Yangwei Deng

Hui Chen

Xinfeng Tao

Fangyi Cao

Sylvain Trépout

Jun Ling
email: lingjun@zju.edu.cn

Min-Hui Li
email: min-hui.li@chimieparistech.psl.eu

Oxidation-Sensitive Polymersomes Based on Amphiphilic Diblock Copolypeptoids

Keywords: polypeptoid, self-assembly, polymersome, oxidation-responsive, light-responsive

INTRODUCTION

Polymersomes are stable and robust vesicles made of amphiphilic block copolymers. [START_REF] Discher | Polymer Vesicles[END_REF] Polymer chemistry enables almost unlimited molecular design of responsive polymersomes whose rupture upon environmental changes can be used for the controlled release of active substances. Moreover, targeted transport can be achieved by taking advantage of the many possibilities to end-functionalize the copolymers. Therefore, polymersomes are good candidates for carriers in drug delivery and bio-imaging, or for micro/nano-reactors to conduct sophisticated and cascade catalytic reactions. [START_REF] Discher | Emerging Applications of Polymersomes in Delivery: From Molecular Dynamics to Shrinkage of Tumors[END_REF][START_REF] Lee | Polymersomes for Drug Delivery: Design, Formation and Characterization[END_REF][START_REF] Marguet | Multicompartmentalized Polymeric Systems: Towards Biomimetic Cellular Structure and Function[END_REF][START_REF] Schoonen | Compartmentalization Approaches in Soft Matter Science: From Nanoreactor Development to Organelle Mimics[END_REF] For the potential applications in drug delivery, controlled release of active species has been studied through the use of copolymers that respond to stimuli like reduction, oxidation, pH changes, temperature variation, shear stress, and light illumination. [START_REF] Li | Stimuli-Responsive Polymer Vesicles[END_REF][START_REF] Du | Advances and Challenges in Smart and Functional Polymer Vesicles[END_REF][START_REF] Meng | Stimuli-Responsive Polymersomes for Programmed Drug Delivery[END_REF][START_REF] Kim | Smart Nanocontainers and Nanoreactors[END_REF][START_REF] Deng | Physical Stimuli-Responsive Liposomes and Polymersomes as Drug Delivery Vehicles Based on Phase Transitions in the Membrane[END_REF][START_REF] Hu | Stimuli-Responsive Polymersomes for Biomedical Applications[END_REF][START_REF] Wang | Concurrent Block Copolymer Polymersome Stabilization and Bilayer Permeabilization by Stimuli-Regulated "Traceless" Crosslinking[END_REF][START_REF] Wang | Reversibly Switching Bilayer Permeability and Release Modules of Photochromic Polymersomes Stabilized by Cooperative Noncovalent Interactions[END_REF][START_REF] García | 13 -Stimuli-Responsive Polymersomes for Drug Delivery Applications[END_REF] Among these stimuli, the reactive oxygen species (ROS) existing in high concentration within tumors and inflammatory tissues present special interests and have been used as effective triggers to develop oxidation-responsive polymer nanostructures for target-specific therapeutic delivery. [START_REF] Lux | Biocompatible Polymeric Nanoparticles Degrade and Release Cargo in Response to Biologically Relevant Levels of Hydrogen Peroxide[END_REF][START_REF] Shim | A Reactive Oxygen Species (ROS)-Responsive Polymer for Safe, Efficient, and Targeted Gene Delivery in Cancer Cells[END_REF][START_REF] Deng | Reactive Oxygen, Nitrogen, and Sulfur Species (RONSS)-Responsive Polymersomes for Triggered Drug Release[END_REF] Hydrogen peroxide (H2O2), singlet oxygen ( 1 O2), superoxide, and hydroxyl radicals are the most well studied ROS in cancer and inflammatory diseases. [START_REF] Liou | Reactive Oxygen Species in Cancer[END_REF][START_REF] Halliwell | Hydrogen Peroxide in the Human Body[END_REF][START_REF] Ohshima | Chemical Basis of Inflammation-Induced Carcinogenesis[END_REF][START_REF] Doskey | Tumor Cells Have Decreased Ability to Metabolize H2O2: Implications for Pharmacological Ascorbate in Cancer Therapy[END_REF] These ROS can also be generated easily with spatiotemporal control by in situ biochemical or photochemical reactions. [START_REF] Ikeda | Installing Logic-Gate Responses to a Variety of Biological Substances in Supramolecular Hydrogel-Enzyme Hybrids[END_REF][START_REF] Vasdekis | Precision Intracellular Delivery Based on Optofluidic Polymersome Rupture[END_REF] For example, H2O2 can be produced by the glucose-oxidase(GOx)/glucose/oxygen system and can induce destabilization of oxidation-sensitive polymersomes. 24 1 O2 and other ROS can be generated under light illumination through photosensitizers incorporated in polymersomes and can eventually cause polymersome rupture and payload release. [START_REF] Vasdekis | Precision Intracellular Delivery Based on Optofluidic Polymersome Rupture[END_REF][START_REF] Xu | 2-(Methylthio)ethyl Methacrylate: A Versatile Monomer for Stimuli Responsiveness and Polymerization-Induced Self-Assembly in the Presence of Air[END_REF][START_REF] He | Photoconversion-Tunable Fluorophore Vesicles for Wavelength-Dependent Photoinduced Cancer Therapy[END_REF] Here, we report the ROS-activated bursting of polymersomes formed from the amphiphilic diblock copolypeptoids that are H2O2-sensitive and also engineered to be light-responsive by the incorporation of a photosensitizer.

The most common oxidation-sensitive polymers [START_REF] Lee | Current Progress in Reactive Oxygen Species (ROS)-Responsive Materials for Biomedical Applications[END_REF][START_REF] Song | Oxidation-Responsive Polymers for Biomedical Applications[END_REF][START_REF] El-Mohtadi | Oxidation-Responsive Materials: Biological Rationale, State of the Art, Multiple Responsiveness, and Open Issues[END_REF] include sulfur-containing aliphatic polymers (polythioethers), [START_REF] Vo | Polymers and Sulfur: What are Organic Polysulfides Good for? Preparative Strategies and Biological Applications[END_REF][START_REF] Sobotta | Oxidation-Responsive Micelles by a One-Pot Polymerization-Induced Self-Assembly Approach[END_REF][START_REF] Zhang | Synthesis of Pegylated Alternating Copolymer Bearing Thioether Pendants for Oxidation Responsive Drug Delivery[END_REF][START_REF] Ke | Therapeutic Polymersome Nanoreactors with Tumor-Specific Activable Cascade Reactions for Cooperative Cancer Therapy[END_REF] selenium/tellurium-based polymers, [START_REF] Cao | Selenium/Tellurium Containing Polymer Materials in Nanobiotechnology[END_REF][START_REF] He | Oxidation-Responsive and Aggregation-Induced Emission Polymeric Micelles with Two-Photon Excitation for Cancer Therapy and Bioimaging[END_REF] aryl oxalate-containing polymers, [START_REF] Lee | Hydrogen Peroxide-Responsive Copolyoxalate Nanoparticles for Detection and Therapy of Ischemia-Reperfusion Injury[END_REF] and phenylboronic ester-containing polymers. [START_REF] Deng | Engineering Intracellular Delivery Nanocarriers and Nanoreactors from Oxidation-Responsive Polymersomes via Synchronized Bilayer Cross-Linking and Permeabilizing Inside Live Cells[END_REF][START_REF] Tan | Anti-Inflammatory Polymersomes of Redox-Responsive Polyprodrug Amphiphiles with Inflammation-Triggered Indomethacin Release Characteristics[END_REF] In recent years, there has been an emerging interest in polythioethers of both main-chain and side-chain types, such as main-chain poly(propylene sulfide) (PPS), [START_REF] Napoli | Oxidation-Responsive Polymeric Vesicles[END_REF] polyethylene glycol with pendent thioethers, [START_REF] Herzberger | Oxidation-Responsive and "Clickable" Poly(ethylene glycol) via Copolymerization of 2-(Methylthio)ethyl Glycidyl Ether[END_REF] and polypeptides with pendent thioethers (poly(L-methionine) and S-substituted poly(L-cysteine)). [START_REF] Rodriguez | Enzyme-Triggered Cargo Release from Methionine Sulfoxide Containing Copolypeptide Vesicles[END_REF][START_REF] Kramer | Reversible Chemoselective Tagging and Functionalization of Methionine Containing Peptides[END_REF][START_REF] Yu | Injectable Bioresponsive Gel Depot for Enhanced Immune Checkpoint Blockade[END_REF][START_REF] Fu | Oxidation-Responsive OEGylated Poly-L-cysteine and Solution Properties Studies[END_REF][START_REF] Liu | Conformation-Directed Micelle-to-Vesicle Transition of Cholesterol-Decorated Polypeptide Triggered by Oxidation[END_REF] The hydrophobic thioether groups can be transformed into more polar sulfoxide or sulfone groups under the oxidation by ROS, leading to a significant increase of the water solubility of polymers. This solubility change results in a modification of the hydrophobic/hydrophilic balance of amphiphilic copolymers containing thioethers and causes the disassembly or dissolution of their polymersomes [START_REF] Napoli | Oxidation-Responsive Polymeric Vesicles[END_REF] or polymer micelles. [START_REF] Fu | Oxidation-Responsive OEGylated Poly-L-cysteine and Solution Properties Studies[END_REF][START_REF] Yan | Facile Synthesis of ROS-Responsive Biodegradable Main Chain Poly(carbonate-thioether) Copolymers[END_REF] In the present work, a bio-sourced side-chain polythioether was synthesized as a Polypeptoids, as the derivatives of polypeptides without hydrogen on the amide groups, avoid the formation of intra-and intermolecular hydrogen bonds. Consequently, polypeptoids possess generally a random coil conformation in solution similar to common polymers and show much better solubility in organic solvents compared to polypeptides. [START_REF] Chan | Polypeptoid Polymers: Synthesis, Characterization, and Properties[END_REF] On the other hand, polypeptoids, similar to polypeptides, maintain good biocompatibility and biodegradability. [START_REF] Lahasky | Thermoresponsive Poly(alpha-peptoid)s: Tuning the Cloud Point Temperatures by Composition and Architecture[END_REF][START_REF] Fetsch | Self-Assembly of Amphiphilic Block Copolypeptoids with C-2-C-5 Side Chains in Aqueous Solution[END_REF][START_REF] Secker | Poly(alpha-Peptoid)s Revisited: Synthesis, Properties, and Use as Biomaterial[END_REF][START_REF] Li | Synthesis and Characterization of Cleavable Core-Cross-Linked Micelles Based on Amphiphilic Block Copolypeptoids as Smart Drug Carriers[END_REF][START_REF] Ulbricht | On the Biodegradability of Polyethylene Glycol, Polypeptoids and Poly(2-Oxazoline)s[END_REF] PMeSPG and PSar (also named as poly(N-methyl glycine)) have a shared polyglycine backbone with, respectively, methyl-S-propyl and methyl as the side chain substituting for the hydrogen on the nitrogen atom. These compounds show the common properties of polypeptoids. Moreover, PSar is a water soluble polypeptoid based on a natural amino acid, which has been investigated for biomedical applications. [START_REF] Heller | Combining Reactive Triblock Copolymers with Functional Cross-Linkers: A Versatile Pathway to Disulfide Stabilized-Polyplex Libraries and Their Application as PDNA Vaccines[END_REF][START_REF] Birke | Polypeptoid-block-Polypeptide Copolymers: Synthesis, Characterization, and Application of Amphiphilic Block Copolypept(o)ides in Drug Formulations and Miniemulsion Techniques[END_REF][START_REF] Klinker | Secondary-Structure-Driven Self-Assembly of Reactive Polypept(o)ides: Controlling Size, Shape, and Function of Core Cross-Linked Nanostructures[END_REF] With good protein resistance, [START_REF] Maurer | Antigenicity of Polypeptides (Poly Alpha Amino Acids)[END_REF][START_REF] Sela | Immunological Studies with Synthetic Polypeptides[END_REF] PSar is considered to be a potential alternative to poly(ethylene glycol) (PEG). [START_REF] Huesmann | A Head-to-Head Comparison of Poly(sarcosine) and Poly(ethylene glycol) in Peptidic, Amphiphilic Block Copolymers[END_REF][START_REF] Birke | Polysarcosine-Containing Copolymers: Synthesis, Characterization, Self-Assembly, and Applications[END_REF] Thus, PSar and PMeSPG represent interesting and useful alternatives to PEG and poly(L-methionine)/poly(L-cysteine)/PPS as copolymer building blocks to prepare oxidation-responsive self-assemblies for controlled drug delivery applications. Up to now, reports of polymersomes based on polypeptoid-containing copolymers have been scarce. [START_REF] Tanisaka | Near-Infrared Fluorescent Labeled Peptosome for Application to Cancer Imaging[END_REF][START_REF] Deng | Poly(epsilon-caprolactone)-block-Polysarcosine by Ring-Opening Polymerization of Sarcosine N-Thiocarboxyanhydride: Synthesis and Thermoresponsive Self-Assembly[END_REF][START_REF] Gaitzsch | Peptoidosomes as Nanoparticles from Amphiphilic Block Alpha-Peptoids Using Solid-Phase-Synthesis[END_REF][START_REF] Fetsch | Self-Assembly of Amphiphilic Block Copolypeptoids -Micelles, Worms and Polymersomes[END_REF][START_REF] Varlas | Poly(sarcosine)-Based Nano-Objects with Multi-Protease Resistance by Aqueous Photoinitiated Polymerization-Induced Self-Assembly (Photo-PISA)[END_REF][START_REF] Jiang | Resolving the Morphology of Peptoid Vesicles at the 1 nm Length Scale Using Cryogenic Electron Microscopy[END_REF] Here we report the first oxidation-responsive polymersomes made of amphiphilic block copolypeptoids. As a typical ROS, H2O2 is produced at a rate of 5 nmol per hour by ten thousand units of cancer cells. [START_REF] Szatrowski | Production of Large Amounts of Hydrogen-Peroxide by Human Tumor-Cells[END_REF] The relatively high H2O2 concentration in tumor and inflammatory tissues has been utilized in targeted drug delivery studies. [START_REF] Wilson | Autonomous Movement of Platinum-Loaded Stomatocytes[END_REF][START_REF] Tu | Redox-Sensitive Stomatocyte Nanomotors: Destruction and Drug Release in the Presence of Glutathione[END_REF] In our work, H2O2

