Lakshmeesha Upadhyaya

Beatriz Oliveira

Vanessa J Pereira

Maria T Barreto Crespo

João G Crespo

Damien Quemener

Mona Semsarilar
email: mona.semsarilar@umontpellier.fr.

†king Abdullah

Ethanol Acva

Ethanol Cta Acva

H Mma

Cpdb Cooh

Ethanol Va

Nanocomposite membranes from nano-particles prepared by polymerization induced self-assembly and their biocidal activity

ABSTRACT

Silver ions have been widely used because of their antimicrobial properties. This study describes the production of novel nanocomposite membranes from a block copolymer and silver nanoparticles (NPs). These composite membranes display properties from both polymeric and inorganic materials along with the biocidal features obtained due to the presence of silver ions.

The spin coating technique is employed to synthesize the nanocomposite membrane consisting of positively charged inorganic NPs and negatively charged polymeric NPs. Polymeric NPs of spherical, wormicular, and vesicular morphologies were synthesized using Reversible additionfragmentation chain transfer (RAFT) polymerization using poly(methacrylic acid)-b-(methyl methacrylate) diblock copolymer. The silver NPs coated with poly(methacrylic acid)-bpoly(quaternized 2-(dimethylamino)ethyl methacrylate), were synthesized using a nanoprecipitation method. The silver NPs act as the bridging entity between the polymeric NPs, as well as conferring the antimicrobial activity to the composite membranes. To test their antimicrobial properties, membranes were incubated with Enterococcus hirae. Comparison with the controls shows a 2 to 3 log decrease in the bacterial count for a contact time of 24 h. Furthermore, membrane filtration experiments conducted with phosphate buffer saline solutions spiked with bacteria indicated the importance of incorporating silver NPs in the nanocomposite membrane to achieve considerable rejection of bacteria as well as biocidal activity. KEYWORDS. Nanocomposite membranes, block copolymers, silver nanoparticles, biocidal activity

INTRODUCTION

Block copolymers are one of the remarkable contenders for the fabrication of polymeric membranes due to their ability to form well organized porous structures with exceptional mechanical stability [START_REF] Upadhyaya | Filtration membranes from self-assembled block copolymersa review on recent progress[END_REF][START_REF] Upadhyaya | Influence of Magnetic Nanoparticles on PISA Preparation of Poly(Methacrylic Acid)-b -Poly(Methylmethacrylate) Nano-Objects[END_REF][START_REF] Rubio | Nanostructured Membranes from Soft and Hard Nanoparticles Prepared via RAFT-mediated PISA[END_REF][START_REF] Upadhyaya | Nano-structured magneto-responsive membranes from block copolymers and iron oxide nanoparticles[END_REF][START_REF] Upadhyaya | Porous membranes from acid decorated block copolymer nanoobjects via RAFT alcoholic dispersion polymerization[END_REF][START_REF] Upadhyaya | Nanostructured Mixed Matrix Membranes from Supramolecular Assembly of Block Copolymer Nanoparticles and Iron Oxide Nanoparticles[END_REF][START_REF] Upadhyaya | Block copolymer based novel magnetic mixed matrix membranes-magnetic modulation of water permeation by irreversible structural changes[END_REF][START_REF] Nehache | Nano-porous structures via self-assembly of amphiphilic triblock copolymers: influence of solvent and molecular weight[END_REF][START_REF] Nehache | Negatively Charged Porous Thin Film from ABA Triblock Copolymer Assembly[END_REF][START_REF] Nehache | Self-assembly of PS-PNaSS-PS triblock copolymers from solution to the solid state[END_REF][START_REF] Nehache | Translocation across a self-healing block copolymer membrane[END_REF]. There are many approaches available for the preparation of block copolymer-based membranes, like spin coating, extrusion, and bulk evaporation [START_REF] Upadhyaya | Filtration membranes from self-assembled block copolymersa review on recent progress[END_REF][START_REF] Nunes | From Charge-Mosaic to Micelle Self-Assembly: Block Copolymer Membranes in the Last 40 Years[END_REF]. Several different research groups have developed various routes for the fabrication of asymmetric isoporous membranes from block copolymers via traditional non-solvent induced phase separation technique [START_REF] Upadhyaya | Nano-structured magneto-responsive membranes from block copolymers and iron oxide nanoparticles[END_REF][START_REF] Upadhyaya | Porous membranes from acid decorated block copolymer nanoobjects via RAFT alcoholic dispersion polymerization[END_REF][START_REF] Upadhyaya | Nanostructured Mixed Matrix Membranes from Supramolecular Assembly of Block Copolymer Nanoparticles and Iron Oxide Nanoparticles[END_REF][START_REF] Nehache | Nano-porous structures via self-assembly of amphiphilic triblock copolymers: influence of solvent and molecular weight[END_REF][START_REF] Nehache | Negatively Charged Porous Thin Film from ABA Triblock Copolymer Assembly[END_REF][START_REF] Nehache | Self-assembly of PS-PNaSS-PS triblock copolymers from solution to the solid state[END_REF][START_REF] Nunes | Ultraporous Films with Uniform Nanochannels by Block Copolymer Micelles Assembly[END_REF][START_REF] Nunes | From Micelle Supramolecular Assemblies in Selective Solvents to Isoporous Membranes[END_REF][START_REF] Nunes | Switchable pH-Responsive Polymeric Membranes Prepared via Block Copolymer Micelle Assembly[END_REF].

As previously reported [START_REF] Upadhyaya | Influence of Magnetic Nanoparticles on PISA Preparation of Poly(Methacrylic Acid)-b -Poly(Methylmethacrylate) Nano-Objects[END_REF][START_REF] Upadhyaya | Nano-structured magneto-responsive membranes from block copolymers and iron oxide nanoparticles[END_REF][START_REF] Upadhyaya | Porous membranes from acid decorated block copolymer nanoobjects via RAFT alcoholic dispersion polymerization[END_REF][START_REF] Upadhyaya | Nanostructured Mixed Matrix Membranes from Supramolecular Assembly of Block Copolymer Nanoparticles and Iron Oxide Nanoparticles[END_REF][START_REF] Upadhyaya | Block copolymer based novel magnetic mixed matrix membranes-magnetic modulation of water permeation by irreversible structural changes[END_REF][START_REF] Farooq | pH-responsive nanostructured membranes prepared from oppositely charged block copolymer nanoparticles and iron oxide nanoparticles[END_REF], we have developed a new strategy to make membranes from block copolymer NPs of different morphologies (spherical, wormicular, and vesicular). The morphologies mentioned above were synthesized via an alcoholic RAFT dispersion polymerization [START_REF] Semsarilar | Green" reversible addition-fragmentation chain-transfer (RAFT) polymerization[END_REF]. The membranes from spherical NPs were more effective due to the arrangement of particles in a well-organized fashion, providing a uniform pore size across the membrane compared to membranes from other morphologies. To enhance the mechanical stability [START_REF] Upadhyaya | Nanocomposite Membranes with Magnesium , Titanium , Iron and Silver Nanoparticles -A Review[END_REF][START_REF] Upadhyaya | Chemical modification of membrane surfaceoverview[END_REF] of the membranes and confer them magnetic properties, iron oxide NPs bearing opposite surface charges were used to link the negatively charged polymeric NPs together. This strategy resulted in the successful preparation of nanocomposite magnetic membranes with a pore size that could be tuned via the application of magnetic fields of different strength [START_REF] Upadhyaya | Block copolymer based novel magnetic mixed matrix membranes-magnetic modulation of water permeation by irreversible structural changes[END_REF]. Also, the use of the magnetic field successfully reduced protein fouling [START_REF] Upadhyaya | Block copolymer based novel magnetic mixed matrix membranes-magnetic modulation of water permeation by irreversible structural changes[END_REF].

Membrane-based filtration techniques are up-and-coming in the area of water filtration for removal of microorganisms, organic pollutants, and inorganic compounds. Membrane processes like ultrafiltration and microfiltration have been successfully employed for water treatment [START_REF] Le | Materials and membrane technologies for water and energy sustainability[END_REF][START_REF] Laîné | Status after 10 years of operationoverview of UF technology today[END_REF]. In water-based separation processes, biofouling may be one of the significant drawbacks due to the deposition of microorganisms like bacteria. After deposition, they may multiply at the membrane surface and produce extracellular material forming a biofilm and thereby affecting the membrane performance. The deposited materials will block the pores and thus decrease flux in a dramatic fashion [START_REF] Meng | Fouling in membrane bioreactors: An updated review[END_REF][START_REF] Shen | Ultrafiltration hollow fiber membranes of sulfonated polyetherimide/polyetherimide blends: preparation, morphologies and antifouling properties[END_REF].

