLA and PMeOx, both approximated to 3.5 Å (0.35 nm). Values are reported in Table

- 1 :

 1 PLA1200-b-PMeOx8200-b-PLA1200 (f = 77 %), PLA2200-b-PMeOx8200-b-PLA2200 (f = 67 %), PLA4200-b-PMeOx8200-b-PLA4200 (f = 50 %) and three copolymers having a hydrophilic middle block of 2.2×10 3 g.mol -1 : PLA1200-b-PMeOx2200-b-PLA1200 (f = 48 %), PLA2200-b-PMeOx2200-b-PLA2200 (f = 34 %), PLA4200-b-PMeOx2200-b-PLA4200 (f = 21 %). Self-assembly of PLA-b-PMeOx-b-PLA triblock copolymers was triggered by nanoprecipitation in water from ACN solution but also by dialysis from ACN (see experimental part) for some of the samples, a slower solvent mixing process, in order to compare nano-structures obtained from both techniques.The suspensions were firstly characterized by DLS at 173°. The results showed nano-objects with hydrodynamic radii between 14 nm and 30 nm depending on the composition as well as objects of several hundred nanometers (as shown in the Figure7.A for the distribution based on PLA2200-b-PMeOx8200-b-PLA2200 by nanoprecipitation).

Figure 7 .

 7 Figure 7. (A) DLS size distribution and (B) cryo-TEM micrograph of nano-objects of PLA2200-b-PMeOx8200-b-PLA2200 (f = 67 %) obtained by nanoprecipitation.

Figure 8 .

 8 Figure 8. DLS size distribution of nano-objects of PLA2200-b-PMeOx8200-b-PLA2200 (f = 67 %) obtained by dialysis.

Figure 9 .

 9 Figure 9. Scattered intensity as a function of scattering vector q for nano-objects of PLA-b-PMeOx-b-PLA obtained by nanoprecipitation (the solid black lines are the fitting curves).

  b-P(MeOx) 2200 -b-PLA 4200 PLA 2200 -b-P(MeOx) 2200 -b-PLA 2200 PLA 1200 -b-P(MeOx) 2200 -b-PLA 1200 PLA 4200 -b-P(MeOx) 8200 -b-PLA 4200 PLA 2200 -b-P(MeOx) 8200 -b-PLA 2200 PLA 1200 -b-P(MeOx) 8200 -b-PLA 1200

1 Table 4 . 2 Copolymers

 142 Characteristics of PLA-b-PMeOx-b-PLA nano-objects

(a) Method 1 is nanoprecipitation, method 2 is dialysis 3 ( 6 (d) Aggregation number. 7 *

 367 b) Hydrodynamic radii (Rh) and σDLS values determined by DLS 4 (c) Determined by fitting the SANS curves using a polydisperse sphere or a core multishell 5 model. These values are not accessible by DLS measurement, the correlogram is not correct due to the large presence of the aggregates

  diblock and triblock amphiphilic copolymers PMeOx-b-PLA and PLA-b-PMeOx-b-PLA were successfully synthesized via CROP and copper-catalyzed Huisgen 1,3dipolar cycloaddition. In that way, two series of copolymers with varying PLA as well as PMeOx chain length were prepared allowing reaching hydrophilic weight ratio (f) in a wide range (21 to 77 %). These copolymers were self-assembled in water via solvent displacement methods and in all cases, "irregular" micelles with quite high aggregation numbers where obtained due to the frozen nature of the PLA blocks. For the diblock copolymers, core-shell micellar structures with a dense PLA core and a hydrated PMeOx shell were obtained in the whole range of f values. Although vesicular structures were reported in parent diblock copolymers such as PEO-b-PLA, their absence in the present study was attributed to the fast preparation method by nanoprecipitation. An original association behavior was evidenced for the triblock copolymers. At concentration as low as 1 g.L -1 , they formed spherical micellelike structure with a few aggregates of finite sizes made of bridged micelles, the micelles being connected by some of the PLA end-blocks. This finding has been reported for the parent PLA-b-PEO-b-PLA copolymers but it occurred only at much larger concentrations than in the present study. Moreover, the micelles displayed original nanostructures with the formation of core-multishell structures in a specific range of f values (close to 48 %) never

Table 1 .