was first employed to assess the oxidation-sensitive property of the polymersomes. A photosensitizer, tetraphenylporphyrin (TPP), was also introduced into the system of polymersomes, which generated in situ ROS under illumination of visible light, and provoked the rupture of polymersome. Our system may be useful in target-specific therapeutic delivery and in the combination of chemotherapy and photodynamic therapy (PDT). [START_REF] Dolmans | Photodynamic Therapy for Cancer[END_REF][START_REF] Zhou | Reactive Oxygen Species Generating Systems Meeting Challenges of Photodynamic Cancer Therapy[END_REF] Other applications in biosensing, biodetection, and nano-/micro-reactors might be possible.

EXPERIMENTAL Monomer, Homopolymer and Block-Copolymer Synthesis

To synthesize the N-substituted thioether-bearing glycine, MeSPG, the purchased D,L-methionine (Sigma Aldrich) was firstly decarboxylated by reflux at 195 °C with the catalysis of R-(-)-carvone and then with hydrochloric acid. The aqueous solution of decarboxylated 3-(methylthio)propylamine (MeSPA) hydrochloride was then reacted with glyoxylic acid under reflux. The MeSPG hydrochloride was obtained after the removal of water. Sarcosine and MeSPG hydrochloride were, respectively, reacted with S-ethoxythiocarbonylmercaptoacetic acid (XAA), and followed by PBr3-catalyzed cyclization, to obtain the monomers Sar-NTA and MeSPG-NTA. Homopolymerization of MeSPG-NTA was conducted in THF at 60 °C, initiated by benzylamine with a controlled feeding ratio. The block copolymers PMeSPG-b-PSar were synthesized by sequential ROP of MeSPG-NTA and Sar-NTA using benzylamine as initiator. The detailed procedures and characterizations are described in Supporting Information.

Vesicle Preparation

In nanoprecipitation method, typically, the copolymer was dissolved in 1 mL of DMF (at 0.5 wt %). A total of 2 mL of deionized milli-Q water was injected slowly to the organic solution under mild shaking (2-3 μL of water per minute to 1 mL of polymer solution). The process of nanoprecipitation was carried out at 25 °C. The turbid mixtures were then dialyzed against water for 3 days to remove DMF using a Spectra/Por regenerated cellulose membrane with a molecular weight cutoff (MWCO) of 3500. In double emulsion method, copolymer was firstly dissolved in chloroform at a concentration of 5 g/L, together with Nile Red at 0.05 g/L (if applicable). Water (10 μL) was added to 1 mL of the above solution. The mixture was then treated by the homogenizer ULTRA-TURRAX T25, at the rate of 20000 rpm for 5 min, to prepare W/O inverse emulsion. W/O emulsion (300 μL) was transferred to 3 mL water, and dispersed by the homogenizer at 5000 rpm for 1 h. The obtained W/O/W double emulsion was heated at 50 °C with stirring (around 500 rpm) for 36 h to remove chloroform by evaporation, resulting in the vesicle sample with polymer concentration of 0.5 g/L. For the vesicle sample in D2O for 1 H NMR analysis, the copolymer/chloroform solution was prepared at 50 g/L, and 400 μL heavy W/O emulsion was prepared and transferred to 2.5 mL heavy water (D2O). About 2 mL vesicle-in-D2O was finally obtained after chloroform evaporation, with concentration at 10 g/L. For photo-activated experiments, TPP/dioxane solution (in the case of 1 H NMR analysis) or TPP/THF solution (in the case of CLSM observation) was added to the vesicle samples (volume ratio of 1 : 100) to obtain the "vesicle + TPP" blend. The molar ratio was typically at [TPP] : [copolymer] = 2.00 : 1 and [TPP] :

[MeSPG] = 1 : 30.

Cryo-Electronic Microscopy (cryo-EM)

Samples (5 μL) were deposited onto a 200 mesh holey copper grid (Ted Pella Inc., U.S.A.)

and flash-frozen in liquid ethane cooled down at liquid nitrogen temperature. Cryo-EM images were then acquired on a JEOL 2200FS energy-filtered (20 eV) field emission gun electron microscope operating at 200 kV using a Gatan ssCCD 2048 × 2048 pixels.

Confocal Laser Scanning Microscopy (CLSM)

For CLSM sample preparation, the coverslips were first treated with N-hydroxysuccinimide (NHS). The NHS esters grafted on the surface of coverslips can trap PMeSPG-b-PSar vesicles, through the reaction between NHS esters and amino groups at the end of PSar on the polymersome surface. This allows the immobilization of polymersomes during microscopic analysis. A narrow chamber between a slide and a surface-modified coverslip was formed using two plastic paraffin film (Parafilm M) strips as spacers. The vesicle dispersion was introduced inside the chamber by capillarity. In oxidation experiments with H2O2, H2O2 aqueous solution (10 μL, 35 mM ) was added by capillarity to the chamber together with vesicle dispersion (30 μL) to obtain a mixture with a final H2O2 concentration at 8.8 mM.

CLSM images were recorded with Leica TCS SP5 confocal scanning system equipped with a 63× oil immersion objective. HeNe 543 nm laser (maximum working power at 1.5 mW) was employed as the excitation light to obtain the fluorescent images.

Cell Viability Test

Human vein endothelial cells and mouse embryonic fibroblast (NIH 3T3) cells were purchased from the Center for Typical Culture Collection, Chinese Academy of Sciences (Shanghai, China). Cells were maintained in high-glucose DMEM (Gibco), supplemented with 10% fetal bovine serum (FBS, Sijiqing Co., Ltd., Hangzhou, China), 100 μg/mL penicillin, and 100 μg/mL streptomycin, and cultured at 37 °C in a 5% CO2 humidified environment. 1.0 x 10 4 cells were seeded in a 96-well plate and cultivated over night to allow cell attachment. The medium was replaced with fresh medium containing varying concentrations of polymersomes C2. After 24 h incubation, the cells were carefully washed with PBS and their viability was quantified using the MTT assay. [START_REF] Yu | A Discrete Organoplatinum(II) Metallacage as a Multimodality Theranostic Platform for Cancer Photochemotherapy[END_REF][START_REF] Bi | Realizing a Record Photothermal Conversion Efficiency of Spiky Gold Nanoparticles in the Second Near-Infrared Window by Structure-Based Rational Design[END_REF][START_REF] Zhu | Supramolecular Peptide Constructed by Molecular Lego Allowing Programmable Self-Assembly for Photodynamic Therapy[END_REF] The viability of cells treated with polymersomes-free medium was denoted as 100%. 4 replicates were used for cell experiments.

RESULTS AND DISCUSSION

Synthesis of Oxidation-sensitive Homopolymer PMeSPG and Diblock Copolymer

PMeSPG-b-PSar

As poly(amino acid)s, polypeptoids can be synthesized by solid phase synthesis, [START_REF] Zuckermann | Efficient Method for the Preparation of Peptoids Oligo(N-substituted glycines) by Submonomer Solid-Phase Synthesis[END_REF] ring opening polymerization (ROP) of N-carboxyanhydrides (NCA), [START_REF] Kricheldorf | Polypeptides and 100 years of chemistry of alpha-amino acid N-carboxyanhydrides[END_REF] and ROP of N-thiocarboxyanhydride (NTA). [START_REF] Tao | α-Amino Acid N-Thiocarboxyanhydrides: A Novel Synthetic Approach toward Poly(α-amino acid)s[END_REF] The N-substituted glycine N-thiocarboxyanhydrides (NNTAs) have recently emerged as promising monomers for polypeptoid preparation because they can be prepared in open air, have a long shelf life in an inert atmosphere, and can conduct well-controlled polymerization. [START_REF] Kricheldorf | Primary Amine-Initiated Polymerizations of Alpha-Amino Acid N-Thiocarbonic Acid Anhydrosulfide[END_REF][START_REF] Tao | Controlled Polymerization of N-Substituted Glycine N-Thiocarboxyanhydrides Initiated by Rare Earth Borohydrides toward Hydrophilic and Hydrophobic Polypeptoids[END_REF][START_REF] Tao | Are N-Substituted Glycine N-Thiocarboxyanhydride Monomers Really Hard to Polymerize?[END_REF][START_REF] Cao | Interfacial Ring-Opening Polymerization of Amino-Acid-Derived N-Thiocarboxyanhydrides Toward Well-Defined Polypeptides[END_REF][START_REF] Siefker | Organic Acid Promoted Controlled Ring-Opening Polymerization of α-Amino Acid-Derived N-thiocarboxyanhydrides (NTAs) toward Well-defined Polypeptides[END_REF] In this work, the synthesis of a new NNTA was introduced, as N-3-(methylthio)propyl glycine NTA (MeSPG-NTA) bearing an oxidizable thioether group (Scheme 1). The N-substituted thioether-bearing glycine, MeSPG, was firstly synthesized from the natural amino acid methionine, through a sequential one-pot "decarboxylation-N-carboxymethylation" procedure (Scheme S1). We implemented this method, for the first time, to transfer the substituting group R of the amino acid from the C-substituted position (C-R) to the N-substituted position (N-R). A number of N-R glycines can potentially be synthesized from natural amino acids using this approach. The monomers MeSPG-NTA and sarcosine-NTA (Sar-NTA) were then prepared from MeSPG and sarcosine, respectively, as described previously (Section 3 in SI). [START_REF] Tao | Controlled Polymerization of N-Substituted Glycine N-Thiocarboxyanhydrides Initiated by Rare Earth Borohydrides toward Hydrophilic and Hydrophobic Polypeptoids[END_REF] The homopolymer PMeSPG84 was synthesized by benzylamine-initiated ring-opening polymerization of MeSPG-NTA (Scheme S2). The block copolymers PMeSPG-b-PSar were synthesized by feeding in sequence MeSPG-NTA and Sar-NTA in amine-initiated polymerizations (Scheme 1, more details in Section 4, SI). [START_REF] Tao | Are N-Substituted Glycine N-Thiocarboxyanhydride Monomers Really Hard to Polymerize?[END_REF] The structures of the homopolymer and block copolymers were confirmed by NMR, SEC, and MALDI-TOF (Figure S2-S5). The compositions of two block copolymers PMeSPG45-b-PSar65 (C1) and PMeSPG60-b-PSar50 (C2) used in this study are summarized in Table S2.

