Gewu Bu
email: gewu.bu@lip6.fr

Thanh Son

Lam Nguyen

Maria Potop-Butucaru
email: maria.potop-butucaru@lip6.fr

Kim Loan Thai
email: kim.thai@lip6.fr

HyperPubSub: Blockchain based Publish/Subscribe

Keywords: Subscribe, Online Trading, Blockchain, Hyperledger Fabric

In this paper we describe the architecture and the implementation of a broker based publish/subscribe system where the broker role is played by a private blockchain, Hyperledger Fabric. We show the effectiveness of our architecture by implementing and deploying a photo trading plateform. Interestingly, our architecture is generic enough to be adapted to any digital asset trading.

I. INTRODUCTION

Publish/subscribe (e.g. [START_REF] Birman | Exploiting virtual synchrony in distributed systems[END_REF]) is a communication primitive designed for large scale dynamic networks due to the loosely coupled interaction between the publishers and subscribers. In this framework, publishers produce events and subscribers express their interests through subscriptions. Any event matching the subscription is delivered to the corresponding subscriber. The matching procedure is executed by brokers, which are also responsible for the event delivery. In this way, publishers and subscribers are completely desynchronized in time and space. Publish/subscribe systems design follow two main research lines: topic-based and content-based systems. In topic-based systems events published on a specific topic are forwarded to all clients subscribed to this topic. In content-based systems subscribers specify their interests using filters. Many publish/subscribe systems are based on a fixed infrastructure of reliable brokers. The broker in a publish/subscribe system solves the following problem: instead of communicating to each other directly, both publishers and subscribers will connect to the broker. The broker receives informations sent by publishers then it filters and transfers received information to corresponding subscribers. One of the filtering mode is topic based filtering. In this case, generated information may have different topic labels defined by publishers. Subscribers will chose topics they like to follow. The broker filters received information by topic and delivers it to suitable subscribers.

Even though publish/subscribe systems have been designed as an alternative communication primitive for large scale churn prone systems their design philosophy can go beyond the sending/receiving messages. That is, in recent years, more and more digital assets are tradable products. Publishers and subscribers of these valuable digital assets can be therefore considered as producers and respectively potential customers on a e-market place that will trade using a broker based publish/subscribe architecture.

It should be noted that classic publish/subscribe system do not aim at trading. Therefore, they cannot be easily adapted to varying industrial requirement including security and privacy of both clients and providers. That is, broker based publish/subscribe systems make easy the coupling between publishers and subscribers during the information delivery however, they still suffer from many drawbacks, some of them listed below:

• Delivery failure. During the delivery, if some subscribers are not available for receiving data, they will lost the information. The broker has to decide whether and when it has to re-transfer the information. That requires brokers to keep connected with subscribers and learn their availability. • Delivery slowdown. When a hot topic having a lot of subscribers is published, the broker has to transfer this information to all its subscribers. That will increase the traffic load on the network and the delivery delay for individual subscribers will increase due to the network capacity limitation. • Security risks. Lacking of a complete secure design, the broker becomes the critical single-failure point. Additional security protocols are needed to ensure the reliability of the system. In order to circumvent some of the above mentioned drawbacks we propose a blockchain based publish/subscribe architecture where the role of the broker is played by a private blockchain Hyperledger Fabric.

II. BASICS OF HYPERLEDGER FABRIC

Hyperledger Fabric [START_REF] Cachin | Architecture of the hyperledger blockchain fabric[END_REF], [START_REF] Androulaki | Hyperledger fabric: A distributed operating system for permissioned blockchains[END_REF] is an open-source private blockchain framework started at Linux Foundation, designed for business purpose and now managed by Digital Asset and IBM. As a private blockchain, Hyperledger Fabric shares common property with traditional blockchains such as Bitcoin [START_REF] Nakamoto | Bitcoin: A peer-to-peer electronic cash system[END_REF]: information stored on the chain is safe and traceable. Moreover, all interactions history are permanent and cannot be changed unless an adversary owns more computation power than all honest participants.

Hyperledger Fabric is composed of the following building blocks:

• Membership Service Provider. As a private chain, participants in the Hyperledger Fabric can have certain trust between each others. The Membership Service Provider (M SP ) module defines relations between participants and gives them different access rights. Only the nodes registered via M SP can connect to Hyperledger Fabric. MSP provides a basic security indentification for the system. • Channel. Instead of having one single blockchain, Hyperledger Fabric maintains simultaneously many blockchains, different types of information stored in different blockchains are independent. Participants can join into one or multi channels according to the business logic design. So that they can access to the blockchain corresponding to the channels they belong to. • Chaincode. For each channel, many Chaincodes (also called smart contracts) can be installed. Chaincodes are programs that can be invoked by all participants in the same channel to interact with the information stored into the channel. Note that interactions such as: creation, modification or delete the information will be recorded in blockchain of the channel. • Ordering Service. Ordering Service is a consensus module. It connects to all the channels. Ordering Service receives incoming invocations belonging to the same channel and order them into blocks. Then it diffuses these blocks to the participants of the channel, so that they can append the block into their local chain and update stored information according to the invoked chaincode. Execution phase. When invoking a chaincode of a channel, a user (called client) having access to this channel can send the proposal to the endorser peers (the network participants who have the chaincode installed) and waits for their reply. When receiving the proposal, endorsers execute individually the program according to the invoked chaincode. Endorser peers sign this endorsement with the result, send it back to the client as a proposal response. When the client receives enough responses, it creates an official invoke requirement (called transaction) that assembles these responses for the next phase.

