Yoanh Moratille

Muhammad Arshad

Celine Cohen

Abdelhamid Maali

Elisabeth Lemaire

Nathalie Sintes-Zydowicz
email: nathalie.sintes@univ-lyon1.fr

Eric Drockenmuller
email: eric.drockenmuller@univ-lyon1.fr

Cross-linked polymer microparticles with tunable surface properties by the combination of suspension free radical copolymerization and Click chemistry

Keywords: Functionalized microparticles, suspension copolymerization, Click chemistry, fluorescent microparticles, non-Brownian suspensions, rheology, frictional contacts, adhesion

We propose a general, versatile and broad in scope two-steps approach for the elaboration of cross-linked polymer microparticles (µPs) with tunable surface properties and functionalities. Surface-functionalized cross-linked polymer µPs with diameter in the 80 μm range are prepared by the combination of: 1) suspension free radical copolymerization of styrene, propargyl methacrylate and 1,6-hexanediol dimethacrylate, 2) subsequent covalent tethering of a variety of azide-functionalized moieties by copper(I)-catalyzed azide-alkyne cycloaddition (i.e. rhodamine B fluorescent dye or poly(ethylene glycol) brushes) and, 3) optional N-alkylation of the 1,2,3-triazole groups followed by anion exchange reactions to afford covalently-tethered 1,2,3-triazolium ionic liquids with iodide or cresol red as counter-anions. The resulting µPs are characterized by laser diffraction, differential scanning calorimetry, as well as by optical, confocal fluorescence, scanning electron and atomic force microscopies. Finally, the rheological properties of concentrated suspensions (volume fractions of 0.40 and 0.44) of the different synthesized µPs dispersed in a 1:1 (vol/vol) mixture of polalkylene glycol and water are studied.

The modification of particles surface properties contributes not only to change the stability of the suspensions against flocculation, but also to significantly modify their rheological behavior at high shear stresses. This last result highlights the importance of non-hydrodynamic contact forces in the rheology of non-Brownian suspensions.

Introduction

Polymer particles are attractive for a wide array of cutting-edge applications such as paints, fillers, cosmetics, coatings, and biomedical devices [1,2]. Polymer microparticles (Ps) with diameters above tens of microns are commonly used as model systems for the study of the rheological properties of non-Brownian suspensions (NBSs) [3][4][5][6][7]. Until the 2000s, it was thought that NBSs exhibited universal behavior as long as the particles were spherical and the residual colloidal forces could be neglected when compared to the induced flow forces.

Non-Brownian and non-colloidal suspensions were also very often considered to be Newtonian. This claim was supported by the idea that the rheology of suspensions was controlled by hydrodynamic forces which are expected to be proportional to the shear rate. This was tantamount to saying that there was no other characteristic time in the system than the inverse of the shear rate and that therefore the viscosity should not depend on the shear rate (Newtonian behavior). This view was contradicted first by numerous experiments [4,[7][START_REF] Singh | Experimental measurements of the normal stresses in sheared Stokesian suspensions[END_REF][START_REF] Dai | Viscometric functions for noncolloidal sphere suspensions with Newtonian matrices[END_REF] which clearly showed that non-Brownian suspensions could exhibit shear-thinning behavior and then by the realization of the importance of the role played by direct contact forces between particles in the rheology of NBSs [START_REF] Guazzelli | Rheology of dense granular suspensions[END_REF][START_REF] Boyer | Unifying suspension and granular rheology[END_REF][START_REF] Gallier | Rheology of sheared suspensions of rough frictional particles[END_REF][START_REF] Peters | Rheology of non-Brownian suspensions of rough frictional particles under shear reversal: A numerical study[END_REF][START_REF] Chèvremont | Quantitative study of the rheology of frictional suspensions: Influence of friction coefficient in a large range of viscous numbers[END_REF]. These contacts, which may be frictional, have several important consequences on rheology. First, friction between particles considerably increases the viscosity of suspensions (about 1 order of magnitude for a 50% concentrated suspension) [START_REF] Guazzelli | Rheology of dense granular suspensions[END_REF][START_REF] Gallier | Rheology of sheared suspensions of rough frictional particles[END_REF][START_REF] Chèvremont | Quantitative study of the rheology of frictional suspensions: Influence of friction coefficient in a large range of viscous numbers[END_REF]. Second, recent studies suggest that shear-thinning can be explained by a stress-dependent friction [START_REF] Chatté | Shear thinning in non-Brownian suspensions[END_REF][START_REF] Lobry | Shear thinning in non-Brownian suspensions explained by variable friction between particles[END_REF][START_REF] More | Effect of roughness on the rheology of concentrated non-Brownian suspensions: A numerical study[END_REF]. It is therefore of utmost importance to control the surface properties of particles, both for applications involving suspension flows and, from a more fundamental point of view, to be able to conduct quantitative studies on the links between rheology and surface properties. However, most studies on the rheological properties of NBS commonly use rather ill-defined commercial µPs which prevent the precise control over their bulk and surface properties. A better understanding of the rheological properties of NBSs and a deepened investigation of the role of involved contact forces requires the access to µPs having tailored chemical, physical and structural properties such as: size, size distribution, shape, stiffness, surface roughness and surface chemistry. Suspension (co)polymerization of functionalized (co)monomers is one of the most suitable and versatile heterogeneous polymerization methods for the elaboration of functional polymer µPs in the 10-1000 µm size range with easily tunable composition as well as dimensional, thermal, mechanical, bulk and surface properties [2,[START_REF] Chaudhary | Suspension polymerization technique: parameters affecting polymer properties and application in oxidation reactions[END_REF][START_REF] Brooks | Suspension polymerization processes[END_REF][START_REF] Dowding | Suspension polymerisation to form polymer beads, Colloids Surfaces A Physicochem[END_REF][START_REF] Alroaithi | Suppressing Coalescence and Improving Uniformity of Polymer Beads in Suspension Polymerization Using a Two-Stage Stirring Protocol[END_REF][START_REF] Cordoví | Influence of stirring speed on the suspension copolymerization of styrene with methyl methacrylate[END_REF][START_REF] Mane | Effect of Chemical Crosslinking on Properties of Polymer Microbeads: A Review[END_REF][START_REF] Mane | Effect of Porogens (Type and Amount) on Polymer Porosity: A Review[END_REF][START_REF] Mane | Functional Polymers: A Review[END_REF]. However, the scope in terms of chemical functionalities of this fast one-pot approach is limited to functionalities compatible with a radical polymerization process in aqueous dispersed media (i.e. concerns with solubility and tolerance to free radicals). However, a broader range of functional groups can be introduced at the surface of the µPs obtained by the suspension copolymerization involving functionalized comonomers (e.g. alcohol, halide, amine, epoxy, vinyl or alkyne-functionalized comonomers) followed by post-polymerization chemical modification of residual surface functionalities [START_REF] Chaudhary | Suspension polymerization technique: parameters affecting polymer properties and application in oxidation reactions[END_REF][START_REF] Brooks | Suspension polymerization processes[END_REF][START_REF] Mane | Functional Polymers: A Review[END_REF][START_REF] Theato | Functional Polymers by Post-Polymerization Modification, First Edit[END_REF]. Robust, efficient and orthogonal ligation reactions, also coined as Click chemistry reactions [START_REF] Kolb | Click Chemistry: Diverse Chemical Function from a Few Good Reactions[END_REF][START_REF] Xi | Click chemistry in materials science[END_REF][START_REF] Castro | CuAAC: An Efficient Click Chemistry Reaction on Solid Phase[END_REF][START_REF] Arslan | The emerging applications of click chemistry reactions in the modification of industrial polymers[END_REF][START_REF] Tăbăcaru | Recent advances in click chemistry reactions mediated by transition metal based systems[END_REF], such as the copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), the ring-opening of epoxide groups (ERO), the hetero Diels-Alder as well as thiol-ene and thiol-yne reactions have been applied to the post-polymerization surface modification of polymer particles with diameters ranging from a few nanometers to 10 µm obtained by dispersion [START_REF] Anwar | RGD-decorated conjugated polymer particles as fluorescent biomedical probes prepared by Sonogashira dispersion polymerization[END_REF], precipitation [START_REF] Goldmann | Surface modification of poly(divinylbenzene) microspheres via thiol-ene chemistry and alkyne-azide click reactions[END_REF][START_REF] Nebhani | Efficient surface modification of divinylbenzene microspheres via a combination of RAFT and hetero diels-alder chemistry[END_REF],