Silver ions have been widely utilized as water disinfectant since their antimicrobial activity against a broad range of microorganisms has been well studied [START_REF] Upadhyaya | Nanocomposite Membranes with Magnesium , Titanium , Iron and Silver Nanoparticles -A Review[END_REF][START_REF] Upadhyaya | Chemical modification of membrane surfaceoverview[END_REF][START_REF] Basri | Silverfilled polyethersulfone membranes for antibacterial applications -Effect of PVP and TAP addition on silver dispersion[END_REF][START_REF] Cao | Immobilization of silver nanoparticles onto sulfonated polyethersulfone membranes as antibacterial materials[END_REF][START_REF] Diagne | Polyelectrolyte and Silver Nanoparticle Modification of Microfiltration Membranes To Mitigate Organic and Bacterial Fouling[END_REF]. The antimicrobial property [START_REF] Kim | Towards antibiofouling ultrafiltration membranes by blending silver containing surface modifying macromolecules[END_REF][27]of silver is mainly due to their interaction with protein thiol groups, which prevents DNA replication and affects the structure as well as the permeability of the bacteria cell membranes [START_REF] Gupta | Molecular basis for resistance to silver cations in Salmonella[END_REF][START_REF] Prathna | Biomimetic synthesis of silver nanoparticles by Citrus limon (lemon) aqueous extract and theoretical prediction of particle size[END_REF][START_REF] Kim | Antimicrobial effects of silver nanoparticles[END_REF][START_REF] Li | Antimicrobial nanomaterials for water disinfection and microbial control: Potential applications and implications[END_REF][START_REF] Madhavan | Silver-Enhanced Block Copolymer Membranes with Biocidal Activity[END_REF]. The incorporation of silver in membranes used for water treatment, to control biofouling, was previously studied. [START_REF] Basri | Silverfilled polyethersulfone membranes for antibacterial applications -Effect of PVP and TAP addition on silver dispersion[END_REF][START_REF] Cao | Immobilization of silver nanoparticles onto sulfonated polyethersulfone membranes as antibacterial materials[END_REF][START_REF] Madhavan | Silver-Enhanced Block Copolymer Membranes with Biocidal Activity[END_REF] Silver has been incorporated in membranes made from cellulose acetate, polyamide, poly(2-ethyl-2-oxazoline), polylactic acid [START_REF] Upadhyaya | Chemical modification of membrane surfaceoverview[END_REF][START_REF] Basri | Silverfilled polyethersulfone membranes for antibacterial applications -Effect of PVP and TAP addition on silver dispersion[END_REF][START_REF] Cao | Immobilization of silver nanoparticles onto sulfonated polyethersulfone membranes as antibacterial materials[END_REF][START_REF] Diagne | Polyelectrolyte and Silver Nanoparticle Modification of Microfiltration Membranes To Mitigate Organic and Bacterial Fouling[END_REF][START_REF] Dasari | Antifouling membranes prepared by electrospinning polylactic acid containing biocidal nanoparticles[END_REF][START_REF] Deng | Antimicrobial nano-silver non-woven polyethylene terephthalate fabric via an atmospheric pressure plasma deposition process[END_REF][START_REF] Jeon | The Effect of Membrane Material and Surface Pore Size on the Fouling Properties of Submerged Membranes[END_REF][START_REF] Park | Versatile surface charge-mediated anti-fouling UF/MF membrane comprising charged hyperbranched polyglycerols (HPGs) and PVDF membranes[END_REF], and Polyvinylidene fluoride (PVDF) [START_REF] Kim | Towards antibiofouling ultrafiltration membranes by blending silver containing surface modifying macromolecules[END_REF]. Nunes et al. [START_REF] Madhavan | Silver-Enhanced Block Copolymer Membranes with Biocidal Activity[END_REF] used the polystyrene-b-poly(4vinylpyridine) block copolymer for the fabrication of microporous membranes where the silver ions were deposited on the membrane surface and pore walls via chelation with the pyridine groups, facilitating the even distribution of the silver ions in the membrane. The NPs were deposited on the surface of the membrane at different pH values, and the ionic concentration of silver and its biocide activity was investigated using Pseudomonas aeruginosa. Chung et al. [START_REF] Kim | Towards antibiofouling ultrafiltration membranes by blending silver containing surface modifying macromolecules[END_REF] blended the silver containing surface modifying macromolecule (SMMs) in PVDF forming PVDF membrane with surface migrated SMMs. Biofouling filtrations were conducted using E.coli spiked in synthetic wastewater as feed at a pressure of 50 psig. After 25 h of operation, the membranes were flushed with distilled water to recover the membranes. Due to the smaller pore size, the solutes have been deposited on membranes contributing to flux reduction as well the membrane has shown greater antimicrobial properties due to the presence of silver ions on the surface.

In this work, we illustrate the fabrication of nanocomposite membranes from block copolymers in their spherical, wormicular, and vesicular morphologies and colloidally stable silver NPs. These membranes were prepared via spin-coating a blend of polymeric NPs and inorganic NPs (Silver particle coated with quaternized poly(2-(dimethylamino) ethyl methacrylate) in solution directly on a porous nylon support. The biocidal activity of selected membranes (from spherical polymeric NPs) was studied at different time intervals using a gram-positive bacterium, Enterococcus hirae.

EXPERIMENTAL

A detailed description of block copolymer synthesis along with materials and equipment used for experimentation are provided in supporting information (SI). The synthesis of silver NPs is explained below. Silver NPs were synthesized using sodium borohydride (NaBH4) as a reducing agent and 10 w/w% PMAA47-b-PQDMAEMA50 diblock copolymer as a stabilizing agent. The reduction procedure of silver nitrate (AgNO3) was carried out at room temperature with the block copolymer as the stabilizing agent. A typical process is as follows: 1 mL of 10 w/w % aqueous solution of PMAA47b-PQDMAEMA50 was added to 10 mL of distilled water under stirring. To this preparation, sodium borohydride in the molar ratio of 2:1 corresponding to silver nitrate was added. After the addition of the reducing agent, an aqueous solution of silver nitrate in the molar ratio of 3:1 corresponding to the diblock copolymer was added dropwise. During the addition of the silver nitrate solution, initially, the color of the reaction mixture changed to yellow and then browned.

The reaction mixture was stirred for 30 min at room temperature followed by dialysis against water to remove unreacted reactants. Scheme 1. Synthesis of polymeric NPs and silver NPs followed by nanocomposite membrane preparation via a spin coating technique.

Antimicrobial properties of membranes

Contact tests with bacterial solutions

The membranes were sterilized with absolute ethanol for 25 s followed by cleaning with sterile water before the contact experiments. Contact experiments (Scheme 2) were performed using 6 well cell culture plates where the three different membranes (nylon support, membrane without silver and membrane with silver) were immersed into 7 mL of sterile phosphate-buffered saline (PBS) solution spiked with a final concentration of 1x10 6 CFU/mL of Enterococcus hirae (PBS).

Controls containing only the bacteria spiked into sterile PBS were also performed to verify the

Membrane filtration of bacterial solutions

For bacterial filtrations (Scheme 2), the cross-flow mode was selected and was conducted in a were carried out at 1.5 bar of transmembrane pressure. Initially, the cross-flow cell, pump, tubings, and membranes were sterilized with absolute ethanol. 50 mL of phosphate-buffered saline (PBS) solution spiked with 1x10 6 CFU/mL Enterococcus hirae was used as a feed solution. Feed, permeate, and retentate samples were analyzed for the different membrane filtration experiments (using the nylon support and the membrane immobilized with silver). 3 mL of each sample was used to prepare serial dilutions for quantification of E. hirae, in duplicate samples, using the pour plate technique and tryptone soy agar media.

RESULTS AND DISCUSSION

Previously, we demonstrated that nanocomposite membranes could be prepared from block copolymer NPs of different morphologies such as spheres, worms, and vesicles synthesized via RAFT dispersion polymerization (Scheme 1) and iron oxide NPs coated with positively charged poly(quaternized DMAEMA) [START_REF] Upadhyaya | Porous membranes from acid decorated block copolymer nanoobjects via RAFT alcoholic dispersion polymerization[END_REF][START_REF] Upadhyaya | Nanostructured Mixed Matrix Membranes from Supramolecular Assembly of Block Copolymer Nanoparticles and Iron Oxide Nanoparticles[END_REF]. It was demonstrated that the membranes made from a mixture of the diblock copolymer and iron oxide NPs had higher flux and constant permeability compared to membranes made from solely diblock copolymer particles. These nanocomposite membranes had excellent mechanical stability, as well as magnetic properties, overbear block copolymer membranes due to the presence of the magnetic iron-oxide NPs. In this work, we employ a similar strategy to prepare nanocomposite membranes containing silver NPs. The presence of silver NPs might confer antimicrobial properties to the membranes and help to suppress biofouling, which is the major problem associated with membrane processing in the form of fouling (partial or complete blockage of pores leading to flux reduction).

3.1.Silver NPs synthesis and characterization

Well defined silver NPs coated with the diblock copolymer (PMAA47-b-PQDMAEMA50) were synthesized by reduction of silver nitrate at room temperature. This is one of the first kind where the PISA formed polymer shells were decorated with block copolymer coated silver NPs. As shown in Fig. 1A, the hydrodynamic diameter of the silver NPs was 28.5 nm, as measured by DLS.

The particle size distribution of 0.087 suggested the presence of near monodisperse particles. The calculated particle diameter at dry state (from TEM images) was 6.9 nm (Fig. 1B). TGA analysis (Fig. 1C) indicated a weight ratio of 16% silver to 84% of the diblock copolymer. The transmission electron microscopy images of these particles showed only spherical particles (no other morphologies such as hexagonal or cubic were observed). The positive zeta potential (+32.29 mV) confirmed the attachment of the diblock copolymer chains on the surface of the silver NPs via the negatively charged PMAA block.

Nanocomposite membrane synthesis and performance

The pure phases of spheres (PMAA47-b-PMMA195; Đ = 1.03, Mn = 21.3 kg/ mol), worms (PMAA47-b-PMMA273; Đ = 1.11, Mn = 28.6 kg/ mol) and vesicles (PMAA47-b-PMMA368; Đ = 1.21, Mn = 33.3 kg/ mol) with 17.5 wt.% of total solids content were used for the preparation of membranes (full characterization of the polymeric NPs could be found in Table S1). The selection of total solids content was random from the phase diagram from our previous work. The prepared membranes from 3 different particle morphologies were used in a cross-flow water filtration setup.

During the filtration experiments, the following observations were made:

 For membranes from spheres, there was a decrease in the flux values as the pressure increased above 2 bar (Fig. S2A).

 For membranes from worms, a decrease in flux was not observed as pressure increased, but permeability has shown a decline phase (Fig. S3A), suggesting the movement of the top wormicular active layer into the sublayer, blocking the available pores.

 For membranes from vesicular NPs, the flux has shown a declining trend after applying 3 bar of pressure difference across the membrane (Fig. S4A).

This decrease is due to mechanical instability of the deposited polymer layer since, at high pressures, the polymeric particles get pushed into the nylon support layer, blocking the pores. This could be easily seen in the SEM images (Fig. S1), taken after filtration. The membranes from worms show larger pores at the top surface compared to others (Fig. 2C) but exhibit a lower flux.

This may be due to their entangled substructure making the pores smaller compared to others.

To increase the cohesion between the particles and the mechanical stability of the deposited layer, positively charged silver NPs coated with PMAA47-b-PQDMAEMA50 were added to the formulation. To prepare the casting solution with a maximum amount of silver NPs, the isoelectric point (where all the negative charges available on the polymeric NPs have been neutralized with positive charges of the silver NPs) of each dope solution was identified. For the polymeric colloidal solution of spheres, vesicles, and wormicular NPs (with a concentration of 6.7 mg/mL), 4.1, 3.1 and 2.1 mL of silver NPs (at 5.1 mg/mL) were required respectively to reach the isoelectric point.

To avoid precipitation in the casting solution, the added volume of silver NPs was kept below the volume corresponding to the isoelectric point. For membranes containing spheres, initially, 1.3 mL of silver NPs was added to 1 mL of polymeric NPs. After water filtration tests, the SEM images suggested partial intrusion of the top active layer into the nylon support. This volume was then increased to 1.7 mL resulting in a stable top layer after filtration cycles up to 4 bar of transmembrane pressure (Fig. 2A &B).

Membranes from worms showed higher mechanical stability compared to those of spheres or L.m -2 .h -1 at 4 bar of TMP. When comparing this value with a similar magneto-responsive membrane [START_REF] Upadhyaya | Block copolymer based novel magnetic mixed matrix membranes-magnetic modulation of water permeation by irreversible structural changes[END_REF] it could be seen that these fluxes are about 54-57% higher for both 1 bar and 4 bar TMP. The same trend (10-37% and 21-25% higher) was observed with membranes made from wormicular and vesicular NPs (Table 1). The reason behind the increase in water flux is probably due to changes in hydrophilicity or due to the change in thickness of the membrane. Both the thickness and contact angle comparison have been depicted in table 2 for the membrane containing silver NPs and iron oxide NPs. The thickness is lower for membranes with silver compared to Iron oxide NPs. This change was not significant for membranes from spheres. So both thickness and contact angle may play a pivotal role for the higher fluxes observed in membranes containing silver, compared to those containing iron oxide NPs.

