Laura Etchenausia

Aurélie Malho Rodrigues

Simon Harrisson

Elise Deniau-Lejeune

Maud Save
email: maud.save@univ-pau.fr

RAFT copolymerization of vinyl acetate and N-vinylcaprolactam: kinetics, control, copolymer composition and thermoresponsive self-assembly

Well-defined thermoresponsive amphiphilic statistical copolymers based on Nvinylcaprolactam (VCL) and vinyl acetate (VAc) were successfully synthesized by RAFT/MADIX polymerization mediated by O-ethyl-S-(1ethoxycarbonyl)ethyldithiocarbonate chain transfer agent at 65°C. We achieved the synthesis of a series of copolymers with controlled molar masses and low dispersities over a wide range of monomer feed ratios (0 < fVac,0 < 1). Both conventional linearization methods and a nonlinear least square methods (NLLS) were applied to estimate reliable values of reactivity ratios for VAc and VCL radical polymerization in regards to the disparate values previously reported in the literature. The highest measurement precision was observed for the NLLS method based on the integrated form of the copolymerization equation developed by Skeist. The calculated values of the reactivity ratios (rVAc = 0.33  0.10 and rVCL = 0.29  0.15) perfectly fitted the drift in monomer feed ratio versus conversion. The VCL and VAc monomer units are distributed homogeneously along the copolymer chains. Both the cloud point temperatures and glass transition temperatures evolve linearly with the copolymer composition. Analyses of the aqueous solutions of the amphiphilic copolymers by means of dynamic and static light scattering show that P(VAc-co-VCL) copolymers undergo a reversible temperature-induced conformational change between large aggregates (T > Tcloud point) and either unimers for FVAc inferior to 0.3 (Nagg = 1, Dh  7 nm) or small aggregates for FVAc = 0.53 (Nagg = 3, Dh  14 nm).

INTRODUCTION

Thermoresponsive polymers are polymers that undergo a conformation phase transition in response to a change of temperature. [START_REF] Roy | New directions in thermoresponsive polymers[END_REF][START_REF] Vancoillie | Thermoresponsive poly(oligo ethylene glycol acrylates)[END_REF] Among the several well-known thermoresponsive polymers, poly(N-vinylcaprolactam) (PVCL) has raised an increasing interest over the last years as reported in recent reviews. [START_REF] Ramos | Temperature-sensitive nanogels: poly(Nvinylcaprolactam) versus poly(N-isopropylacrylamide)[END_REF][START_REF] Liu | N-vinylcaprolactam): a thermoresponsive macromolecule with promising future in biomedical field[END_REF][START_REF] Cortez-Lemus | Poly(N-vinylcaprolactam), a comprehensive review on a thermoresponsive polymer becoming popular[END_REF] This biocompatible polymer with lower critical solution temperature (LCST) in water ranging between 30 -40 °C, [START_REF] Ramos | Temperature-sensitive nanogels: poly(Nvinylcaprolactam) versus poly(N-isopropylacrylamide)[END_REF][START_REF] Vihola | Cytotoxicity of thermosensitive polymers poly(N-isopropylacrylamide), poly(N-vinylcaprolactam) and amphiphilically modified poly(N-vinylcaprolactam)[END_REF] is attractive for different applications such as delivery systems, [START_REF] Liu | N-vinylcaprolactam): a thermoresponsive macromolecule with promising future in biomedical field[END_REF] or particle stabilization. [START_REF] Liu | Gold nanorods coated with a thermo-responsive poly(ethylene glycol)-b-poly(Nvinylcaprolactam) corona as drug delivery systems for remotely near infrared-triggered release[END_REF] An effective means of tuning the temperature in which the change of PVCL-chain solvation state occurs is to control both the polymer chain length, [START_REF] Tager | Thermodynamic study of poly(n-vinyl caprolactam) hydration at temperatures close to lower critical solution temperature[END_REF][START_REF] Beija | Thermoresponsive poly(N-vinyl caprolactam)coated gold nanoparticles: sharp reversible response and easy tunability[END_REF][START_REF] Peng | Water-Soluble Reactive Copolymers Based on Cyclic N-Vinylamides with Succinimide Side Groups for Bioconjugation with Proteins[END_REF][START_REF] Peng | Temperature and pH dual-responsive poly(vinyl lactam) copolymers functionalized with amine side groups via RAFT polymerization[END_REF] and the introduction of co-monomers in the polymer chain. [START_REF] Peng | Water-Soluble Reactive Copolymers Based on Cyclic N-Vinylamides with Succinimide Side Groups for Bioconjugation with Proteins[END_REF][START_REF] Peng | Temperature and pH dual-responsive poly(vinyl lactam) copolymers functionalized with amine side groups via RAFT polymerization[END_REF][START_REF] Zhao | Effect of copolymer composition of RAFT/MADIX-derived N-vinylcaprolactam/N-vinylpyrrolidone statistical copolymers on their thermoresponsive behavior and hydrogel properties[END_REF][START_REF] Kermagoret | One-pot controlled synthesis of double thermoresponsive N-vinylcaprolactambased copolymers with tunable LCSTs[END_REF] Among the different co-monomers that can be used to tune the phase transition temperature of PVCL chains in water, vinyl acetate (VAc) is an attractive co-monomer due to the wide range of applications of poly(vinyl acetate), which is used as a binder for wound dressing, in latex paints, in the paper industry and as a versatile adhesive. [START_REF] Harrisson | RAFT Polymerization of Vinyl Esters: Synthesis and Applications[END_REF][START_REF] Amann | Biodegradability of Poly(vinyl acetate) and Related Polymers[END_REF] Moreover, PVAc is a precursor of poly(vinyl alcohol) which is a biocompatible and biodegradable polymer of interest for biomedical applications, as a stabilizer of colloidal systems or as a superabsorber. [START_REF] Amann | Biodegradability of Poly(vinyl acetate) and Related Polymers[END_REF][START_REF] Sionkowska | Current research on the blends of natural and synthetic polymers as new biomaterials: Review[END_REF] During the last decades, controlled radical polymerization (CRP), also recently named reversibledeactivation radical polymerization (RDRP), [START_REF] Jenkins | Terminology for reversible-deactivation radical polymerization previously called "controlled" radical or "living" radical polymerization (IUPAC Recommendations[END_REF] has been extensively investigated to precisely control molecular weight, dispersity, architecture and microstructure of a wide range of (co)polymers. [START_REF] Braunecker | Controlled/living radical polymerization: Features, developments, and perspectives[END_REF] The most developed CRP techniques are nitroxide mediated polymerization (NMP), [START_REF] Nicolas | Nitroxidemediated polymerization[END_REF] atom transfer radical polymerization (ATRP), [START_REF] Matyjaszewski | Atom Transfer Radical Polymerization (ATRP): Current Status and Future Perspectives[END_REF] copper(0)-mediated radical polymerization, [START_REF] Wang | Reversible-Deactivation Radical Polymerization in the Presence of Metallic Copper. Comproportionation-Disproportionation Equilibria and Kinetics[END_REF][START_REF] Konkolewicz | End of controversy?[END_REF] reversible addition-fragmentation chain transfer (RAFT) [START_REF] Chiefari | Thiocarbonylthio Compounds (SC(Z)S-R) in Free Radical Polymerization with Reversible Addition-Fragmentation Chain Transfer (RAFT Polymerization). Effect of the Activating Group Z[END_REF][START_REF] Chong | Thiocarbonylthio Compounds [SC(Ph)S-R] in Free Radical Polymerization with Reversible Addition-Fragmentation Chain Transfer (RAFT Polymerization). Role of the Free-Radical Leaving Group (R)[END_REF] including macromolecular design via the interchange of xanthates (MADIX), [START_REF] Destarac | Xanthates as Chain-Transfer Agents in Controlled Radical Polymerization (MADIX): Structural Effect of the O-Alkyl Group[END_REF] reversible iodine transfer polymerization (RITP) [START_REF] Boyer | Reverse Iodine Transfer Polymerization (RITP) of Methyl Methacrylate[END_REF] and organometallic-mediated radical polymerization (OMRP). [START_REF] Allan | Organometallic mediated radical polymerization[END_REF] Despite the interest of poly(vinyl esters) and poly(vinyl amides) for a range of applications, the controlled radical polymerization of unconjugated monomers has long been recognized as a challenge due to the difficulty in controlling the equilibrium between the unstabilized radical and the dormant species. [START_REF] Cortez-Lemus | Poly(N-vinylcaprolactam), a comprehensive review on a thermoresponsive polymer becoming popular[END_REF][START_REF] Harrisson | RAFT Polymerization of Vinyl Esters: Synthesis and Applications[END_REF] In the recent years, improvements in RAFT/MADIX polymerization, [START_REF] Beija | Thermoresponsive poly(N-vinyl caprolactam)coated gold nanoparticles: sharp reversible response and easy tunability[END_REF][START_REF] Stenzel | Xanthate Mediated Living Polymerization of Vinyl Acetate: A Systematic Variation in MADIX/RAFT Agent Structure[END_REF][START_REF] Favier | A Detailed On-Line FT/NIR and 1H NMR Spectroscopic Investigation into Factors Causing Inhibition in Xanthate-Mediated Vinyl Acetate Polymerization[END_REF][START_REF] Wan | Controlled radical polymerization ofNvinylcaprolactam mediated by xanthate or dithiocarbamate[END_REF][START_REF] Shao | RAFT polymerization of N-vinylcaprolactam and effects of the end group on the thermal response of poly(N-vinylcaprolactam)[END_REF][START_REF] Guinaudeau | Facile Access to Poly(N-vinylpyrrolidone)-Based Double Hydrophilic Block Copolymers by Aqueous Ambient RAFT/MADIX Polymerization[END_REF][START_REF] Shanmugam | Photoinduced Electron Transfer-Reversible Addition-Fragmentation Chain Transfer (PET-RAFT) Polymerization of Vinyl Acetate andN-Vinylpyrrolidinone: Kinetic and Oxygen Tolerance Study[END_REF][START_REF] Girard | RAFT/MADIX (co)polymerization of vinyl trifluoroacetate: a means to many ends[END_REF] OMRP, [START_REF] Kermagoret | One-pot controlled synthesis of double thermoresponsive N-vinylcaprolactambased copolymers with tunable LCSTs[END_REF][START_REF] Debuigne | Synthesis of End-Functional Poly(vinyl acetate) by Cobalt-Mediated Radical Polymerization[END_REF][START_REF] Miao | Photo-induced cobaltmediated radical polymerization of vinyl acetate[END_REF][START_REF] Kermagoret | Double thermoresponsive di-and triblock copolymers based on N-vinylcaprolactam and N-vinylpyrrolidone: synthesis and comparative study of solution behaviour[END_REF] RITP [START_REF] Tonnar | Synthesis of poly(vinyl acetate)-b-poly(dimethylsiloxane)-b-poly(vinyl acetate) triblock copolymers by iodine transfer polymerization[END_REF] and ATRP, [START_REF] Jiang | A novel poly(N-vinylcaprolactam)-based welldefined amphiphilic graft copolymer synthesized by successive RAFT and ATRP[END_REF][START_REF] Yang | High-efficiency synthesis of welldefined cyclic poly(N-vinylcaprolactam) and its solution properties[END_REF] offered opportunities to control the polymerization of the so-called less activated monomers (LAM) such as vinyl acetate, Nvinylcaprolactam (VCL) or N-vinylpyrrolidone (VP). RAFT/MADIX polymerization afforded the possibility to synthesize various LAM-based copolymers such as block copolymers (PVAcb-PVCL, [START_REF] Wan | Controlled radical polymerization ofNvinylcaprolactam mediated by xanthate or dithiocarbamate[END_REF] , PVCL-b-PVP [START_REF] Liang | Synthesis and self-assembly of thermosensitive double-hydrophilic poly(N-vinylcaprolactam)b-poly(N-vinyl-2-pyrrolidone) diblock copolymers[END_REF] ) or statistical copolymers (P(VP-stat-VCL) [START_REF] Zhao | Effect of copolymer composition of RAFT/MADIX-derived N-vinylcaprolactam/N-vinylpyrrolidone statistical copolymers on their thermoresponsive behavior and hydrogel properties[END_REF][START_REF] Maji | Thermoresponsive polymer coated gold nanoparticles: from MADIX/RAFT copolymerization of N-vinylpyrrolidone and N-vinylcaprolactam to salt and temperature induced nanoparticle aggregation[END_REF] ). Among controlling the molar mass and dispersity via RDRP, copolymerization kinetics and associated effect on copolymer composition assist in the design of statistical copolymers with suitable performances. Indeed, both copolymer composition and monomer distribution of statistical polymer sequences can affect properties such as phase transition temperature, [START_REF] Vancoillie | Thermoresponsive poly(oligo ethylene glycol acrylates)[END_REF][START_REF] Zhao | Effect of copolymer composition of RAFT/MADIX-derived N-vinylcaprolactam/N-vinylpyrrolidone statistical copolymers on their thermoresponsive behavior and hydrogel properties[END_REF][START_REF] Kermagoret | One-pot controlled synthesis of double thermoresponsive N-vinylcaprolactambased copolymers with tunable LCSTs[END_REF] blockstatistical copolymer self-assembly, [START_REF] Bendejacq | Double-polyelectrolyte, like-charged amphiphilic diblock copolymers: Swollen structures and pH-and salt-dependent lyotropic behavior[END_REF][START_REF] Lejeune | Amphiphilic Diblock Copolymers with a Moderately Hydrophobic Block: Toward Dynamic Micelles[END_REF] cyclization [START_REF] Moskowitz | Semibatch RAFT copolymerization of acrylonitrile and N-isopropylacrylamide: Effect of comonomer distribution on cyclization and thermal stability[END_REF] and rheological properties of copolymers self-assembled either in water [START_REF] Charbonneau | Controlling the Dynamics of Self-Assembled Triblock Copolymer Networks via the pH[END_REF] or in bulk. [START_REF] Cherifi | Synthetic methodology effect on the microstructure and thermal properties of poly(n-butyl acrylate-co-methyl methacrylate) synthesized by nitroxide mediated polymerization[END_REF][START_REF] Mok | Microphase separation and shear alignment of gradient copolymers: Melt rheology and small-angle X-ray scattering analysis[END_REF] It requires the most accurate values of the reactivity ratios. However, the discrepancy between the values of reactivity ratios reported in the literature for VAc and VCL copolymerization (rVAc = 0.63 and rVCL = 0.31 (bulk), [START_REF] Kirsh | Water Soluble Poly-N-Vinylamides: Synthesis and Physicochemical Properties[END_REF][START_REF] Cortez-Lemus | Poly(N-vinylcaprolactam), a comprehensive review on a thermoresponsive polymer becoming popular[END_REF] rVAc = 0.36 and rVCL = 1.06 (bulk) [START_REF] Kermagoret | One-pot controlled synthesis of double thermoresponsive N-vinylcaprolactambased copolymers with tunable LCSTs[END_REF], 50 rVAc = 0.35 and rVCL = 2.5 (media not reported), 51 rVAc = 0.3 and rVCL = 2.2 (ethanol), 49 rVAc = 0.3 and rVCL = 1.3 (butanol) [START_REF] Kirsh | Water Soluble Poly-N-Vinylamides: Synthesis and Physicochemical Properties[END_REF] provides uncertainty in the monomer sequence along the chain. This might limit the development of more complex materials based on VAc and VCL copolymers. It is thus of interest to explore how effective is RAFT/MADIX polymerization at controlling the copolymerization of VAc and VCL monomers for various initial monomer feed compositions. Special attention is dedicated to the copolymer compositions. Non-linear least square methods and linearization methods are examined to accurately determine the reactivity ratio of VAc and VCL. The influence of the P(VAc-co-VCL) copolymer composition on glass transition temperature, phase transition temperature, and copolymer self-assembly is investigated.