(micro)emulsion [START_REF] Ouadahi | Synthesis of azide-functionalized nanoparticles by microemulsion polymerization and surface modification by click chemistry in aqueous medium[END_REF][START_REF] Evans | Click chemistry as a route to surface functionalization of polymer particles dispersed in aqueous media[END_REF][START_REF] Breed | Functionalization of polymer microspheres using click chemistry[END_REF] or seeded [START_REF] Bayraktar | Click-chemistry for surface modification of monodisperse-macroporous particles[END_REF] polymerization techniques [START_REF] Goldmann | Orthogonal ligation to spherical polymeric microparticles: Modular approaches for surface tailoring[END_REF]. However, there is a rather limited amount of work related to the chemical modification of the surface of polymer µPs with diameters above tens of microns. Besides the work of Shipp et al. who reported cross-linked functional µPs (average size 50 µm) from thiol-ene radical suspension photo-polymerization [START_REF] Durham | Functional polymer particles via thiol-ene and thiol-yne suspension "click" polymerization[END_REF], they mainly consist in the combination microfluidic processes with post-polymerization surface chemical modification using thiol-yne or thiol-ene reactions [START_REF] Prasath | Thiol-ene and thiol-yne chemistry in microfluidics: A straightforward method towards macroporous and nonporous functional polymer beads[END_REF][START_REF] Gokmen | Revealing the nature of thio-click reactions on the solid phase[END_REF][START_REF] Kaufmann | Bifunctional Janus beads made by "sandwich" microcontact printing using click chemistry[END_REF][START_REF] Kaufmann | Chemically orthogonal trifunctional Janus beads by photochemical "sandwich" microcontact printing[END_REF].

For instance, Du Prez et al. reported the general synthesis of thiol or alkyne-functionalized cross-linked polymer µPs (average size 400 µm) by photopolymerization using a wide range of thiol, alkene and alkyne monomers [START_REF] Prasath | Thiol-ene and thiol-yne chemistry in microfluidics: A straightforward method towards macroporous and nonporous functional polymer beads[END_REF]. The versatility of these µPs was further investigated by thiol-X or CuAAC post-polymerization modification of alkyne or thiol surface functionalities [START_REF] Gokmen | Revealing the nature of thio-click reactions on the solid phase[END_REF]. Du Prez, Ravoo et al. introduced bifunctional Janus µPs, particles with different functionalities on opposing poles, in a two-step process consisting of: 1) microfluidic to prepare epoxy, azide or thiol-functionalized polymer µPs (average size 170 µm) and 2) surface modification by "sandwich" microcontact printing employing either ERO, CuAAC or thiol-yne reactions [START_REF] Kaufmann | Bifunctional Janus beads made by "sandwich" microcontact printing using click chemistry[END_REF][START_REF] Kaufmann | Chemically orthogonal trifunctional Janus beads by photochemical "sandwich" microcontact printing[END_REF]. However, to the best of our knowledge surface functionalization of polymer µPs prepared by suspension (co)polymerization using Click chemistry processes has not been reported yet.

Herein, we report free radical suspension copolymerization for preparing alkyne-functionalized cross-linked polymer µPs, with diameter in the 80 μm range, bearing alkyne groups both within and at the surface of the µPs so that they may react with azide-terminated compound through subsequent CuAAC Click reactions. We demonstrate facile, robust and versatile surface modification of these polymer µPs through grafting of azideterminated polymer brush precursors and azide-functionalized fluorescent dyes. 1,2,3-Triazoliumfunctionnalized µPs were also demonstrated by N-alkylation of the 1,2,3-triazole moieties. Finally, the rheological properties of the concentrated NBSs including these surface-functionalized µPs were investigated.

Materials and methods

Materials

Styrene (Merck, 99%), 1,6-hexanediol dimethacrylate (HDDMA, Tokyo Chemical Industry, 98%), propargyl methacrylate (PMA, Alfa Aesar, 98%), lauroyl peroxide (LPO, Fisher Scientific, 95%), poly(vinyl alcohol) (PVA, Acros Organics, Mw = 88 000 g mol -1 , degree of hydrolysis = 88%), 4-methoxyphenol (MEHQ, Merck, 98%), sodium hydroxide (NaOH, Carlo Erba, 97%), L-ascorbic acid (Merck, 99%), copper(II) sulfate pentahydrate (CuSO4.5H2O, Merck, 98%), rhodamine B (Merck, 95%), 4-dimethylaminopyridine (DMAP, Merck, 99%), N,N-dicyclohexylcarbodiimide (DCC, Merck, 99%), iodomethane (CH3I, Merck, 99%), cresol red sodium salt (NaCR, Merck, 95%), dichloromethane (DCM, Merck, 99%), N,N-dimethylformamide (DMF, Carlo Erba, 99.9%), acetonitrile (CH3CN, Merck), toluene (Carlo Erba, 99.9%), tetrahydrofuran (THF, Carlo Erba, 99.9%), ethyl acetate (EtOAc, Carlo Erba, 99.8%), methanol (MeOH, Fisher Scientific, 99.8%) and ethanol (EtOH, Carlo Erba, 96%) were used as received. 2-[2-(2-Azidoethoxy)ethoxy]ethanol 1 and α-methoxy-ωazidoethoxy-poly(ethylene glycol) 3 (Mw = 37 000 g mol -1 ) were synthesized according to previously published procedures [START_REF] Gao | Synthesis of molecular brushes by "grafting onto" method: Combination of ATRP and click reactions[END_REF].

Synthesis of alkyne-functionalized cross-linked µPs MP1

Spherical cross-linked µPs MP1 were prepared by suspension free radical copolymerization in a 250 mL jacketed glass reactor equipped with a six-bladed impeller, an argon gas inlet, a condenser and a thermometer.

The continuous aqueous phase containing PVA (0.64 g, 0.40 wt%) dissolved in distilled water (160 mL) and the dispersed organic phase containing styrene (28.5 g, 285 mmol), HDDMA (4.26 g, 16.8 mmol), PMA (4.16 g, 33.5 mmol) and LPO (1.35 g, 3.39 mmol) were degassed by argon bubbling during 15 min. The monomer phase was introduced dropwise into the aqueous phase maintained at room temperature (r.t.) while stirring at 820 rpm under argon atmosphere. The stirring was maintained for 30 min at r.t. after which the reactor temperature was raised to 80 °C. Polymerization was allowed to proceed for 3 h before the reaction mixture was cooled down. µPs were isolated by several centrifugation and redispersion cycles using ethanol (3 × 100 mL) and deionized water (5 × 100 mL) in order to remove residual monomers and adsorbed PVA. Finally, µPs MP1 were freeze dried to yield a redispersable white powder (25.8 g, 67.6 %).