Water contact angles of the membranes could be used for the estimation of changes in surface hydrophilicity [START_REF] Li | The double effects of silver nanoparticles on the PVDF membrane: Surface hydrophilicity and antifouling performance[END_REF]. The contact angle of the membranes without any silver NPs was found to be 48°, which was decreased to 45° when Iron oxide NPs were incorporated in the membranes. This number further changed to 39° when silver NPs were integrated. The possible explanation for changes in hydrophilicity may be due to the following reasons. In aqueous solution, due to oxidation, silver ions will be released by silver NPs. These released ions will be adsorbed on the surface of silver NPs forming a layer of hydrated silver ions, thereby changing the hydrophilicity of the layer. The dissolved oxygen concentration in water will also facilitate the release of hydrated silver ions during the process [START_REF] Li | The double effects of silver nanoparticles on the PVDF membrane: Surface hydrophilicity and antifouling performance[END_REF][START_REF] Liu | Ion Release Kinetics and Particle Persistence in Aqueous Nano-Silver Colloids[END_REF]. The properties of silver NPs are shown in table S2 (SI). For antibacterial and filtration studies, two dosages of silver ions were used one with lower concentration (1 mL of polymeric NPs: 1.3 mL of silver NPs) and one with a higher level (1mL of polymeric NPs: 1.7 mL of silver NPs). The membranes with 2 dosages (lower and higher concentration of silver were filtered using water at pH 7.1 at 4 bar of upstream pressure (dead end mode) for 80 h. Permeate collected during filtration was analyzed using Atomic Absorption Spectroscopy (AAS), and the concentration of silver leached out is depicted in Fig. 3. After 6 h of permeation, about 61 and 98 µg/L of silver ions were detected in permeate from membranes with lower and higher concentrations of silver. This number further reduced to 38 and 45 µg/L when the filtration time increased to 8 h (see inset of Fig. 3), which is within the range of toxicity threshold [START_REF] Cao | Silver nanoparticles for antibacterial devices : biocomparibility and toxicity[END_REF]. For all antibacterial tests and filtration studies, the membranes were compacted for 8 h prior to use making sure that the antibacterial effect observed is due to the attached silver NPs in the matrix of polymeric NPs forming the membrane. The permeate was also analyzed by DLS (Dynamic light scattering), which confirmed no traces of silver or polymeric NPs.

To estimate the amount of silver NPs present on the membrane after leaching, a mass balance was carried out for membrane with lower and higher silver concentration. The concentration of leached silver in µg/h was plotted against time (h), and the area under the curve was estimated using trapezoidal rule (integration method) [START_REF] Schrag | Pharmacokinetics and Toxicokinetics[END_REF]. The area under the curve estimates the amount of silver NPs in µg lost from the membranes. The estimation confirmed the loss of about 4.42% of silver NPs for the 1 st 8 h of operation under 4 bar of transmembrane pressure followed by further 4.07% loss of silver NPs, for the membrane with a lower concentration of silver confirming the presence of 91.51% of silver NPs remaining in membrane (Fig. 3A). For the membrane with higher concentration of silver (Fig. 3B), this loss was 5.7% in the 1 st 8 h followed by a further 3.72% loss of silver NPs assuring the presence of 90.58% of silver NPs in the membrane.

3.3.Antibacterial properties of the membrane

Antibacterial properties of pristine membranes from spheres, worms, and vesicles without any silver NPs revealed no biocidal activity. To test the biocidal activity of the nanocomposite membranes, only membranes made from spheres containing different dosages of silver NPs were selected using a gram-positive bacteria Enterococcus hirae as a model system. The inactivation kinetics of the bacteria with lower and higher concentrations of the silver NPs incorporated within the membranes was evaluated for various contact times (Fig. 4).

The concentration of the bacteria reduced from 1.15*10 6 CFU/mL to 5.93*10 5 CFU/mL at the end of 8 th h, which was further decreased to 1.26*10 4 CFU/mL during 24 h of contact time for membrane with a lower concentration of silver NPs (Fig. 4A). This decrease in number corresponds to 98.9%, which corresponds to almost 2 log decrease (Fig. 4C) in the bacterial concentration after 24 h of contact time. When the silver concentration was increased to a higher level, the bacterial count decreased from 1.02*10 6 CFU/mL to 2.0*10 3 CFU/mL after 24 h of contact between the membranes and the solution with bacteria (Fig. 4B). This decrease in number corresponds to a 99.8% reduction, which is about 3 log decrease in the bacteria count after 24 h of contact (Fig. 4C).

When the concentration was additionally increased by 0.3 mL (yielding 2 ml of silver NPs), a 3 log decrease was achieved within 19 h of contact time. However, just 1.7 mL of silver NPs is enough to provide the required mechanical stability to the active layer. The duration of contact could be further decreased to 15 h when the concentration was also increased to 2.15 mL showing the importance of silver concentration in the membrane and the contact time.

3.4.Bacterial filtration studies

The cross-flow mode was selected over dead-end filtration since the cross-flow mode is less prone to fouling due to the direction of the fluid flow tangential to the membrane surface. The bacterial filtration studies using the membrane made from spherical NPs containing a lower and higher amount of silver NPs are shown in Fig. 5.

In this filtration assay, nylon membranes were used as a control, and all the experiments were carried out at 1.5 bar of TMP. The rejection factor of the reference nylon membranes for bacteria

A B C

was about 38-40%. The membrane containing a lower amount of silver NPs showed a rejection of 81%, and the membrane with a higher amount of silver NPs had a rejection of 96.3%. The rejection percentage is a clear indication that the bacteria has passed through the membrane due to the formation of larger pores since not enough silver NPs were present to keep the polymeric NPs together under the applied pressure (refer to the results obtained for water filtration). When the silver NPs added to the casting solution increased to 2.15 mL, the rejection of bacteria increased to 99.6%, indicating the control over the pore size.

The collected permeate showed a 1 log decrease (93% reduction) in the bacterial count for the membrane containing the lower amount of silver NPs and approximately 2 log decrease (98.4% reduction) for the membrane containing a higher amount of silver NPs. The retentate, as well as the feed stream, showed a slight decrease in the bacteria concentration for the membrane with a higher amount of silver NPs. We believe that this modest decrease in the bacterial count is due to insufficient contact time as well as the amount of silver NPs. During the experiment, about 50 mL of solution was filtered during 25 min with membranes with silver, not providing enough time for deactivation. The performance can be enhanced by increasing the silver concentration as well as keeping the bacterial solution in contact with the membrane for specific residence time. Since the membrane rejection is about 96.3% for higher silver NPs concentration, most of the bacteria will be retained in the retentate, leading to higher contact time with the membrane and, consequently, to a higher inactivation. To estimate the amount of silver NPs lost from membrane after compaction followed by bacterial filtration studies, mass balance of silver NPs carried out and shown in SI. For the bacterial filtration studies, the transmembrane pressure was reduced to 1.5 bar and filtration carried out. If the filtration is carried out continuously for 72 h ( 0-8 h for compaction at 4 bar followed by bacterial filtration 8-80 h), the loss of silver NPs estimated to be 5.41% and 6.49% for the membrane with the lower and the higher silver NPs respectively suggesting the presence of significant amount of silver NPs in membranes. As aimed, the technique proposed for the preparation of membranes with an effective bactericide effect was successful. A promising proof-of-concept was achieved.

It should be emphasized that during the operating periods used in this work, no biofouling was observed. However, long-term studies (at least 1 to 2 years of operation) are required before claiming an effective solution. The contact time between the nanocomposite membranes and E. hirae to achieve 2-3 log inactivation depends on the concentration of silver NPs incorporated in the membranes. Membrane filtration of solutions spiked with bacteria also revealed that the silver incorporation within the membranes made from spherical polymeric NPs is crucial to increase the bacteria rejection due to their smaller pore size. Due to the high membrane retention rate, microorganisms will have high contact time with the membrane surface, which will lead to high inactivation. This work also needs further investigation on process effectiveness in real matrices, with natural organic matter and turbidity interference.

CONCLUSION

SUPPORTING INFORMATIONS

The supporting information contains Polymeric NPs characterization, flux and permeability data, mass balance of silver NPs in membrane and SEM images of membranes containing different proportions of polymeric and inorganic NPs.

Notes

The authors declare no competing financial interest.

Scheme 2 .

 2 Scheme 2. Analysis of antimicrobial properties and membrane filtration of solutions spiked with

  homemade cross-flow cell (Area of the membrane = 2.3 cm x 1.2 cm). The filtration experiments

Fig. 1 .

 1 Fig. 1. Characterization of silver NPs: (A) Particle size distribution by DLS (B) Particle size

  vesicles due to their entangled structure. At 4 bar of transmembrane pressure, a slight change in permeability was observed. The addition of an extra 0.5 mL of silver NPs to the casting solution was enough to obtain a stable top layer that could stand 4 bar of pressure. In the case of membranes from vesicles, 1.3 mL of silver NPs were needed to prepare a stable layer. The SEM images of the tested membranes, after two cycles with a maximum transmembrane pressure of 4 bar, are shown in Fig.2. The permeability and flux data obtained for these nanocomposite block copolymer membranes are shown in Fig.S2, S3& S4 indicate that the strategy of adding silver NPs with opposite charge enhances the mechanical stability (tested up to 4 bar of transmembrane pressure) of the prepared membranes and is entirely dependent on the concentration of added silver NPs.

Fig. 2 .

 2 Fig. 2. SEM Images of thin-film block copolymer membranes top surface and cross-section

Fig. 3 .

 3 Fig. 3. Leaching out of silver ions from nanocomposite membranes containing (A) lower

Fig. 4 .

 4 Fig. 4. Inactivation kinetics of Enterococcus hirae due to the incorporation of silver NPs in

Fig. 5 .

 5 Fig. 5. Bacterial filtration studies with membranes made from spheres containing silver NPs with

  In this work, we have used silver NPs as a bridging entity between the polymeric NPs made from PMAA47-b-PMMAy(where y = 195-spheres, 273-worms, and 368-vesicles) block copolymer synthesized using RAFT polymerization. These NPs have been used to make nanocomposite membranes. The nanocomposite membranes prepared showed excellent mechanical stability containing different amounts of inorganic NPs linking the polymeric NPs for all three different morphologies studied. At 4 bar of transmembrane pressure, the membranes from spheres exhibited the highest flux value (589.6 L.h -1 .m -2 ), whereas the membranes from vesicles and worms showed a maximum flux of 411.9 L.h -1 .m -2 and 277.5 L.h -1 .m -2 , respectively.