 1 Diblock copolymers PMeOx-b-PLA: molecular characteristics and weight fractions of PLA.

	Copolymers	D E	FDGHI	D E	FJK	D E LMNLONMPGQ QRSNTLU	PLA wt. %	PLA wt. %	PLA wt. %	f
		(g.mol -1 )	(g.mol -1 )	(g.mol -1 )	expected	NMR	TGA	(%)
	PMeOx2200-b-PLA2800 2.2×10 3	2.8×10 3	5.0×10 3	56	58	57	44
	PMeOx2200-b-PLA5200 2.2×10 3	5.2×10 3	7.4×10 3	70	75	76	30
	PMeOx5500-b-PLA2800 5.5×10 3	2.8×10 3	8.3×10 3	34	34	32	66
	PMeOx5500-b-PLA5200 5.5×10 3	5.2×10 3	10.7×10 3	48	52	50	52

Acknowledgement

We are grateful to Sena Hamadi for SEC analysis and ICP-OES measurements as well as Mohamed Guerrouache for Raman spectroscopy. We would like to thank the LLB-Orphe é large-scale facility for giving us access to SANS measurements and Fabrice Cousin for discussions and help during experiments. We would like to thank Nathalie Jarroux, Laboratoire Analyse et Modélisation pour la Biologie et l'Environnement (LAMBE), Université d'Evry Val d'Essonne for her help for SEC analysis in DMF.

The dialysis method brought about similar results as from the nanoprecipitation method, except that RSANS, Nagg were generally larger in the former method. Figure 10 shows the overlay of the SANS plots obtained from the 2 methods for a same sample (PLA2200-b-PMeOx2200-b-PLA2200). Whereas these two samples showed very similar SANS signals, the aggregation was stronger in the sample prepared from dialysis method as the scattered intensity at low q increased more steeply. Indeed, when the self-assembly took place more slowly, here via a dialysis process, the micelles which were close enough, could form more bridges between them.

The self-assembly of triblock copolymers PLA-b-PMeOx-b-PLA, by two relatively rapid processes compare to other common methods such as direct solubilization or slow addition of water in organic solution for example, thus allowed the formation of micelles. Average


Clément Belletier
email: clement.belletier@unifr.ch.

Alice Normand

Valérie Camos

Pierre Barrouillet

Pascal Huguet

RUNNING HEAD: PROACTIVE CONTROL AND SOCIAL PRESENCE Choking under experimenter's presence: impact on proactive control and practical consequences for psychological science

Keywords: choking, experimenter presence, working-memory, proactive control, social facilitation

Evidence accumulated for more than a century on audience effects shows that being watched by others typically impairs performance on difficult tasks. However, recent research under the label of « choking under pressure » suggests that this performance impairment is, ironically, specific to the individuals who are the most qualified to succeed-those with a high working memory capacity (WMC). Here, we predicted and found that being watched by evaluative others such as the experimenter undermines proactive control on which the high-WMC individuals rely the more. These results refine our understanding of both audience and choking effects, and lead to innovative, practical recommendations for psychological science.