Monitoring of monomer conversion during synthesis of µPs MP1 by suspension polymerization

The monomer conversion during the synthesis of µPs MP1 by suspension copolymerization was monitored by gravimetry. The continuous aqueous phase containing PVA (0.64 g, 0.40 wt%) dissolved in distilled water (160 mL) and the dispersed organic phase containing styrene (28.5 g, 285 mmol), HDDMA (4.26 g, 16.8 mmol), PMA (4.16 g, 33.5 mmol) and LPO (1.35 g, 3.39 mmol) were degassed by argon bubbling during 15 min. The monomer phase was introduced dropwise into the aqueous phase maintained at room temperature (r.t.) while stirring at 820 rpm under argon atmosphere. After stirring for 30 min, the emulsion was divided into ca. 6 mL aliquots and filled into glass vials under argon atmosphere. The polymerization was carried out at 80 °C under constant stirring at 820 rpm. At polymerization times ranging from 0 to 3 h, vials were cooled down in an ice bath and quenched by the addition of ca. 6 mL of a 1 wt% solution of MEHQ inhibitor in THF. Each sample was then dried in a vacuum oven at 60 °C until reaching a constant weight. Monomer conversion X was calculated using equation 1:

𝑋 = 𝑚 𝑝 𝑚 𝑚 (1)
with mp the mass of dried polymer and mm the initial mass of comonomers in the aliquot.

Synthesis of azide-functionalized rhodamine B 2

A solution of DCC (4.05 g, 19.7 mmol) in CH2Cl2 (10 mL) was added dropwise to a solution of 2-[2-(2azidoethoxy)ethoxy]ethanol 1 (1.38 g, 7.90 mmol), rhodamine B (3.95 g, 8.25 mmol) and DMAP (192 mg, 1.57 mmol) in CH2Cl2 (20 mL) maintained at 0 °C. After stirring in the dark at room temperature for 24 h, the crude reaction mixture was purified by column chromatography using a 1:4 mixture of EtOAc and MeOH to yield after evaporation of solvents the rhodamine B azide 2 as a violet solid (2.64 g, 52.5%). 1 (m, 2H, Ha), 1.28 (t, 12H, J = 7.1Hz, Hv). 13

General procedure for the CuAAC functionalization of µPs. Preparation of rhodamine-functionalized µPs

MP2

Alkyne-functionalized µPs MP1 (1.0 g, ca. 1.2 × 10 -5 mmol of surface alkyne functionalities), rhodamine B azide 2 (1.5 mg, 2.4 × 10 -3 mmol), AscNa (200 µL of a 2.4 × 10 2 mM aqueous solution, 4.8 × 10 -2 mmol) and CuSO4 (200 µL of a 12 mM aqueous solution, 2.4 × 10 -3 mmol) were added to a 1:1 (vol/vol) mixture of H2O and EtOH (10 mL) and stirred in the dark for 72 h at 60 °C. The µPs were submitted to several centrifugation/dispersion cycles using 1:1 (vol/vol) H2O/EtOH mixtures (5 × 50 mL), then H2O (3 × 50 mL) and were finally freeze-dried to yield cross-linked µPs MP2 as a pink powder (991 mg, 99.1%).

Preparation of poly(ethylene glycol)-grafted µPs MP3

The general procedure for CuAAC functionalization was applied to µPs MP1 (1.0 g, ca. 1.2 × 10 -5 mmol of surface alkyne functionalities), azide 3 (44 mg, 1.2 × 10 -3 mmol), AscNa (100 µL of a 2.4 × 10 2 mM aqueous solution, 2.4 × 10 -2 mmol) and CuSO4 (100 µL of a 12 mM aqueous solution, 1.2 × 10 -3 mmol) to afford µPs MP3 as a white powder (992 mg, 99.2%).

Preparation of tri(ethylene glycol)-grafted µPs MP4

The general procedure for CuAAC functionalization was applied to µPs MP1 (1.0 g, ca. 1.20 × 10 -5 mmol of surface alkyne functionalities) with azide 1 (2.1 mg, 1.2 × 10 -2 mmol), AscNa (1.0 mL of a 2.4 × 10 2 mM aqueous solution, 2.4× 10 -1 mmol) and CuSO4 (1.0 mL of a 12 mM aqueous solution, 1.2 × 10 -2 mmol) to afford µPs MP4 as a white powder (995 mg, 99.5%).

Preparation of (1,2,3-triazolium iodide)-functionalized µPs MP5 by N-alkylation

µPs MP4 (1.0 g, ca. 1.2 × 10 -5 mmol 1,2,3-triazole groups), CH3I (1.5 mL, 2.4 × 10 -2 mmol) in CH3CN (10 mL) was stirred for 72 h at 40 °C. After evaporation of solvent and excess iodomethane under reduced pressure, µPs were submitted to several centrifugation/dispersion cycles using CH3CN (2 × 50 mL), 1:1 (vol/vol) H2O/ CH3CN mixtures (2 × 50 mL), then H2O (2 × 50 mL) and were finally freeze-dried to yield cross-linked µPs MP5 as a slightly yellow powder (931 mg, 93.1%).

Preparation of (1,2,3-triazolium cresol red)-functionalized µPs MP6 by anion exchange

µPs MP5 (500 mg, ca. 6.0 × 10 -6 mmol of 1,2,3-triazole groups) and cresol red sodium salt (2.4 mg, 6.02 × 10 -3 mmol) in H2O (10 mL) was heated at 60 °C for 72 h. µPs were then submitted to several centrifugation/dispersion cycles using H2O (5 × 25 mL) and were finally freeze-dried to yield µPs MP6 as a dark brown powder (989 mg, 98.9%).

Characterization

Size and size distribution of the µPs were measured by laser light diffraction (LD, Mastersizer 2000, Malvern Instruments) in ethanol, as well as dynamic laser light scattering (DLS, Nanosizer ZS, Malvern Instruments).

Particle size distribution is defined by the span = (d90-d10)/d50, where the d10, d50 and d90 values represent the particle diameter corresponding to the 10%, 50% and 90% cumulative percentage. µPs were observed as 0.02 wt% dispersions in water by optical microscopy (OM, BX41, Olympus) with a 10× objective, in transmission mode. The morphology of the µPs was characterized by scanning electron microscopy (SEM, Merlin Compact, Zeiss) at the Centre Technologique des Microstructures (University of Lyon). The samples were mounted on the sample holder with carbon SEM tape and coated with a thin layer of copper. An accelerating voltage of 1.00 kV was used to be sensitive to the extreme surface morphology. The surface morphology and roughness of the µPs were measured using an atomic force microscope (Bioscope AFM equipped with Controller IIIa, Bruker company). A glass slide was cleaned using ultra-pure water and ethanol. It was then coated with a thin layer of epoxy glue (Araldite, Hutsman Advanced Materials). The µPs were then manually dispersed on the surface of the epoxy glue. After a few minutes, pressurized nitrogen was thrown onto the surface, so that the non-sticked µPs were removed. Several samples were prepared for both µPs MP1 and MP3. The AFM was operated in tapping mode using rectangular cantilever (CSG30, NT-MDT Spectrum Instruments) with a tip radius of curvature < 10 nm. After adjusting the laser at the AFM tip, the cantilever was centered on the probed µP using an optical microscopy setup. The µPs were scanned at scan rate of 1 Hz and scan size of 20×20 and 1×1 µm 2 .