EXPERIMENTAL PART

Materials

Vinyl acetate (VAc, Sigma Aldrich, 99% + ) was mixed with inhibitor removers (Sigma Aldrich, 0.1 g of inhibitor remover for 50 mL of VAc) for 30 minutes prior to be filtered and used for polymerization. N-vinylcaprolactam (VCL, Sigma Aldrich, 98%) was distilled under reduce pressure (P = 0.05 mmHg) in the presence of hydroquinone (Sigma Aldrich, 99% + ). Azobisisobutyronitrile (AIBN, Sigma Aldrich, 98%), 1,3,5-trioxane (Sigma Aldrich, ≥ 99%), diethyl ether (Sigma Aldrich, ≥ 99.8%) were used as received. The O-ethyl-S-(1ethoxycarbonyl)ethyldithiocarbonate molecular transfer agent was synthesized according to a previously described procedure [START_REF] Bernard | Poly(vinyl ester) Star Polymers via Xanthate-Mediated Living Radical Polymerization: From Poly(vinyl alcohol) to Glycopolymer Stars[END_REF] (purity: 93 %).

General procedure for the synthesis of P(VAc-co-VCL) statistical copolymer

Polymerizations were performed with a total of 5 g of monomers, varying the initial molar fraction of VAc and VCL from fVAc,0 = 0 to 1. The initial concentration of xanthate agent was ranging from 1.6 to 2.4 mol.L -1 and the ratio between monomer concentration and xanthate agent concentration ranged between 420 and 460 (Table S.1. in Supporting Information).

In a typical experiment (Expt 6 in Table 1), xanthate agent (36.5 mg, 1.10  10 -4 mol), AIBN (5.4 mg, 3.30  10 -5 mol), VCL (2.00 g, 1.44  10 -2 mol), 1,3,5-trioxane (0.887 g, 9.86  10 -3 mol) and VAc (3.04 g, 3.53  10 -2 mol) were introduced in a 10 mL round bottom flask and stirred magnetically. The mixture was degased by nitrogen bubbling during three minutes in an ice bath. The flask was weighed before and after degasing to ensure the absence of VAc evaporation during this step. A sample was withdrawn under nitrogen at time t=0. The round bottom flask was placed into an oil bath previously heated to 65°C. Samples were withdrawn every hour until the reaction was stopped (6 hours) by cooling down in an ice-water bath and introduction of oxygen in the mixture. Individual conversions of monomers were calculated from nuclear magnetic resonance spectra using 1, 3, 5-trioxane as internal standard (equation 1). The weight fraction of VAc in the copolymer (wVAc) was obtained from equation 5, considering the molar fraction of the monomers in the copolymer (Fmono, equation 4) and the molar masses of each monomer (MVCL = 139 g.mol -1 and MVAc = 86 g.mol -1 ). where T is absolute temperature, the viscosity of the solvent, kB the Boltzmann constant and D is the diffusion coefficient. The hydrodynamic diameters of the the P(VAc-co-VCL) copolymers were also measured accurately using the a ALV setup (Langen, Germany). The values of Dh below the cloud point were calculated from the diffusion coefficient using the Stokes-Einstein equation and was extrapolated to zero concentration and zero-q values.

Static Light Scattering (SLS).

Light Scattering measurements were performed on a ALV setup (Langen, Germany), consisted of a 22 mW He-Ne laser, operating at a wavelength of λ = 632.8 nm, an ALV CGS/8F goniometer, an ALV High QE APD detector, and an ALV 5000/EPP multibit, multiautocorrelator. All the measurements were carried out at temperature below the cloud point, for scattering angles, θ, ranging from 30° to 150°. For P(VAc-co-VCL) copolymers with FVAc inferior to 0.3, the measurements were performed at polymer concentrations ranging between 1 g.L -1 and 20 g.L -1 while for the P(VAc-co-VCL) copolymer with FVAc of 0.53, the temperature of measurement was set at 10°C at polymer concentrations range of 1 -4 g.L -1 . The weight average molar mass of the scatterers (Mw) was determined using the Zimm equation (equation 6). The average scattered intensity of the sample (Isample) is measured in relation to the average scattered intensity of the solvent (Isolvent) and a standard (Istandard). The Rayleigh ratio (R) of the sample, which corresponds to the normalized contribution of the sample to the scattering intensity, is then determined based on the known Rayleigh ratio of the standard (R,standard, here we used toluene with R,Toluene = 2.79 x 10 -5 cm -1 ), equation 7. K.c/R can then be determined using equations 6 to 9, taking into account the wavelength of the laser (), the refractive index of the standard (nstandard), the refractive index increment (dn/dc) of the sample and Avogadro's number (NA).

𝐾.𝑐

𝑅 𝜃 = 𝑞 2 𝑅 𝑔 2 3𝑀 𝑤 + 1 𝑀 𝑤 + 2𝐴 2 𝑐 Equation 6 𝑅 𝜃 = 𝐼 𝑠𝑎𝑚𝑝𝑙𝑒 -𝐼 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝐼 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑅 𝜃,𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 Equation 7 𝐾 = 4𝜋 2 𝑛 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 2 (𝑑𝑛 𝑑𝑐 ⁄ ) 2
𝑁 𝐴 𝜆 [START_REF] Liu | N-vinylcaprolactam): a thermoresponsive macromolecule with promising future in biomedical field[END_REF] Equation 8

𝑞 = 4𝜋𝑛 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 𝜆 𝑠𝑖𝑛 𝜃 2 Equation 9
For all of the samples, the resulting distribution of relaxation times obtained via DLS was bimodal. To account for the presence of the larger aggregates in the SLS experiments, the relative scattered intensity contributions from fast and slow mode of relaxation were determined from the DLS data, which allowed for the calculation of the Rayleigh ratio for the fast mode only (Rθ,fast). [START_REF] Chassenieux | Association of Hydrophobically End-Capped Poly(ethylene oxide)[END_REF][START_REF] Lejeune | pH Induced Desaggregation Of Highly Hydrophilic Amphiphilic Diblock Copolymers[END_REF][START_REF] Patterson | Structural Characterization of Amphiphilic Homopolymer Micelles Using Light Scattering, SANS, and Cryo-TEM[END_REF] 𝑅 𝜃,𝑓𝑎𝑠𝑡 = 𝐴 𝑓𝑎𝑠𝑡 (𝜃) The specific refractive index increment values (dn/dc) of the aqueous solutions of copolymers were determined at 20°C with a T-rEX (Wyatt) differential refractometer, operating at a wavelength of λ = 632 nm, at concentration ranging form 0.2 to 1 g.L -1 . The following values were measured: dn/dc = 0.177 ± 0.001 mL.g -1 (FVAc = 0.28), dn/dc = 0.171 ± 0.005 mL.g -1

(FVAc = 0.19 ) and dn/dc = 0.163 ± 0.008 mL.g -1 (PVCL). According to these values, the dn/dc of the copolymer with FVAc = 0.53 was extrapolated at 0.186 mL.g -1 (Figure S.3, Supporting Information).