Table 1 .

 1 Comparison of water flux at pH 7.1 for membranes containing iron oxide and silver NPs

	Pressure			Flux (L.m -2 .h -1 )
	(Bar)	Membranes from Spheres Membranes from Worms	Membranes from
						Vesicles
		Iron oxide	Silver NPs	Iron oxide	Silver NPs Iron oxide	Silver NPs
		NPs[6]		NPs[6]		NPs[6]
	1	96.5	148.6	55.6	61.3	85.3	103.3
	4	375.3	589.6	152.7	209.3	328.3	411.9

Table 2 .

 2 The thickness and contact angle for Iron and silver NPs containing membranes

	Thickness (µm)	Contact Angle (°)

Permeate Feed Retentate Feed Permeate Retentate 10 0 10 1 10 2 10 3 10 4 10 5 10 6 Nylon Support Nanocomposite membrane CFU/mL A B Permeate Feed Retentate Feed Permeate Retentate

ACKNOWLEDGEMENTS

The doctorate of L.U. has been completed at the Institut Europeen des Membranes and carried out in three universities: ́ Universite ́ de Montpellier (France), Universidad de Zaragoza (Spain), and Universidade Nova de Lisboa (Portugal), and financed by a scholarship of the European Commission Education, Audiovisual and Culture Executive Agency (EACEA), under the program Erasmus Mundus Doctorate in Membrane Engineering, EUDIME (FPA No. 2011-0014, Edition III). D.Q. and M.S. acknowledge financial support from the "Agence Nationale pour la Recherche" (ANR-13-JS08-0008-01). Financial support from Fundação para a Ciência e a Tecnologia through the PhD fellowship SFRH/BD/111150/2015 is gratefully acknowledged. iNOVA4Health-UID/Multi/04462/2013, a program financially supported by Fundação para a Ciência e Tecnologia/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement, is also gratefully acknowledged. This work was also supported by the Associate Laboratory for Green Chemistry LAQV which is financed by national funds from FCT/MCTES (UIDB/50006/2020).


Enrique Folgado

Qiao Song

Sebastien Perrier

Vincent Ladmiral

Mona Semsarilar

Fluorinated Nanotubes: Synthesis and self-assembly of cyclic peptidepoly(vinylidene fluoride) conjugates

The synthesis of cyclic peptide-poly(vinylidene fluoride) (CP-PVDF) conjugates comprising (D-alt-L)-cyclopeptides as building blocks and their self-assembly into tube-like structures is described. By growing two PVDF polymeric chains from opposite sides of a preassembled cyclic-peptide macro-chain transfer agent, a PVDF-CP-PVDF conjugate was prepared. This "grafting-from" strategy, allowed the synthesis of the conjugate with high purity and using facile purification steps. The controlled self-assembly of the conjugate from DMF or DMSO solutions was carried out by addition of THF. This triggered the aggregation process that led to formation of tube-like structures. The mean length and width of the PVDF-CP-PVDF tubes were measured using atomic force microscopy (AFM) and transmission electron microscopy (TEM). Surprisingly, the self-assembly of the CP-PVDF conjugates in DMF/THF allowed the preparation of long (up to 25µm) tube-like structures. The formation of such long tubular peptide-polymer aggregates, based on the stacking of cyclopeptides, is unprecedented and is believed to rely on synergetic effects between the stacking of the cyclic peptide and the interactions of the fluoropolymer-peptide conjugates.

INTRODUCTION

Recently, peptide-polymer conjugates have attracted special attention for their application in a wide range of fields, including therapeutics and separation technologies. [START_REF] Shu | Peptide-Polymer Conjugates: From Fundamental Science to Application[END_REF][START_REF] Kim | Oriented Self-Assembly of Cyclic Peptide Nanotubes in Lipid Membranes[END_REF][START_REF] Ruiz | Tailoring the Water Structure and Transport in Nanotubes with Tunable Interiors[END_REF][START_REF] Hartlieb | Stimuli-Responsive Membrane Activity of Cyclic-Peptide-Polymer Conjugates[END_REF][START_REF] Rodriguez-Vazquez | Membrane-Targeted Self-Assembling Cyclic Peptide Nanotubes[END_REF] A fascinating class of peptides that are known to self-assemble into supramolecular nanotubes (NTs) are cyclic peptides (CPs) comprising an even number of alternating D-and L-amino acids. [START_REF] Granja | Self-Assembling Organic Nanotubes Based on a Cyclic Peptide Architecture[END_REF][START_REF] Chapman | Design and Properties of Functional Nanotubes from the Self-Assembly of Cyclic Peptide Templates[END_REF] The pioneering work on CP nanotubular structures was carried out by Ghadiri and co-workers. [START_REF] Granja | Self-Assembling Organic Nanotubes Based on a Cyclic Peptide Architecture[END_REF] The alternating chirality of the amino acids in the macrocycle leads to amide bonds that alternate in orientation perpendicular to the plane of the CP rings. [START_REF] De Santis | Conformational Analysis of Regular Enantiomeric Sequences[END_REF] As a result, a contiguous intermolecular hydrogen-bonded network arises, resulting in the formation of hollow and extended cylindrical structures. In these structures, all of the amino acid side chains are directed towards the outside of the cycle. Because of this side chain arrangement, the interior of the resulting assemblies remain empty, thus creating a channel along the axis of the cyclic peptide nanotubes. In addition, both the functional groups and the diameter of the nanotube can be precisely controlled by changing the sequence and the number of amino acids in the cyclic peptide. [START_REF] Rodriguez-Vazquez | Membrane-Targeted Self-Assembling Cyclic Peptide Nanotubes[END_REF][START_REF] Dehn | Synthetic Strategies for the Design of Peptide/Polymer Conjugates[END_REF] Despite the great progress made with CP NTs in applications such as ion sensing, [START_REF] Motesharei | Diffusion-Limited Size-Selective Ion Sensing Based on SAM-Supported Peptide Nanotubes[END_REF] transmembrane ion channels [START_REF] Kim | Oriented Self-Assembly of Cyclic Peptide Nanotubes in Lipid Membranes[END_REF][START_REF] Binfield | Imaging Proton Transport in Giant Vesicles through Cyclic Peptide-Polymer Conjugate Nanotube Transmembrane Ion Channels[END_REF] and drug delivery systems, [START_REF] Larnaudie | Cyclic Peptide-Polymer Nanotubes as Efficient and Highly Potent Drug Delivery Systems for Organometallic Anticancer Complexes[END_REF] limitations with respect to their solubility, functionality and lack of control over their length restrict the expansion of applications.

Polymer conjugation allows some degree of control over the tube length, and the nature of the grafted polymer influences the solubility of the CP-polymer NTs. [START_REF] Chapman | Modular Design for the Controlled Production of Polymeric Nanotubes from Polymer/Peptide Conjugates[END_REF][START_REF] Koh | Study of (Cyclic Peptide)-Polymer Conjugate Assemblies by Small-Angle Neutron Scattering[END_REF] To a great extent, CP-polymer conjugates, whereby the CP has been used as a supramolecular template, have addressed these issues. [START_REF] Chapman | Modular Design for the Controlled Production of Polymeric Nanotubes from Polymer/Peptide Conjugates[END_REF][START_REF] Couet | Polymer-Wrapped Peptide Nanotubes: Peptide-Grafted Polymer Mass Impacts Length and Diameter[END_REF][START_REF] Poon | Pushing the Limits of Copper Mediated Azide-Alkyne Cycloaddition (CuAAC) to Conjugate Polymeric Chains to Cyclic Peptides[END_REF][START_REF] Chapman | Structure Elucidation and Control of Cyclic Peptide-Derived Nanotube Assemblies in Solution[END_REF][START_REF] Cate | Self-Assembling Peptide-Polymer Conjugates Comprising (D-Alt-L)-Cyclopeptides as Aggregator Domains[END_REF] Typically, CP-polymer conjugates are synthesized via either a grafting-from (divergent) or a grafting-to (convergent) approach. [START_REF] Dehn | Synthetic Strategies for the Design of Peptide/Polymer Conjugates[END_REF][START_REF] Gauthier | Peptide/Protein-Polymer Conjugates: Synthetic Strategies and Design Concepts[END_REF][START_REF] Larnaudie | Cyclic Peptide-Polymer Conjugates: Grafting-to vs Grafting-From[END_REF] In the grafting-from approach, [START_REF] Ruiz | Tailoring the Water Structure and Transport in Nanotubes with Tunable Interiors[END_REF][START_REF] Larnaudie | Cyclic Peptide-Polymer Conjugates: Grafting-to vs Grafting-From[END_REF] the polymer chains are grown from the peptide using a variety of polymerization techniques [START_REF] Couet | Surface-Initiated ATRP of N-Isopropylacrylamide from Initiator-Modified Self-Assembled Peptide Nanotubes[END_REF][START_REF] Larnaudie | Amphiphilic Core-Shell Supramolecular Polymer Brushes from Cyclic Peptide-Polymer Conjugates[END_REF] while in the grafting-to approach the polymers are synthesized separately and then grafted to the peptide using highly efficient coupling reactions. [START_REF] Cate | Self-Assembling Peptide-Polymer Conjugates Comprising (D-Alt-L)-Cyclopeptides as Aggregator Domains[END_REF][START_REF] Danial | Janus Cyclic Peptide-Polymer Nanotubes[END_REF] However, despite the use of these highly efficient reactions, such as copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC), [START_REF] Chapman | Modular Design for the Controlled Production of Polymeric Nanotubes from Polymer/Peptide Conjugates[END_REF] or activated ester-mediated ligations, [START_REF] Larnaudie | Cyclic Peptide-Polymer Conjugates: Grafting-to vs Grafting-From[END_REF] the grafting-to approach often requires an excess of polymer and time-consuming laborintensive purification steps to remove the unreacted polymer. [START_REF] Rho | Probing the Dynamic Nature of Self-Assembling Cyclic Peptide-Polymer Nanotubes in Solution and in Mammalian Cells[END_REF] In addition, the grafting-from synthetic strategy is not limited by monomer side chain functionalities orthogonal to the chain end group used for the conjugation, as in the grafting-to route. [START_REF] Chapman | Modular Design for the Controlled Production of Polymeric Nanotubes from Polymer/Peptide Conjugates[END_REF][START_REF] Rho | Probing the Dynamic Nature of Self-Assembling Cyclic Peptide-Polymer Nanotubes in Solution and in Mammalian Cells[END_REF] In recent years, many polymers have been grown from or grafted to cyclic peptides using different chemistries. [START_REF] Shu | Peptide-Polymer Conjugates: From Fundamental Science to Application[END_REF][START_REF] Rodriguez-Vazquez | Membrane-Targeted Self-Assembling Cyclic Peptide Nanotubes[END_REF][START_REF] Binfield | Imaging Proton Transport in Giant Vesicles through Cyclic Peptide-Polymer Conjugate Nanotube Transmembrane Ion Channels[END_REF][START_REF] Cate | Self-Assembling Peptide-Polymer Conjugates Comprising (D-Alt-L)-Cyclopeptides as Aggregator Domains[END_REF][START_REF] Larnaudie | Cyclic Peptide-Polymer Conjugates: Grafting-to vs Grafting-From[END_REF][START_REF] Couet | Surface-Initiated ATRP of N-Isopropylacrylamide from Initiator-Modified Self-Assembled Peptide Nanotubes[END_REF][START_REF] Rho | Probing the Dynamic Nature of Self-Assembling Cyclic Peptide-Polymer Nanotubes in Solution and in Mammalian Cells[END_REF][START_REF] Song | Supramolecular Switching of the Self-Assembly of Cyclic Peptide-Polymer Conjugates: Via Host-Guest Chemistry[END_REF] The first report of a "grafting-from" approach was made by Couet et al in 2006, who described the use of preassembled CP initiators to prepare CP-PNIPAM (Poly(N-isopropylacrylamide)) conjugates, using surfaceinitiated atom transfer radical polymerization (SI-ATRP). [START_REF] Couet | Surface-Initiated ATRP of N-Isopropylacrylamide from Initiator-Modified Self-Assembled Peptide Nanotubes[END_REF] In 2016, Larnaudie et al. expanded the approach to reversible addition-fragmentation chain-transfer (RAFT) polymerization, by functionalising a CP using a trithiocarbonate RAFT agent bearing an N-hydroxysuccinimide (NHS)moiety. [START_REF] Larnaudie | Cyclic Peptide-Polymer Conjugates: Grafting-to vs Grafting-From[END_REF] They explored the polymerization of a variety of monomers, including butyl acrylate, N-isopropylacrylamide, N-acryloylmorpholine, 2-hydroxyethyl acrylate and polyethyleneglycol acrylate. They also compared the "graftingfrom" and the "grafting-to" approaches and concluded that the "grafting-to" strategy is more flexible in terms of choice of solvents and polymers. However, the "grafting-from" approach affords purer conjugate in a shorter time. This approach, however, relies on the availability of a solvent that can solubilize the peptide, the monomer and the resulting conjugate at the same time.