The images were processed and analyzed using the "Nanoscope_Analysis" software (Bruker). Confocal fluorescence microscopy was performed at the Centre Technologique des Microstructures (University of Lyon) using a confocal laser scanning microscope (CLSM LSM 800 Zeiss), equipped with 40× and 63× oil immersion objectives and suitable filter to detect rhodamine B probe (λem = 565 nm, λex = 543 nm). 1 H NMR (400 MHz) and 13 C NMR (100 MHz) spectra were recorded on a Bruker Avance 400 spectrometer in CDCl3. Chemical shifts (δ) are given in ppm in reference to the signal of residual CHCl3 for 1 H NMR (δ = 7.26 ppm) and CDCl3 for 13 C NMR (δ = 77.0 ppm). The signals of the different proton and carbon atoms were assigned through additional 1D and 2D NMR experiments (DEPT, COSY, HSQC, HMBC). Abbreviations for peak multiplicity are s for singlet, d for doublet, t for triplet, dd for doublet of doublet, dt for doublet of triplet and m for multiplet. HRMS experiments were carried out on a MicrOTOF-Q-II spectrometer (Bruker Daltonics, Bremen). Differential Scanning Calorimetry (DSC) was performed using a DSC Q200 (TA Instrument) calibrated with an indium standard. The samples were prepared using sealed pans and the experiments were conducted under a nitrogen purge of 25 mL min -1 on ca. 10 mg. The sample was first heated to 120 °C at a rate of 10 °C min -1 . Then, the temperature was decreased to -50 °C at a rate of 10 °C min -1 followed by a second heating to 200 °C at a rate of 10 °C min -1 . The glass transition temperature (Tg) was measured at the mid-point of the transition (on the second heating cycle) using the TA Thermal Analysis software. X-ray photoelectron spectroscopy (XPS) experiments were carried out using a K-alpha+ X-ray Photoelectron Spectrometer from Thermo Fisher Scientific equipped with a micro-focused monochromatic Al Kα X-ray beam source (1486.6 eV, 400 × 400 µm² spot size) and a flood gun to compensate for the static charge build-up. The samples were mounted in well generated by metal flat washer glued on the sample holder with tape. All data were acquired at the normal with respect to the plane of the surface. High-resolution core-level spectra of C1s, N1s and I3d were recorded with a 19, 18 and 30 eV pass energy, respectively. The peaks were referenced to C1s at 284.6 eV. The Fityk software was used for data processing. During peak fitting, the N1s curve was split into two groups of three peaks with a ratio of 1:1:1, for the 1,2,3-triazole and 1,2,3-triazolium groups. The shear viscosity of suspensions made either with µPs MP1, MP3 or MP5 was compared at two particle volume fractions: = 0.40 and = 0.44. The µPs were dispersed in a Newtonian fluid made of a 1:1 (vol/vol) mixture of water and polyalkylene glycol (Ucon Lubricant 75-H-90,000, Dow). Polyalkylene glycol is added to water in order to increase the viscosity of the suspending liquid which allows to explore a wide range of shear stress values at low Reynolds numbers. The viscosity of the mixture is 0.96±0.05 Pa.s at 22 °C and the density is the same as that of the µPs: 1.045 g cm -3 . The suspensions were mixed with a spatula until they become homogeneous and then the air bubbles trapped during mixing were removed by placing the suspension containers in an ultrasonic bath for 1 h and then in an oven at 60 °C for 12 h. Viscosity measurements were then carried out in torsional parallel plate geometry, using a controlled-stress rheometer Mars II (Thermo-Scientific). The bottom disk is stationary while a torque ( is applied to the top disk.

The suspension was poured on the bottom disc maintained at 22 °C by means of a Peltier element and the gap was set to 1.8 mm. The shear cell was covered with a Plexiglas lid in order to minimize evaporation of the suspending liquid. Parallel-plate geometry has the advantage that shear-induced particle migration is very limited. Indeed, in many cases, shear-induced particle migration gives rise to particle concentration heterogeneities that affect rheometric measurements. This is, for example, the case in a wide-gap cylindrical Couette geometry in which the particles tend to migrate towards the outer cylinder [START_REF] Phillips | A constitutive equation for concentrated suspensions that accounts for shear-induced particle migration[END_REF] or in a cone-plane geometry where particles migrate outwards [START_REF] Chow | Shear-induced particle migration in Couette and parallel-plate viscometers: NMR imaging and stress measurements[END_REF]. On the opposite, it has been shown that there was no [START_REF] Chow | Shear-induced particle migration in Couette and parallel-plate viscometers: NMR imaging and stress measurements[END_REF] or very weak [START_REF] Merhi | Particle migration in a concentrated suspension flowing between rotating parallel plates: Investigation of diffusion flux coefficients[END_REF] migration in a torsional parallel flow so that the apparent viscosity (ηapp) can be deduced from the ratio of the torque( to the angular velocity of the rotating disc() using equation 2:

𝜂 𝑎𝑝𝑝 = 2×ℎ× 𝜋×Ω×R 4 (2)
where R = 30 mm is the radius of the rotating disc and h = 1.8 mm is the gap between the two discs. The gap has been chosen to be wider than 20 times the particle diameter which is the gap width required for slip and structuring effects to be negligible. This high gap value restricts the range over which shear stress ( can be applied. Indeed, it is known that parallel plate flows of suspensions tend to exhibit edge fracture when the magnitude of the second normal stress is larger or of the same order of magnitude as the capillary pressure: |N2c|  5×/h where  is the surface tension of the suspension [START_REF] Keentok | Edge fracture in cone-plate and parallel plate flows[END_REF]. Taking  50 mN m -1 , then |N2c|  100 Pa. As the magnitude of N2 is expected to be in the order of a fraction of the shear stress (N2 ~ /2), the upper limit of the shear stress range is of the order of a few hundred Pa.

The second difficulty of using a rotating parallel plate geometry is that the shear rate is not constant but increases from zero at the center to γṘ = ×R/h at the rim which can be problematic for a fluid whose viscosity varies with shear rate, as is the case for concentrated non-Brownian suspensions. However, this problem can be overcome by introducing a Rabinovitch correction [50]:

𝜂 =  𝑎𝑝𝑝 (1 + 1 4 𝑑 𝑙𝑛 𝑎𝑝𝑝 𝑑 𝑙𝑛 𝑅 ̇) (3)
Finally, it must be checked that the measurements are carried out when the viscosity has reached its steady value. The viscosity of a suspension is closely related to its microstructure and the microstructure only reaches its steady state when the deformation to which the suspension has been subjected has exceeded a few units [START_REF] Blanc | Local transient rheological behavior of concentrated suspensions[END_REF].

Therefore, to meet this requirement, shear stress steps were applied rather than a continuously growing shear stress and to verify that, for each value of the stress, the suspension was sheared over a strain of several units. Scheme 1. Synthesis of alkyne-functionalized cross-linked µPs MP1.