Size Exclusion Chromatography (SEC).

The SEC system operates in THF, at 30°C (flow rate: 1mL.min -1 ). The SEC apparatus is The refractive index increments (dn/dc) of the P(VAc-co-VCL) copolymers synthesized by bulk copolymerization were determined using equation 12.

(dn/dc) copo = w VAc ( dn dc 12The values of dn/dc of PVCL and PVAc homopolymers were respectively taken from references [START_REF] Beija | Thermoresponsive poly(N-vinyl caprolactam)coated gold nanoparticles: sharp reversible response and easy tunability[END_REF] and 51 : (dn/dc)PVCL = 0.109 mL.g -1 and (dn/dc)PVAc = 0.0582 mL.g - (heating/cooling rate: 0.5 °C.min -1 ). Transmittance at 500 nm was plotted versus the temperature. The minimum of the first-order derivative fit of transmittance versus temperature was considered as the temperature of cloud point (Tcp) (see Figure 9).

) PVAc + w VCL ( dn dc ) PVCL Equation

RESULTS AND DISCUSSION

RAFT/MADIX copolymerization of VAc/VCL: evaluation of control

First, the level of control of RAFT/MADIX (co)polymerization of vinyl acetate and Nvinylcaprolactam mediated by the O-ethyl-S-(1-ethoxycarbonyl)ethyldithiocarbonate transfer agent (Scheme 1) was investigated. The polymerizations were carried out in bulk at 65 °C with varying initial monomer feed ratios (fVAc,0) as reported in Table S.1 (Supporting Information).

Both VAc and VCL monomers being liquid at 65°C, the polymerization was performed in bulk in order to limit the use of toxic and/or flammable polar solvents such as 1,4-dioxane, chloroform, N,N-dimethylformamide or anisole, which were previously used as solvents for RAFT (co)polymerization of VCL with other less activated monomers. The semilogarithmic kinetic plots of the overall molar monomer conversion versus time are linear in the initial range of conversions but some plots curve upwards for higher conversions indicating a steady increase in propagating radical concentration (Figure 1). Such an inhibition period of 60 to 180 minutes was observed in the kinetic plots. Such inhibition period was previously observed in xanthate-mediated vinyl acetate polymerization and was ascribed to the vulnerability of the reactive vinyl acetate propagating radical towards oxygen and by-products generated during the CTA synthesis. [START_REF] Stenzel | Xanthate Mediated Living Polymerization of Vinyl Acetate: A Systematic Variation in MADIX/RAFT Agent Structure[END_REF][START_REF] Favier | A Detailed On-Line FT/NIR and 1H NMR Spectroscopic Investigation into Factors Causing Inhibition in Xanthate-Mediated Vinyl Acetate Polymerization[END_REF] The induction period may also be due to initial selective formation of monoadduct but the length of the inhibition period did not show a specific trend with the initial VAc molar feed ratio. A similar range of molar conversion in monomers (40 -60 %) was reached within 6 hours of RAFT polymerization carried out at 65°C for the entire range of the initial VAc feed composition (0 < fVAc < 1, Table 1). Note that a significant increase in the time required to reach 30 % monomer conversion for Co-MRP copolymerization of VAc and VCL was previously reported for fVAc,0 > 0.4 (48 to 115 h). 13 1).

Figure 3. Evolution of the size exclusion chromatograms with monomer conversion for xanthate mediated VAc/VCL copolymerization with fVAc,0 = 0.62 (expt 5 in Table 1).

The experimental Mn are greater than the theoretical ones, which implies that the RAFT agent The perfect overlay of the UV-visible (λ = 355 nm) and RI traces of the SEC chromatograms observed for the series of P(VAc-co-VCL) copolymers depicts the presence of the dithiocarbonate chain-end and so the living character on the entire molar mass distribution (see

Figure S.6, Supporting Information).

Determination of reactivity ratios and P(VAc-co-VCL) microstructure.

Reactivity ratios are most often calculated using a linearization of the Mayo-Lewis equation [START_REF] Mayo | Copolymerization I : A basis for comparing the behavior of monomers in copolymerization, the copolymerization of styrene and methyl methacrylate[END_REF] (either the differential or integrated form). The Kelen-Tüdos (KT) method is one of the most common methods (see details in Supporting Information). [START_REF] Kelen | Analysis of linear methods for determining copolymerization reactivity ratios .1. New improved linear graphic method[END_REF] The authors later modified the method in order to limit large systemic error in calculation due to the low conversion assumption. The extended Kelen-Tüdos method takes into account the individual monomer conversions. [START_REF] Tüdos | Analysis of linear methods for determining copolymerization reactivity ratios. III. Linear graphic method for evaluating data obtained at high conversion levels[END_REF] As reported in Table 2, similar values of reactivity ratios were calculated from either the Kelen-Tüdos method or the extended Kelen-Tüdos method for RAFT copolymerization of VAc and VCL performed in bulk at 65°C (Table 1) (rVAc = 0.43 and rVCL = 0.20 Table 2). Nonlinear least square (NLLS) methods can also be used to determine the reactivity ratios (see details in Supporting Information). [START_REF] Hunley | Nonlinear Method for Determining Reactivity Ratios of Ring-Opening Copolymerizations[END_REF][START_REF] Van Den | Nonlinear regression by visualization of the sum of residual space applied to the integrated copolymerization equation with errors in all variables. I. Introduction of the model, simulations and design of experiments[END_REF][START_REF] Lessard | Styrene/Acrylic Acid Random Copolymers Synthesized by Nitroxide-Mediated Polymerization: Effect of Free Nitroxide on Kinetics and Copolymer Composition[END_REF][START_REF] Guerrero-Sanchez | High-Throughput Method for RAFT Kinetic Investigations and Estimation of Reactivity Ratios in Copolymerization Systems[END_REF] The sum of the squares of the residuals was minimized to set the values of reactivity ratios fitting the experimental copolymer composition, FVAc calculated from the individual monomer conversion (equation 4) with the theoretical copolymer composition calculated from the Mayo-Lewis equation [START_REF] Mayo | Copolymerization I : A basis for comparing the behavior of monomers in copolymerization, the copolymerization of styrene and methyl methacrylate[END_REF] (FVAc, theo, equation 13).

𝐹 A, theo. = 𝑟 A × 𝑓 A 2 + 𝑓 A × 𝑓 B 𝑟 A × 𝑓 A 2 +2 × 𝑓 A × 𝑓 B + 𝑟 B × 𝑓 B 2 Equation 13
The theoretical data of the FVAc = f(fVAc,0) plots fit the experimental data for values of reactivity ratios of rVAc = 0.37 and rVCL = 0.16 (Figure 4). This S profile is in good agreement with the evolution of the individual monomer conversion versus time displayed in Figure 4. Indeed, for initial VAc fraction below the azeotropic composition (𝑓 VAc,0,azeotropic =

(1-𝑟 VCL )

(2-𝑟 VCL -𝑟 VAc ) = 0.57), VAc is more rapidly consumed than VCL while the opposite trend is observed for fVAc,0 higher than fVAc,0, azeotropic (Figure 4). The second nonlinear least squares method is based on the integrated form of the copolymerization equation. [START_REF] Skeist | Copolymerization: the Composition Distribution Curve[END_REF][START_REF] Meyer | Integral and differential binary copolymerization equations[END_REF] Equation 14 accurately describes the drift of the monomer feed ratio (fVAc) over the course of copolymerization in relationship with the values of reactivity ratios. This composition drift model provides an enhanced number of analyzed experimental data and therefore the curve fitting method decreases the uncertainty of reactivity ratio measurements. 14Where Xm is the overall molar monomer conversion,

𝑋 𝑚 = 1 -( 𝑓 A 𝑓 A,0 ) 𝛼 × ( 𝑓 B 𝑓 B,0 ) 𝛽 × ( 𝑓 A,0 -𝛿 𝑓 A -𝛿 ) 𝛾 Equation
= 𝑟 B 1 -𝑟 B , 𝛽 = 𝑟 A 1 -𝑟 A , 𝛾 = 1 -𝑟 A × 𝑟 B (1-𝑟 A ) × (1 -𝑟 B ) and 𝛿 = 1 -𝑟 B 2 -𝑟 A -𝑟 B
. The composition of the monomer feed was calculated from equation 15.

𝑓 A = 𝑓 A,0 × (1-𝑥 A ) 𝑓 A,0 × (1-𝑥 A ) +(1-𝑓 A,0 ) × (1-𝑥 B )

Equation 15

The experimental values of fVAc and Xm were fitted to the integrated form of the copolymer composition equation (equation 14) using the visualization of the sum of squares space method. [START_REF] Van Den | Nonlinear regression by visualization of the sum of residual space applied to the integrated copolymerization equation with errors in all variables. I. Introduction of the model, simulations and design of experiments[END_REF][START_REF] Harrisson | Living spontaneous gradient copolymers of acrylic acid and styrene: one-pot synthesis of pH-responsive amphiphiles[END_REF] We calculated the reactivity ratios as rVAc = 0.33 and rVCL = 0.29. The point estimates and 95% joint confidence interval are displayed in Figure 5. The interval for rVAc (0.24-0.45) is slightly narrower than for rVCL (0.15-0.49). [START_REF] Kermagoret | One-pot controlled synthesis of double thermoresponsive N-vinylcaprolactambased copolymers with tunable LCSTs[END_REF] (open circle: reported reactivity ratios; closed circle and line: best estimate and 95% joint confidence region from NLLS fitting of Skeist equation of their experimental data). Black: circles data from reference [START_REF] Kirsh | Water Soluble Poly-N-Vinylamides: Synthesis and Physicochemical Properties[END_REF] in various solvents and square from references 51 The theoretical plots of fVAc versus Xm perfectly fit the experimental data during the course of polymerization for the whole series of VAc/VCL copolymerizations (Figure 6). The composition drift is rather limited for initial VAc molar fraction above 0.5, but it is slightly more pronounced for lower initial fractions of VAc (fVAc,0 < 0.4). Regarding plots of Figure 6, the formation of statistical copolymers with homogeneously distributed VAc and VCL units is expected as the overall monomer conversion was limited to 70 % for the series of copolymers (Table 1). Table 2 summarizes the values of reactivity ratio of VAc and VCL calculated in the present work with linearization techniques or nonlinear least squares methods. As depicted in Figure 5, the three sets of reactivity ratios calculated from KT, extended KT and Lewis Mayo NLLS method (equation 13) were at the close outer limit of the 95% joint confidence interval (Figure 5). The two first methods take into account the copolymer composition at low conversion. As previously reported by Klumpermann et al., [START_REF] Klumperman | Peculiarities in atom transfer radical copolymerization[END_REF] the RAFT agent can influence the monomer selectivity at the early stages of polymerization. Also, previous studies revealed an impact of the RDRP equilibrium implemented by reversible termination on the copolymer composition for very low monomer conversion. [START_REF] Zapata-Gonzalez | Copolymer Composition Deviations from Mayo-Lewis Conventional Free Radical Behavior in Nitroxide Mediated Copolymerization[END_REF] Not only did the reactivity ratios reported in the literature exhibit discrepancy between their values, their values were out of the range of the present 95% joint confidence interval. One reason for very different reactivity ratios between this work and the study of Kermagoret et al. [START_REF] Kermagoret | One-pot controlled synthesis of double thermoresponsive N-vinylcaprolactambased copolymers with tunable LCSTs[END_REF] might be accounted to the difference between RAFT polymerization and Co-MRP.