Introduction

The presence of observers typically impairs performance on difficult or poorly learned tasks, a phenomenon thought to be due to the energization of dominant but incorrect responses [START_REF] Cottrell | Social facilitation[END_REF][START_REF] Zajonc | Social facilitation[END_REF] for reviews see [START_REF] Bond | Social facilitation: a meta-analysis of 241 studies[END_REF][START_REF] Geen | Drive theory of social facilitation: Twelve years of theory and research[END_REF]. More recent results in humans and nonhuman primates suggest that being watched by others also interferes with executive control required to perform well on difficult tasks [START_REF] Belletier | Does the experimenter presence affect working memory?[END_REF][START_REF] Belletier | Choking under monitoring pressure: being watched by the experimenter reduces executive attention[END_REF][START_REF] Conty | The cost of being watched: Stroop interference increases under concomitant eye contact[END_REF][START_REF] Huguet | Cognitive control under social influence in baboons[END_REF][START_REF] Wagstaff | Some cognitive and neuropsychological aspects of social inhibition and facilitation[END_REF][START_REF] Wühr | The impact of social presence on voluntary and involuntary control of spatial attention[END_REF]; for a review see Belletier, Normand & Huguet, in press). Of particular interest here, [START_REF] Belletier | Choking under monitoring pressure: being watched by the experimenter reduces executive attention[END_REF] showed that the deleterious impact of evaluative observers on executive control (assessed via the Simon task-a popular tool for measuring executive control) is actually restricted to the individuals who are the most qualified to succeed, that is, those with a high working memory capacity (WMC). More generally, there is evidence that high-pressure situations reduce executive resources and cause high-WMC individuals to perform below their actual abilities, a phenomenon called choking under pressure [START_REF] Beilock | When high-powered people fail working memory and "choking under pressure[END_REF]; for a review see [START_REF] Beilock | Choke: What the secrets of your brain reveal about getting it right when you have to[END_REF]. Therefore, it may be that many of audience effects on difficult tasks actually reflect choking. Nonetheless, the reason why high-WMC individuals choke in the presence of an evaluative audience remains poorly understood.

According to [START_REF] Braver | The variable nature of cognitive control: a dual mechanisms framework[END_REF] dual-mechanism of control theory, executive control can be achieved either through a proactive or a reactive mode. Proactive control involves active maintenance of information to prepare and anticipate a given response (the action being goaldriven), whereas reactive control involves a later recruitment of attention (the action being event-driven). There is evidence that high-WMC individuals rely more on proactive control than their low-WMC counterparts [START_REF] Redick | Integrating working memory capacity and contextprocessing views of cognitive control[END_REF][START_REF] Redick | Cognitive control in context: Working memory capacity and proactive control[END_REF][START_REF] Richmond | Remembering to prepare: The benefits (and costs) of high working memory capacity[END_REF]. Proactive control is actually more efficient than reactive control for difficult tasks and thus accounts for the overall cognitive superiority of high-WMC individuals.

Here, we tested whether the reduction of executive control in the high-WMC individuals faced with an evaluative audience [START_REF] Belletier | Choking under monitoring pressure: being watched by the experimenter reduces executive attention[END_REF] can be replicated with a task specifically designed to assess proactive control, a question left unanswered in our previous research and largely neglected in the myriad of works on audience and choking effects.

Method

Participants and design. Eighty young adults (62 females, 18 males; Mage = 19.3, SDage = 1.83) participated in the study in exchange for course-credit. They all had corrected-to-normal vision. A power analysis using g-power [START_REF] Faul | G* Power 3: A flexible statistical power analysis program for the social, behavioral, and biomedical sciences[END_REF] for a linear multiple regression analysis (including three predictors) with an alpha level of .05, a desired power of .80, and f 2 =.15 (a medium size effect, see [START_REF] Belletier | Choking under monitoring pressure: being watched by the experimenter reduces executive attention[END_REF] indicated a needed sample size of 77 participants. The ethical approval was given by the "comité de protection de la personne, 6th committee of the region Auvergne-Rhône-Alpes", under the number IRB00008526.

Materials and procedure.