Results and discussion

Synthesis of alkyne-functionalized cross-linked µPs

Cross-linked alkyne-functionalized µPs MP1 (Scheme 1) were obtained by suspension free radical copolymerization of styrene (85 mol%), PMA (10 mol%) and HDDMA cross-linker (5 mol%) using PVA as stabilizer (0.4 wt% with respect to the water continuous phase) and LPO as initiator (1 mol% with respect to comonomers). The monomers/H2O volume ratio was fixed at 1:4 and the polymerization was carried out at 80 °C for 3 h. Gravimetric monitoring as a function of polymerization time has shown that after a ca. 5 min inhibition period an almost complete conversion of the monomers was reached after 3 h of reaction at 80 °C (Fig. 1). After elimination of the surfactant and monomer residuals by centrifugation/dispersion cycles using ethanol and deionized water followed by freeze-drying, alkyne-functionalized cross-linked µPs MP1 were obtained in 68% yield as a white powder that could be readily redispersed in water, EtOH, and CH3CN. As shown by DSC analysis of µPs MP1 (Fig. S2 in the Supporting Information) no exotherm was found, indicating the absence of residual monomers. Besides, a Tg value of 94 °C was afforded thus confirming a glassy state at room temperature. It is worth mentioning that the obtained Tg value is in good agreement with the value calculated from the Fox's law (Tg = 92 °C) using the molar ratios of each comonomers in the feed and the Tg values previously reported for the corresponding homopolymers (Tg = 100, 55 and 28 °C for homopolymers of styrene, PMA and HDDMA) [START_REF] Chanda | Plastics Technology Handbook, Fifth Edit[END_REF][START_REF] Ishizone | Anionic polymerization of monomers containing functional groups, 11[END_REF][START_REF] Boutevin | Synthesis and thermal properties of bismaleate and bisfumarate telechelic oligomers from hydroxytelechelic polybutadienes[END_REF]. Optical microscopy confirmed the spherical shape of µPs MP1 with diameters ranging from ca. 20 to 160 µm (Fig. 2a-c). This was corroborated by LD (Fig. 2d) as µPs MP1 exhibit d50 of 80 µm with a span of 0.74. Replicas of the synthesis of µPs MP1 showed almost identical size and size distribution (Fig. 2d), attesting for the robust and highly reproducible particle formation process. The spherical shape and low surface roughness of µPs MP1

was also confirmed by SEM (Fig. 3a-b). However, at higher magnification a low amount of adhered nanoparticles (nPs) with ca. 20-200 nm diameter could be observed at the surface of µPs MP1 (Fig. 3c). Such nPs, also coined as "emulsion particles", result from the decomposition of initiator in the aqueous phase into free radicals, reacting with comonomers dissolved in the aqueous phase [START_REF] Jahanzad | On the evolution of particle size average and size distribution in suspension polymerization processes[END_REF]. The formation of these emulsion particles is heavily dependent on the partial water solubility of comonomers and initiator, as well as the concentration of surfactant [START_REF] Jahanzad | Suspension polymerisation in the presence of an inhibitor; Prolonged transition stage and suppressed emulsion particle formation[END_REF][START_REF] Gonçalves | Secondary particle formation in seeded suspension polymerization[END_REF]. Here, methacrylate-based and styrene monomers as well as initiator with very low water solubility limit excessive formation of such secondary particles (Table S1). After polymerization, DLS of the unwashed water phase showed emulsion particles of ca. 60-500 nm diameter (Fig S3). Most emulsion particles were discarded by repeated washings and centrifugations cycles in EtOH and deionized water, however some remained permanently attached to the surface of µPs due to the difunctional nature of the HDDMA cross-linker (Fig 3c). Also, PVA has long been identified as a cause of interparticle bridging and can occur by adsorption between the hydrophobic acetate blocks and the surface of hydrophobic polymer particles [START_REF] Almog | Dispersion Polymerization of Styrene: Effect of Surfactant[END_REF][START_REF] Brooks | Basic aspects and recent developments in suspension polymerisation[END_REF] or covalent bonds, in the presence of free radical, reactive sites may form on PVA leading to subsequent grafting to the particles [START_REF] Kim | Poly(vinyl alcohol) stabilization of acrylic emulsion polymers using the miniemulsion approach[END_REF]. The surface morphology of the different µPs by AFM is further discussed into more details in section 3.2. µPs MP1 appeared redispersible and insoluble in water, EtOH, CH3CN and DCM at room temperature, attesting for the efficient cross-linking of the polymer. The cross-linking degree, and so the swelling, of the µPs is critical as it dictates the accessibility of bulk and surface alkyne groups during subsequent chemical modifications. The swelling of µPs MP1 (S = VS/VW) was thus calculated by comparing the volume of the µPs measured by LD after 72 h of soaking in water (VW) and in different solvents (VS). For poor solvents, the swelling was moderate (S = 112 and 116% for EtOH and CH3CN, respectively) while for DCM, a good solvent for PS, the more significant swelling (S = 239%) without any dissolution of the µPs attested the high cross-linking density of the µPs MP1 (Fig. S4).

Scheme 2. Synthesis of rhodamine-functionalized µPs MP2 and poly(ethylene glycol)-grafted µPs MP3 by

CuAAC post-polymerization chemical modification of alkyne-functionalized µPs MP1. The alkyne-functionalized µPs MP1 were used as clickable scaffolds for their surface chemical modification by CuAAC using rhodamine B azide 2 and α-methoxy-ω-azidoethoxy-poly(ethylene glycol) 3 (Scheme 2). Since 1) alkyne groups are distributed at both the core and the surface of the µPs, 2) azides 2 and 3 are hydrosoluble, and 3) µPs MP1 do not swell in water and in EtOH, the CuAAC surface modifications were carried out under hydro-alcoholic conditions using CuSO4/sodium ascorbate as catalytic system and a 1:1 (vol/vol) H2O/EtOH as dispersing medium. These conditions ensured that the chemical modification almost exclusively occurred at the µPs surface [START_REF] Sharpless | Just click it: Undergraduate procedures for the copper(I)-catalyzed formation of 1,2,3-triazoles from azides and terminal acetylenes[END_REF]. Azides 2 and 3 were introduced in large excess (100 equiv.) with respect to the estimated amount of surface alkyne groups in µPs MP1 estimated using Eq. S3 and S5. Poly(ethylene glycol)-grafted µPs MP3 were also obtained by the CuAAC tethering of α-methoxy-ωazidoethoxy-poly(ethylene glycol) 3 (Mn = 37 kg mol -1 ) at the surface of µPs MP1 (Scheme 2). Both LD (Fig. 2d) and SEM (Fig. S8a-b) analyses of µPs MP3 confirmed that size, size distribution and surface morphology were comparable to those of alkyne-functionalized µPs MP1. The surface morphology of µPs MP1 and MP3 was investigated in more details by AFM (Fig. 5). Besides the presence of previously mentioned emulsion particles of ca. 30 nm in diameter, µPs MP1 presented low surface roughness with a root mean square (RMS) roughness of 2.2 nm (Fig. 5c), consistent with previous SEM observations (Fig. 3c). However, phase images of MP1 showed a surface with distinct domains, suggesting areas with different concentration of PS, PPMA and PHDDMA (Fig. 5e). Conversely, the surface of µPs MP3 showed an increased RMS roughness of 3.6 nm and the apparition of small, globular domains of ca. 20-150 nm in diameter (Fig. 5d). The low phase contrast between the globular-like morphologies and the background suggests that the entire surface is homogeneously covered by PEG brushes (Fig. 5f).These features have been frequently observed in the literature concerning PEG brushes "grafted-to" planar silicon substrates [START_REF] Ostaci | Poly(ethylene glycol) brushes grafted to silicon substrates by click chemistry: Influence of PEG chain length, concentration in the grafting solution and reaction time[END_REF]. A last sequence of surface chemical modifications was applied to alkyne-functionalized µPs MP1 in order to afford cross-linked polystyrene µPs having surface tethered 1,2,3-triazolium-based ionic liquids (Scheme 3).