Table 2. VAc and VCL reactivity ratio values calculated from the different methods.

P(VAc-co-VCL) copolymers with different monomer compositions (expt 5 and 7 in Table 1)

were analyzed by [START_REF] Kermagoret | One-pot controlled synthesis of double thermoresponsive N-vinylcaprolactambased copolymers with tunable LCSTs[END_REF] C NMR and the spectra were compared to PVCL and PVAc spectra. The integrality of the spectra is shown in 1), P(VAc0.53-co-VCL0.47) (expt 5 in Table 1) and P(VAc0.83-co-VCL0.17) (expt 7 in Table 1) (co)polymers in CDCl3: (a) zoom on the PVAc and PVCL carbonyl signal range; (b) zoom on the PVAc methine signal range.

The copolymer compositions can be calculated on the basis of the integrals of the well-resolved carbonyl signals at 170 -180 ppm of each polymer unit of the P(VAc-co-VCL) copolymer (Table S.2, Supporting Information). Such [START_REF] Kermagoret | One-pot controlled synthesis of double thermoresponsive N-vinylcaprolactambased copolymers with tunable LCSTs[END_REF] C NMR compositions match the compositions calculated either from the individual monomer conversions (Equation 4) or from the integrals of the proton NMR spectra of the copolymers (Table S The polar amide group of PVCL creates favorable association with surrounding water molecules by hydrogen bonding below the LCST and hydrophobic interactions (sevenmembered ring) predominates above the LCST. [START_REF] Tager | Thermodynamic study of poly(n-vinyl caprolactam) hydration at temperatures close to lower critical solution temperature[END_REF][START_REF] Kirsh | Structural transformations and water associate interactions in poly-N-vinylcaprolactam-water system[END_REF] The phase transition temperature of PVCL is in the physiological range (30-40 °C) [START_REF] Ramos | Temperature-sensitive nanogels: poly(Nvinylcaprolactam) versus poly(N-isopropylacrylamide)[END_REF][START_REF] Liu | N-vinylcaprolactam): a thermoresponsive macromolecule with promising future in biomedical field[END_REF][START_REF] Cortez-Lemus | Poly(N-vinylcaprolactam), a comprehensive review on a thermoresponsive polymer becoming popular[END_REF] and can be tuned by the polymer concentration [START_REF] Yang | High-efficiency synthesis of welldefined cyclic poly(N-vinylcaprolactam) and its solution properties[END_REF] and its molar mass. [START_REF] Beija | Thermoresponsive poly(N-vinyl caprolactam)coated gold nanoparticles: sharp reversible response and easy tunability[END_REF] The incorporation of a co-monomer unit is an additional parameter to modify the temperature of cloud point (Tcp). As depicted in Figure 9, a dependence of the phase transition temperature with the copolymer composition is observed by turbidimetry analysis.

For low VAc content (FVAc = 0 -0.28), the polymers in water exhibit a sharp and reversible thermal transition between a clear solution and a cloudy solution above a certain temperature (Tcloud point). This transition is broadened for a higher content of hydrophobic VAc (FVAc =0.53) but reversibility is still observed. The hysteresis never exceeds 2 degrees (Figure 9). 3). The increasing incorporation of hydrophobic VAc units into PVCL thermoresponsive chains leads to a decrease of the copolymer cloud point temperature (Figure S.10). As the hydrophobic composition of the copolymers is increased, the hydrogen bonding effect between the copolymer chains and water is lowered while hydrophobic interactions are enhanced, thus lowering the required energy to collapse (i.e., lower Tcp). [START_REF] Peng | Water-Soluble Reactive Copolymers Based on Cyclic N-Vinylamides with Succinimide Side Groups for Bioconjugation with Proteins[END_REF][START_REF] Peng | Temperature and pH dual-responsive poly(vinyl lactam) copolymers functionalized with amine side groups via RAFT polymerization[END_REF] The characteristics of the P(VAc-co-VCL) copolymers dispersed in water were studied by means of dynamic and static light scattering at a temperature below the cloud point temperature (see Table 3). .5 a Cloud point temperature measured by UV-visible spectroscopy of solution of copolymers at 3 g.L -1 in water, the given value is the average of the cloud point temperature values obtained for the first heating, cooling, and second heating; b Cloud point temperature measured by dynamic light scattering of solution of copolymers at 5 g.L -1 in water; c Temperature of the static light scattering measurements; d Data measured on ALV setup (see experimental part, SLS and DLS) at the temperature of measurement reported in this Table 3, which corresponds to a temperature below the cloud point. At all concentrations the DLS analyses of P(VAc-co-VCL) copolymers dispersed in water revealed a main population of small aggregates (fast mode of relaxation) with hydrodynamic diameter (Dh) of 5 to 22 nm, depending on the VAc content, and a minor population of larger aggregates of Dh ∼ 200 nm (slow mode of relaxation). It would be tempting to assign the slow mode of relaxation to aggregates in equilibrium with the unimers. However, the weight concentration of these large aggregates is negligible given that the scattered light intensity of these aggregates varies only between 30 and 50 %. Indeed, the scattered light intensity being proportional to both Mw and the square of the scatter dimension (Rg 2 ), the contribution of large aggregates predominates even at very low fraction. [START_REF] Chassenieux | Association of Hydrophobically End-Capped Poly(ethylene oxide)[END_REF] Similar populations of large aggregates were reported for poly(ethylene oxide) (PEO) end-capped with dodecyl groups and were identified as spurious aggregates. [START_REF] Chassenieux | Association of Hydrophobically End-Capped Poly(ethylene oxide)[END_REF] For P(VAc-co-VCL) amphiphilic copolymers with low VAc content (FVAc < 0.3), the fast mode of relaxation corresponds to a value of the apparent Dh below 7 nm. This suggests that the polymers are present as free polymer chains, which is confirmed by an aggregation number of unity measured by SLS (Table 3). Note that the light scattered by the minor larger aggregates was accounted in order to accurately determine the absolute weight average molar mass (Mw) and aggregation number of the fast mode of relaxation (see experimental section). For higher content of VAc (FVAc = 0.53), self-assembly of the P(VAc-co-VCL) copolymer into aggregates of three polymer chains (Nagg = 3, see Table 3) was achieved. The hydrodynamic diameter of the aggregates, measured from the fast mode of relaxation in DLS, was indeed larger (Dh ∼ 20 nm). The steeper angular dependence of the light scattered by the P(VAc0.53-co-VCL0.47) in comparison with the copolymers with lower VAc content depicts the presence of aggregates for the more hydrophobic copolymer (Figure

S.2. in Supporting Information

). These results show that a level of ca 50 mol-% of VAc is required to induce non-covalent intermolecular hydrophobic interactions at temperature below the could point, leading to supramolecular assembly.

CONCLUSION

In summary, the copolymerization of N-vinylcaprolactam with the hydrophobic vinyl acetate is successfully controlled by RAFT polymerization mediated by the O-ethyl-S-(1ethoxycarbonyl)ethyldithiocarbonate chain transfer agent to provide a series of well-defined thermoresponsive poly(N-vinylcaprolactam-co-vinyl acetate) copolymers with tunable phase transition temperatures and glass transition temperatures. Regardless the initial monomer feed ratio, the synthesis of statistical copolymers fulfills the features of a controlled polymerization with a linear increase of the molar masses and low dispersity values (Ð < 1.3). It should be mentioned that the polymerization control is enhanced by increasing the initial VAc feed ratio.

We applied both conventional linearization methods and nonlinear least square methods to estimate reliable values of reactivity ratios for VAc and VCL radical polymerization in regards to the disparate values previously reported in the literature. The nonlinear least squares (NLLS) method based on the integrated form of the copolymerization equation (Skeist equation) provides values of reactivity ratios of rVAc = 0.33 and rVCL = 0.29 with the azeotropic composition calculated at fVAc,0 = 0.57. It should be mentioned that the reactivity ratio values calculated either from linearization techniques such as Kelen-Tüdos, extended Kelen-Tüdos or from nonlinear least-squares fitting, based on the Mayo-Lewis equation at low conversion, fall on the outside limit of the 95% joint confidence region (0.24 < rVAc < 0.45 ; 0.15 < rVCL < 0.49).

These results are important to provide information on the copolymer microstructure and clearly indicate a limited monomer compositional drift in the synthesized P(VAc-co-VCL) copolymers. Given the nearly constant composition in VAc and VCL along the polymer chains, the [START_REF] Kermagoret | One-pot controlled synthesis of double thermoresponsive N-vinylcaprolactambased copolymers with tunable LCSTs[END_REF] C NMR signal of the PVAc methine group is thus influenced by the monomer fraction and the values of the copolymer glass transition (Tg) are in accordance with the theoretical Tg of statistical copolymers calculated on the basis of the Fox-Flory law. Turbidimetry analyses of the aqueous solutions of the amphiphilic P(VAc-co-VCL) copolymers indicated that the copolymer composition fine tunes the cloud point temperature (Tcp) between 20 and 30 °C. A linear decrease of the cloud point temperature of the aqueous copolymer solution was observed with the increase of VAc molar fraction in the copolymer. It worth emphasizing that a sharp and reversible temperature phase transition was observed for VCL-rich copolymers while a broadening of this transition occurred for the more hydrophobic P(VAc0.53-co-VCL0.47).