We then investigated the thioether-to-sulfoxide modification of the homopolymer PMeSPG84 under a simple oxidation by H2O2. As shown in Figure S11, [START_REF] Discher | Polymer Vesicles[END_REF] H NMR spectra of PMeSPG84 before and after a 90-min treatment by 2.9 M H2O2 (10 wt%) (Section 7 in SI) indicated clearly the reaction from thioether to sulfoxide, in comparison with the spectra of polymethionine sulfoxides reported previously. 82

Nano-Polymersomes and Their Destruction by Oxidation

Two copolymers PMeSPG45-b-PSar65 (C1) and PMeSPG60-b-PSar50 (C2) (Table S2) were chosen for the preparation of polymersomes, since their hydrophilic weight ratios (41% and 29%, respectively) were favorable for vesicle formation. [START_REF] Blanazs | Self-Assembled Block Copolymer Aggregates: From Micelles to Vesicles and their Biological Applications[END_REF] Nanoscale vesicles of diameter of 100-250 nm, together with lamellae and spherical/cylindrical micelles, were obtained by nanoprecipitation (Section 5 in SI). Figure 1A The contour length of the hydrophobic part (PMeSPG45) was calculated as 17.6 nm using the MeSPG unit length of 3.9 Å obtained by modelling an oligomer of 8 repeat units after the MM2 energy minimization with the software Chem3D (Figure S8). We noted that the membrane thickness was much lower than the sum of the lengths of two stretched chains (35.2 nm). Therefore, the conformation of the hydrophobic chain PMeSPG should be coil-like in the membrane and the membrane has a unilamellar bilayer structure where the chain entanglement would be possible between two leaflets.

The C1 self-assemblies were then treated with 2.9 M H2O2 (10 wt%) (more details in Section 7, SI). After the treatment no vesicle was observed under cryo-EM. Instead, only a very few loose irregular clusters were detected (Figure 1B), which were probably the residues left by the oxidized vesicles. The oxidation of the C1 self-assemblies was also followed by the evolution of the count rates measured by dynamic light scattering (DLS) in kilo counts per second (kcps). The count rates represent the scattering intensity and indicate the size and/or concentration of particles. The count rates decreased exponentially upon the addition of H2O2 with a characteristic time τ = 20.2 min (Figure 1C). The decline in count rates can be explained by the decrease in the size and/or concentration of the self-assemblies. After a 60-min or longer treatment, the count rates decreased to less than 100 kcps, the limit below which the size measurement becomes unreliable. The evolution of count rates confirmed the disruption of vesicles and other self-assemblies upon oxidation by H2O2. The variation of count rate was also analyzed with lower concentration of H2O2, such as 8.8 mM and 88 mM (Figure S12). The count rates also declined along with the oxidation time, but more slowly with a characteristic time τ = 288.8 min and 112.8 min for H2O2 concentration of 8.8 mM and 88 mM, respectively. The evolution of count rates of self-assemblies C1 measured by DLS as a function of the time of treatment with 2.9 M H2O2. The symbols are the experimental points. The dashed line is the fit with the exponential decay function c = cmaxe -t/τ , where cmax is the maximum count rate at t = 0, and τ = 20.2 min is the characteristic time for c to decrease to 36.8% of cmax.

Giant Polymersomes and Their Destruction by Oxidation

To address in more detail the oxidation-responsive transformation of the vesicular morphology, we prepared micrometer-sized giant vesicles and then followed their disruption under the oxidation of H2O2 in situ using confocal laser scanning microscopy (CLSM). An emulsion-mediated approach through water-in-oil-in-water (W/O/W) double emulsion [START_REF] Bae | Osmotically Driven Formation of Double Emulsions Stabilized by Amphiphilic Block Copolymers[END_REF][START_REF] Park | Stepwise Drug-Release Behavior of Onion-Like Vesicles Generated from Emulsification-Induced Assembly of Semicrystalline Polymer Amphiphiles[END_REF][START_REF] Wang | Dual-Phase Separation in a Semiconfined System: Monodispersed Heterogeneous Block-Copolymer Membranes for Cell Encoding and Patterning[END_REF] was employed to prepare giant vesicles (see SI and Figure S9). Both block copolymers C1 and C2 can form giant vesicles by this double emulsion method. As the vesicles obtained from C2 have larger sizes compared to those from C1, C2 was chosen to make giant polymersomes in the following studies for their easier optical observation by CLSM. The obtained polymersomes C2 had a wide size distribution from 0.5 to 20 μm in diameter (Figure 2A). The relatively small vesicles among them were trapped successfully in the sample grid by the rapid vitrification for cryo-EM imaging (Figure 2B and Figure S10). Their membrane thickness measured as around 12 nm, which is consistent with a unilamellar bilayer structure of amphiphilic block copolymer C2 as discussed above.

The sample of giant polymersomes labeled with Nile Red (1 wt%) was placed between a slide and a surface-modified coverslip to observe the response of polymersomes to H2O2 oxidation under CLSM (more details in SI for CLSM experiments and surface treatment procedure). The disruption of C2 vesicles was initiated by the addition of H2O2. Typically, 10 μL of 35 mM H2O2 (0.12 wt%) was added into 30 μL of polymersome dispersion (60 wt% of copolymer), resulting in a final concentration of 8.8 mM for H2O2 (0.03 wt%) and a molar ratio of [H2O2] : [MeSPG] = 3.62 : 1. We observed two pathways in the vesicle disruption. To confirm the chemical oxidation of copolymers under the conditions of CLSM observation, the analyses by DLS was performed for giant polymersomes C2 treated by 8.8 mM H2O2.

The ratio of count rates α in Figure 3 represents the count rates at time t normalized with respect to count rates before H2O2 addition. The evolution of α as a function of time (Figure 3A, red curve) supported the tendency of concentration/size of the self-assemblies to decrease through the oxidation. We also examined the cases with higher H2O2 concentrations (88 mM and 880 mM). As shown in Figure 3A (blue and black curves), the value of α decreased more rapidly when treated with higher H2O2 concentration. The decay of α under each condition was fitted with an exponential function, giving the characteristic time τ of 302.6 min, 70.6 min, and 7.4 min for H2O2 concentration at 8.8 mM, 88 mM, and 880 mM, respectively. The polymersome oxidation was also followed by [START_REF] Discher | Polymer Vesicles[END_REF]

Light-Activated Bursting of Giant Polymersomes

Having successfully prepared oxidation-sensitive giant polymersomes, we then searched for a more controllable and more practical way to introduce the oxidation agents rather than the direct addition of H2O2. The generation of ROS such as singlet oxygen ( 1 O2) can be triggered remotely and precisely by light. 1 O2 transformed from the ground state triplet oxygen ( 3 O2) upon light illumination has been reported to destabilize liposomes [START_REF] Riske | Giant Vesicles under Oxidative Stress Induced by a Membrane-Anchored Photosensitizer[END_REF] and polymersomes. [START_REF] Vasdekis | Precision Intracellular Delivery Based on Optofluidic Polymersome Rupture[END_REF][START_REF] Xu | 2-(Methylthio)ethyl Methacrylate: A Versatile Monomer for Stimuli Responsiveness and Polymerization-Induced Self-Assembly in the Presence of Air[END_REF][START_REF] Mabrouk | Photosensitization of Polymer Vesicles: A Multistep Chemical Process Deciphered by Micropipette Manipulation[END_REF][START_REF] Robbins | Photoinitiated Destruction of Composite Porphyrin-Protein Polymersomes[END_REF] In a few examples, [START_REF] Vasdekis | Precision Intracellular Delivery Based on Optofluidic Polymersome Rupture[END_REF][START_REF] Xu | 2-(Methylthio)ethyl Methacrylate: A Versatile Monomer for Stimuli Responsiveness and Polymerization-Induced Self-Assembly in the Presence of Air[END_REF] ROS thus produced reacted with thioether groups and provoked the disassembly of polymersomes. The light-triggered rupture of polymersomes made of PEG-b-PPS with a main-chain polythioether (poly(propylene sulfide)) has been shown to be useful in precision intracellular delivery. [START_REF] Vasdekis | Precision Intracellular Delivery Based on Optofluidic Polymersome Rupture[END_REF] Here, we explored photo-activated rupture of PMeSPG-b-PSar polymersomes by introducing photosensitizers into the system.

The giant polymersomes C2 obtained from double emulsion method were blended with the photosensitizer, tetraphenylporphyrin (TPP). [START_REF] Xu | 2-(Methylthio)ethyl Methacrylate: A Versatile Monomer for Stimuli Responsiveness and Polymerization-Induced Self-Assembly in the Presence of Air[END_REF] TPP is a hydrophobic porphyrin, which emits red fluorescence under visible light excitation and can be activated to transform dissolved oxygen to 1 O2. TPP has a strong absorption band with maximum at 419 nm (so called Soret band) and four weak bands with maxima at 515, 550, 593 and 649 nm (so called Q-bands). [START_REF] Kim | Mechanistic study of the synthesis and spectral properties of meso-tetraarylporphyrins[END_REF] Visible lights and lasers in the Q-bands have been used frequently to activate the generation of 1 O2. [START_REF] Xu | 2-(Methylthio)ethyl Methacrylate: A Versatile Monomer for Stimuli Responsiveness and Polymerization-Induced Self-Assembly in the Presence of Air[END_REF] The mixture of polymersomes C2 and TPP was prepared by adding 10 μL TPP/THF solution to 1 mL vesicle samples. As shown in Figure S13, the CLSM fluorescence image confirmed that TPP molecules could attach on the surface of the polymersomes and might enter by diffusion in the hydrophobic part of the membrane via hydrophobic interaction. The evolution of 1 H NMR spectra was followed of polymersomes C2 loaded with TPP in D2O (10 μL TPP/1,4-dioxane solution was added to 1 mL polymersome C2/D2O sample, resulting in the "vesicle + TPP" blend with the molar ratio of [TPP] : [MeSPG] = 1 : 30) as a function of the illumination time under white LED light with irradiance of 500 mW/cm 2 (Figure S14).

the distribution of TPP molecules around a vesicle was not homogeneous and more concentrated in some places, high concentration of 1 O2 could be generated by TPP in these places, which transformed locally many amphiphilic copolymers to water soluble polymers.