Working Memory Capacity. We first measured participants' WMC using a computerbased version of the classic Reading Span Task (RSPAN; [START_REF] Daneman | Individual differences in working memory and reading[END_REF]. All participants were left alone in the experimental room. Each display included a meaningful or meaningless sentence that the participants read while verifying whether it made sense (e.g., "We were fifty miles out at sea before we lost sight of land"), followed by a to-beremembered letter. The sentences (each composed of 12-17 words, M = 14.4 words, SD = 1.2) were taken from the French version of the RSPAN [START_REF] Desmette | Adaptation en langue française du «Reading Span Test» de Daneman et Carpenter[END_REF]. Meaningless sentences were created by changing only one word (miles to lawns in the previous example). The set size ranged from three to seven sentenceletter problems per trial with 3 trials per set size for a total of 15 trials. At the end of each set, participants had to enter the sequence of the to-be-remembered letters in the correct order. This first measure lasted about 20 minutes, followed by a rest period of 3 minutes.

Proactive control. Participants were then introduced to a task called AXCPT 70, in which most of the trials require proactive control [START_REF] Redick | Cognitive control in context: Working memory capacity and proactive control[END_REF]. This version of the task was the first to be used to investigate the superiority of the high WMC individuals over their low WMC counterparts regarding proactive control [START_REF] Redick | Integrating working memory capacity and contextprocessing views of cognitive control[END_REF]. In each trial, two letters, a cue and a probe, were presented one at a time on a computer display. Participants were instructed to detect the sequences in which the letter A was followed immediately by the letter X. Without sacrificing accuracy, they had to press as fast as possible a "target" button if the probe was X and the previous letter (the cue) was A, and a "non-target" button in any other cases. Half the participants used their right versus left hand to respond with the "target" and "non-target" button respectively, whereas the other half used the opposite response mapping. Each letter was displayed for 500 ms. The interstimulus (cue-probe) interval (ISI) was either short (1000 ms; 50% of the trials) or long (5000 ms; 50% of the trials), as also done by [START_REF] Redick | Integrating working memory capacity and contextprocessing views of cognitive control[END_REF]. In a way to maintain constant the overall duration of trials, the intertrial interval (ITI) was 5000 ms when the ISI was short, and the ITI was 1000 ms when the ISI was long. Long ISI has been shown to exacerbate the differences in terms of proactive control between patients and healthy participants [START_REF] Braver | Context processing and context maintenance in healthy aging and early stage dementia of the Alzheimer's type[END_REF]. As we are the first ones to investigate the effect of social presence on proactive control, we manipulated ISI to explore whether these effects are more important in the long ISI condition, but this manipulation did not lead to specific predictions.

Seventy percent of trials were target trials (i.e., AX trials). On these trials, proactive control (active maintenance of the cue "A") is more efficient than reactive control (late response based exclusively on the target "X"). The remaining 30% trials were non-target trials among which one third were BX trials (incorrect cue but correct probe), one third were AY trials (correct cue but incorrect probe), and the last third were BY trials (incorrect cue and incorrect probe). On the BX trials, proactive control (active maintenance of the incorrect cue "B") is more efficient than reactive control (late response based on the correct target "X"), assuming that reactive control in this case leads to more errors because the incorrect cue was not maintained as such. On the AY trials, proactive control (active maintenance of the correct cue "A") is less efficient than reactive control (late response based on the incorrect target "Y"), assuming that proactive control in this case leads to wrongly anticipate the correct probe. On the BY trials, proactive control (active maintenance of the incorrect cue "B") makes no difference with reactive control (late response based on the incorrect target "Y").