Interestingly, the P(VAc-co-VCL) copolymers with VAc composition below 30 mol-% forms unimers in water below Tcp, as judged by static light scattering measurements (Nagg = 1) and dynamic light scattering (Dh  7 nm). Increasing the VAc content up to 53 mol-% is sufficient to induce P(VAc0.53-co-VCL0.47) copolymer self-assembly into small aggregates (Nagg = 3, Dh  20 nm) via intermolecular hydrophobic interactions at 10 °C (T < Tcp). The knowledge of the minimum hydrophobic fraction resulting in association of P(VAc-co-VCL) chains at room temperature opens perspectives towards the formation of thermoresponsive supramolecular structures such as physically crosslinked colloidal particles synthesized by polymerization induced copolymer self-assembly in aqueous dispersed media. [START_REF] Delaittre | Synthesis by nitroxide-mediated aqueous dispersion polymerization, characterization, and physical core-crosslinking of pH-and thermoresponsive dynamic diblock copolymer micelles[END_REF]

3

 3 corresponds to the integral of one proton of 1,3,5-trioxane (5.1 ppm, 6 H) used as internal standard and I1H,mono corresponds to either the integral of the proton of VCL monomer at 3.6 ppm (I1H,VCL, 2 H) or VAc monomer at 7.1-7.4 ppm (I1H,VAc = I7.1-7.4 ppm -I1H,VCL/2) (see 1 H NMR spectrum of a crude sample in Figure S.1 in Supporting Information). The final overall molar and weight conversions (respectively named Xm and Xw) were calculated from the monomer individual conversions (xmono., equation 1) according to equation 2 and equation 3 (n0 and m0 are respectively the initial number of moles and the initial mass of each monomer). 𝑋 𝑚 = 𝑥 VAc * 𝑛 0,VAc + 𝑥 VCL * 𝑛 0,VCL 𝑛 0,VAc + 𝑛 0,VCL Equation 2 𝑋 𝑤 = 𝑥 VAc * 𝑚 0,VAc + 𝑥 VCL * 𝑚 0,VCL 𝑚 0,VAc + 𝑚 0,VCL Equation The molar fraction of the VAc monomer incorporated into the copolymer (FVAc) was calculated from the monomer individual conversion (xVAc, equation 1) and the initial number of moles of monomers (n0,VAc and n0,VCL), according to equation 4. 𝐹 VAc = 𝑥 VAc * 𝑛 0,VAc 𝑥 VAc * 𝑛 0,VAc + 𝑥 VCL * 𝑛 0,VCL Equation 4

2 ,

 2 𝐼 𝑠𝑎𝑚𝑝𝑙𝑒 (𝜃)-𝐼 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 (𝜃) 𝐼 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 (𝜃) 𝑅 𝜃,𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 Equation 10 Afast (θ) is the scattered intensity contribution from the fast mode of relaxation at a given scattering angle determined by DLS, Isample, Isolvent, and Istandard are the scattered intensities at angle θ, by the sample, solvent, and reference liquid, respectively, and Rθ,standard is the Rayleigh ratio for the toluene used as the standard. The K.c/Rθ,fast values were averaged across the different q-values and plotted versus concentration to determine the absolute weight-average molar mass (Mw, aggregate) (Figure S.Supporting Information). The aggregation number was calculated as follows: Nagg = Mw,aggregate/Mw,polymer, with Mw,polymer corresponding to Mw of the copolymer measured by SEC in THF from MALLS detector.

11 Where

 11 equipped with a Viscotek VE 1122 automatic injector, a set of shodex columns (KF 801, KF 8025, KF 804 et KF 806 from Waters) working in series, a Wyatt Heleos II Multi Angle Laser Light Scaterring detector (MALLS, 18 angles, λ0 = 664.4 nm), a refractive index (RI) detector Viscotek VE 3580 and a Viscotek VE 3210 UV-visible detector. Toluene was used as flow marker. All (co)polymer samples were prepared at 5 g.L -1 concentrations. The number-average molar mass (Mn) and molar mass distribution (Đ) were obtained from MALLS data using the Mi value of each slice of the chromatogram (see equation 6 at zero concentration). The experimental Mn were compared to the theoretical number average molar masses of the copolymers calculated from equation 11. 𝑀 n,theo. = 𝑀 Xanthate + 𝑋 𝑤 (𝑚 0,VAc + 𝑚 0,VCL ) / 𝑉 Total ([Xanthate] 0 +0.6 * [AIBN] 0 * (1-𝑒 -𝑘 d * 𝑡 )) Equation MXanthate corresponds to the molar mass of the molecular xanthate agent (MXanthate = 222 g.mol -1 ), Xw is the final overall weight conversion (see equation 3), kd is the AIBN dissociation constant into dimethylformamide at 60°C (kd = 6.45 × 10 -5 s -1 ), 51 t corresponds to the polymerization time, [Xanthate]0 and [AIBN]0 are respectively the initial concentrations of the chain-transfer agent and the initiator. m0,VAc and m0,VCL are the initial weight of monomers (in g) and VTotal (L) is the initial volume of the reaction mixture.

30 Scheme 1 .

 301 Scheme 1. Synthesis of P(VAc-co-VCL) copolymers by RAFT/MADIX polymerization mediated by O-ethyl-S-(1-ethoxycarbonyl)ethyldithiocarbonate (= xanthate chain transfer agent, CTA) in bulk.

Figure 1 .

 1 Figure 1. Semilogarythmic plots of overall molar monomer conversion (Xm) versus time :  fVAc,0 = 0,  fVAc,0 = 0.15,  fVAc,0 = 0.30, + fVAc,0 = 0.52,  fVAc,0 = 0.62,  fVAc,0 = 0.71,  fVAc,0 = 0.87 and  fVAc,0 = 1 (expt 1-8 in Table1).The experimental molar masses of the series of P(VAc-co-VCL) copolymers increase linearly with the overall monomer weight conversion (Figure2) in accordance with equation 11. The low dispersity (Đ) values of the final (co)polymers which range between 1.25 and 1.04 are consistent with a controlled character of the RAFT/MADIX (co)polymerization mediated by O-ethyl-S-(1-ethoxycarbonyl)ethyldithiocarbonate chain transfer agent (Table1). Moreover,

Figure 2 .

 2 Figure 2. Evolution of Mn,MALLS and dispersity versus the overall weight conversion (Xw) for: fVAc,0 = 0.15,  fVAc,0 = 0.30, + fVAc,0 = 0.52,  fVAc,0 = 0.62,  fVAc,0 = 0.71,  fVAc,0 = 0.87 (expt 2-7 in Table1).

  is not fully consumed during the course of the polymerization. The xanthate CTA exhibits the characteristic absorption band of a dithiocarbonate group at λ = 355 nm (see Figure S.5a in Supporting Information). Thus, the presence of an additional peak at low elution volume (35.5 mL) observed in the UV-visible SEC traces of the final copolymers (recorded at λ = 355 nm) confirms the incomplete consumption of the xanthate chain transfer agent (CTA) (see Figure S.5b, Supporting Information). The efficiency of the xanthate CTA, calculated on the basis of the ratio between theoretical and experimental Mn rises with the increase of the initial VAc molar fraction (

Figure 4 .

 4 Figure 4. Experimental values of FVAc,0 () versus fVAc,0 and evolution of monomer individual conversions with time for different monomer initial molar feed ratios. The black curve corresponds to the non-linear fit of Lewis-Mayo equation with rVAc = 0.37 and rVCL = 0.16. The dotted line is the azeotropic curve. The error bar was set on the basis of the error bar from NMR technique used to determine FVAc,0.

Figure 5 .

 5 Figure 5. Reactivity ratios (points) and 95% joint confidence regions (lines) for copolymerizations of VAc and VCL. Blue: data of the present work. Red: data from reference[START_REF] Kermagoret | One-pot controlled synthesis of double thermoresponsive N-vinylcaprolactambased copolymers with tunable LCSTs[END_REF] (open circle: reported reactivity ratios; closed circle and line: best estimate and 95% joint confidence region from NLLS fitting of Skeist equation of their experimental data). Black: circles data from reference[START_REF] Kirsh | Water Soluble Poly-N-Vinylamides: Synthesis and Physicochemical Properties[END_REF] in various solvents and square from references51 (solvent not

  Figure 5. Reactivity ratios (points) and 95% joint confidence regions (lines) for copolymerizations of VAc and VCL. Blue: data of the present work. Red: data from reference[START_REF] Kermagoret | One-pot controlled synthesis of double thermoresponsive N-vinylcaprolactambased copolymers with tunable LCSTs[END_REF] (open circle: reported reactivity ratios; closed circle and line: best estimate and 95% joint confidence region from NLLS fitting of Skeist equation of their experimental data). Black: circles data from reference[START_REF] Kirsh | Water Soluble Poly-N-Vinylamides: Synthesis and Physicochemical Properties[END_REF] in various solvents and square from references51 (solvent not

Figure 6 .

 6 Figure 6. Variation of the VAc composition in the monomer feed (fVAc) versus the molar conversion. Symbols: plots of experimental data of fVAc (equation 15) versus Xm,  fVAc,0 = 0.15,  fVAc,0 = 0.30, + fVAc,0 = 0.52,  fVAc,0 = 0.62,  fVAc,0 = 0.71,  fVAc,0 = 0.87; The dotted lines correspond to the calculated values of Xm (equation 14) for given theoretical values of fVAc.

Figure S. 7 ( 7 . 29 Figure 7 .

 77297 Figure7.[START_REF] Kermagoret | One-pot controlled synthesis of double thermoresponsive N-vinylcaprolactambased copolymers with tunable LCSTs[END_REF] C NMR spectra of PVAc, PVCL, P(VAc0.19-co-VCL0.81) (expt 2 in Table1), P(VAc0.53-co-VCL0.47) (expt 5 in Table1) and P(VAc0.83-co-VCL0.17) (expt 7 in Table1) (co)polymers in CDCl3: (a) zoom on the PVAc and PVCL carbonyl signal range; (b) zoom on the PVAc methine signal range.

Figure 8 .

 8 Figure 8. Experimental glass transition temperature of the P(VAc-co-VCL) copolymers as a function of the weight fraction of VAc in the copolymer (equation 5); Dotted line: theoretical glass transition temperature of the copolymers calculated from equation 16.

Figure 9 .

 9 Figure 9. Transmittance at λ = 500 nm versus temperature for aqueous solution (3 g.L -1 ) of: P(VAc0.53-co-VCL0.47) copolymer (expt 5 inTable 1), P(VAc0.28-co-VCL0.72) copolymer (expt 3 in Table 1), P(VAc0.19-co-VCL0.81) copolymer (expt 2 Table 1) and P(VCL) homopolymer (expt 1 in Table 1); Full line (-): First heating, Dashed line (˗ ˗ ˗): Cooling, Dotted line (): Second heating; Photos of the solutions below (left) and above (right) the cloud point.

  Figure 9. Transmittance at λ = 500 nm versus temperature for aqueous solution (3 g.L -1 ) of: P(VAc0.53-co-VCL0.47) copolymer (expt 5 inTable 1), P(VAc0.28-co-VCL0.72) copolymer (expt 3 in Table 1), P(VAc0.19-co-VCL0.81) copolymer (expt 2 Table 1) and P(VCL) homopolymer (expt 1 in Table 1); Full line (-): First heating, Dashed line (˗ ˗ ˗): Cooling, Dotted line (): Second heating; Photos of the solutions below (left) and above (right) the cloud point.

  dn/dc calculated using equation 12 (dn/dccalc. = 0.082 mL.g -1 ). Therefore, for the series of P(VAc-co-VCL) copolymers, dn/dc values were calculated from equation 12.

	Differential Scanning Calorimetry (DSC). DSC measurements were carried out using the
	Q100 apparatus from TA instruments. Analysis were conducted at a heating rate of 10°C/min,
	under nitrogen gas flow, from 0°C to 200°C. The measurements method included a first heating
	step from 0 to 200°C at 10°C/min, a cooling step from 200 to 0°C at 10°C/min and a second
	heating step from 0 to 200°C at 10°C/min. Glass transition temperatures (Tg) of the
	(co)polymers were measured during the second heating step. (Co)polymers were dried under
	vacuum for 24h prior analysis.