Surface modification of alkyne-functionalized µPs

Initially, CuAAC reaction between 2-[2-(2-azidoethoxy)ethoxy]ethanol 1 and alkyne functionalities from the surface of µPs MP1 afforded 1,2,3-triazole-functionnalized µPs MP4. Subsequent N-alkylation of 1,2,3-triazole groups in acetonitrile for 72 hours at 40 °C using a large excess of methyl iodide (i.e. 2000 equiv. according to initial surface alkyne groups) yielded (1,2,3-triazolium iodide)-functionalized µPs MP5. SEM of MP4 and MP5 (Fig. S8c-f) showed that the surface morphology remained unchanged after the sequential chemical modifications. The occurrence of this surface chemical modification sequence was demonstrated by XPS analysis of (1,2,3-triazolium iodide)-functionalized µPs MP5. First, high-resolution I3d spectra of µPs MP1 and MP5 provided a clear evidence of the formation of surface tethered 1,2,3-triazolium iodides through the appearance of I3d5/2 and I3d3/2 peaks (at 618.1 and 629.4 eV, respectively) characteristic of iodine anions (Fig. 6a). This was corroborated by the high-resolution N1s spectra with the appearance of a main peak at 401.4 eV and a shoulder peak at 398.8 eV typical of 1,2,3-triazolium and 1,2,3-triazole groups, respectively (Fig. 6b) [START_REF] Moulder | Handbook of X-ray Photoelectron Spectroscopy: A Reference Book of Standard Spectra for Identification and Interpretation of XPS Data[END_REF]. Decomposition of the averaged high resolution N1s spectrum into three peaks with 1:1:1 ratio for 1,2,3-triazole and 1,2,3triazolium groups allowed to quantify the presence of ca. 20 mol% of 1,2,3-triazole groups remaining after the N-alkylation step (Fig. 6c). This appeared rather surprising according to the large excess of methyl iodide and the Counts (a.u.)

Binding Energy (eV) long reaction time. This could not be explained by higher swelling of the µPs during the CuAAC step (in hydroalcoholic medium) than the N-alkylation step (in acetonitrile) (Fig. S4). A more likely explanation is the de-N-alkylation reaction of the 1,2,3-triazolium groups and the reactivity of iodide groups during XPS analysis which has been previously reported [START_REF] Saad | X-ray induced degradation of surface bound azido groups during XPS analysis[END_REF]. Finally, anion exchange reaction between the surface 1,2,3-triazolium iodide groups of µPs MP5 and cresol red sodium salt fluorescent dye afforded fluorescently labeled µPs MP6.

The presence of cresol red fluorescent counter-anion was again confirmed by confocal fluorescence microscopy and appeared as a wide yellow fluorescent ring of ca. 10 µm surrounding µPs MP6 (Fig. 4b). The apparent surface localization of fluorescence suggests that the anion exchanged occurred and attests for the efficiency of the Nalkylation step. A possible reason for the additional width may arise from the size difference of the selected particles, smaller local radius of curvature of the particle leads to a wider fluorescence glare intensity.

Influence of surface functionalization on the rheological properties of NBSs of µPs MP1, MP3 and MP5

In this section, the effect of µPs surface properties on the rheology of NBS was investigated. Numerical simulations carried out over the last decade have shown the crucial role played by direct particle contact forces in the rheology of NBS [START_REF] Gallier | Rheology of sheared suspensions of rough frictional particles[END_REF][START_REF] Seto | Discontinuous shear thickening of frictional hard-sphere suspensions[END_REF][START_REF] Clavauda | Revealing the frictional transition in shearthickening suspensions[END_REF]. All the results of numerical simulations show that the viscosity of concentrated NBSs increases significantly when the coefficient of interparticle friction increases in the range [0-1]. On the contrary, these simulations show a very small impact of particle roughness on the viscosity of the suspensions.

This result is in apparent contradiction with the experimental results of Tanner & Dai [START_REF] Moon | The effect of sphere roughness on the rheology of concentrated suspensions[END_REF][START_REF] Tanner | Particle roughness and rheology in noncolloidal suspensions[END_REF][START_REF] Hoyle | Effect of particle roughness on the rheology of suspensions of hollow glass microsphere particles[END_REF] who studied the viscosity of suspensions of rough PS, PMMA or glass µPs. These results have shown a significant increase in viscosity with the increase of the particles surface roughness. These contradictory results may stem from the fact that it is quite experimentally challenging to vary independently the friction coefficient and roughness. To decouple the influence of these two parameters, we have chosen here to compare the behavior of NBSs with µPs having equivalent size, size distribution and roughness but strikingly different surface functionalities. Fig. 7 shows the variation of the shear viscosity with shear stress for suspensions composed of either µPs MP1, MP3 or MP5 for two particle volume fractions: = 0.40 and 0.44. The influence of surface chemistry on suspension viscosity is clearly visible, especially for the most concentrated suspensions (= 0.44, Fig. 7b).

Recent studies have shown that shear-thinning observed in concentrated NBSs was due either to the presence of adhesion forces between particles [START_REF] Richards | The role of friction in the yielding of adhesive non-brownian suspensions[END_REF] or to the decrease in the interparticle friction coefficient when the shear stress increases [START_REF] Chatté | Shear thinning in non-Brownian suspensions[END_REF][START_REF] More | Effect of roughness on the rheology of concentrated non-Brownian suspensions: A numerical study[END_REF] or both [START_REF] Papadopoulou | Journal of Non-Newtonian Fluid Mechanics On the shear thinning of non-Brownian suspensions : Friction or adhesion ?[END_REF]. These two effects lead to variations in viscosity with shear stress that have very different patterns. In particular, in the case of shear-thinning induced by the presence of adhesive forces between particles, the viscosity decrease is expected to be all the more marked the smaller the stress. Inspection can probably be explained by the promotion of interparticle contacts as the shear stress is ramped up from zero.

This scenario has been proposed to explain discontinuous shear-thickening (DST) in stabilized suspensions [START_REF] Mari | Shear thickening, frictionless and frictional rheologies in non-Brownian suspensions[END_REF].

In DST, the viscosity jumps across non-shear-thickened to shear-thickened regime can be of several orders of magnitude. The shear-thickening here observed is much less spectacular, but one has to keep in mind that DST is only observed for very concentrated suspensions (≳ 0.54). For lower particle volume fractions, DST turns out to CST with a continuous -and rather limited-increase of viscosity with stress [START_REF] Guy | Towards a Unified Description of the Rheology of Hard-Particle Suspensions[END_REF][START_REF] Singh | A constitutive model for simple shear of dense frictional suspensions[END_REF]. So the shear-thickening here observed can be interpretated in terms of the competition between the flow-induced forces which push the particles against each other and the repulsive forces (here of electrostatic nature) which tend to prevent contacts and is then the evidence that the (1,2,3-triazolium iodide)-functionalization of the µPs enabled to stabilize the suspension.

Beyond a typical stress of a about 10 Pa, the rheograms obtained either with MP1 or MP5-based suspensions converge and show a similar shear-thinning trend that is controlled by interparticle frictional forces.

The MP3-based suspension behaves in a different way with a continuous regular shear-thinning over more than three stress decades. Moreover, the viscosity is significantly lower than that of the other two suspensions.