Each experimental block of trials included 56 AX trials (28 short-ISI, 28 long-ISI), 8 AY trials (4 short-ISI, 4 long-ISI), 8 BX trials (4 short-ISI, 4 long-ISI), and 8 BY trials (4 short-ISI, 4 long-ISI). Cues and probes for non-target trials were randomly picked among the letters R, S, T and U. All types of trials were randomly presented. The task was run on a Dell PC using E-prime 2.3. Social presence. After 15 practice trials alone in the experimental room, participants completed two experimental blocks (112 AX trials, 16 BX trials, 16 AY trials and 16 BY trials) while being randomly assigned to one of two social presence conditions. In the "alone" condition, the experimenter (who was one of the two female experimenters) left the experimental room before task completion. In the "experimenter presence" condition, the experimenter remained in the experimental room and was positioned opposite the participant so as not to see the participants' ongoing task, but watched them 60% of the time; an efficient social presence condition (see [START_REF] Huguet | Social presence effects in the Stroop task: further evidence for an attentional view of social facilitation[END_REF][START_REF] Sharma | Exploring the temporal dynamics of social facilitation in the Stroop task[END_REF]Spatola et al., 2018, in press).

Expectations. [START_REF] Redick | Integrating working memory capacity and contextprocessing views of cognitive control[END_REF] found that high-WMC individuals performed better than their low-WMC counterparts on AX and BX trials on which proactive control is required. However, the recent advances on audience and choking effects questions the superiority of the high-WMC individuals under the presence of evaluative observers. Here, we expected to replicate this superiority when the participants performed the AX-CPT in isolation, but not when being watched by an experimenter, i.e., an evaluative audience potentially leading to choking.

Post-experimental questionnaires. Self-reports of stress, evaluation feeling, discomfort and perceived difficulty as well as demographic information were collected at the end of the session [START_REF] Normand | Does evaluative pressure make you less or more distractible? Role of top-down attentional control over response selection[END_REF]. See Text S2 for the questionnaire data.

Results

Because two participants produced extremely high error rates on the AX-CPT (> 57%), all the analyses on accuracy reported below were performed on 78 participants. Concerning the analyses of reaction times (RTs), some participants failed to answer to BX trials with a long ISI (N = 1) or with a short ISI (N = 1), to all BX trials (N = 1) or to all BX and BY trials (N = 1). These participants (N = 4) were discarded in the corresponding analyses.

Working memory capacity. An item was scored as "correct" when it was recalled in the correct serial position. WM span represented the percentage of correct answers at each trial weighted by the number of sentences in the trial (M = .686, SD = .161) and was used as a continuous variable rather than as a dichotomous one (e.g., [START_REF] Wicherts | Comment on "Poverty impedes cognitive function[END_REF].

AX-CPT Error Rates and RTs.

Mean error rate was 11%, which comes close to what has been previously observed [START_REF] Redick | Integrating working memory capacity and contextprocessing views of cognitive control[END_REF]. Outliers RTs were identified for each participant, and rejected according to a 2.5 absolute deviation around the median (MAD) analysis eliminating 5.3% of the data. MAD analysis is a more robust analysis than the "+/-3 standard deviations from the mean" to detect abnormal values since this method is not influenced by sample size or the value of the mean [START_REF] Leys | Detecting outliers: Do not use standard deviation around the mean, use absolute deviation around the median[END_REF]. Mean correct RT was 554 ms.

Trials requiring proactive control. Proactive control leads to a better performance on the AX and BX trials. RTs and errors were therefore averaged for these two types of trials in order to obtain an index of proactive control. As the number of trials is much higher for the AX trials than for the BX trials in the version of the AX-CPT that we used, the average between the two types of trials was weighted by the number of trials. The percentage of errors and correct RTs were then submitted to linear regression analyses with WM Span (centered; [START_REF] Aiken | Multiple regression: Testing and interpreting interactions[END_REF], Social presence (isolation: -0.5; experimenter presence: +0.5) and their interaction as predictors. The regression analysis on errors was non-significant, F(3,71)=1.76, p=.16, and none of the predictors reached significance, ps > .13. The results of the regression analysis 1 on RTs indicated that the predictors explained 19% of the variance F(3,71)=5.63, p=.002, R 2 =.19. The effect of Social presence was significant, ß= .29, p = .008, η 2 p=.09. Participants working alone responded faster (M=502ms, SE=13ms) than participants working in presence of the experimenter (M=569ms, SE=16ms). More importantly, this effect was qualified by a Social presence x WM interaction, ß= .35, p = .004, η 2 p=.11. Simple slopes analyses [START_REF] Dawson | Moderation in management research: What, why, when, and how[END_REF] indicated that for participants in isolation, the higher their span the faster their RTs, t(73)= -2.00, p=.049, whereas this relationship was reversed in the presence of the experimenter t(73)=2.22, p=.03 (see Figure 1).