The subsequent removal of the water soluble chains from the membrane was so substantial and rapid that the big pores left could not be healed by decreasing the vesicle surface and diameter. Eventually, the vesicle burst rapidly after only 118 s of illumination. This is the case of the pathway I L . In contrast, in the case of the pathway II L , TPP should be distributed more uniformly at the vesicle surface; copolymers in the membrane were oxidized and withdrawn at a rather constant and slower rate. Consequently, the membrane had time to heal the pores by decreasing its surface area, leading to the slow and progressive shrinking of the vesicle (over 14 min). The photo-activated bursting of polymersomes can also be controlled locally with the precision of micrometers. Figure 4D shows the CLSM images of a large area (I) before the observation and (II) after the observation of around 15 min with two focused spots marked by circles (conditions as described in Figure 4C). The vesicles under the focus area disappeared, while those in other regions remained. During the photo capture and video recording, these two focused regions received more laser dose and produced more ROS leading to the vesicle disruption. This observation suggests that the oxidation-responsive PMeSPG-b-PSar vesicles can be opened or destroyed selectively in a controllable way through the manipulation of laser irradiation.

Cell Viability Test

For the potential biomedical applications of PMeSPG-b-PSar polymersomes, we assessed their biocompatibility using cell viability test with MTT as the indicator. Two kinds of cells, human vein endothelial cells (ECs) and mouse embryonic fibroblast cells (MEFs), were incubated, respectively, with the polymersomes C2 obtained from double emulsion. Different concentrations of polymersomes such as 31.25, 62.50, 125.0, 250.0, 500.0 and 1000 μg/mL were tested. As presented by the histogram in Figure 5, the tested cells maintained the relative viability above 80% after 24 h of incubation, even with the polymersome suspension at a concentration as high as 1 mg/mL. These observations confirmed reliable biocompatibility of PMeSPG-b-PSar copolymers and their polymersomes. Even though the H2O2 concentration used here is on the order of millimole and greatly exceeds the micromolar level of H2O2 in vivo, encapsulation of oxidant-generating enzymes can be envisioned, such as glucose-, lactose-, or uric acid-oxidase, [START_REF] Ikeda | Installing Logic-Gate Responses to a Variety of Biological Substances in Supramolecular Hydrogel-Enzyme Hybrids[END_REF] to produce in situ high concentration of H2O2 at the targeted sites where the corresponding substrate is present in sufficient quantity. For the light-sensitive vesicle rupture, with judicious choice of the photosensitizer, the light source can be extended to the near-infrared range. All these possibilities are very promising for controlled drug delivery without damaging surrounding cells or organelles. It will also be possible to combine chemotherapy and photodynamic therapy with the use of photosensitizers. These systems can also be used for the design of polymersome-based micro/nano-reactors, biosensors and biodetectors.

ASSOCIATED CONTENT

Scheme 1 .

 1 Scheme 1. Synthesis of PMeSPG-b-PSar block copolymers and their self-assembly into

  and FigureS6show the images of cryogenic electronic microscopy (cryo-EM) of the C1 self-assemblies, where the process of vesicle formation was detectable with the semi-closed vesicle (as shown by the arrow). The C1 self-assemblies obtained by nanoprecipitation exhibited non-ergodic characteristics and some intermediate morphologies, like the semi-closed vesicle in the form of a pulled bag from the closure of the lamellae, were kinetically frozen (detailed discussion in Section 5, SI). The thickness of the hydrophobic part of the membrane was estimated to be 12 ± 1 nm, based on high-resolution cryo-EM image showing the electron-dense (darker) edges of the membrane.

Figure 1 .

 1 Figure 1. (A) Cryo-EM images of self-assemblies C1 obtained by nanoprecipitation before

Figure

  Figure 2D and Movie S1 (in SI) present the pathway I O (the superscript O indicate the trigger

Figure 2 .

 2 Figure 2. (A) Bright-field optical microscopy image of polymersomes C2 prepared by double

Figure 3 .

 3 Figure 3. (A)The ratio of count rates α (α = (count rates at the time t)/(count rate at t = 0

  H NMR. For this analysis, polymersomes C2 was prepared by double emulsion using deuterium oxide (D2O) instead of water. The mixture of polymersomes C2 and H2O2 has a concentration of H2O2 at 176 mM, but with the molar ratio of [H2O2] : [copolymer] = 217 : 1, and of [H2O2] : [MeSPG] = 3.62 : 1 (the same molar ratio as used in CLSM experiment). The evolution of 1 H NMR spectra as a function of oxidation time is shown in Figure 3B (only the characteristic interval of chemical shift is displayed). In general, the intensities of many signals tended to increase with time, either because of the rise of mobility of polymers in the polymer assemblies upon oxidation such as the signal c and signal d, or because of the appearance of oxidized signals such as a O , b O and c O . The signals assigned to the thioether eventually disappeared after sufficient oxidation time (here 12 h). The photographs of the NMR sample tube beside the 1 H NMR spectra show clearly the turbidity changes of the polymersome dispersion. After 12 h of H2O2 treatment, the initial turbid dispersion became transparent, confirming the transformation from giant vesicles to solution of free polymers or of tiny aggregates.

Figure 4 .

 4 Figure 4. (A) The schematic illustration of the disruption process of PMeSPG60-b-PSar50

Figure 5 .

 5 Figure 5. Relative cell viability of polymersomes C2 obtained from double emulsion, for

ACKNOWLEDGMENTS

We acknowledge the French National Research Agency (project ANR-16-CE29-0028) and the National Natural Science Foundation of China (project 21528402 and 21674091) for financial support. Y.D. thanks the China Scholarship Council for funding his Ph.D. scholarship in France. We thank Yixian Wu and Patrick Keller for fruitful discussions.

The sample appearances in NMR tube before illumination and after 300 min of illumination are also shown in Figure S14. Similar to the case of giant polymersomes treated by H2O2, the transformation from thioether to sulfoxide, triggered here by light in the presence of photosensitizer TPP, also led to the disassembly of giant polymersomes.

The in situ and real-time observation of the photo-activated polymersome opening was performed by CLSM using the light source of the microscope as both observation and activation tools. Giant polymersomes C2 labeled by Nile Red were first prepared and then mixed with TPP solubilized in a tiny volume of THF (10 μL TPP/THF was added to 1 mL polymersome C2/D2O sample, resulting in the "vesicle + TPP" blend with the molar ratio of

Notes

The authors declare no competing financial interest.

Table of Contents

Oxidation-Sensitive Polymersomes Based on Amphiphilic Diblock Copolypeptoids

Amphiphilic diblock copolypeptoids with oxidation-responsive properties were synthesized, which self-assembled into unilamellar vesicles. These vesicles can burst under the action of reactive oxygen species (ROS) like the hydrogen peroxide, or the singlet oxygen produced by light-activation of photosensitizer with spatiotemporal control.


Shenghong Yao

Aikaterini Bethani

Nadia Ziane

Cyril Brochon

Guillaume Fleury

Georges Hadziioannou

Philippe Poulin

Jean-Baptiste Salmon

Eric Cloutet

Synthesis of a conductive copolymer and phase diagram of its suspension with single-walled carbon nanotubes by microfluidic technology

Keywords: Carbon nanotube, Conductive inks, Copolymers, Microfluidics, Phase diagram

Amphiphilic block copolymers composed of a poly(3-hexylthiophene) (P3HT) segment and a sulfonated polystyrene (PS-stat-PSS) sequence with well-defined and easily tunable structure were synthesized through Grignard metathesis polymerization (GRIM), RAFT polymerization and sulfonation of PS. Due to the hydrophilic nature and ionic conductivity of the PSS segment, such copolymer shows good solubility in water and high conductivity ~1 S/m in form of dry film. Conductivity can be further enhanced with the addition of single-walled nanotubes (SWNTs). The present amphiphilic block copolymer enables efficient unbundling and stabilization of SWNTs in water. With the help of an original microfluidic technique referred to as microfluidic pervaporation, we investigated the concentration process of SWNT/copolymer suspensions up to dry films and obtained a complete phase diagram which reveals the aggregation of SWNT during the concentration process in a given concentration range. High conductivity of about 370 S/m is achieved for SWNT/copolymer nanocomposites at high concentrations of SWNTs. The microfluidic pervaporation method is also shown to provide a direct determination of the CNT percolation threshold.

Introduction

Flexible electronics and printed organic electronics are considered nowadays as key technologies in academy and obviously in industry. Their development will be based on the availability of new organic materials which combine deformability and suitable electronic properties. 1 These new materials include conductive polymers and films of conducting nano-objects such as carbon nanotubes and graphene among others. Such materials are often assembled into films, fibers and coatings via liquid processing which include spin coating, roll to roll coating, ink-jet printing, wet fiber spinning, etc. Achievement of optimal properties requires the complex formulations of inks in which the electroactive matters are solubilized or dispersed. The stabilization of electronic inks is critical and generally requires the tedious and empirical formulation of mixtures of varied concentrations. Electronic inks made of Carbon Nanotubes (CNTs) have been widely explored. However, the conductivity of CNT films remains significantly lower than that of individual CNTs. 2 Their poor dispersibility in most solvents, their ability to aggregate easily and the high contact resistance between CNTs are the main reasons for this deviation. In the past decades, one of the most effective and nondestructive way to enhance the dispersibility of CNT is the non-covalent modifications of the CNT surface with organic mediating molecules, ranging from low molecular weight molecules to supramolecular polymers. [3][4][5] Nevertheless, insulating polymers are not desirable with respect to their low conductivity, because they annihilate the excellent electrical properties of CNTs by acting as interfacial resistance. 6 Thus, conjugated polymers (CPs), such as poly(3-hexylthiophene) 7,8 , polypyrroles 9 , polycarbazole 5,10,11 , polyfluorene 11,12 and poly(m-phenylene vinylene) 13 , provide alternative strategies to disentangle CNTs while preserving their electronic properties. The strength of π-π interactions between CPs and CNT outmost walls was proven to be much stronger than the interaction between nonconjugated polymers like polystyrene and CNT based on theoretical molecular dynamics studies. 14 Such an excellent interfacial binding is pivotal in determining the charge transfer of nanotube-based polymer electronic devices. 15 Although the inter-tube van der Waals interactions is weakened by the wrapping of CPs, wrapped CNTs are difficult to be dispersed in most common solvents due to the poor solubility of CPs, particularly in water. Thus, a structure which consists of a conjugated rod-block and a soluble coil block was recently explored to improve the dispersibility of functionalized CNTs. 3,6,7 These materials exhibit similar optical and electrical properties as the π-CPs. Zou et al. reported that the dispersibility of CNTs wrapped with P3HT-b-PS copolymer (2.5 mg/ml for SWNTs and 3.0 mg/ml for MWNTs) is more than two times higher to that of wrapped CNTs by P3HT homopolymer with a lower percolation threshold in chloroform. 7,16 However, among the block copolymers investigated, little work has been devoted to water-soluble CPs 17 and their ability to individualize SWNTs. Furthermore, the electrical performance (e.g. conductivity) of such CNT-CPs materials, attributed to the assembly of CPs and dispersed CNTs, has not yet been investigated in depth.