Poly(vinylidene fluoride) (PVDF), a semi-crystalline polymer presenting excellent physicochemical properties as well as electroactive properties (piezoelectricity, pyroelectricity and ferroelectricity) is used in very diverse fields. [START_REF] Ameduri | Organometallic Mediated Radical Polymerization of Vinylidene Fluoride[END_REF][START_REF] Ribeiro | Electroactive Poly(Vinylidene Fluoride)-Based Structures for Advanced Applications[END_REF][START_REF] Wu | Flexible Composite-Nanofiber Based Piezo-Triboelectric Nanogenerators for Wearable Electronics[END_REF][START_REF] Najjar | Biocompatible Silk/Polymer Energy Harvesters Using Stretched Poly (Vinylidene Fluoride-Cohexafluoropropylene) (PVDF-HFP) Nanofibers[END_REF][START_REF] Marino | The Formation of Polyvinylidene Fluoride Membranes with Tailored Properties via Vapour / Non-Solvent Induced Phase Separation[END_REF][START_REF] Wang | A Silane-Based Interfacial Crosslinking Strategy to Design PVDF Membranes with Versatile Surface Functions[END_REF] In addition to its electroactive properties, PVDF is also often use in filtration membranes. CP-PVDF conjugates self-assembled nanotubes could offer a way to design and fabricate PVDFbased membranes endowed with well-defined functionalized channels.

We report herein the fabrication of the first CP-PVDF conjugates, as illustrated in Scheme 1, constructed via a divergent synthetic approach using a CP(-Xanthate) 2 building block via Macromolecular Architecture Design by Interchange of Xanthates (MADIX). In appropriate solvents, self-assembly is possible, resulting in well-defined PVDF nanotubular structures. The use of a CP containing eight alternating D-and L-amino acids permits a facile templated approach for the formation of well-defined PVDF NTs featuring sub nanometer channels within their cores.

Experimental section

Materials. N-hydroxysuccinimide (98%), triethylamine (TEA) (>99.5%), 2-bromopropionyl bromide (97%), magnesium sulfate anhydrous (>99.5%), potassium ethyl xanthogenate (96%), 4-dimethylaminopyridine (DMAP, 99 %), 2-chloro-4,6-dimethoxy-1,3,5-triazine (97 %), triisopropylsilane (TIPS, 99 %), and aluminum oxide were purchased from Sigma-Aldrich. 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP, 99 %) and iodine were purchased from Acros Organics. N-methylmorpholine (NMM, 99 %) and piperidine were purchased from Alfa Aesar. Sodium hydroxide pellets, sodium thiosulfate pentahydrate and anhydrous magnesium sulfate (MgSO 4 ) were purchased from Fisher. N,N-diisopropylethylamine (DIPEA, 99 %), was purchased from Merck. Fmoc-D-Leu-OH, Fmoc-L-Lys(Boc)-OH, Fmoc-L-Trp(Boc)-OH, O-(1H-6-chlorobenzotriazole-1yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2-chlorotrityl chloride resin (100-200 mesh) were purchased from Iris Biotech and used as received. Tert-amyl peroxy-2-ethylhexanoate (Trigonox 121, purity 95%) was purchased from AkzoNobel (Chalons en Champagne, France). Deuterated solvents for NMR were purchased from Euristop. All solvents were bought from commercial sources and used as received. VDF was kindly supplied by ARKEMA. The cyclization coupling agent 4-(4,6-dimethoxy-1,3,5-triazin-2yl)-4-methylmorpholinium tetrafluoroborate (DMTMM•BF4) was synthesized according to an established literature method. [START_REF] De Santis | Conformational Analysis of Regular Enantiomeric Sequences[END_REF] Nuclear Magnetic Resonance (NMR). The NMR spectra were recorded on a Bruker AV III HD Spectrometer (400 MHz for 1 H and 376 MHz for 19 F). Coupling constants and chemical shifts are given in hertz (Hz) and parts per million (ppm), respectively. The experimental conditions for recording 1 H and [START_REF] Gauthier | Peptide/Protein-Polymer Conjugates: Synthetic Strategies and Design Concepts[END_REF] F NMR spectra were as follows: flip angle, 30°; acquisition time, 4s; pulse delay, 1 s; number of scans, 16 (or 32 for 19 F); and pulse widths of 9.25 and 11.4 μs for 1 H and [START_REF] Gauthier | Peptide/Protein-Polymer Conjugates: Synthetic Strategies and Design Concepts[END_REF] F NMR respectively.

Dynamic light scattering (DLS). DLS measurements of polymer solutions were carried out with an Anton Paar Litesizer TM 500 using a quartz cuvette at 25°C.

Transmission electron microscopy (TEM). TEM studies were conducted using a JEOL 1400+ instrument equipped with a numerical camera, operating with a 120 kV acceleration voltage at 25 °C. To prepare TEM samples, a drop (10.0 μL) of micellar solution was placed onto a Formvar/Carbon or Lacey/Carbon coated copper grid for 30 s, blotted with filter paper and dried under ambient conditions.

Scanning electron microscopy (SEM). SEM analyses were conducted using a Hitachi S-4500 instrument operating at spatial resolution of 1.50 nm at 15 kV energy. The samples were prepared by spin coating of 50 µL of the solution on a silicon wafer. The samples were then placed on a flat mount after being coated with an ultrathin layer of electrically conducting Platinum deposited by high-vacuum evaporation.

Atomic force microscopy (AFM). AFM samples were prepared by spin coating of 50 µL (diluted 10 times in acetone in the case of PVDF-CP crude of polymerization reaction) of the solution on a freshly cleaved mica wafer. AFM images were acquired with a Pico SPM II provided by Molecular Imaging. The imagery was controlled by the PicoView 1.10 software. The experiments were all carried out in tapping mode. The types of tips used were PPS-FMR purchased from Nanosensors with a frequency resonance between 45 and 115 kHz and a force constant between 0.5 and 9.5 N/m. Gwyddion 2.25 software was used to treat the images. Samples were prepared by spin coating 50 µL of the solution on the surface of freshly cleaved mica wafers (Sample concentration 0.1 mg mL -1 in DMF: THF (1:9)).

Mass spectrometry. Measurements were performed on a Bruker MicroToF for ESI ToF and on an Agilent 6130B Single Quad for ESI.

Gel Permeation Chromatography (GPC): GPC was measured using an Agilent PL50 instrument with a differential refractive index detector. The instrument contained two PolarGel H columns (300 mm × 7.5 mm) and a PolarGel 5 μm guard column. DMF with 0.1% LiBr additive was used as the eluent. The system ran at 1 mL min -1 (50 °C), with an injection volume of 100 μL. The samples were prepared by filtering them through 0.22 μm pore size PTFE membranes, before injection. Agilent EasyVial poly(methyl methacrylate) standards were used to calibrate the instrument and output data were analyzed using Agilent GPC/SEC software.

Synthesis. Synthesis of N-succinimidyl bromoacetate, (Scheme 1, 1). N-hydrosuccinimide (NHS) (6.33 g; 55 mmol) was placed in a 250 mL round bottom flask and dissolved in 80 mL of DCM under magnetic stirring. The flask was placed in an ice bath and TEA (8.1 mL, 58 mmol) in 16 mL of DCM was added dropwise. After stirring for 30 min, 2-bromopropionyl bromide (6.08 mL; 58 mmol) in 16 mL of DCM was added dropwise over a period of 1 h. The reaction was left for 24 h at room temperature (25 °C). The mixture was then washed with brine and the organic phase was collected and dried over anhydrous magnesium sulfate. The solvent was removed by rotary evaporation. The brownish solid was dissolved in isopropanol at 75 °C and a few drops of DCM were added. The product was left to crystallize in the fridge and was filtered to yield 92%.

Synthesis of NHS-CTA-XA, (Scheme 1, 2). N-succinimidyl bromoacetate (6 g; 24 mmol) was placed in a 100 mL round bottom flask and dissolved in 45 mL of absolute ethanol. The flask was placed in an ice bath and potassium ethyl xanthogenate (4.8 g; 29 mmol) was added with a spatula over a period of 45 min. The heterogeneous solution was stirred 3 h at room temperature, then filtered over Celite and the solvent removed by rotary evaporation. The product was dissolved in 90 mL of DCM and washed with pure water (4 x 150 mL) and dried over magnesium sulphate then solvent was removed by rotary evaporation yielding a crystalline yellow powder (65 %).