This result was anticipated since brush-modified surfaces are known to exhibit a much lower friction coefficients than bare surfaces (see [START_REF] Mocny | Tribology of surface-grafted polymer brushes[END_REF] for a review). The typical values of friction coefficient for PEG-grafted surfaces are in the range [0.01-0.1] while the friction coefficient of bare polystyrene is of the order of [0.3-0.5]. Thus, grafting polymer brushes on particle surface is an efficient way to decrease NBS viscosity. Therefore, rheometric measurements have shown significant impact of surface properties of particles on rheology even though, given the particle size (80 µm), the studied suspensions are deemed "non-colloidal". These observations support the idea that a fine characterization of particle properties is necessary to both predict and understand the rheological behavior of NBS. From a practical point of view, the sensitivity of NBS rheology to particle surface properties opens perspectives in application fields where it is necessary to optimize the rheological properties.

Conclusions

This study demonstrates the successful synthesis of large alkyne-functionalized cross-linked µPs via suspension copolymerization and their subsequent surface modification by Click chemistry. Using this general surface grafting strategy poly(ethylene glycol)-grafted µPs were first fabricated. The visualization of the µPs carried out with SEM showed spherical shape and smooth surface. Well-defined and homogeneous surface-tethered PEG brushes observed by AFM and changes to the rheological properties of the resulting dense suspensions demonstrated successful surface modification. In an additional approach, cross-linked µPs with surface-tethered 1,2,3-triazolium ionic liquids were obtained by CuAAC of azide-functionalized tri(ethylene glycol) and subsequent N-alkylation of the 1,2,3-triazole groups. Their surfaces were characterized by XPS, confocal fluorescence microscopy and rheological measurement of surface-modified µPs. This represents a general, straightforward and effective method to afford cross-linked polymer µPs with a broad range of surface properties.

We have shown that the grafting of hydrophilic polymers or ionic liquid to initially hydrophobic cross-linked polymer µPs is a facile approach to provide the tuning of the rheological properties of the resulting Non-Brownian suspensions. This idea has been long established in the field of colloidal Brownian suspensions but is here extended to the case of concentrated non-Brownian suspensions and should be useful for the design of NBS in many applications where rheology is crucial. Our current work focuses on expanding the range of monomers used in suspension copolymerization to afford polymer µPs with strikingly different compositions and thermomechanical properties. This may be particularly useful for the study of particle suspension dynamics.

Suspension copolymerization led to µPs with alkyne groups distributed between the particle core and the surface and might therefore open the way to dually functionalized core-shell µPs. In hydroalcoholic medium, coupling through CuAAC occurs on the surface and performing a second functionalization step in organic medium should involve the alkyne groups located within particle core. Finally, better control of the µPs dimensions and dispersity using other emulsification processes is currently undergoing. here V is the volume of the layer formed by PEG (cm -3 ), ρ is the volumetric mass density of PEG and Mw is the molar mass of PEG. The number of surface alkyne functionalities n2 is ca. 1.2 × 10 -5 mmol g -1 equivalent to ca. 0.1 chain nm -2 .

Figure captions

From these methods, the estimated quantity of surface alkyne functionalities was fixed at n2 and surface modification was carried out with an excess of azide reactant compared to n2 (100 equiv.).

Scheme S1. Synthesis of azide-functionalized rhodamine B 2. * No value in the literature, considered value is based on N-propyl methacrylate [5]

  H NMR (400 MHz, CDCl3, δ): 8.29 (dd, 1H, J1 = 1.1 Hz, J2 = 7.9 Hz, Hi), 7.77 (dt, 1H, J1 = 1.4 Hz, J2 = 7.5 Hz, Hk), 7.69 (dt, 1H, J1 = 1.4 Hz, J2 = 7.7 Hz, Hj), 7.24 (dd, 1H, J1 = 1.0 Hz, J2 = 7.5 Hz, Hl), 7.02 (d, 2H, J = 9.5 Hz, Hp), 6.86 (dd, 2H, J1 = 2.5 Hz, J2 = 9.5 Hz, Hq), 6.75 (d, 2H, J = 2.4Hz, Hs), 4.13 (m, 2H, Hf), 3.56 (m, 16H, Hb, Hc, Hd, He, Hu), 3.31

Fig. 1 .

 1 Fig. 1. Monomers conversion as a function of polymerization time at 80 °C during the synthesis of µPs MP1 by suspension polymerization (The solid line is intended only as a guide to the eye).

Fig. 2 .

 2 Fig. 2. (a-c) Optical microscopy images of alkyne-functionalized µPs MP1. (d) LD size distribution of µPs MP1 (solid line) as well as µPs MP1 replica (dotted line) and poly(ethylene glycol)-grafted µPs MP3 (dashed line).

Fig. 3 .

 3 Fig. 3. (a-c) SEM images of alkyne-functionalized µPs MP1 at different magnifications.

4 .Fig. 4 .

 44 Confocal fluorescence microscopy of equatorial cross-sections (5 µm thick) of (a) rhodaminefunctionalized µPs MP2 and (b) cresol red-functionalized µPs MP6.

A

  first proof of the CuAAC chemical modification of the surface of µPs MP1 was provided by confocal fluorescence microscopy of µPs MP2 after tethering fluorescent azide-functionalized rhodamine B 2. Azidefunctionalized fluorescent dye 2 was synthesized through DCC-mediated esterification of rhodamine B and 2-[2-(2-azidoethoxy)ethoxy]ethanol 1 (Scheme S1). The structure and purity of 2 was assessed by NMR spectroscopy (Fig.S5and Fig.S6) and by electrospray ionization high-resolution mass spectrometry. The absorption and emission spectra of 2 (λabs = 560 nm, λem = 586 nm in water) was obtained by UV-vis spectroscopy (Fig.S7).Azide-functionalized fluorescent dye 2 was tethered at the surface of µPs MP1 by CuAAC to afford µPs MP2 (Scheme 2). After thorough removal of non-reacted azide 2, analysis of equatorial cross-section of µPs MP2 by confocal fluorescence microscopy showed the presence of a yellow fluorescent ring, demonstrating an effective surface anchoring of azide 2 (Fig.4a). Due to the low swelling of the µPs in the EtOH and water, the width of the fluorescent ring (ca. 3 µm) is most probably due to a "fluorescence glare" stemming from structures above and below the focal plane rather than to the actual depth of the grafting reaction. The Z-stacks obtained by confocal fluorescence microscopy were used for the reconstruction and segmentation of µPs MP2 (Video S1).

Fig. 5 .

 5 Fig. 5. AFM (a-b) (20×20 µm²) 3D surface reconstruction, (c-d) (1×1 µm²) height and (e-f) (1×1 µm²) phase images of (a,c,e) alkyne-functionalized µPs MP1 and (b,d,f) poly(ethylene glycol)-grafted µPs MP3.

Scheme 3 .Fig. 6 .

 36 Scheme 3. Synthesis of 1,2,3-triazolium-functionalized µPs MP5 and MP6 by N-alkylation and ion exchange reaction.

Fig. 7 .

 7 Fig. 7. Shear viscosity vs shear stress for suspensions composed of alkyne-functionalized µPs MP1 (red dots), poly(ethylene glycol)-grafted µPs MP3 (blue dots) and (1,2,3-triazolium iodide)-functionalized µPs MP5 (black dots) for particle volume fractions of (a) 0.40 and (b) 0.44. The viscosity of the suspending liquid is 0.96 Pa.s.

of

  Fig. 7b shows the presence of two distinct shear-thinning regimes for the MP1-based suspension: one at low shear stress (typically observed for < 5 Pa) and another regime where the viscosity decreases less rapidly for > 10 Pa. By comparing this curve with that obtained for the MP5-based suspension, it is clear that the addition of a surface charge leads to the suppression of the first shear-thinning regime (at low stress values). The electrical charge carried by the particles results in an electrostatic force barrier which prevents particles from coming into contact due to van der Waals forces. The weak shear-thickening that is observed at low stress values (≲ 3 Pa)

Fig. 1 .