In the present work, we propose a well-defined strategy to synthesize P3HT-b-PSS copolymer by adding sulfonated groups on the PS part of P3HT-b-PS through a controlled sulfonation reaction, which is much more simpler than the reported one in reference 18 . The presence of an ionic coil block (PSS) both enhances the electrical properties of the π-conjugated backbone (the so-called self-doped polymers) and leads to a good solubility in polar solvents. 19 Thus the obtained P3HTb-(PS-stat-PSS) copolymer exhibits excellent solubility in water and superior conductivity (~1 S/m) as compared to P3HT (~10 -6 S/m 20 or ~3.8×10 -3 S/m 21 ) and P3HT-b-PS (~2×10 -5 S/m in this work or less than10 -7 S/m 7 ). Its formulation with SWNTs demonstrates its efficiency to debundle and disperse SWNTs in water. Individualization of SWNTs is demonstrated by photoluminescence in Raman spectra. The highest concentration obtained of stable SWNT dispersion is around 3.8 mg/mL using a 4:1 copolymer to SWNT mass ratio. This concentration is higher than that reported for P3HT-b-PS dispersed CNTs in organic solvents. 7 In order to examine the stability of the P3HT-b-(PS-stat-PSS)/SWNT aqueous dispersions, we used a recent microfluidic tool to monitor in situ the concentration process of dilute dispersions up to completely dry SWCNTs/CPs films. 22 Such experiments lead to the phase diagram for the SWNT/P3HT-b-(PS-stat-PSS) aqueous dispersion, which reveals a micro-phase concentration in a well-defined concentration range. The ultimate conductivity of the final dry composite achieved is as high as 370 S/m which is four orders of magnitude higher than the case of SWNT/PS nanocomposite with SWNT dispersed by P3HT-b-PS 7 , although the percolation threshold observed for P3HT-b-(PS-stat-PSS) dispersed SWNTs nanocomposite approaches 15vol%.

Experimental

Synthesis of P3HT-b-(PS-stat-PSS).

Our strategy includes three main steps: (i) synthesis of ω-functional P3HT as a macro-RAFT initiator, (ii) polymerization of styrene and (iii) controlled sulfonation of the PS block.

(1) Synthesis of P3HTmacroinitiator: Allyl terminated regioregular P3HT homopolymer was synthesized via a Nickel-catalyzed chain growth polymerization with the end groups functionalized using Grignard metathesis polymerization (GRIM) according to the literature procedure from McCullough et al. 23 and Yokozawa et al. 24 . The allyl end group of the polymer was further treated with 9-borabicyclo(3.3.1)nonane (9-BBN) followed by the addition of hydrogen peroxide and aqueous NaOH solution resulting in a hydroxypropyl end group. 25 Then, hydroxyl terminated P3HT (0.3g, 0.03 mmol) was dissolved in distilled dichloromethane at 40°C. The mixture was stirred followed by addition of 3benzylsulfanylthiocarbonylsulfanylpropionic acid (RAFT agent) (0.041g, 0.15 mmol),4-(dimethylamino) pyridinium-p-toluene sulfonate(DPTS) (0.01g, 0.03 mmol) and N,N'diisopropylcarbodiimide (DIPC) (0.03 mL, 0.195 mmol) under nitrogen atmosphere. 26 The reaction mixture was stirred for 3 days at 30°C. The resulting P3HT macroinitiator was precipitated in methanol and filtered.

(2) RAFT polymerization ofP3HT-b-PS: P3HT macroinitiator (0.3 g, 0.03 mmol) was added in freshly distilled styrene (4 mL, 34.8 mmol) under N2 protection. The reaction mixture was stirred at 45°C until P3HT macroinitiator was completely dissolved, and three freeze-pump-thaw cycles proceeded. Then, the reaction mixture was immersed in a thermostated oil bath at 120°C and the molecular weight of polystyrene was controlled by the time of reaction. Finally, the reaction was terminated by freezing the mixture in liquid N2. The resulting P3HT-b-PS copolymer was precipitated in methanol, filtered and dried in vacuum oven.

(3) Sulfonation of P3HT-b-PS: P3HT-b-PS copolymer (1g, mol(styrene)=8.26 mmol) was dissolved in 15 mL degassed dichloroethane under Ar atmosphere when a acetyl sulfate was prepared. Acetic anhydride (1.6 mL, 16.52 mmol) was added in dichloroethane (4 mL), and the mixture was cooled down to 0°C followed by addition of sulfuric acid (0.66 mL, 12.39 mmol) under Ar protection. The obtained acetyl sulfate was then added in the copolymer solution under

Ar and the reaction mixture was heated to 60°C for 3 days. Deposition process called the HiPco process (High Pressure Carbon Monoxide). 29 As-produced materials contain iron nanoparticles with a weight fraction of about 32.6 wt%. SWNTs were used as received (in a form of a wet cake) without further purification or chemical modification. Such a protocol yields black, homogeneous nanotube dispersions.

Microfluidic investigation of the phase diagram of SWNT/copolymer dispersions and microfluidic printing of electrodes

We used two almost similar microfluidic devices to (i) investigate the phase diagram of the SWNT/copolymer dispersions, and (ii) to print electrodes starting from dilute dispersions. These devices both integrate a thin poly(dimethylsiloxane) PDMS membrane, across which water can permeate from a microchannel containing the dispersion, see Fig. 1a. Similar microfluidic devices were used recently to manufacture micro-scaled materials 30,31 , but also to investigate the phase diagrams of different complex fluids at the nanoliter scale. 22,32 The detailed functioning of these devices can be found in the above cited references and is briefly summarized below.

A microfluidic channel (typical dimensions h=5-30 µm, w=100 µm) is sealed by a thin PDMS membrane (e=35-60 µm) on a length L0=10-15 mm (see Fig. 1a). Such devices are made using standard photolithography techniques: channels are made within a PDMS matrix and sealed by a glass slide. Pervaporation of water through the PDMS membrane (at a typical rate qe~1 µm 2 /s per unit length) drives a flow within the channel, see arrows in Fig. 1b andc. We then used two different protocols depending on our applications.

(1) For the screening of the ternary phase diagram SWNT/copolymer/water, we monitor the shrinkage of a drop containing the dilute dispersions due to the water pervaporation along the channel (see Fig. 1b). Typical shrinkage kinetics are given by τe= L0hw/(L0qe) ≈ 1500 s. Details on the protocol to make such a drop (thanks to the gentle flow of an inert oil, Fluorinert FC40, in surrounding channels) can be found in reference 22 . In this particular regime, the pervaporationinduced flow within the drop prevents from the formation of concentration gradients during the drop's shrinkage (see 22 for a complete demonstration of this result). Concentration within the drop thus follow (t)L(t) = 0L0 where 0 is the initial solute concentration (SWNT or copolymer) and L0 is the initial drop length (15 mm). We used a stereo-microscope (Olympus) coupled with a CCD camera to check for the possible de-mixing during the concentration of both SWNTs and copolymers, as shown in section 3.2. Such observations also reveal that no concentration gradients indeed develop during the shrinkage of the drop (revealed using the colored adsorption of the copolymer solution and/or the adsorption of the dispersed SWNT).

Custom-made image analysis programs (Matlab) lead to precise estimates of the drop's length L(t) (accuracy ± 5 µm) and thus to precise estimates of the concentrations within the drop (t).

(2) For making electrodes, we proceeded as follows. The channel is connected to a reservoir (a simple hole punched in the PDMS matrix, ~50 µL) containing the dilute complex fluid under study (SWNT/copolymer dispersion or polymer solutions, see section 3.2). The pervaporation of water along the channel drives a flow from the reservoir which concentrates slowly the nonvolatile solutes up to the tip of the channel, see Fig. 1c. After a transient time (typically ~2 hrs depending on the concentration and the geometrical parameters), a dense material starts to invade the channel, see Fig. 1c. The detailed mechanisms of such a nucleation and growth scenario were investigated in references 30,32 for the case of nanoparticles, polymers, and surfactants. After the complete growth of a composite in the channel, the elastomeric mold is carefully peeled off from the substrate leaving thus printed microstructures on the substrate. Such a process works well for the cases investigated in the present work: pure PSS (Sigma-Aldrich, Mw~75 KDa, 18 wt% in water) and P3HT-b-(PS-stat-PSS) solutions and SWNT/P3HT-b-(PS-stat-PSS) dispersions leading to printed composites. This process makes also possible the printing of composites on flexible substrates as demonstrated in Fig. 1d, where we sealed the channels using another PDMS film deposited on a flexible PET film.

Electrical characterization of the printed electrodes

The conductivity of the electrodes printed by the method explained above was measured by impedance spectroscopy (7260 Impedance Analyzer, MaterialsMates Italia). For improving the measurements, the reservoir containing the solutions/dispersions is connected to multiple channels in parallel. We thus obtained up to 29 printed microstructures and we measured the conductivity of these parallel electrodes using silver paste deposited on each side of the strips, see in Fig. S1 in SI. To get values of conductivity, we also measured the widths and heights of the printed electrodes using mechanical profilometry (Veeco). Significant deviations from the channel dimensions were observed (e.g. h=30, w=100 µm leading to electrodes with 15×90 µm 2 ). These deviations mainly come from the deformations of the PDMS matrix during the concentration process, and the corresponding mechanisms were investigated in 31 . S2). Then the allyl end group is further treated with 9-BBN followed by the addition of hydrogen peroxide and aqueous NaOH solution in order to obtain a hydroxypropyl end group (see 1 in scheme 1), which reacts with the RAFT agent to yield a ω-thiocarbonate P3HT used as a macroinitiator for RAFT of PS (see 2 in scheme 1). The molecular weight of PS segments is strictly controlled by the reaction time of RAFT polymerization(see 3 in scheme 1).The achieved degree of polymerization (DPn of ~630 and Mn of 65 kDa) has been calculated from the presence of phenyl ring protons at 6.58 ppm in the 1 H NMR spectrum shown in Fig. 2 (a). To better understand the electrical features of our copolymer and to clarify their origin, the conductivity of each component in our copolymer was determined separately (see Fig. 3). We thus used the microfluidic technology detailed in Section 2.3(2) to print microstructured dry films on a glass substrate starting from aqueous solutions of the synthesized copolymer, of pure PSS and of P3HT-b-PS (see Fig. 1c). For these three cases, microfluidic pervaporation makes it possible to print homogeneous strips playing the role of electrodes and Fig. 3 displays their electrical conductivity as a function of frequency. Regioregular P3HT is a semi-conducting polymer which is broadly used as an electron donor in the active layer of solar cell with PCBM as electron acceptor. However, as expected from a non-doped semi-conducting p-type polymer, P3HT itself exhibits a relative low electrical conductivity, ~10 -6 S/m in the observed frequency range of interest for most flexible electronic applications. 20 The block copolymer P3HT-b-PS synthesized in this work, which has been reported to disperse CNTs in organic solvent 7 , shows a relatively higher conductivity of ~2×10 -5 S/m but still far from that of P3HT-b-(PS-stat-PSS), ~1 S/m. Meanwhile, commercial PSS presents a conductivity that compares with that of our copolymer. We thus conclude that the ionic conductivity of PSS segments dominates the resulting electrical property of the copolymer films in the investigated range of frequency.