[START_REF] Roy | New directions in thermoresponsive polymers[END_REF]

. In order to confirm the dn/dc calculation from equation 12, the dn/dc value was also measured experimentally by SEC via the plot of the RI area of the chromatogram versus the copolymer concentration (see Figure S.4, Supporting Information). The experimental dn/dc value of P(VAc0.63-co-VCL0.37) copolymer (expt 6 in Table

1

) (dn/dcexp. = 0.084 mL.g -1 ) was in good agreement with the Turbidimetry analysis. Turbidimetry measurements of the (co)polymers solutions (0.3 wt% in water) were performed with a Shimadzu UV-2450PC spectrophotometer, from 13 °C to 33°C

Table 1 )

 1 . Such enhancement of CTA efficiency is supported by the analysis of the UV-visible traces of the SEC chromatograms of the final copolymers which depict a decrease in the relative height of the residual CTA peak for higher initial VAc molar fractions copolymers by Co-MRP,[START_REF] Kermagoret | One-pot controlled synthesis of double thermoresponsive N-vinylcaprolactambased copolymers with tunable LCSTs[END_REF] the present work shows a better control of RAFT VCL/VAc copolymerization by increasing the initial VAc feed ratio.

	(see Figure S.5b in Supporting Information). In summary, both the increase of xanthate
	efficiency and the decrease of dispersity values (Table 1) by increasing fVAc,0 from 0 to 1
	converge to show that the chain transfer constant to xanthate agent (Ctr = ktr/kp) is higher for
	RAFT polymerization of VAc than of VCL. A value of Ctr of 25 was reported for O-ethyl-S-
	(1-ethoxycarbonyl)ethyldithiocarbonate mediated VAc polymerization,

[START_REF] Dufils | Phosphonate-terminated poly(vinyl acetate) synthesized by RAFT/MADIX polymerization[END_REF]

but no value of Ctr has been reported for the RAFT polymerization of VCL mediated by this xanthate agent. While an increase of dispersity values for higher levels of VAc was reported for the synthesis of P(VAc-co-VCL)

Table 1 .

 1 Final compositions and macromolecular features of the P(VAc-co-VCL) copolymers synthesized by RAFT/MADIX copolymerization in bulk at 65°C. is the VAc molar fraction in the initial mixture; b Xm is the overall monomer molar conversion at 6h, equation 2; c Xw is the overall monomer weight conversion at 6h, equation 3; d FVAc is the molar fraction of the monomer into the copolymer, equation 4; e Refractive index increment of the copolymer in THF determined from equation 12; f Theoretical number-average molar mass from equation 11; g Values obtained from the SEC analysis with a MALLS detector.

	Expt fVAc,0 a Xm b	Xw c	FVAc d dn/dcpolymer e	Mn,theo f	Mn,MALLS g	ÐMALLS g	Mn,theo/Mn,MALLS
		%	%		mL.g -1	g.mol -1	g.mol -1
	0	56 56 0	0.109	30 519 45 330	1.12	0.67
	0.15	41 41 0.19 0.103	20 510 48 190	1.19	0.43
	0.30	51 52 0.28 0.099	25 237 51 530	1.25	0.49
	0.52	56 57 0.49 0.090	23 669 32 840	1.11	0.72
	0.62	63 66 0.53 0.088	25 504 35 370	1.10	0.72
	0.71	56 59 0.63 0.083	23 029 28 650	1.08	0.80
	0.87	59 60 0.83 0.071	21 695 25 660	1.08	0.85
	1	63 63 1	0.058	21 085 23 380	1.04	0.90
	a fVAc,0

see Figure 8 and Table S.3,

						.2 and Figure S.8 in Supporting
	Information). The P(VAc-co-VCL) final (co)polymers exhibit a unique glass transition
	temperature (Tg) characterized by differential scanning calorimetry (Table S.3, Supporting
	Information). The good agreement between the experimental TgS of copolymers and the
	theoretical ones calculated from Fox-Flory equation (equation 16) (Supporting Information) confirms the homogeneous distribution of VAc and VCL monomer in
	the P(VAc-co-VCL) chain.
	1 𝑇 g,theo P(VAc-𝑐𝑜-VCL)	=	𝑤 VAc 𝑇 g,PVAc	+	𝑤 VCL 𝑇 g,PVCL	Equation 16

Table 1 )

 1 , P(VAc0.28-co-VCL0.72) copolymer (expt 3 in

Table 1 )

 1 , P(VAc0.19-co-VCL0.81) copolymer (expt 2

Table 1 )

 1 and P(VCL) homopolymer (expt 1 in

Table 1

 1

Table 3 .

 3 Characteristics of P(VAc-co-VCL) copolymers dispersed in water.

	Expt FVAc	Tcp, UV-vis a °C	Tcp, DLS b °C	T c °C	Mw, SLS d g.mol -1	Nagg. d Dh,DLS d nm
	2	0.19 27.4 ± 0.4	32.7	25	52 100	1	6.9

ACKNOWLEDGEMENTS

The French ministery of research and the University of Pau and Pays de l'Adour are acknowledged for funding L.E PhD under co-tutella regime with UPV. CNRS is acknowledged for M.S. and S.H. funding. S.H. acknowledges funding from the Agence National de Recherche (Project ASYMCOPO, ANR-15-CE08-0039). The authors are grateful to A. Khoukh for his support to NMR technique.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available as separate file.

AUTHOR INFORMATION

Corresponding Author *E-mail: maud.save@univ-pau.fr (M.S.).

Notes

The authors declare no competing financial interest.

TOC


Laurent Tranchant

M Billon

Save

Dr Mohamed Boularas

Dr Emilie Gombart

Dr Jean-François F Tranchant

Laurent Billon

Dr Maud Save

Pr L Billon

Design of Smart Oligo(ethylene glycol)-Based Biocompatible Hybrid Microgels loaded with Magnetic Nanoparticles

Design of smart oligo(ethylene glycol)-based biocompatible hybrid microgels loaded with magnetic nanoparticles

Introduction

"Smart" stimuli-responsive microgels dispersed in aqueous media are fascinating crosslinked polymeric colloids which undergo a volume phase transition in response to different external stimuli. [1] Thermoresponsive microgels are attractive materials for different emerging fields. [2] Due to their propensity to uptake and release active species, they proved to be suitable candidates for biomedical applications. [3] Their unique properties were also exploited in the field of materials to develop photonic crystals, [4] stabilizers of pickering emulsions [5] or responsive CO 2 -absorbents. [6] Hybrid microgels composed of inorganic nanoparticles (NPs) widen the microgel properties by combining the shrinking properties of the polymer network with the intrinsic properties of the inorganic NPs (magnetic, optical or catalytic properties).

Promising materials were prepared on the basis of thermoresponsive microgels loaded with iron oxide, [7] gold, [8] silver [9] or quantum dot NPs. [7d] So far, the most studied hybrid microgels are based on poly(N-isopropylacrylamide) (PNiPAM) [10] and a few example on poly(N-vinylcaprolactam) (PVCL). [7b, 9a] Nevertheless the potential toxicity of the acrylamidebased microgels might limit their application range. [11] Recently, there has been a growing interest for copolymers based on oligo(ethylene glycol) methacrylates as suitable alternatives for the design of thermoresponsive polymers. [12] These copolymers exhibit relevant features such as enhanced biocompatibility, tunable and sharp transition temperature. Whilst these thermoresponsive linear copolymers have been widely investigated for different purposes since 2006, [12] their microgel analogues have been scarcely investigated, [13] and no attention was paid to the preparation of oligo(ethylene glycol) methacrylate-based hybrid microgels.

The present work reports the synthesis and characterization of oligo(ethylene glycol) methacrylate-based magnetic thermoresponsive hybrid microgels. It should be mentioned that two different procedures have been proposed in the literature for the synthesis of hybrid microgels in aqueous dispersed media. The first one is based on the in-situ synthesis of NPs from the microsphere template while the second one consists in inserting the pre-formed nanoparticles into the interior of the microgel. Even though the first method has been widely investigated, [7b, 7d] the second one has been less explored due to difficulties in achieving high concentration of NPs while limiting their aggregation and release. [14] Nevertheless, this method deserves our attention as it represents a far more efficient way to preliminarily control the colloidal features of each separate particle and to ease material handling. Herein, we aim to address these issues by showing that multi-responsive methacrylic acid functionalized oligo(ethylene glycol) methacrylate-based microgels with fine tuned homogeneous microstructure can be relevant candidates to design stable monodisperse thermoresponsive hybrid microgels loaded with a high concentration of pre-formed magnetic NPs via a simple method.

Results and Discussion

The microgels are synthesized by surfactant-free precipitation polymerization in an environmentally friendly aqueous continuous phase (see details in Supporting Information).

The overlay of the individual conversions of the monomers (di(ethylene glycol) methyl ether methacrylate (MEO 2 MA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and methacrylic acid (MAA)) and the oligo(ethylene glycol) diacrylate (OEGDA) crosslinker indicates a homogeneous distribution of all units within the microgel (Figure S3, Supporting Information). It is interesting to note that the value of the apparent acid dissociation constant pK a of the P(MEO 2 MA-co-OEGMA-co-MAA) microgels exhibits a constant value as a function of the degree of ionization α of MAA (see Figure S6 in Supporting Information and equations ( 1) and ( 2

)). ] [ ] [ ] [ MAA Na MA Na MA + = + - + - α (1) ) 1 log( α α - - = pH pK a ( 2
)
The plateau is consistent with the absence of the polyelectrolyte effect, [15] hence supporting the homogeneous distribution of isolated carboxylic acid units from the outer to the inner of the P(MEO 2 MA-co-OEGMA-co-MAA) microgel. The multiresponsiveness of the P(MEO 2 MA-co-OEGMA-co-MAA) microgels was investigated by dynamic light scattering (DLS) at different temperatures and pH values. The pH value of the aqueous dispersion was adjusted between pH 4 and pH 9, thus scanning a degree of ionization of MAA from zero to one (Figure 1). The profile of the hydrodynamic diameter (D h ) versus temperature confirms the thermoresponsiveness of the P(MEO 2 MA-co-OEGMA-co-MAA) microgels with their ability to collapse with increasing temperature (Figure 1a). The biocompatible microgels proved to be concomitantly pH-responsive as the volume phase transition temperature (VPTT) increases from 34 -36 °C at pH 4-5 to 51 -56 °C at pH 7-9 (Figure 1b). Such increase of VPTT with pH is assigned to an increase of the degree of ionization of the methacrylic acid units which counterbalances the polymer collapse (Figure 1b). It is noteworthy that in contrast to P(NiPAM-co-AA) or P(VCL-co-MAA) microgels which displayed a broadening of thermal transition by increasing pH value, [16] the present P(MEO 2 MA-co-OEGMA-co-MAA) thermoresponsive microgels possess a sharp collapse-to-swelling transition (∆T < 10 °C) for all pH values (Figure 1b). The increase of the hydrodynamic diameter of the multiresponsive microgels from 0.5 µm to ca 1.0 µm is observed by raising pH from 4.9 to 8.0 (Figure 2, 9-fold increase of particle volume at 20°C). Interestingly, this profile is observed at both temperatures (20 °C and 60 °C), which shows that the increase of both electrostatic repulsions and internal osmotic pressure induced by sodium carboxylate units occurs in both swollen and collapsed states. [17] The evolution of the microgel swelling ratio (Φ, see equation ( 3)) with pH value shows three distinguished intervals (Figure 2).