Synthesis of the Cyclic Peptide CP(-NH 2 ) 2 . Standard Fmocdeprotection solid-phase peptide synthesis was used to first synthesize the protected linear peptide. Using a coupling agent, the cyclization was completed in dilute conditions to avoid intermolecular reactions. The insolubility of the stacked cyclic peptides in methanol was used to isolate the pure cyclic peptide. The cyclic peptide was then deprotected using TFA to reveal the amines of the lysine residues and azoles on the tryptophan residues. [START_REF] Larnaudie | Cyclic Peptide-Polymer Conjugates: Grafting-to vs Grafting-From[END_REF] Synthesis of the Linear Peptide H2N-L- Scheme 1, 3). Fully protected linear octapeptide was prepared via solid phase peptide synthesis (SPPS) on a Prelude Automated Peptide Synthesizer TM (Protein Technologies Inc.) using 2-chlorotrityl chloride resin as the solid support. The first Fmoc-protected amino acid was coupled to the resin using DIPEA (4 eq.) in DMF, followed by capping of unreacted resin sites using a solution of MeOH:DIPEA:DCM (7:1:2, v/v/v). Deprotection of the Fmoc group of the amino acids was done using 20% piperidine in DMF. Subsequent amino acids were coupled using Fmoc-amino acids (5 eq.), HCTU (5 eq.) and NMM (10 eq.) in DMF. In the last step, the linear octapeptide was cleaved from the resin (while keeping protecting groups on) by a solution of 20 vol % 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) in DCM. Yield quantitative.

Lys(Boc)-D-Leu-L- Trp(Boc)-D-Leu-L-Lys(Boc)-D-Leu-L-Trp(Boc)-D-Leu- COOH,

Protected cyclic peptide Cyclo(-L-Lys(Boc)-D-Leu-L-Trp(Boc)-D-Leu-L-Lys(Boc)-D-Leu-L-Trp(Boc)-D-Leu-),

Scheme 1, 4). Linear peptide (200 mg, 0.127 mmol) was dissolved in DMF (20 mL) and N 2 was bubbled through the solution for 20 min. DMTMM•BF 4 (1.2 eq., 51 mg, 0.152 mmol) was dissolved in DMF (5 mL), N 2 bubbled through the solution for 20 min, then this solution was added dropwise to the linear peptide solution. The mixture was stirred under an atmosphere of N 2 for 5 days. The DMF solution was reduced to a volume of ~ 1 mL under reduced pressure, and methanol (20 mL) was added. Aliquots of the suspension were distributed into 2 mL Eppendorf tubes and centrifuged at 10000 rpm for 4 minutes using a benchtop centrifuge. After removal of the supernatant, the pellets were redispersed in methanol. The Eppendorf tubes were centrifuged once more and the supernatant discarded. The pellets were redispersed in methanol and the solvent was evaporated under reduced pressure to yield the Boc-protected cyclic peptide in the form of a white powder. Yield 73 % (138 mg, 0.093 mmol). MS (ESI) [M+Na] + calculated: 1503.89, found: 1503.8.

Deprotected Cyclic Peptide Cyclo(-L-Lys-D-Leu-L-Trp-D-Leu-L-Lys-D-Leu-L-Trp-D-Leu-), (Scheme 1, 5). Boc groups were removed using a deprotection solution of TFA/TIPS/H 2 O (18:1:1 vol, 5 mL). The protected cyclic peptide was stirred for 2 hours in the deprotection solution, then precipitated using chilled diethyl ether and washed twice more with chilled diethyl ether. The off-white powder was collected and dried under vacuum. Yield: quantitative Synthesis of the Cyclic Peptide Chain Transfer Agent, (Scheme 1, 6). The desired cyclic peptide chain transfer agent CP-(XA) 2 was obtained by coupling the chain transfer agent (NHS-CTA-XA) to the lysine residues of CP (Scheme 1). The CP (120 mg; 0.11 mmol; 1 eq.) was dissolved in 6 mL DMSO. Complete dissolution was reached after 10 min in ultrasound bath. Then NHS-CTA-XA (64.68 mg; 0.222 mmol, 2 eq.) and NMM (0.074 mL; 0.666 mmol, 3eq.) were added and the solution was stirred at room temperature for 3 days. Mass spectrometry monitoring indicated that the coupling reaction was quantitative, as no residual CP or mono-functionalized product was detected, affording CP-(XA) 2 in high yield (89 %). The product was precipitated twice in chilled diethyl ether and dried under vacuum. MS (ESI) (Figure S4) [M+Na] + calculated: 1433.91, found: 1433.95.

VDF RAFT/MADIX polymerization using NHS-CTA-XA in acetone. RAFT polymerization was carried out in a thick Carius tube containing NHS-CTA-XA (38 mg, 13.0 10 -2 mmol), acetone (7 ml) and the initiator (Trigonox-121) (3.3 mg, 13.0 10 -1 mmol) were mixed and the tube was degassed with three freeze-pump-thaw cycles to remove any trace of oxygen. The gaseous VDF monomer (0.5 g, 7.81 mmol) was transferred into the Carius tube and cooled in liquid nitrogen. The tube was then sealed, before being placed horizontally in a shaking water bath thermostated at 73 °C. After 24 hours, the tube was frozen in liquid nitrogen, opened and left to return to ambient temperature. Once room temperature was reached, the crude sample was precipitated twice in a tenfold excess of chilled pentane. The NHS-PVDF polymer was recovered by centrifugation at 4000 rpm for 15 min in 10 mL conical centrifuge tubes. The polymer was dried overnight under vacuum at 25 °C (Yield = 60 %). Yield was used as conversion since conversion is very difficult to calculate accurately for gaseous monomers.

VDF RAFT/MADIX grafting-from polymerization using CP-(XA) 2 , (Scheme 1, 7). RAFT polymerization was carried following the same protocol described above. CP-(XA) 2 (93 mg, 6.51 10 -2 mmol), acetone (7 ml) and the initiator (Trigonox-121) (3 mg, 13.0 10 -1 mmol) were sonicated for 10 min or until complete CP-(XA) 2 dispersion before degassing and introducing the gaseous VDF monomer (0.5 g, 7.81 mmol). Conversion was not estimated since the study of the aggregates in solution required no further purification. However, by weighing the Carius tube before and after polymerization (after breaking the glass tube to allow unreacted VDF to evaporate) a yield of 50% was estimated.

Preparation of CP-(PVDF) 2 solutions. Stock solutions of 1 mg mL -1 of CP-(PVDF) 2 were prepared in DMF or DMSO, using ultrasounds and heating at 60°C until full solubilisation.

Preparation of the self-assembled nanotubes. 0.1 mL of the solutions described above were placed in stirring plates with magnetic stirring bars. Then, 0.9 or 9.9 mL of THF were added dropwise using a syringe pump at a fixed rate of (4 mL h -1 ) (Final conjugate concentration and solvent ratios of 0.1 or 0.01 mg mL -1 and 1:9 or 1:99 respectively). The solutions were let stirred slowly for 24h. 10 µL and 50 µL were taken to prepare TEM and AFM samples respectively.

RESULTS AND DISCUSSION

Synthesis of the Cyclic Peptide Chain Transfer Agent.

The initial cyclic peptide (CP(-NH 2 ) 2 ) (sequence of amino acids:

L-Lys-D-Leu-L-Trp-D-Leu-L-Lys-D-Leu-L-Trp-D-Leu) (see Scheme 1) was synthesized according to previously reported procedures. [START_REF] Larnaudie | Cyclic Peptide-Polymer Conjugates: Grafting-to vs Grafting-From[END_REF] The desired cyclic peptide chain transfer agent CP-(XA) 2 was obtained by coupling the chain transfer agent (NHS-CTA-XA) to the lysine residues of the CP. Mass spectrometry indicated that the reaction proceeded quantitatively in 72 hours. Although aminolysis of the xanthate moiety by the lysine residues is a potential side reaction, no evidence of such corresponding O-thiocarbamate formation was found.

Study of the Suitable Polymerization Conditions. Recent studies show that xanthate RAFT agents, peroxide initiators and dimethyl carbonate (DMC) as solvent are the best conditions for the RAFT/MADIX polymerization of VDF. [START_REF] Guerre | Deeper Insight into the MADIX Polymerization of Vinylidene Fluoride[END_REF] VDF polymerization in DMC proceeds faster than in other organic solvents, affording high yields, and also leads to relatively small quantities of -CH 2 -CF 2 -H terminated dead chains (mainly formed by radical transfer from -CF 2 • radicals to DMC). [START_REF] Guerre | Deeper Insight into the MADIX Polymerization of Vinylidene Fluoride[END_REF][START_REF] Asandei | Mild-Temperature Mn 2(CO) 10-Photomediated Controlled Radical Polymerization of Vinylidene Fluoride and Synthesis of Well-Defined Poly(Vinylidene Fluoride) Block Copolymers[END_REF] Unfortunately, the prepared CP(-XA) 2 is not soluble in DMC therefore preventing its use as solvent. Whilst DMF and DMSO are both suitable solvents for the CP and PVDF and the solvent of choice for most polymerizations initiated from CP macro CTA, [START_REF] Larnaudie | Cyclic Peptide-Polymer Conjugates: Grafting-to vs Grafting-From[END_REF][START_REF] Larnaudie | Amphiphilic Core-Shell Supramolecular Polymer Brushes from Cyclic Peptide-Polymer Conjugates[END_REF] both solvents act as chain transfer agents in the polymerization of VDF, thus leading to poor conversion. [START_REF] Asandei | Mild-Temperature Mn 2(CO) 10-Photomediated Controlled Radical Polymerization of Vinylidene Fluoride and Synthesis of Well-Defined Poly(Vinylidene Fluoride) Block Copolymers[END_REF] We therefore turned our attention to an alternative solvent as compromise, acetone, which is a suitable solvent for the polymerization of VDF, although more prone to H-abstraction than DMC. [START_REF] Guerre | Deeper Insight into the MADIX Polymerization of Vinylidene Fluoride[END_REF] While the unmodified CP was insoluble in acetone, the modified CP macroCTA (CP(-XA) 2 ) formed a stable dispersion in acetone. This milky appearance of the solution was attributed to the strong tendency of the CP to selfassemble into nanotubes (See S20). A test polymerization of VDF using NHS-CTA-XA as RAFT agent was performed in acetone to assess the control of the polymerization. [START_REF] Shu | Peptide-Polymer Conjugates: From Fundamental Science to Application[END_REF] H NMR spectroscopy confirmed that PVDF was formed albeit at the cost of loss of end-group functionality, due to the expected increase in transfer events to the solvent (i.e., higher amount of dead chains : -CH 2 -CF 2 -H and -CF 2 -CH 3 , signals at 6.3 and 1.7 ppm (Figure S5), -92.00, -114.5 and -107.7 ppm (Figure S6) respectively). The molar fractions of the different end-groups were determined using equations S13 to S16 and data from [START_REF] Gauthier | Peptide/Protein-Polymer Conjugates: Synthetic Strategies and Design Concepts[END_REF] F NMR spectrum (Figure S8), estimated to be: -CF 2 -XA (0 %), -CH 2 -XA (40.4 %), -CF 2 -CH 3 (6.2 %) and -CF 2 H (49.4 %). It is noteworthy that in comparison, a typical VDF polymerization in DMC leads to up to 15 % of -CF 2 H and 85 % of -CH 2 -XA for a polymer of DP=50, [START_REF] Asandei | Mild-Temperature Mn 2(CO) 10-Photomediated Controlled Radical Polymerization of Vinylidene Fluoride and Synthesis of Well-Defined Poly(Vinylidene Fluoride) Block Copolymers[END_REF] although irreversible transfer reactions tend to prevail at high conversions. Thus, higher DP with high functionality can only be obtained at low conversions due to a progressive loss of xanthate groups. [START_REF] Guerre | Limits of Vinylidene Fluoride RAFT Polymerization[END_REF] It is therefore clear that the RAFT polymerization of VDF does do not follow the typical molecular weight control and kinetics expected from a RAFT system. The conditions used here are however, a compromise based on the solubility of the CTA, and provide a PVDF that retains its -chain end group, attached to the cyclic peptide. These results suggests that the grafting-from RAFT polymerization of VDF using acetone as solvent is a good compromise if low DPs are targeted and the xanthate -end group functionality can be sacrificed.