Furthermore, the conductivity of copolymers could be slightly varied by controlling the length of PSS segments, as shown in Fig. S3). typical tip sonication treatment. The stability of the SWNT/copolymer dispersions was also examined through a centrifugation test. After being subjected to a centrifugation treatment at 13200 rpm for 30 min, most SWNTs remained dispersed, and the highest SWNT concentration obtained in the supernatant is around 3.8 mg/mL with 4:1 copolymer to SWNT mass ratio. This concentration is much higher than the CNT dispersions with its analogue P3HT-b-PS in organic solvent 7 , indicating that our copolymer is an efficient dispersant for SWNTs in aqueous solutions. As mentioned earlier, such an efficient dispersion is attributed to the good solubility of the PSS block in water. Furthermore, as shown in Figure 4, the difference of intensity background in Raman spectra of dispersed nanotubes and wet-cake materials reveals unambiguously the presence of individualized semiconducting SWNTs in the SWNT/copolymer suspensions. Indeed, the observed difference, specified by PL in the spectra of Figure 4, corresponds to photoluminescence emission by semiconducting nanotubes with an excitation wavelength of 785 nm. Photoluminescence of nanotubes is quenched when the particles are under the form of bundles. 33 A detailed determination of the chirality of the dispersed and individualized nanotubes would require combined optical absorption measurements, Raman spectroscopy and photoluminescence mapping 34 , which is out of the scope of the present work.

Investigation of the SWNT/copolymer dispersions

As shown in Fig. 4, the characteristic peaks of the suspension, radial breathing modes (RBM) at low frequencies between 250 and 350 cm -1 , D and G′ bands around 1280 and 260 cm -1 , and G bands in the range of 1550-1600 cm -1 , appear indeed at the same wave number as that of nanotubes bundles in wet-cakes except the additional broad photoluminescence (PL) lines. 27 The Raman spectrum of the copolymer solution is also presented in Fig. 4 without any PL. All these investigations show that our synthesized copolymer is a promising candidate for dispersing SWNTs in aqueous solutions for its use in flexible electronics. Fig. 5 shows the concentration kinetics of a SWNT/copolymer dispersion used as prepared in a microfluidic channel sealed by a PDMS membrane across which water pervaporates. This figure

shows more precisely typical snapshots at the tip of the channel (the complete video can be found in SI-V1) during the continuous concentration of both CNTs and copolymers due to the pervaporation-induced flow (see Experimental Section). The channel appears initially transparent when filled with the dilute dispersion, and then becomes progressively more and more opaque due to the concentration increase of CNTs (Fig. 5a-c). The typical rate of concentration is both controlled by the time scale of this pervaporation process (τe~1500 s) and the initial concentration of the dispersion. At a well-defined time (Fig. 5d), aggregation of CNTs shows up with black strip patterns oriented perpendicular to the channel. These black strips keep progressing and accumulating towards the tip of channel (Fig. 5e-f). Finally, a dense and dry material invades progressively the whole channel towards the reservoir on a typical time scale ~70 hrs. This process leads finally to printed electrodes within the channel, and we discuss later their electrical properties as a function of the SWNT concentrations. The above scenario is observed for dispersions with SWNT and copolymer volume ratio higher than 1:20, however for lower ratios (down to pure copolymer solutions), we do not observe any demixing during the pervaporation-induced drying of the solutions/dispersions. Deeper insights into this microphase separation were investigated with the help of the microfluidic technique shown in Fig. 1b and detailed in reference 22 . A droplet of a dilute dispersion at a given volume fractions of CNTs and copolymer is left to pervaporate within a long channel (length 15mm). The uniform pervaporation of water along the droplet across the thin PDMS membrane imposes a simple relation between the shrinkage of the drop length and the concentration of SWNTs and copolymer described by:

(t)L(t)=(0)L(0),
where (0), L(0) are the initial concentrations and the initial drop length respectively, and (t), L(t) that at time t. More detailed information about the design of this microfluidic chip leading to homogeneous concentration during drying can be found in reference 22 . Thanks to this technique, we could quantitatively determine the concentrations of each component of the ternary phase diagram during the evaporation process. It should be noted that, a very thin layer of copolymer is deposited on the channel PDMS surface owing to the amphiphilic property of the copolymer, and this may induce uncertainties in the 5-10% range. Nevertheless, as shown in Fig. 6, a generalized phase diagram is obtained by detecting the demixing of samples with various initial concentrations of SWNTs and copolymer. Fig. S4 presents typical snapshots taken during the shrinkage of droplet (Fig. S4a), the demixing (zoom at the tip of channel, Fig. S4b), and the complete video can be found in SI-V2 (only the tip of channel is observed for sake of clarity). It is interesting to find that this demixing could not be observed for the samples with (CNT)< 5 vol% (values estimated in the final dry composite shown as sample 2 in Fig. 6), thus a quite uniform composite film is obtained as shown in video in SI-V3 (only the tip of channel is observed for sake of clarity).

The detailed information about the concentration of each sample corresponding to the initial, the demixing and the final evaporation process are shown in Table S1. It is found that stable dispersions (sample 1 and 2) without demixing during the concentration are the samples with the higher copolymer concentrations. For sample 2, 31.2 mg copolymer is used for dispersing1.68 mg SWNTs. At this concentration a sufficient amount of polymers is present to provide an effective stabilization of the nanotubes. The involved mechanisms of stabilization include presumably both steric and electrostatic interactions considering that the PSS groups are exposed towards the solvent. The large size of the adsorbed polymer chains are key advantages for efficient stabilization of SWNTs in water but it retards the percolation of the CNTs in dry films. The above argument is confirmed by the variation of the conductivity of the SWNT/copolymer composites with the SWNT concentration shown in Fig. 7. Note that continuous films could still be obtained from the samples despite the demixing leading to the aggregation of the CNTs. Although the samples with (CNT)≤5 vol% remains uniform during the concentration process, the corresponding conductivities are similar to that of the pure copolymer film (~1 S/m). A significant increase of the conductivity shows up around 15 vol%, up to a very high conductivity of about 370 S/m at (CNT)=36 vol%. Such a behavior suggests a percolation threshold around 15 vol% with a critical exponent t=0.2 according to σc ∝ (fSWNT-fc) t , where σc, t are the conductivity of SWNT/copolymer nanocomposites and the critical exponent in the conductive region respectively. Similar values were reported for MWNT/PVDF nanocomposites, however, with a rather low conductivity ~10 -4 S/m at 1Hz. 35 For such SWNT, the theoretical percolation threshold is expected to be much smaller because of their extreme high aspect ratio. However, the percolation threshold of SWNT-based composites depends both on the size distribution of the CNTs but also on other factors including interactions, correlation of orientations, waviness, and inter-tube tunneling distance between CNTs. 36 This leads to two competing requirements: (i) the need for a good dispersant, and (ii) the smallest contact resistance between CNTs in the final composite. For very well dispersed CNTs in a insulating matrix, the conductivity is dominated by the matrix even for concentrations above the theoretical threshold, which may explain the low conductivity reported in the literature for CNTs composites (fc= 0.03 wt% with σc~10 -3 S/m 7 , fc =0.095 wt% with σc~10 -4 S/m 37 ). In our system, we do not observe any CNT aggregation during the drying process for concentration below (CNT)≤5 vol%, leading to conductivities dominated by the copolymer matrix as explained above. However, we observe a huge increase of the conductivity suggesting the exposure of CNTs surface for (CNT)>5 vol%, which progressively becomes the main interactions between CNTs. We believe that this effect comes from the observed aggregation of CNTs during the drying process leading to the formation of CNTs-rich clusters embedded in a copolymer-rich matrix (see Fig. 4e). The percolation of these clusters may yield a very high conductivity as the contact resistances between CNTs are no more dominated by the copolymer matrix in the CNTrich cluster. Such a high conductivity of our system exhibits a strong potential for future electronics applications. From a more general point of view the microfluidic pervaporation method is shown to be a valuable tool to optimize the formulation of electronic inks using in particular environment friendly aqueous solvents and minute amounts of materials.

Amphiphilic P3HT-b-(PS-stat-PSS) block copolymer was synthesized through a simple and well-controlled method including GRIM polymerization of P3HT, RAFT polymerization of PS and sulfonation of PS. This route makes it possible to easily tune the size of each segment to adapt the solvent applied from nonpolar to polar. In this case, P3HT60-b-(PS63-stat-PSS567) with large PSS segments exhibits excellent solubility in water which is essential for CNT water-based dispersion. Microfluidic tools were then used to both investigate the phase diagram of the dispersions and make microstructured films playing the role of electrodes. Our block copolymer presents a promising high conductivity, ~1 S/m, arising from the ionic conductivity of the dominant PSS segment. Furthermore, Raman spectroscopy revealed unambiguously the presence of individualized semiconducting SWNTs in the SWNT/copolymer dispersions which proves the efficiency of our copolymer at dispersing SWNTs. Demixing is observed during the concentration of the SWNT/copolymer dispersions with low mass ratio copolymer/CNT. This demixing promotes a high conductivity of 370 S/m at high concentration of CNT, (CNT)=36vol%. The microfluidic pervaporation method is shown thereby to be a valuable tool for optimizing the formulation of electronic inks and for finding the most suitable compositions to achieve targeted properties. Our future work will be focused on the implementation of such nanocomposites made with SWNT/copolymer dispersions within electronic devices and on the characterization of their performances.

ASSOCIATED CONTENT

*Supporting Information

The Supporting Information is available free of charge on the ACS Publications website at DOI:

Fig 1 .3 Results and discussion 3 . 1

 131 Fig 1.(a) 3D and top (schematic) views of microfluidic pervaporation. A microfluidic channel (typical dimensions w=100µm, h=5-30µm and L0=15mm) is embedded in a thin PDMS membrane (e=35-60µm). Water pervaporates trough the PDMS membrane at a rate qe (~1 µm 2 /s, per unit length). (b) The shrinkage of a drop due to the pervaporation of water makes possible the investigation of the ternary phase diagram SWNT/copolymer/water. Colors represent the different concentrations. The pervaporation-induced flow (see arrows) prevents from the development of concentration gradients within the drop. (c) Similar mechanisms lead to the formation of a dense printed composite when the channel is continuously connected to a reservoir containing the dilute SWNT/copolymer/water dispersion. Colors represent the different concentrations. (d) Picture of an array of printed electrodes on a flexible substrate made of a thin PMDS film deposited on a flexible substrate. The logo (Labex Amadeus) was printed using a standard printer on the PET sheet.

Figure 2 . 1 HFigure 3 .

 213 Figure 2. 1 H NMR spectra of (a) P3HT-b-PS and (b) P3HT-b-(PS-stat-PSS) block copolymer. The inset picture shows a copolymer/water solution in a vial.

Figure 4 .

 4 Figure 4. Raman spectra of a copolymer/water solution with 1.22 wt% copolymer, SWNT wetcake and a SWNT/copolymer dispersion with 0.3 wt% SWNT and 1.22 wt% copolymer in water. The incident laser wavelength is 785 nm. The inset image shows the corresponding SWNT/copolymer dispersion in a vial.

Figure 5 .

 5 Figure 5. Series of snapshots obtained from the tip of a single channel in microevaporator during the concentration process of a SWNT/copolymer solution with initial concentration: SWNT= 0.02 vol%, copolymer= 0.1 vol%. The elapsed times are noted above. Contrasts of e, d and f are increased to show the CNT aggregation. The channel width is 100 µm.

Figure 6 .

 6 Figure 6. Phase diagram for SWNT/copolymer solution. Blue dots indicate initial concentration of the SWNTs in droplet, red dots that at phase separation and green dashed line that in final dry SWNT/copolymer composite from 0 to 100%. The black lines and blue dotted ones in between are for guiding eyes.

Figure 7 .