 1 Fig. 1. Monomers conversion as a function of polymerization time at 80 °C during the synthesis of µPs MP1 by suspension polymerization (The solid line is intended only as a guide to the eye).

Fig. 2 .

 2 Fig. 2. (a-c) Optical microscopy images of alkyne-functionalized µPs MP1. (d) LD size distribution of µPs MP1 (solid line) as well as µPs MP1 replica (dotted line) and poly(ethylene glycol)-grafted µPs MP3 (dashed line).

Fig. 3 .

 3 Fig. 3. (a-c) SEM images of alkyne-functionalized µPs MP1 at different magnifications.

Fig. 4 .Fig. 5 .Fig. 6 .

 456 Fig. 4. Confocal fluorescence microscopy of equatorial cross-section (5 µm thick) of (a) rhodaminefunctionalized µPs MP2 and (b) cresol red-functionalized µPs MP6. Fig. 5. AFM (a-b) (20×20 µm²) 3D surface reconstruction, (c-d) (1×1 µm²) height and (e-f) (1×1 µm²) phase images of (a,c,e) alkyne-functionalized µPs MP1 and (b,d,f) poly(ethylene glycol)-grafted µPs MP3. Fig. 6. High resolution (a) I3d and (b) N1s of spectra of alkyne-functionalized µPs MP1 (black line) and (1,2,3triazolium)-grafted µPs MP5 (red line). (c) Peak fitted high resolution of N1s spectrum of µPs MP5. The N1s curve (black line) was split into 3 signals with 1:1:1 area ratio for the 1,2,3-triazole groups (blue dashed lines) and 3 signals with 1:1:1 area ratio for the 1,2,3-triazolium groups (pink dashed lines). The red line corresponds to the sum of the different contributions.

Fig. 7 .Scheme 1 .Scheme 2 .Scheme 3 .

 7123 Fig. 7. Shear viscosity vs shear stress for suspensions composed of either µPs alkyne-functionalized MP1 (red dots), poly(ethylene glycol)-grafted MP3 (blue dots) or (1,2,3triazolium iodide)-functionalized MP5 (black dots) =0.40 and (b) 0.44. The viscosity of the suspending liquid is 0.96 Pa s.

Fig. S2 .Fig. S3 .

 S2S3 Fig. S2. DSC data of alkyne-functionalized µPs MP1 obtained from the suspension copolymerization of styrene/HDDMA/PMA (85/5/10 mol%).

Fig. S5. 1 H

 1 Fig. S5. 1 H NMR spectrum (25 °C, CDCl3) of azide-functionalized rhodamine B 2 ( * : peak assigned to CHCl3).

Fig. S6. 13 C

 13 Fig.S6.13 C NMR spectrum (25 °C, CDCl3) of azide-functionalized rhodamine B 2.

  Fig. S8. SEM images of (a-b) poly(ethylene glycol)-grafted µP MP3, (c-d) tri(ethylene glycol)-grafted µPs MP4 and (e-f) (1,2,3-triazolium iodide)-functionalized µP MP5 at different magnifications.

Table .

 . S1. Water solubility of the monomers and initiator used for the preparation of alkyne-functionalized MP1 µPs and estimation of their respective mass fraction contained in the aqueous phase.

		Water solubility	Mass fraction in the	Reference
		(g L -1 )	aqueous phase (wt%)
	Styrene	0.27 (25 °C)	0.15	[4]
	PMA	< 1.0 (20 °C)	3.9	*
	HDDMA	0.023 (20 °C)	0.16	[6]
	LPO	2.0 × 10 -10	2.4 × 10 -8	[7]

* * 0.85 0.05 0.10

Acknowledgments

The authors gratefully acknowledge Veronica La Padula, Pierre Alcouffe and Valentin Cinquin and the CTµ microscopic center at University Lyon 1 for confocal fluorescence microscopy and SEM images. This work was supported by the Agence Nationale de la Recherche (ANR-18-CE06-0009).

Appendix A. Supplementary material

Supplementary data associated with this article can be found, in the online version, at …

Supporting Information for

Cross-linked polymer microparticles with tunable surface properties by the combination of suspension free radical copolymerization and Click chemistry

Determination of critical micelle concentration (CCMC)

Surface tensions (γ) of aqueous solutions were measured at 22 °C by the Wilhelmy plate method using a KRUSS tensiometer K100 and a platinum plate of 10 × 19.9 × 0.2 mm. Prior to each experiment, the instrument was calibrated by measuring the surface tension of distilled water (γ = 72.7 mN m -1 at 22 °C). A 10.0 g L -1 stock solution was prepared by dissolving PVA (100 mg, Mw = 88 000 g mol -1 ) in distilled water (10 mL) at 60 °C and was used to prepare 500, 200, 100, 75, 50, 10 and 1 mg L -1 solutions. Before surface tension was measured, each solution was allowed to stand for 5 min and the platinum plate was always cleaned with distilled water followed by ethanol and heating with a burner. Each experiment was repeated three times and average values of the surface tension data were reported. Surface tension of solutions was plotted against surfactant concentration Cs (mol L -1 ). The critical micelle concentration value CCMC (mol L -1 ) was determined by the intersection of the linear regression lines (Fig. S1). The plot of surface tension versus surfactant concentration was further used to estimate surface excess, Γs (mol m -2 ) using the Gibbs relation S1:

and from that, assuming that all the surfactant is at the interface, it is possible to determine ms the minimum mass of surfactant required to stabilize a particular emulsion size using equation S2:

where R (J mol -1 K -1 ) is the universal gas constant, T (K) is the temperature and Cs is the bulk molar surfactant concentration and d(γ)/d(ln Cs) is the slope of the surface tension plot; Mw (g mol -1 ) is the molar mass of PVA, Vw (L) is the volume of water in the emulsion and Aw/o (m 2 ) is the water/oil interfacial area within the emulsion calculated from the d50 diameter of the emulsion droplets.

Fig. S1. Surface tension as a function of PVA concentration

Calculation of the number of surface alkyne functionalities

Considering the grafting density of azide-terminated PEG brushes on alkyne-functionalized polymer particles ranges from 0.017 to 0.5 chains nm -2 , the number of surface alkyne functionalities n1(mmol g -1 ) at the surface of the µPs was determined using equation S3 [1][2][3]:

where D is the grafting density (chains nm -2 ), A is the surface area of a µP with a diameter equal to the d50 diameter (80 µm), Na (mol -1 ) is Avogadro's number; N is the number of µPs in the sample, m is the mass of the sample and ρ is the volumetric mass density of the µPs (1.045 g cm -3 ). Estimated number of surface alkyne functionalities n1 lies between ca. 2.0 × 10 -6 and 6.0 × 10 -5 mmol g -1 .

Another estimation of the number of surface alkyne functionalities (n2) can be made by considering the number of alkyne functionalities that can react with 2-[2-(2-azidoethoxy)ethoxy]ethanol 1 (Mw = 37 000 g mol -1 ) to form a ca. 10 nm thick dense layer of PEG brushes at the surface of the µPs using equation S5 : )

PVA concentration (wt%)

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Declaration of Interest Statement