1 The regression remained significant when all the participants were included (F(3,75)=5.43 ; p<.01 ; R 2 =.18) with similar effects of Social presence (ß= .35; p = .002) and Social presence x WM span interaction (ß= .24; p = .04). Likewise, our findings were unchanged when using a +/-3 SD cut off (.5% of trials were discarded).

Working Memory Span

Isolation

Experimenter Presence

(squares), with regression lines (full and dotted line for experimenter presence and isolation, respectively).

Effect of ISI.

The same analyses on proactive, reactive and neutral trials were conducted on the long and short ISI trials. The same pattern of results as before was observed, indicating that the effect of the experimenter presence was the same on both ISI (See Text S1

for the full analyses).

Comparison between proactive and reactive trials. Because the AXCPT 70 contains much more proactive trials than reactive trials, it is not well adapted to a direct comparison between these two types of trials. However, it is worth noting that the results of the same regression analysis as before on the difference between proactive and reactive trials (rather than proactive trials alone) were also consistent with our approach. The regression was not significant on accuracy (p=.20), but was significant on RTs: F(3,71)=3.25, p=.03, R 2 =.12, with a significant Social presence x Working memory span interaction, ß=.26, p=.04, η 2 p=.06.

Simple slopes analyses indicated that, under isolation, the higher span the higher the advantage of proactive trials over reactive trials t(73)=-2.25, p=.03. This relationship was not found in the presence of the experimenter t(73)=1.04, p=.30. Thus, in this critical experimenter's presence condition, working memory span did no longer predict the performance difference between proactive and reactive trials.

Discussion

There is evidence that high-Working Memory Capacity (WMC) individuals rely more on proactive control (i.e., on the active maintenance of information to anticipate a given response) than their low-WMC counterparts [START_REF] Redick | Cognitive control in context: Working memory capacity and proactive control[END_REF][START_REF] Redick | Integrating working memory capacity and contextprocessing views of cognitive control[END_REF][START_REF] Richmond | Remembering to prepare: The benefits (and costs) of high working memory capacity[END_REF]. This central feature of high-WMC individuals accounts for their superiority over the low-WMC individuals on difficult tasks [START_REF] Braver | The variable nature of cognitive control: a dual mechanisms framework[END_REF][START_REF] Engle | Working memory capacity as executive attention[END_REF]. However, previous results showed that the relationship between Working Memory (WM) and executive control in a Simon Task is reversed in the presence of an evaluative audience [START_REF] Belletier | Choking under monitoring pressure: being watched by the experimenter reduces executive attention[END_REF]. Therefore, we extended these results to one task known to be a sound measure of proactive control, the AX-CPT70, with the idea that this type of control would be impaired by the presence of others, thereby reducing specifically the performance of high-WMC individuals.

Our results showed that when participants worked alone (experimenter outside the experimental room), the higher was their WM span the faster they were on proactive control trials (AX and BX trials). This relationship was reversed when participants were watched by the experimenter (inside the experimental room), similarly to what was observed on RTs in the Simon task [START_REF] Belletier | Choking under monitoring pressure: being watched by the experimenter reduces executive attention[END_REF]. The presence of the experimenter made no difference on AY trials measuring reactive control. These findings indicate that being watched by evaluative others such as the experimenter undermines proactive control but does not seem to impact reactive control. However, the version of the AXCPT that we used here is known to require essentially proactive control [START_REF] Redick | Cognitive control in context: Working memory capacity and proactive control[END_REF]. Future research should therefore make use of different variations of the AX-CPT to test whether the presence of an evaluative audience-especially the experimenter presence-undermines proactive control more than reactive control, and whether reactive control is or is not immune to social presence effects and why. Moreover, we used a version of the task in which short and long ISI are mixed, which could have increased the executive demand and therefore have made participants more vulnerable to the executive cost of the experimenter presence. Besides that, the effects of social context were the same with both ISI, providing evidence that these effects are robust and did not necessitate a long delay between cue and target to be observed.