 7 Figure 7. Conductivity as a function of SWNT concentration for SWNT/copolymer dry composite films

  250A equipped with a 13 mm step disruptor horn and a 3 mm tapered microtip) for 15 min at a frequency of 20 kHz, and centrifuged at 4000 rpm for 30 min to separate extra copolymer pieces. Then the supernatant was used to disperse SWNTs at different concentrations

	Size Exclusion Chromatography (SEC, PL-GPC 50 Plus (Agilent Technologies)) were used to
	indicate the composition and the dispersity of the copolymer.
	2.2 SWNT/copolymer dispersions
	Synthesized P3HT-b-(PS-stat-PSS) was dissolved in water by tip sonication treatment (Branson
	Sonifier S-
	The resulting P3HT-b-(PS-stat-PSS)
	was withdrawn by filtration and dried. 1 H-NMR spectrum (Bruker AVANCE I, 400MHz) and

using sonication during 90 min with the same frequency and cold water protection according to related experiments on similar materials.

27,28

Raw SWNTs bundles were obtained from Unidym (Sunnyvale,CA, batch number R0513). These nanotubes are synthesized by a Chemical Vapor

ACKNOWLEDGMENTS

This work was supported by the LabEx AMADEus (ANR-10-LABX-42) in the framework of IdEx Bordeaux (ANR-10-IDEX-03-02) i.e. the Investissements d'Avenir programme of the French government managed by the Agence Nationale de la Recherche. Authors are grateful to Dr. J. Leng and future Dr. C. Laval for microfluidic chips design and Matlab code, and Dr. J. Jolly for Raman investigation.

AUTHOR INFORMATION

Corresponding Authors: Dr. J-B. Salmon and Dr. E. Cloutet * E-mail: jean-baptiste.salmon-exterieur@solvay.com; cloutet@enscbp.fr

The authors declare no competing financial interest.

TOC

Synthesis of conductive copolymer and phase diagram of its suspension with SWNT for printing by microfluidic technology

Shenghong Yao, † , ‡ , § Aikaterini Bethani, † Nadia Ziane, ‡ Cyril Brochon, † Guillaume Fleury, † Georges Hadziioannou, † Philippe Poulin, § Jean-Baptiste Salmon, ‡ ,* Eric Cloutet, † ,* † Laboratoire de Chimie des PolymèresOrganiques (LCPO), CNRS (UMR5629), ENSCPB, 16 avenue PeyBerland, 33607 Pessac Cedex, France ‡ Laboratoire du Futur (LOF), CNRS (UMR5258), Université de Bordeaux, 178 Avenue du Docteur Schweitzer, 33608 Pessac Cedex, France § Centre de Recherche Paul Pascal(CRPP), Université de Bordeaux, 115 avenue Schweitzer, 33600 Pessac Cedex, France


domain: chim phys

text:
Ge Xu

Jiawei Li

Jianqiao Wu

Hao Zhang

Jun Hu

Min-Hui Li

Tough Polymeric Hydrogels Formed by Natural Glycyrrhetinic Acid-Tailored Host-Guest Macro-Crosslinking Toward Biocompatible Materials

Keywords: polymeric hydrogel, host-guest pairs, cyclodextrins, glycyrrhetinic acid, natural product

ABSTRACT: Host-guest polymeric hydrogels often suffer from poor biocompatibility and weak mechanical strength owing to the low complexation degree between host-and guest-polymers. In contract to reported works, here a biocompatible host-guest macro-crosslinker (HGMC) was synthesized beforehand by complexation of poly(β-cyclodextrin) with natural glycyrrhetinic acid-derivative functionalized with acrylates at two extremities. After crosslinking polyacrylamide three polymeric hydrogels were obtained, the swelling ratios and micro-pore sizes of which could be adjusted effectively by HGMC content. Moreover, they exhibited robust anti-compressing and elastic features. These biocompatible tough polymeric hydrogels hold the promise for potential applications as biomaterials.

Host-guest polymeric hydrogels, consisting of 3D networks crosslinked by host-guest interactions, have attracted considerable attention by virtue of their distinct stimuli responsiveness and inherent processability. [1][2][3] Currently, one of the most common strategies is to prepare host-polymers and guest-polymers by grafting host and guest functional groups on polymer main chains, respectively. [4][5][6] Unfortunately, low grafting degrees are often obtained because of the steric hindrance of polymer backbones, thus leading to the weak mechanical strength. In addition, these hydrogels also suffer from poor biocompatibility as nonrenewable host-guest pairs are frequently used, which further restricts their applications in biocompatible materials. [7][8][9] Thus, it is of great importance to develop a simple yet useful method for constructing host-guest polymeric hydrogels with high mechanical strength from biocompatible host-guest pairs. Cyclodextrins (CDs), cyclic oligomers of D-(+)-glucose linked via α-1,4-glucose bond, are produced from enzymatic processing of starch with good biocompatibility and availability. [10] More importantly, the hydrophobic cavity of CDs makes them typical host molecules to bind with various guest molecules like adamantane, [11,12] azobenzene, [13,14] ferrocene, [15,16] cholic acid, [17,18] and glycyrrhetinic acid, [19,[START_REF] Hao | Natural-product-tailored polyurethane: size-dictated construction of polypseudorotaxanes with cyclodextrin-triterpenoid pairs[END_REF] according to the size and hydrophobicity. As firstly reported by us in 2016, natural glycyrrhetinic acid (GA) has a mild binding affinity with β-cyclodextrin (β-CD) (Ka = 1.59 × 10 3 M -1 ) and great biocompatibility. [19] GA is a 30 C-containing plant metabolite in licorice root and possesses a variety of remarkable biocompatibility and biological activities. [START_REF] Zhang | Doxorubicin-loaded glycyrrhetinic acidmodified alginate nanoparticles for liver tumor chemotherapy[END_REF] The fused pentacyclic structure offers GA a rigid molecular backbone of nanometric dimensions. Besides, the presence of hydroxyl and carboxyl groups with several chiral centers makes GA an interesting building block for chiral assemblies and materials. [START_REF] Wan | Long-lived and thermoresponsive emulsion foams stabilized by self-assembled saponin nanofibrils and fibrillar network[END_REF][START_REF] Gao | Supramolecular helical nanofibers assembled from a pyridinium-functionalized methyl glycyrrhetate amphiphile[END_REF][START_REF] Gao | Natural triterpenoid-tailored phosphate: in situ reduction of heavy metals spontaneously to generate electrochemical hybrid gels[END_REF] In our previous works, we found that the GA/β-CD pair adjusted the LCST (lower critical solution temperature) behavior of copolymers composed of GA-based methacrylate and N,N'dimethylacrylamides, [START_REF] Hao | Thermosensitive triterpenoid-appended polymers with broad temperature tunability regulated by host-guest chemistry[END_REF] dictated the construction of polypseudorotaxanes of GA-tailored polyurethane, [START_REF] Hao | Natural-product-tailored polyurethane: size-dictated construction of polypseudorotaxanes with cyclodextrin-triterpenoid pairs[END_REF] and drove the formation of self-healable supramolecular hydrogels by grafting GA and β-CD to N,N'-dimethylacrylamide copolymers. [19] Obviously, GA/β-CD is an ideal biocompatible host-guest pair for constructing the host-guest polymeric hydrogels.

In order to circumvent the weak mechanical strength of GA/β-CD hydrogels caused by the low grafting degree of GA and β-CD on polymer backbones, in this research we reported a simple strategy for preparing tough host-guest polymeric hydrogels by using a macro-crosslinker. In this strategy, the GA-derivative with two terminal acrylates (GADA) and poly(β-cyclodextrin) (PCD) were used as biocompatible guest and host to fabricate the host-guest macro-crosslinker (HGMC) beforehand. After that, the rotaxane-type hydrogels were produced by crosslinking acrylamide (AAm) in the presence of HGMC via a UV photo-polymerization (Scheme 1). There were several advantages in this strategy: 1) the host-guest complexation was formed before the polymerization, which avoided the issues of steric hindrance of polymer backbones; 2) besides the crosslinking between GADA and polyacrylamide, the inclusion between PCD and GADA provided additional crosslinking sites, which endowed the hydrogels better mechanical performance; 3) when external forces were applied to the hydrogels, the host-guest interactions between PCD and GADA in these rotaxane-type hydrogels could dissipate energy by sliding β-CD from GA backbone, consequently making the hydrogels very tough. The results showed that all the hydrogels with different molar ratios of HGMC and AAm were anti-compressing, nonfragile and elastic. Moreover, they could easily recover to their original states on account of the host-guest interactions. This work provided an effective way to construct biocompatible polymeric hydrogels with good mechanical strength for potential applications as biomaterials in soft-tissue repair.

Scheme 1. Schematic illustration of (A) glycyrrhetinic acid-derivative functionalized with acrylates at two extremities (GADA), (B) poly(β-cyclodextrin) (PCD), and (C) polymeric hydrogels formed by host-guest macro-crosslinking.