Synthesis of the CP-PVDF conjugate via "Grafting-from"

RAFT Polymerization of VDF. The RAFT polymerization of VDF was carried out in the milky suspension of CP(-XA) 2 in acetone. This system is presumably more akin to a graftingfrom system due to the suboptimal solubility of the macroCTA Scheme 1. Synthetic route to the cyclic peptide-PVDF conjugate employing a "grafting-from" approach. in acetone. Visually, no changes were observed in terms of solubility or colloidal stability at the end of the polymerization. The GPC trace (Figure 1) of CP(PVDF) 2 suggests that all the PVDF chains have grown from the CP(CTA) 2 since no shoulders are observed at lower molar masses. The dispersity of this CP(PVDF) 2 (1.26) was comparable to the typical dispersity of PVDF obtained by RAFT polymerization. [START_REF] Guerre | Deeper Insight into the MADIX Polymerization of Vinylidene Fluoride[END_REF][START_REF] Guerre | Limits of Vinylidene Fluoride RAFT Polymerization[END_REF] The PVDF segments grown from the CP appear to have an average DP of 45 from NMR calculations (using equation S10), higher than the estimated theoretical DP of 30 (calculated from equation S9, see supporting information). The results suggest that due to the partial solubility of the CP(XA) 2 macroCTA in acetone, all the xanthate sites were not available for VDF polymerization leading to longer PVDF chains.

Interestingly, this polymerization also led to PVDF chains possessing large amounts of regular end-groups (-CH 2 CF 2 -XA). The molar fractions of the end-groups determined from the [START_REF] Gauthier | Peptide/Protein-Polymer Conjugates: Synthetic Strategies and Design Concepts[END_REF] F NMR spectrum (Figure S8) were found to be -CF 2 -XA (36.0 %), -CH 2 -XA (9.7 %), -CF 2 -CH 3 (8.2 %) and -CF 2 H (46.1 %). These values are surprising as previous studies, including the RAFT polymerization conducted here using NHS-CTA-XA, had shown that the RAFT polymerization of VDF quickly leads to the accumulation of the reversely terminated end-groups (-CF 2 -CH 2 -XA) due to their lower reactivity towards the majority radicals -CF 2 • . [START_REF] Guerre | Deeper Insight into the MADIX Polymerization of Vinylidene Fluoride[END_REF][START_REF] Asandei | Mild-Temperature Mn 2(CO) 10-Photomediated Controlled Radical Polymerization of Vinylidene Fluoride and Synthesis of Well-Defined Poly(Vinylidene Fluoride) Block Copolymers[END_REF][START_REF] Boyer | Iodine Transfer Polymerization (ITP) of Vinylidene Fluoride (VDF). Influence of the Defect of VDF Chaining on the Control of ITP[END_REF] In any case, -CF 2 -XA chains were not present at an advanced polymerization stage. [START_REF] Guerre | Deeper Insight into the MADIX Polymerization of Vinylidene Fluoride[END_REF][START_REF] Guerre | Limits of Vinylidene Fluoride RAFT Polymerization[END_REF] Self-Assembly. The alternating D-and L-amino acid conformation of the peptide leads to the stacking of the CP via H-bonds perpendicular to the plane of the CP ring between the amide groups to form cylindrical structures. [START_REF] Granja | Self-Assembling Organic Nanotubes Based on a Cyclic Peptide Architecture[END_REF][START_REF] Ghadiri | Self-Assembled Nanoscale Tubular Ensembles[END_REF][START_REF] Scanlon | Self-Assembling Peptide Nanotubes[END_REF] The L-lysines positioned on the opposite sides of the peptide ring provide useful primary amines to anchor functional groups such as the xanthates chain transfer agent used here. Initially, the crude product resulting from the polymerization of VDF was examined by electron microscopy to observe the structures present in the acetone suspension (Figure 2a). TEM images (Figure 2b) show that the CP-(PVDF) 2 spontaneously self-assembled in acetone to form aggregates of various sizes that resemble short rods and twisted ribbons (Figure 2b, c andS18). The width of these objects was not homogeneous (30 to 100 nm) and exceeded, in most cases, the diameter of the CP-(PVDF) 2 . These aggregates were stable even at 50 °C and after sonication (Figure S17).

These PVDF-based tubular aggregates can be directly deposited on a substrate by spin-coating creating a porous network (Figure 2c). Such material might be interesting for the preparation of thin-film membranes.

To study the self-assembly properties of the CP-(PVDF) 2 conjugates, we attempted to first disassemble these assemblies, and then to re-assemble them in a controlled manner. The disassembly was investigated by dynamic light scattering (DLS) in presence of solvents that are known to disrupt Hbonds between the CP, trifluoroacetic acid (TFA), dimethylsulfoxide (DMSO) and N,N-dimethylformamide (DMF), which are also good solvents for PVDF. [START_REF] Kim | Oriented Self-Assembly of Cyclic Peptide Nanotubes in Lipid Membranes[END_REF][START_REF] Chapman | Modular Design for the Controlled Production of Polymeric Nanotubes from Polymer/Peptide Conjugates[END_REF][START_REF] Poon | Pushing the Limits of Copper Mediated Azide-Alkyne Cycloaddition (CuAAC) to Conjugate Polymeric Chains to Cyclic Peptides[END_REF][START_REF] Scanlon | Self-Assembling Peptide Nanotubes[END_REF] However, solutions of CP-(PVDF) 2 in TFA turned black within 1h, probably due to the degradation of the CP moieties. Light scattering measurements of the DMF and DMSO solutions of the CP-(PVDF) 2 conjugates at room temperature suggested the presence of large aggregates, in contrast to what is typically observed for other types of polymer-CP conjugates. The crystallinity of PVDF likely prevented complete disassembly of the conjugates, despite the disruption of H-bonds between CPs. To disrupt this crystallinity-driven assembly, the solutions were heated to 60°C (in DMF) and 80 °C (in DMSO) for 1h and sonicated for 20 min. Under these conditions, DLS analyses indicated complete dissolution of the aggregates (Figure S19). The controlled re-assembly of the CP-(PVDF) 2 conjugates was then carried out by slow addition, to these solutions, of THF (a good solvent of low molar mass PVDF). This procedure help to limit the disruption of H-bonds by solvent and promote assembly between CPs. [START_REF] Cate | Self-Assembling Peptide-Polymer Conjugates Comprising (D-Alt-L)-Cyclopeptides as Aggregator Domains[END_REF] In the THF / DMF solution, the CP(-PVDF) 2 self-assembled into cylindrical aggregates as observed by SEM (Figure 3), which revealed structures with lengths up to 250 nm. TEM analyses were also carried out (Figure 4), to show a more detailed structure of the aggregates. Initial analyses showed a high concentration of cylindrical structures (Figure 4a and4b), which were further imaged after diluting the solution ten times with pure THF and depositing the solution on a Lacey/Carbon TEM grid (Figure 4c and4d). This approach resulted in better contrast and higher resolution, and revealed that the cylindrical structures were in fact nanotubes (with length and diameter ranging from 200 to 400 nm (see Figure S20) and from 30-40 nm respectively), which inner channel can be seen on Figure 5d. Further study of the self-assembled objects was performed using AFM. (Figure 5), after spin-coating a solution of conjugates onto a mica substrate. Figure 5a-c shows an extended area of fiber like aggregates, which length and diameter, extracted from the topographic image (Figure . 5a) compare well with the data obtained from TEM images. The obtained average results (Figure 5f) were used to determine the aggregation number (N agg , number of stacked CPs). Considering the distance between each CP, reported to be 0.47 nm [START_REF] Song | Supramolecular Switching of the Self-Assembly of Cyclic Peptide-Polymer Conjugates: Via Host-Guest Chemistry[END_REF] and the data extracted from the TEM and AFM (length = 200-300 nm), N agg was estimated between 425 and 638. The width of these aggregates is dictated by the CP diameter and the DP of PVDF. PVDF likely crystallized in its α-phase. In such a crystal PVDF segments of 45 units are estimated to have an approximate length of 9 nm. [START_REF] Folgado | Preparation of Well-Defined 2D-Lenticular Aggregates by Self-Assembly of PNIPAM-b-PVDF Amphiphilic Diblock Copolymers in Solution[END_REF] The CP diameter is about 1nm (with an internal diameter of 0.7-0.8 nm). These estimations lead to a CP(-PVDF) 2 conjugate of ca. 19 nm, which is smaller than the width observed by TEM images (40-45 nm). This discrepancy may be explained by analytical errors on the resolution from the TEM measurements in this size range and on the DP of the PVDF that may be underestimated if not all the xanthate moieties generated a PVDF chain. The assemblies formed from the CP(-PVDF) 2 solution in DMSO (Figure 6) revealed to be much longer cylindrical structures, of up to 25 µm in length (Figure 6a) which appeared to be composed of laterally aggregated peptide-polymer nanotube (Figure 6b). Bundles of laterally stacked peptides in deuterated DMSO have been reported before. [START_REF] Koh | Study of (Cyclic Peptide)-Polymer Conjugate Assemblies by Small-Angle Neutron Scattering[END_REF] Koh et al. observed bundles of short tubes from unconjugated CPs stacked laterally in deuterated DMSO. [START_REF] Koh | Study of (Cyclic Peptide)-Polymer Conjugate Assemblies by Small-Angle Neutron Scattering[END_REF] This type of assemblies was also observed in the case of partially conjugated CP-polymer with polybutylacrylate (PBA), due to the poor compatibility between PBA and DMSO. [START_REF] Koh | Study of (Cyclic Peptide)-Polymer Conjugate Assemblies by Small-Angle Neutron Scattering[END_REF] We hypothesize that since some of the polymerization sites were not accessed during the VDF polymerization in the acetone milky suspension of CP(XA) 2 , the resulting partially conjugated CPs may laterally stacked into the observed tubes, similar to what was observed in the case of CP-PBA.