Altogether, our results refine our understanding of both audience and choking effects.

More than a century of research on audience effects [START_REF] Guerin | Social facilitation[END_REF] shows that being watched by others typically impairs performance on difficult tasks. Previous findings indicated that being watched by others reduced both the range of cue utilization [START_REF] Baron | Distraction-conflict theory: Progress and problems[END_REF][START_REF] Huguet | Social presence effects in the Stroop task: further evidence for an attentional view of social facilitation[END_REF][START_REF] Normand | Does evaluative pressure make you less or more distractible? Role of top-down attentional control over response selection[END_REF] and executive attention [START_REF] Wagstaff | Some cognitive and neuropsychological aspects of social inhibition and facilitation[END_REF][START_REF] Wühr | The impact of social presence on voluntary and involuntary control of spatial attention[END_REF]. However, this long tradition failed to consider that social impairment effects may actually be restricted to high-WMC individuals who typically choke under pressure [START_REF] Beilock | When high-powered people fail working memory and "choking under pressure[END_REF][START_REF] Gimmig | Choking under pressure and working memory capacity: When performance pressure reduces fluid intelligence[END_REF]. Not only do the present findings strengthen our confidence that the deleterious impact of evaluative audience (when any) reflects choking, but also help specifying which operative mode of control is impaired. Likewise, past research on choking showed that cognitive control and underlying prefrontal activity are reduced in high-WMC individuals under evaluative pressure [START_REF] Beilock | When high-powered people fail working memory and "choking under pressure[END_REF][START_REF] Decaro | Choking under pressure: multiple routes to skill failure[END_REF][START_REF] Lee | Out of control: Diminished prefrontal activity coincides with impaired motor performance due to choking under pressure[END_REF]; for reviews see [START_REF] Beilock | Choke: What the secrets of your brain reveal about getting it right when you have to[END_REF][START_REF] Yu | Choking under pressure: the neuropsychological mechanisms of incentiveinduced performance decrements[END_REF], but did not specify which component of control is altered. The present findings show that choking induced by evaluative audiences may reflect reduced proactive control.

Finally, the present research also leads to practical recommendations for psychological science. The large majority of psychological experiments imply the presence of an experimenter at some points in time (either intermittently or throughout participants' task performance) and space (close to or distant from the participants), with varied behaviors (more or less evaluative, cold or warm, etc.). However, the potential impact of the experimenter's presence and behavior on cognitive processes has been largely overlooked. If simply being watched by the experimenter leads the individuals with higher WMC to choke on tasks relying on proactive control, then even subtle variations in the experimenter's presence and behaviors from one study to another (if not within the same study from one participant to another) may cause dramatic changes in performance, resulting in potentially contradictory findings. These changes reflecting variations in the experimenter's presence and behaviors may occur in all fields of psychology whenever proactive control is at stake, from research on executive functions or intelligence in cognitive or developmental psychology to research on emotion and personality traits [START_REF] Braver | The variable nature of cognitive control: a dual mechanisms framework[END_REF]. Also consistent with this view, there is evidence that a third party observer in the context of neuropsychological testing may be associated with poorer performance on measures of verbal learning, memory, fluency, attention, and executive functions [START_REF] Eastvold | Does a third party observer affect neuropsychological test performance? It depends[END_REF]. More generally, experimenter effects are not new (for recent examples, see [START_REF] Doyen | Behavioral priming: it's all in the mind, but whose mind?[END_REF]Foroughi, Montfort, Paczynski, McKnight & Greenwood, 2016).