For the preparation of host-guest macro-crosslinker HGMC, GADA and PCD were firstly synthesized as shown in Scheme 1A and 1B. GA reacted with succinic anhydride to give the intermediate (GAA) with proton signals at 2.65 and 2.69 ppm assigning to methylene groups (Ha, Hb, Figure 1A). 2-[2-(2-chloroethoxy)ethoxy]ethanol was then conjugated to GAA via an ester reaction, affording another intermediate (GA-OEG) with peaks at 3.60~3.73 and 4.18~4.37 ppm belonging to methylene groups of OEG moiety (Hc, Hd, He, Figure 1B). Lastly, the guest molecule GADA was obtained by reacting GA-OEG with acryloyl chloride, and the classic proton signals from terminal acrylates at 5.85, 6.15 and 6.40 ppm (Hg, Hh, Hi) were observed clearly (Figure 1C), strongly indicating the successful synthesis of GADA. On the other hand, PCD was synthesized through copolymerizing epichlorohydrin with β-CD. As can be seen from 13 C NMR spectrum (Figure S1), the peaks at 62 and 72 ppm were assigned to the characteristic methylene carbon formed in the polycondensation of epichlorohydrin with β-CD. [START_REF] Yang | Light-switchable self-Healing hydrogel based on host-guest macro-crosslinking[END_REF][START_REF] Renard | Preparation and characterization of water soluble high molecular weight βcyclodextrin-epichlorohydrin polymers[END_REF] Besides, the size exclusion chromatography (SEC) showed that PCD had a molecular weight (Mn) 7300 Da (calibrated with polystyrene standard, Figure 2A). Subsequently, the macro-crosslinker HGMC was prepared by mixing the oil-like GADA into an aqueous solution of PCD. As shown in Scheme 1C, after stirring for 24 h, the light yellow oil of GADA floating on the up-layer of PCD aqueous solution disappeared and a homogeneous dispersed solution was generated, because the hydrophobic interior cavity of PCD had trapped GADA. To clarify the host-guest interactions between GADA and PCD, 1 H NMR, SEC and X-ray diffraction (XRD) measurements were performed. No signal of methyl groups on GA moiety was observable in 1 H NMR spectrum of GADA in D2O (Figure 2B, up), which indicated the formation of aggregates due to the hydrophobic nature of GA. Upon addition of PCD signals for methyl protons (positions 23-29) appeared on account of the complexation of GA with PCD (Figure 2B, bottom) [19,[START_REF] Hao | Natural-product-tailored polyurethane: size-dictated construction of polypseudorotaxanes with cyclodextrin-triterpenoid pairs[END_REF][START_REF] Hao | Thermosensitive triterpenoid-appended polymers with broad temperature tunability regulated by host-guest chemistry[END_REF] . It was consistent with the SEC results, where a larger molecular weight (Mn) 9400 Da of HGMC was detected in comparison with that of PCD (Figure 2A). According to Formula S1 in Supporting Information, the molar ratio between β-CD and GADA was calculated to be 2:1. More evidence came from XRD diffraction patterns. As shown in Figure S2, a certain crystalline structures of PCD were demonstrated by two peaks at 2θ = 12.7 and 18.8 o in the diffractogram, corresponding to repeating distances of 0.70 and 0.47 nm. Conversely, there was only a broad scattering signal in the range of 6-50 0 for HGMC, because the complexation of PCD with GADA prevented the crystallization of PCD. [START_REF] Li | Self-healing supramolecular self-assembled hydrogels-based on poly(L-glutamic acid)[END_REF] Having macro-crosslinker HGMC in hands, the hydrogels were prepared by polymerizing AAm with the presence of HGMC and photo-initiator I2959 via a UV photopolymerization (Scheme 1C). As illustrated in Table 1, three hydrogels, Gel-1, Gel-2 and Gel-3, were successfully prepared with different contents of HGMC. The swelling results showed that Gel-1, Gel-2 and Gel-3 in deionized (DI) water swelled more than 100% in the first 1 h, and then the volume continued to increase steadily. After 24 h, the equilibrium state was achieved with an order of swelling ratio Gel-1 > Gel-2 > Gel-3 (Figure 3A, Table 1). The reason was that as the concentration of HGMC increased, the crosslinking density was enhanced while the flexibility of networks was decreased, thus leading to a reduction in the swelling ratio. Moreover, scanning electron microscopy (SEM) images were used to reveal their microstructures. Before subjecting to SEM analysis, hydrogels at swelling equilibrium were lyophilized under vacuum at -60°C and then coated by gold. As shown in Figure 3B-D, the microporous structures of Gel-1, Gel-2 and Gel-3 were observed, and the pore sizes tended to be smaller from Gel-1 to Gel-3, which was consistent with the swelling results. Apparently, the hydrogels had the 3D crosslinked networks with micro-pore structures, and their swelling behaviors and pore sizes could be adjusted effectively by the content of macro-crosslinker HGMC. Oscillatory rheology technique was used to measure the mechanical properties of Gel-1, Gel-2 and Gel-3. The frequency sweep results showed that all the hydrogels exhibited a higher storage modulus (G', in the order of 10 4 Pa) compared to the loss modulus (G'', in the order of 10 3 Pa) in the range from 1 to 100 Hz, revealing their viscoelastic behaviors (Figure 4A). Moreover, with the increase in concentration of HGMC, the G' value was increased significantly, that is, 9770 Pa for Gel-1, 11830 Pa for Gel-2, and 14990 Pa for Gel-3 (at frequency = 10 Hz), which was mainly attributed to the enhanced crosslinking density of hydrogel networks. The strain amplitude sweeps were also employed to determine the deformation limit. As shown in Figure 4B, the transition of G' and G'' occurred at the strain of 130%, 540%, and 1200% for Gel-1, Gel-2 and Gel-3 under a frequency of 1 Hz, which implied the beginning of destruction of hydrogel networks at such a strain. [START_REF] Haque | Lamellar bilayers as reversible sacrificial bonds to toughen hydrogel: hysteresis, self-recovery, fatigue resistance, and crack blunting[END_REF] Furthermore, the compression, cyclic compression and tensile tests of all the hydrogels were performed to investigate their mechanical properties. As shown in Figure 5A, all of them had the good mechanical strength, and no yield or breaking point was observed for each hydrogel during the compression process until pressure of 0.81, 1.20 and 1.63 kPa for Gel-1, Gel-2 and Gel-3, respectively. This observation clearly revealed their nonfragile nature. More-over, as the concentration of macro-crosslinker HGMC increasing, the crosslinking density of networks was enhanced, thus resulting in an increase in the modulus from 1.28 to 1.71 kPa (Figure 5B). In addition, after three loadingunloading compression tests, the hydrogel was still intact keeping the original state and nearly identical strength at maximum deformation, although there was a hysteresis (Figure 5C). It should be noted that this hysteresis was normal in the hydrogel systems with the presence of hostguest interactions, which meant that the compression energy was dissipated effectively by sliding β-CD from GA backbone in hydrogels. [START_REF] Nakahata | Self-healing materials formed by cross-linked polyrotaxanes with reversible bonds[END_REF] Besides, the maximal stretching strain of Gel-1, Gel-2 and Gel-3 reached 454%, 297%, and 131%, respectively, with fairly elasticity in the stretched stats (Figure 5D). All the above results indicated that our hydrogels were anti-compressing, nonfragile and elastic, and could recover to their original states on account of the non-covalent host-guest interactions. In summary, to address the issue of low mechanical strength of previously reported polymeric hydrogels made of host-group-grafted polymers and guest-group-grafted polymers, a biocompatible host-guest macro-crosslinker HGMC was synthesized by complexation of natural GAderivative with terminal acrylates (GADA) with poly(β-cyclodextrin) (PCD), which was confirmed by NMR, SEC and XRD studies. Three polymeric hydrogels were prepared via a UV photo-polymerization of AAm with different contents of HGMC. The swelling tests and SEM images revealed that all the hydrogels had 3D crosslinked structures with micro-pores, and their swelling ratios and pore sizes could be adjusted effectively by the content of HGMC. In addition, they exhibited the great anti-compressing, nonfragile and elastic features, which were mainly attributed to the host-guest macro-crosslinker HGMC. This work provided an effective way to prepare biocompatible tough polymeric hydrogels, which might be applied as biomaterials in soft-tissue repair.

ASSOCIATED CONTENT

Supporting Information. Materials and methods; synthesis of GADA and its intermediates; synthesis of PCD; fabrication of HGMC inclusion complex and preparation of hydrogels with different contents of HGMC; 13 C NMR spectrum of PCD in D2O; X-ray diffraction patterns of PCD, GADA and HGMC;

Table of Contents

Figure 1 . 1 H

 11 Figure 1. 1 H NMR spectra of (A) GAA, (B) GA-OEG and (C) GADA (400 MHz, CDCl3). *represents the solvent peak.

Figure 2 .

 2 Figure 2. (A) SEC spectra of PCD and HGMC with refractometer as detector (DMF as eluent, polystyrene standards for molecular weights calibration). (B) 1 H NMR spectra of GADA (up), PCD (middle) and HGMC (bottom) in D2O (400 MHz). *represents the solvent peak.

Figure 3 .

 3 Figure 3. (A) Swelling behaviors and (B, C, D) SEM images of Gel-1, Gel-2 and Gel-3. Scale bar is 2 μm for (B), (C) and (D).

Figure 4 .

 4 Figure 4. (A) Oscillatory frequency sweep of Gel-1, Gel-2 and Gel-3 at a strain 1%; (B) G' and G'' of Gel-1, Gel-2 and Gel-3 as a function of strain at a frequency of 1 Hz.

Figure 5 .

 5 Figure 5. (A) Mechanical compression curves of Gel-1, Gel-2 and Gel-3, and (B) their responding compression modulus. (C) Cyclic compression curves of Gel-3. (D) Mechanical tensile curves of Gel-1, Gel-2 and Gel-3.

Table 1 . Formulations and swelling properties of Gel- 1, Gel-2 and Gel-3.

 1

	Gels	HGMC (mmol)	AAm (mmol)	Molar Ratio a	Water Content	Swelling Ratio b
	Gel-1	0.12	12	0.010	52%	487%
	Gel-2	0.18	12	0.015	46%	339%
	Gel-3	0.24	12	0.020	41%	249%

a [HGMC]/[AAm]; b Gels were swollen in DI-water for 24 h.

H-,13 C-NMR and MS spectra of GAA, GA-OEG and GADA, respectively.

ACKNOWLEDGMENT

This work is supported by NSFC (no. 21604085, 21604001) and National Key R&D Program of China (no. 2017YFD0200302).

AUTHOR INFORMATION

Corresponding Author *E-mail: jhu@mail.buct.edu.cn *E-mail: min-hui.li@chimieparistech.psl.eu

Notes

The authors declare no competing financial interest.

Supporting Information

Tough Polymeric Hydrogels Formed by Natural Glycyrrhetinic Acid-Tailored Host-Guest Macro-Crosslinking Toward Biocompatible Materials

Ge Xu, † Jiawei Li, † Jianqiao Wu, † Hao Zhang, † Jun Hu* , † and Min-Hui Li* , †, ‡ † Beijing Advanced Innovation Center for Soft Matter Science and Engineering, Beijing University of Chemical Technology, Beijing 100029, China ‡ Chimie ParisTech, PSL University Paris, CNRS, Institut de Recherche de Chimie, Paris 75005, France Email: jhu@mail.buct.edu.cn; min-hui.li@chimieparistech.psl.eu Materials and methods. Glycyrrhetinic acid, β-cyclodextrin, I2959, succinic anhydride, 2-[2-(2chloroethoxy)ethoxy]ethanol, epichlorohydrin, trimethylamine, acryloyl chloride, acrylamide, potassium carbonate, potassium iodide, potassium hydroxide and other reagents were local commercial products. Acrylamide was recrystallized from chloroform. Dialysis bag was purchased from Viskase. All organic solvents were dried and distilled before use.

NMR spectra were recorded on a Bruker AV400 spectrometer operating at 400 MHz. HRMS-ESI were performed on Ultra High Definition(UHD) Accurate-Mass Q-TOF LC-MS.

Molecular weight and polydispersity index (PDI) of polymers were determined by size exclusion chromatography (SEC) with an Agilent Technologies 1260 infinity LC equipped with quaternary gradient pump and a refractive-index detector (G1362A). DMF was used as eluent at a flow rate of 1.0 mL/min at 23℃, and polystyrene standards were employed for molecular weights calibration.

The swelling properties of hydrogels was estimated by the swelling ratio (Q) of each sample during the interval measured at room temperature by weighting swollen hydrogels in water (mw), followed by recording their dried mass (md) using the following equation:

The molar ratio between β-CD and GADA in host-guest macro-crosslinker HGMC can be calculated as Formula S1: = 𝑀𝑀 𝑛𝑛 (PCD) 𝑀𝑀 𝑤𝑤 (𝛽𝛽-CD+10epichlorohydrin-10 HCl) / 𝑀𝑀 𝑛𝑛 (HGMC)-𝑀𝑀 𝑛𝑛 (PCD)

Where Mn(PCD) and Mn(HGMC) were obtained from SEC results, Mw(GADA) was afforded by ESI-MS (+), and Mw(β-CD + 10epichlorohydrin -10HCl) was calculated according to the feed ratio of β-CD and epichlorohydrin in preparation of PCD.

XRD was conducted with an Ultima IV Goniometer from Rigaku by using CuKa radiation, and scanning was performed from 5 to 90°. SEM images were obtained using a field emission scanning electron microscope (JSM-7610F with 10 kV acceleration voltage). Square gels (3 mm × 2 mm) at the swelling equilibrium were lyophilized under vacuum at -60 °C. Before subjecting to SEM analysis, gold was coated on dry gels.

S4

Rheological characterization of hydrogels was done with an AR 2000 rheometer (TA Instruments) equipped with a 2° steel cone geometry of 20 mm diameter and solvent trap. They were monitored by time sweep, frequency sweep, and strain sweep experiments.

Compression tests were performed on an electronic universal testing machine (SANS, CMT4000, CN), and the hydrogels had the cylindrical shape (height 10.24 mm, diameter 18.71 mm) for compression and cyclic compression tests. While tensile tests were performed on a universal test machine (UTM, Instron 3345, USA). The specimens were of 20 mm width and 2 mm thickness with a testing gauge length of 5 cm.