        = °°C h C h D D φ (3)
In interval (I) corresponding to a degree of ionization of MAA equal to zero, lowering pH by adding HCl conveys an increase of salt concentration inducing a decrease of the swelling ability of the P(MEO 2 MA-co-OEGMA-co-MAA) microgels due to salting-out effect [17b] hydrophobic behavior of the microgels under acidic conditions. Interval (II) shows the maximum value of swelling ratio (Φ = 3), indicating that pH 5.0 -6.0 is the optimum pH range to promote the highest volume transition of microgels. For a degree of ionization of MAA above 24 % (pH > 6.0), the third interval corresponds to a decrease of the size change magnitude as the swelling ratio decreases from 3.0 to 1.7. This limited swelling is ascribed to the presence of repulsive electrostatic interactions between the homogeneously distributed sodium carboxylate units. In summary, the carboxylic acid-functionalized oligo(ethylene glycol)-based microgels represent novel promising multiresponsive biocompatible colloids.

We highlighted their well-defined inner microstructure with a homogeneous distribution of both methacrylic acid and crosslinker. Noteworthy, this feature is an important point to enable the further design of stable magnetic hybrid thermoresponsive microgels.

The preparation of the hybrid microgels is based on a straightforward strategy. The first incorporation of the aqueous dispersion of pre-formed iron oxide NPs (γ-Fe 2 O 3 ) (D h = 18 nm) at pH 3.0 is driven by polar interactions between the carboxylic acid units of microgels and the cationic charges of the NPs. The magnetic NPs are further destabilized inside the microgel by adjusting the pH at 7.0 corresponding to the point of zero charge of the maghemite NPs. [18] It is important to emphasize that this strategy offers the unique opportunity to load high content of pre-formed magnetic NPs (up to 33 wt-% vs microgel) while maintaining the hybrid microgel stability in aqueous solution (see Figure 3 and4). The loading of NPs is quantitative (see results of thermogravimetric analysis in Table S3, Supporting Information). Moreover, Figure 3 highlights the homogeneous distribution of the inorganic NPs into each microgel. Unlike the example of itaconic acid-functionalized PNiPAM microgels showing extensive desorption of magnetic NPs during the uptake step, [14] the present γ-Fe 2 O 3 -loaded biocompatible microgels do not exhibit any NP release (Figure 3). For the present system, it is noteworthy that the methacrylic acid units play a crucial role to promote interaction with magnetic NPs and to ensure the stability of hybrid microgels. Indeed, when using MAA-free P(MEO 2 MA-co-OEGMA) microgels, NPs were expelled outside (see Figure S10, Supporting Information) along with microgel flocculation. On the other hand, the hybrid γ-Fe 2 O 3 -P(MEO 2 MA-co-OEGMA-co-MAA) microgels loaded with maghemite were highly stable and monodisperse (Figure 3 and4c). An important observation from Figure 4a is that the magnetic hybrid microgels still exhibit a swelling-deswelling behavior with a reduction of the D h value from 1.0 µm at 20 °C to 0.4-0.5 µm at 60 °C. Starting from a similar hydrodynamic diameter at swollen state (D h,20°C ∼ 1.05 µm), the ability of the magnetic γ-Fe 2 O 3 -P(MEO 2 MA-co-OEGMA-co-MAA) microgels to collapse is significantly higher than the one of the initial P(MEO 2 MA-co-OEGMA-co-MAA) microgels (Φ hybrid microgel = 25 > Φ microgel = 2). This water expulsion can be an important point for future release of active species. Furthermore, the presence of magnetic nanoparticles induces a noticeable decrease of the microgel VPTT from 52 °C to 37 °C, a suitable temperature range for the fields of cosmetics and health care. This transition temperature is moreover constant whatever the NP content (Figure 4). Both of these considerations illustrate the homogeneous distribution of NPs as the volume phase transition behavior of the γ-Fe 2 O 3 -P(MEO 2 MA-co-OEGMA-co-MAA) hybrid microgels is different from PNIPAM-or PVCLbased composite microgels exhibiting a heterogeneous distribution of magnetic nanoparticles. [7b, 7c] Interestingly, the well-separated magnetic nanoparticles, interacting with the homogeneously distributed carboxylic acid functions in the present oligo(ethylene oxide)based network, does not hinder the change of copolymer conformation. The polar interactions more likely limits the ionization of the acid functions at pH 7, hence lowering both the VPTT value and the hydrodynamic diameter of the collapse hybrid microgels in comparison with the precursor microgels (Figure 4a, ∆ VPTT = 15 °C, ∆ Dh,60 °C = 400 nm).

Conclusions

In conclusion, we designed for the first time smart oligo(ethylene glycol)-based biocompatible hybrid thermoresponsive microgels containing a high fraction of magnetic NPs. The significance of the present work is to use a simple method for the preparation of such microgels by loading a high content of pre-formed magnetic nanoparticles, up to 33 wt-% of γ-Fe 2 O 3 versus microgels, into the biocompatible microgels while maintaining simultaneously perfect colloidal stability, monodisperse particles, sharp thermoresponsiveness, intense collapse-to-swelling transition and quantitative nanoparticle encapsulation. The homogeneous distribution in MAA of the multiresponsive oligo(ethylene glycol)-based microgels proved to be the key parameter to overcome the difficulties which usually arise from this method. Interestingly, a robust system is proposed here as the volume phase transition temperature of the hybrid thermoresponsive microgels is not influenced by the fraction of magnetic nanoparticles. As the collapse-to-swelling ability of the microgel is maintained, the present system could offer the opportunity to tune the VPTT of the hybrid microgels by selecting the initial molar ratio of OEGMA and MEO 2 MA. In summary, we successfully prepared stable monodisperse oligo(ethylene glycol)-based hybrid colloids combining multiple properties: biocompatibility, thermo-dependent swelling ability with tunable transition temperature and magnetic properties. The proposed strategy via the rational design of smart multi-functional polymeric vehicles by simple association of two distinct preformed aqueous colloidal dispersions with controlled features, offers interesting perspectives for different fields of applications such as cosmetics or health care.

Experimental section

The details concerning microgel synthesis, monitoring of individual monomer conversion, potentiometric and conductometric titration, evolution of pKa as a function of the ionization degree, TGA analysis, TEM images of magnetic nanoparticles and hydrid microgels are gathered in Supporting Information. The microgels were then purified by 5 centrifugation/redispersion cycles (10 000 rpm, 30 min) with milliQ grade water and finally stored at room temperature.Table S1 Table S1:

Table of Content

Monomer Conversion Measurement

In order to monitor the individual monomer conversion (x), the surfactant free precipitation polymerization was performed as described above but adding trioxane as internal standard (n trioxane = 0.1 (n OEGMA + n MEO₂MA + n OEGDA + n MAA ). For that purpose, samples were withdrawn at different time intervals and deuterated water (10 v-%) was added in each sample for proton NMR analysis (Figure S1 and Figure S2). The conversions of (MEO 2 MA +, OEGMA), MAA and OEGDA were calculated from the ratio of their respective peak integration with the integration of trioxane, according to equation Eq. S1 and monomer conversion versus time is represented in Figure S3.

) 0 ( ) 0 ( ) ( ) ( 0 0 0 / / 1 ] [ / ] [ ] [ / ] [ 1 ] [ ] [ 1 Trioxane M t Trioxane t M t t t I I I I Trioxane M Trioxane M M M x - = - = - =

Eq. S1

With I M and I Trioxane are the values of the peak integration of monomer M and internal standard (trioxane) at initial time t 0 and sampling time t. being peptized by adding pure water. A stable aqueous ferrofluid solution composed of 6 wt% of positively charged maghemite nanoparticles is recovered.

Morphology and hydrodynamic diameter of the maghemite nanoparticles were characterized respectively by transmission electron microscopy (TEM) and dynamic light scattering (DLS).

It was observed from TEM images (Figure S7) that maghemite particles are in "rock-like" shape and polydisperse with number average diameter ranging from 5 to 20 nm. Dynamic light scattering indicated an average hydrodynamic diameter of 18 nm in aqueous dispersion at pH of 2.

These ferrofluid dispersions are stable in acidic media but start to flocculate at a pH above 4, with an isoelectric point (or point of zero charge) at pH 7.2. 5 The nanoparticle destabilization observed at pH above 4 is mainly due to the neutralization of the cationic hydroxyl surface groups (OH 2 + ). The hybrid microgels were prepared by loading the cationic γ-Fe 2 O 3 nanoparticles into the stimuli-responsive P(MEO 2 MA-co-OEGMA-co-MAA) microgels (Table S3). For experiment 1 of Table S3 as example, 40 mL of the aqueous dispersion of P(MEO 2 MA-co-OEGMA-co-MAA) microgels at a concentration of 1.45 g.L -1 (pH 6.5) was introduced into a round bottom flask and the pH was adjusted to 3.0 by addition of nitric acid solution (HNO 3 0.1M). The cationic ferrofluid solution (10 mL at 1.34 g.L -1 ) was added dropwise to the aqueous microgel dispersion under continuous stirring at room temperature and was kept overnight under stirring. The pH of the aqueous solution of microgels was then adjusted to 7.0 by addition of alkaline solution (KOH, 0.5M) in order to destabilize the magnetic nanoparticles into the microgel structure while promoting the stability of the final hybrid microgel by the potassium The aqueous microgel dispersions at a concentration of 0.1 mg.mL -1 were cast onto the TEM copper grids and dried at room temperature for 24 hours before TEM analysis.

A3

B3 C3

1 µm

Figure 1 .

 1 Figure 1. (a) Hydrodynamic diameter of P(MEO 2 MA-co-OEGMA-co-MAA) microgels vs temperature measured at different pH; (b) Evolution of VPTT and α vs pH.

Figure 2 .

 2 Figure 2. (a) Hydrodynamic diameter at 20°C (squares), 60°C (circles) and swelling ratio (diamonds) of P(MEO 2 MA-co-OEGMA-co-MAA) microgels as a function of pH.

  (Φ = 3 at pH 5.0, Φ = 1 at pH 3.0). The absence of shrinkage at pH 3.0, together with the close values of the hydrodynamic diameter (D h = 510 nm, Figure2) and the number-average diameter measured at dry state (D TEM = 460 nm, FigureS9a), are both features of a

Figure 3 .

 3 Figure 3. Transmission Electron micrographs of γ-Fe 2 O 3 -P(MEO 2 MA-co-PEGMA-co-MAA) hybrid microgels loaded with 16 wt-% (a and b) or 33 wt-% (c) of magnetic NPs.

Figure 4 .

 4 Figure 4. Features of γ-Fe 2 O 3 -P(MEO 2 MA-co-OEGMA-co-MAA) hybrid microgels: (a) D h (at pH 7) vs temperature for various contents of γ-Fe 2 O 3 ; (b) dispersion of hybrid microgels under permanent magnet ; (c) particle size distribution at 55°C (PSD).