However, past research in this area focused on the effect of experimenter's expectations (and sometimes participants' beliefs about the experimenter's expectations), while we focus here specifically on the effects of experimenter's presence.

As such, our results suggest to systematically include details about the experimenter's presence and behavior in the method section of research articles, which seems just as important as other details related to the task itself (distance to the computer screen, visual angle relative to stimuli, SOA, etc). All these indications are crucially needed to better standardize the conditions both within the same study from one participant to another and from one study to another, to ensure replicability.

Fig 1 .

 1 Fig 1. Reaction times on the proactive trials (AX and BX) as a function of working memory

  Trials requiring reactive control. The percentage of correct responses and the RTs in the AY trials were submitted to the same regression models. Both regressions were non-significant (on errors: F(3,74)=.77, p=.52; on RTs: F(3,73)=1.18, p=.25).

	Neutral trials. The same regressions were conducted on the BY trials. The regression on the
	percentage of errors was non-significant, F(3,74)=1.39, p=.25. However, the regression on the
	RTs was significant F(3,73)=3.75, p=.02, R 2 =.13. Only Social presence reached significance,
	ß= .31; p = .006, η 2	p=.10) (ps>.22 for WM span main effect and Social presence x WM span
	interaction). Participants working in isolation were faster (M=489 ms, SE=18 ms) than
	participants working in the presence of the experimenter (M=562 ms, SE=19 ms).

Supplemental Material for

Choking under experimenter's presence: Impact in Proactive control and practical consequences for psychological science

This file contains:

Analyses on ISI and Questionnaire

Text S1: Analyses on ISI.

Short vs Long ISI in proactive trials. The percentage of errors and correct RTs for long and short ISI proactive trials were submitted separately to a linear regression with the WM Span (centered; [START_REF] Aiken | Multiple regression: Testing and interpreting interactions[END_REF], the Social Presence (isolation: -0.5; experimenter presence: +0.5) and their interaction as predictors. Text S2: Analyses on Questionnaire.

The final questionnaire was made of 4 items [START_REF] Normand | Does evaluative pressure make you less or more distractible? Role of top-down attentional control over response selection[END_REF] measuring Stress ("I felt rather stressed during the task"), Evaluation Feeling ("I felt evaluated during the task"), Discomfort ("I was quite comfortable during the task", reversed) and Perceived Difficulty ("I had difficulties during the task"). Participants answered all these items according to a 7-point Likert scale anchored from "strongly agree" to "strongly disagree". We first assessed the internal consistency of an index of perceived anxiety combining the 4 items (α =.71, M = 3.38, SD = 1.19). [START_REF] Belletier | Choking under monitoring pressure: being watched by the experimenter reduces executive attention[END_REF] showed that participants' level of perceived anxiety was unaffected by the presence of the experimenter. To test if it was also the case in the present data, we ran a linear regression with the WM Span (centered), Social Presence (isolation: -0.5; experimenter presence: +0.5) and their interaction as predictors. The regression model was significant F(3,76)=3.22, p=.03, R 2 =.11. The effect of Working memory span was significant (ß= -.30, p = .01, η 2 p=.08). This effect was qualified by a significant interaction (ß= -.31, p = .01, η 2 p=.08) between working memory span and social presence. As indicated by simple slopes analyses, in isolation, the relationship between perceived anxiety and working memory was not significant t(76)=.10, p=.92. In the presence of the experimenter, the higher the span the lower the level of perceived anxiety t(76)=-3.06, p=.003. Thus, consistent with earlier findings [START_REF] Belletier | Choking under monitoring pressure: being watched by the experimenter reduces executive attention[END_REF], it seems that the deleterious effect of the experimenter presence observed on proactive control was not related to higher anxiety, quite the contrary.