The lengths observed by TEM (25 µm) suggest a N agg of up to 53,200, which is greater than any values reported for similar cyclic peptide systems. Such long assemblies have not been reported for other CP-polymer conjugates, suggesting that PVDF polymer chains have an important effect on the assembly of these conjugates.

Conclusions

The synthesis of a cyclic peptide macroCTA bearing two xanthate moieties (CP(-XA) 2 ) was successfully achieved by the coupling of a NHS-functionalized RAFT agent onto the lysine residues of the CP. The RAFT/MADIX polymerisation of VDF in the presence of this CP(-XA) 2 difunctional macroCTAs was carried out in acetone, although CP(-XA) 2 formed a stable suspension in this solvent rather than a solution. The success of the polymerisation was however confirmed by [START_REF] Gauthier | Peptide/Protein-Polymer Conjugates: Synthetic Strategies and Design Concepts[END_REF] F NMR. The signal of the first VDF unit directly connected to the R-group of the macroCTA confirmed the growth of PVDF from the CP macroCTA. Also, the presence of VDF-XA signals confirms that the polymerisation was controlled by a RAFT mechanism. Nevertheless, these polymerization conditions led to a significant loss of xanthate termini due to a high extent of transfer (three times higher than in DMC). After its disassembly in DMSO or DMF, the formation of tubular structures of different lengths was triggered using THF as the PVDFselective solvent. These PVDF-based tubular aggregates might also find applications in separation and membrane science due to the porosity brought by the CP nanotubes.

ASSOCIATED CONTENT Supporting Information

The Supporting Information is available free of charge on the ACS Publications website 1H, 13C and, 19F NMR spectra, equations for DP calculations and chain end proportion determination, mass spectrometry, GPC traces, supplementary SEM images (PDF)

TOC for

Fluorinated Nanotubes: Synthesis and self-assembly of cyclic peptidepoly(vinylidene fluoride) conjugates

Enrique Folgado, a,b, ‡ Qiao Song, c, ‡ Sebastien Perrier, c,d,e* Vincent Ladmiral, a Mona Semsarilar b* a Institut Charles Gerhardt Montpellier, ICGM, Univ Montpellier, CNRS, ENSCM, Montpellier, France.

Figure 1 .

 1 Figure 1. Normalized SEC chromatograms (RI detector) of a concentrated Cyclic peptide (CP; red trace), a diluted Cyclic peptide solution in DMF (CP; red discontinuous trace) and a solution of CP-(PVDF) 2 (CP-PVDF; blue trace).

Figure 2 .

 2 Figure 2. a) Photograph of the Carius tube containing the acetone suspension after polymerization of VDF in the presence of CP-(XA) 2 , b) TEM image of the diluted suspension shown in (a), c) SEM image of thin films prepared from the crude CP-(PVDF) 2 conjugate (without further purification or dilution) by spin coating of 50 µL of suspension on a silicon wafer.

Figure 3 .

 3 Figure 3. SEM image of CP(-PVDF) 2 prepared from a 1 mg mL -1 DMF solution by adding THF (addition rate = 4 mL h -1 ). Final concentration 0.1 mg mL -1 (DMF 10% v/v in the final solution).

Figure 4 .

 4 Figure 4. TEM image of CP(-PVDF) 2 prepared from a 1 mg mL -1 DMF solution by adding THF (addition rate was 4 mL h -1 ). Final concentration (a, b) 0.1 mg mL -1 and (c, d) 0.01 mg mL -1 (DMF 10 and 1% v/v respectively in the final solutions). Sample prepared in Formvar/Carbon coated copper TEM grid (a and b). Sample prepared in Lacey/Carbon coated copper TEM grid (c and d) scales bars are 200 nm (a, b, and c) and 50 nm (d).

Figure 5 .

 5 Figure 5. AFM images of the nanotubes formed by CP(-PVDF) 2 deposited onto mica substrate. The sample was prepared from a 1:9 DMF:THF solution (conjugate concentration 0.1 mg mL-1). (a) Topography, (b) Amplitude, (c) Phase, (d) Height profile and width measurement, (e) width from TEM image, (f) values extracted from the analysis TEM and AFM images.

Figure 6 .

 6 Figure 6. SEM image of CP(-PVDF) 2 prepared from a 1 mg mL -1 DMF solution by adding THF (addition rate was 4 mL h -1 ). Final concentration 0.1 mg mL -1 (DMF 10% v/v in the final solution). Scale bars are 10 µm (a) and 200 nm (b).

b

  Institut Européen des Membranes, IEM, Univ Montpellier, CNRS, ENSCM, Montpellier, France. c Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK. d Faculty of Pharmacy, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia e Warwick Medical School, The University of Warwick, Coventry CV4 7AL, UK. Fluorinated hollow nanotubes were prepared from pre-assembled cyclic peptides bearing xanthate moieties by RAFT/MADIX of VDF 1 H NMR (400 MHz, CDCl 3 , ppm, Figure S2) δ = 4.70 (dq, J = 7.1, 2.3 Hz, 1H), 4.64 (q, J = 7.4 Hz, 2H), 2.86 (s, 4H), 1.73 (d, J = 7.4 Hz, 3H), 1.44 (t, J = 7.1 Hz, 3H).

Figure S2. 1 H

 1 Figure S2. 1 H NMR spectrum (CDCl 3 , 400 MHz) of NHS-CTA-XA.

13 C

 13 {1H} DEPT-135 NMR (100 MHz, CDCl 3 , Figure S3) δ = 13.52 (-O-CH 2 -CH 3 ), 16.50 (-CH(CH 3 ), 25.60 (-CH 2 -CH 2 -), 44.19 (-CH(CH 3 ), 70.91 (-O-CH 2 -CH 3 ).

Figure S3. 13 C

 13 Figure S3. 13 C DEPT 135 NMR spectrum (CDCl 3 , 101 MHz) of NHS-CTA-XA.

Figure S4 .

 S4 Figure S4. Mass spectrometry of the purified CP-(XA) 2 .

Figure S5. 1 H

 1 Figure S5. 1 H NMR spectrum in (CD 3 ) 2 CO of a PVDF homopolymer synthesized by RAFT polymerization with NHS-CTA-XA (400 MHz)

Figure S6. 19 F

 19 Figure S6. 19 F NMR spectrum in (CD 3 ) 2 CO of a PVDF homopolymer synthesized by RAFT polymerization with NHS-CTA-XA. (376 MHz)

Figure S7. 1 H

 1 Figure S7. 1 H NMR spectrum in C 3 D 7 NO of a CP-(PVDF) 2 conjugate synthesized by surface initiated RAFT polymerization with CP(-XA) 2 .

Figure S8. 19 F

 19 Figure S8. 19 F { 1 H} NMR spectrum in C 3 D 7 NO of a CP-(PVDF) 2 conjugate synthesized by surface initiated RAFT polymerization with CP(-XA) 2 .

Figure S12 .

 S12 Figure S12. Synthetic route to the cyclic peptide-PVDF conjugate employing a »grafting-to » approach.

Figure

  Figure S18. a) SEM image of twisted ribbon structures observed in the diluted suspension shown in Figure 2a. b) size distribution of the assemblies observed in Figure 2b.

Figure S19 .

 S19 Figure S19. DLS intensity-average hydrodynamic diameter distribution of CP(PVDF) 2 conjugates (1 mg mL -1 ) in (a) DMF after heating at 60 °C (1h) and sonication (20 min) and (b) DMSO after heating at 80 °C (1h) and sonication (20 min).

Table S9 . M n , M w , M p and Ð .

 S9

	Theoretical DP:
					DP theo =	1 2 [CP(XA) 2 ] 0 [VDF] 0 x Conversion =	1 7.81 mmol 2 6.51 10 -2 mmol	x 0.5 = 30
	(Equation S10)
	DP of each arms of CP-(PVDF) 2 estimated from 19 F NMR spectrum (Figure S8) using equation S11:
	DP
	=	(∫	-71.0 -70.8 + ∫	-74.9 -74.7 + ∫	-112.9 -112.7 𝐶𝐹 2 𝑆(Z end group)) + ∫ (∫ -71.0 -70.8 + ∫ -107.2 -106.8 𝐶𝐹 2 𝐶𝐻 3 + ∫ -74.9 -74.7 + ∫ -112.9 -112.7 𝐶𝐹 2 𝑆(Z end group)) + ∫ -114.1 -113.6 𝐶𝐹 2 𝐻 + (∫ -94.0 -90.9 + ∫ -107.2 -113.2 -112.8 + ∫ -106.8 𝐶𝐹 2 𝐶𝐻 3 + ∫ -115.8 𝐶𝐹 2 ((HT), (HH), (R end group) and (H termination))) -115.2 -114.1 -113.6 𝐶𝐹 2 𝐻
	(Equation S11)
						M n (g/mol) M w (g/mol) M p (g/mol) Ð
					CP	3600	5400	3500	1.49
					CP-dilute	3700	7000	3300	1.86
					CP-PVDF	4500	5700	4300	1.26

Polymer Chemistry

ACKNOWLEDGMENT

The authors thank the Royal Society for funding (Grant IE161841) and Arkema for providing VDF.

Funding Sources

The authors thank Institut Carnot Chimie Balard Cirimat, LabEx CheMISyst (ANR-10-LABX-05-01), IEM, ICGM (E.F.; V.L.; M.S.), CNRS-Osez l'Interdisciplinarité 2017 (MS), Monash-Warwick Alliance (Q.S.; S.P.), European Research Council (TUSUPO 647106; Q.S.; S.P.). S. P and M. S. also thank the Royal Society for financial support (Royal Society International Exchanges grant IE161841).

AUTHOR INFORMATION

Corresponding Author * Sebastien Perrier (S.Perrier@warwick.ac.uk) * Mona Semsarilar (mona.semsarilar@umontpellier.fr) Present Addresses †If an author's address is different than the one given in the affiliation line, this information may be included here.

Author Contributions

The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript. -

Supporting information

Fluorinated Nanotubes: Synthesis and self-assembly of cyclic peptide-poly(vinylidene fluoride) conjugates Fluorinated hollow nanotubes were prepared from pre-assembled cyclic peptides bearing xanthate moieties by RAFT/MADIX of VDF