  Smart biocompatible oligo(ethylene glycol)-based hybrid microgels loaded with a high content of homogeneously distributed pre-formed magnetic nanoparticles are synthesized by a simple two step method. The monodisperse thermoresponsive magnetic microgels exhibit a constant value of the volume phase transition temperature up to high NPs content (33 wt-%) thanks to the homogeneous microstructure of the initial biocompatible microgels Mohamed Boularas, Emilie Gombart, Jean-François Tranchant, Laurent Billon* and Maud Save* Design of Smart Oligo(ethylene glycol)-Based Biocompatible Hybrid Microgels loaded with 272 g, 0.573 mmol) and OEGDA (0.029 g, 0.117 mmol) were dissolved in 57.5 mL of water. The reaction mixture was first introduced in a 250 mL 3-neck round-bottom flask equipped with a mechanical stirrer set at 150 rpm) and purged with nitrogen for 45 minutes at room temperature to remove oxygen. A solution of MAA (0.026 g, 0.305 mmol dissolved in 2 mL of water) was then introduced into the round bottom flask. The mixture was heated up to 70 °C prior to introduce the KPS solution (14.3 mg dissolved in 2.5 mL of degassed water) into the reactor in order to initiate the polymerization. The reaction mixture became turbid in few minutes and was kept at 70 °C for 6 hours (150 rpm) to complete the reaction. The individual conversions of MAA, OEGDA and ethylene glycol-based methacrylates were monitored by analyzing the polymerization medium by off-line 1 H NMR in D 2 0/H 2 0 (1:9 v/v).

  Synthesis of P(MEO 2 MA-co-OEGMA-cofeed ratio: [MEO 2 MA:OEGMA:MAA:OEGDA] = [84:9:5:2] mol-%., solid content at 100 % of monomer conversion = 2.1 wt-%; T = 70°C.

Figure S1: 1 H

 1 Figure S1: 1 H NMR spectra of polymerization medium at initial state

Figure S5 :

 S5 Figure S5: Degree of ionization α( 20°C) of MAA units as a function of pH at 20°C.

Figure S7 :

 S7 Figure S7 : TEM micrographs of γ-Fe 2 O 3 maghemite nanoparticles at scale of a. 100 nm and b. 20 nm.

  Finally the dispersion was purified by 5 centrifugation/redispersion (5 000 rpm, 20 min each).

Г

  microgel and Г γ-Fe2O3 are respectively the solid content in microgels and in iron oxide nanoparticles calculated just after addition of both aqueous dispersions. The theoretical value of introduced nanoparticles (γ-Fe 2 O 3 theoretical = Г γ-Fe2O3 /(Г γ-Fe2O3 + Г microgel )) is compared to the experimental fraction of inorganic nanoparticles (= γ-Fe 2 O 3 experimental) measured experimentally by thermogravimetric analysis (FigureS8).

Figure

  Figure S8 : Thermogravimetric plots of the hybrid γ-Fe 2 O 3 -P(MEO 2 MA-co-OEGMAco-MAA)/ microgelsloaded with two different amount of ferrofluid.

Figure S9 :

 S9 Figure S9: TEM micrographs of A/ P(MEO 2 MA-co-OEGMA-co-MAA) microgels ; B/ γ-Fe 2 O 3 -P(MEO 2 MA-co-OEGMA-co-MAA) hybrid microgels loaded with 15.9 wt% of magnetic NPs.and C/ γ-Fe 2 O 3 -P(MEO 2 MA-co-OEGMA-co-MAA) hybrid microgels loaded with 32.6 wt% of magnetic NPs.

Figure

  Figure S10: TEM micrographs of acid-free P(MEO 2 MA-co-OEGMA) microgels loaded with 4.7 wt% of magnetic NPs. Methods Nuclear Magnetic Resonance (NMR) spectroscopy. 1 H NMR spectra were recorded using a Brüker 400 MHz spectrometer at 25 °C. Conductometric and potentiometric techniques. A graphite electrode connected to a CDM230 conductivity meter (Radiometer Analytical, France) and SenTix 81 pH-electrode connected to

Table S2 : Molar content of carboxylic acid functions per gram of microgel (in mmol.g microgel -1 ) determined by titration using conductometry data.

 S2

	[COOH] theoretical	[COOH] exp
	mmol.g -1	mmol.g -1
	0.233	0.169 ± 0. 001)

Table S3 : Synthesis of hybrid γ-Fe 2 O 3 -P(MEO 2 MA-co-OEGMA-co-MAA) microgels. Expt Г microgel Г γ-Fe 2 O 3 γ-Fe 2 O 3 theoretical γ-Fe 2 O 3 experimental

 S3

		g.L -1	g.L -1	wt%	wt%
	1	1.49	0.299	16.6	15.9
	2	1.5	0.599	28.5	32.6

H NMR (H

O:D 2 O 90:10, 400 MHz): MEO 2 MA: δ (ppm): 1.85 (s,

3H, (C=C-CH 3 )), 3.3 (s, 3H, O-CH 3 ), 3.54 (d, 2H, O-CH 2 -CH 2 -O-CH 3 ), 3.64 (d, 2H, O-CH 2 -CH 2 -O-CH 3 ), 3.74 (d, 2H, C=C-(OC)-O-CH 2 -CH 2 -O-), 4.26 (d, 2H, C=C-(OC)-O-CH 2 -CH 2 -O-), 5.65 (dd, 1H, (H trans -HC=C(CH 3 )-C(=O)O-)), 6.1 (dd, 1H, (H cis -HC=CH(CH 3 )-C(=O)O-)). OEGMA : δ (ppm): 1.85 (s, 3H, (C=C-CH 3 )), 3.3 (s, 3H, O-CH 3 ), 3.54 (d, 2H, O-CH 2 -CH 2 -O-CH 3 ), 3.64 (d, 26-30H, O-CH 2 -CH 2 -O-CH 3 ), 3.74 (d, 2H, C=C-(OC)-O-CH 2 -CH 2 -O-), 4.26 (d, 2H, C=C-(OC)-O-CH 2 -CH 2 -O-), 5.92 (dd, 1H, (H trans -HC=C(CH 3 )-C(=O)O-)), 6.1 (dd, 1H, (H cis -HC=C(CH 3 )-C(=O)O-)), MAA: δ (ppm): 1.81 (s, 3H, C=C-(CH 3 )). 5.55 (dd, 1H, H trans -HC=C(CH 3 )-C(=O)OH)). 5.93 dd, 1H, H cis -HC=C(CH 3 )-C(=O)OH)), OEGDA : δ (ppm): 3.64 (d, 16-12H, O-CH 2 -CH 2 -O-), 3.74 (d, 4H, C=C-(OC)-O-CH 2 -CH 2 -O-), 4.26 (d, 4H, C=C-(OC)-O-CH 2 -CH 2 -O-), 5.92 (dd, 1H, (H trans -HC=CH-C(=O)O-)), 6.1 (dd, 1H, (H 2 C=CH-C(=O)O-)), 6.36 (dd, 1H, (H cis -HC=CH-C(=O)O-)), trioxane: δ(ppm)= 5.1 (s, 3H, (CH 2 )).

Acknowledgements:

The authors are grateful to LVMH for funding MB PhD, CNRS and UPPA for MS and LB. The authors would like to acknowledge T. Pouget (LVMH) for TEM, A. Khoukh (IPREM EPCP) for NMR, J. Rieger and L. Bouteiller (UMR8232) for supplying Nanosizer90 for DLS of hybrid microgels.

Supporting Information

Supporting Information is available from the Wiley Online Library or from the author EG repeat units, number average weight M n = 475 g.mol -1 , Aldrich), oligo(ethylene glycol)diacrylate (OEGDA, number average weight M n = 250 g.mol -1 , Aldrich) and potassium persulfate (KPS, 99%, ABCR) were used as received for the synthesis of P(MEO 2 MA-co-OEGMA-co-MAA) microgels. The water used for the microgel synthesis and characterization was purified using a Millipore Milli-Q system.

Synthesis of P(MEO 2 MA-co-OEGMA-co-MAA) microgels (Table S1)

The microgels are synthesized by surfactant-free precipitation polymerization in an aqueous continuous phase. Starting from a monophasic solution, the microgels are prepared by precipitation copolymerization of di(ethylene glycol) methyl ether methacrylate (MEO 2 MA), oligo(ethylene glycol) methyl ether methacrylate (OEGMA) and methacrylic acid (MAA) in the presence of oligo(ethylene glycol)diacrylate (OEGDA) as crosslinker and potassium persulfate (KPS) as initiator. At the initial stage, all reactants are soluble in water and the polymer chains precipitate during polymerization carried out at 70°C to form crosslinked colloidal particles stabilized by the sulfate charges (2.1 wt-% of solids content). The initial feed composition is [MEO 2 MA:OEGMA:MAA:OEGDA] = [84:9:5:2] mol-%. Crosslinker OEGDA and initiator KPS were kept at 1.92 mol% and 0.86 mol% versus the total vinylic molecules (MEO 2 MA + OEGMA + OEGDA). Briefly, MEO 2 MA (0.966 g, 5.14 mmol),

Salt-free base-into-acid titration of the P(MEO 2 MA-co-OEGMA-co-MAA) microgels by potentiometric and conductometric characterizations

Carboxylic functions titration. Base-into-acid titrations were performed at 20°C by coupling conductometric and potentiometric techniques as previously described elsewhere. 4 Samples were prepared by suspending 50 mg of freeze-dried microgels into 30 mL of MilliQ water.

The pH of the aqueous dispersion was adjusted below 3.0 with hydrochloride acid (0.1 M solution of HCl) in order to protonate all carboxylic acid groups. A freshly prepared solution of sodium hydroxide ([NaOH] = 1.55 mM) was used for titration.

The molar content of COOH units into the microgels was calculated from the conductivity versus NaOH volume curves using the standard extrapolation/intersection method to determine the titration endpoints (represented in Figure S4). The concentration of COOH units into the microgel was calculated using equation Eq. S2. The results are summarized in Table S2:

Eq. S2

V eq1 and V eq2 are respectively the volume of NaOH added to neutralize the excess of HCl and the volume of NaOH added to neutralize COOH units of the microgel (see Figure S4).

The degree of ionization of carboxylic acid functions α and their acidity constant pK a (presented in Figure S5 and Figure S6) were calculated from potentiometric titration data using the following equations: and particle size distribution (PSD) is given by the cumulant analysis method (a sample is considered monodisperse for values below 0.1). All calculations were performed using the NanoQ software.

Particle size of the hybrid microgels were measured using a Zetasizer Nano S90 from Malvern working at an angle of 90°, using a 4mW He-Ne laser at 633 nm. All calculations were performed using the Nano DTS software. Where k B is the Boltzmann constant, T is the absolute temperature, η is the viscosity of the medium, r is the hydrodynamic radius, and D Z is the apparent diffusion coefficient.

For measurements of the hydrodynamic diameter performed at different temperatures, the change of the water viscosity with temperature was taken into account. Samples were equilibrated for 15 min at each temperature before analysis.