Ordering phase. A client sends its transaction to the ordering service after finishing the execution phase. The ordering service orders all submitted transactions per channel and combines these transactions into blocks.

Validating phase. The ordering service broadcasts blocks to all the peers in a channel. Then each peer will verify each transaction in the block. Transactions failing the verification will be marked as invalid. After the checking, each peer appends the whole block (including invalid transactions) to its local blockchain. Note that invalid transaction can be distinguished, and will be considered as not existent, so that invalid invoke requirement will not affect the stored information.

Secure membership management, independent multichannel structure and flexible chaincodes make Hyperledger Fabric become a powerful business logic oriented framework.

III. HYPERPUBSUB DESIGN

In this section we propose a new broker based Publish/Subscribe system, HyperPubSub, designed on top of Hyperledger Fabric. HyperPubSub is specifically design to support trading of digital assets. In order to validate our architecture we designed a prototype of a online photo trading plateform.

In HyperP ubSub, publishers and subscribers are two types of clients in Hyperledger Fabric framework: one generates digital products, the other express their interest to consume them. The core of Hyperledger Fabric will be the broker in charge to connect publishers and subscribers matching to the same topic by joining them into the corresponding channel. By combining chaincode and multi-blockchain structure, various topics can be managed safety and independently. In this way, the Delivery failure and the Delivery slowdown problem in classic Publish/Subscribe system can be avoided: subscribers don't need to receive the information passively. That is, they can query the information by invoking the chaincode proactively and individually.

When subscribers decide to buy a digital product published by publishers, the online trading can be done safely in trading dedicated channels, isolated from the other channels. All the trading history and details are stored into a blockchain, so no one can cheat during the trading.

A. Use Case: an Online Photo Trading Platform

In the following we instantiate HyperP ubSub in order to respond to a practical use case1 : an online photo trading platform that will bring together professional photographers and press agencies. Photographers (publishers in our architecture) upload their photos and set a price for their products; customers (subscribers in HyperP ubSub) can review photos according to different categories (topics) and can choose to buy those they wish. This platform makes use of blockchain technology in order to guarantee that the metadata of a photo cannot be altered, in the same time it ensures the security of the online trading and the integrity of the photographers and customers identities.

In Fig. 2 we represent our architecture. The network topology is transparent to the clients. We have several gateways (F1-F2-F3-F4) that share the connections with the clients. Two database servers (B) are designed to store the photographs, one master and one slave for backup. The information of the clients, their photos, transactions, trades, etc. are managed by Hyperledger Fabric network (A). This blockchain network contains orderer nodes (C) which run Raft consensus [START_REF] Ongaro | In search of an understandable consensus algorithm (extended version)[END_REF]. In our prototype we consider only one organization with six endorser peers nodes (D) and four channels (from E1 to E4). Each channel contains and runs one chaincode. There are four independent blockchains: E1 channel stores the information of the clients; E2 channel contains the meta information of all photos; E3 channel is dedicated to all trades from customers to photographers; E4 channel stores extra information that facilitates the administration of this environment. Some basic behaviors of two kinds of clients are defined and programmed. Each of them can create a new account and login. A photographer can upload a photo from his device to data base (B), set this photo with three different prices for three different copyrights. This photo can be set in multiple categories (topics) (e.g. nature, sport, human, animal).

A customer can see all of the photos and additional information concerning the photographers so that the customer can choose one photo and buy it using the HyperPubsub coins (the crypto money in our system). A customer must posse an amount of coins superior or equal to the price value of the photo he wants to trade. Therefore, we created an application for coins administration. The idea is that, a customer will use fiat to transfer money to the owner of the photo. After receiving the money, the owner of the platform use this application to provide a customer with coins. Here below is the description of a process to buy a photo:

• A customer uses the application to login. This application will then connect to the gateway to transfer this cus-tomer's login information. Gateway then query to the our blockchain system to verify the user. In case the verification succeeds, the gateway queries the list of the photos from the blockchain system and sends the result to the customer. • After logging in, the customer can choose to show a specific photo category and then can chose the photo to buy. After validation, the application will connect and send a query to the gateway. The gateway then connect to channel E1, E3, E4 and sends corresponding chaincode queries. After this step, the walled of the customer will be decreased and atomically the wallet of the photographer who owns that photo will be increased. • Data base server will receive the request for downloading the original photo. It will find that photo in its data base then send it back to the gateway such that the customer can download it.

B. Implementation Environment for our prototype.

After having the architecture design above, we begin to implement in a real environment. We implemented a prototype where some parts of the above described architecture have been omitted. We have only one gateway for all photographers and customers and one database. We deployed our prototype in two cloud instances, one in Germany (Heidelberg University) and one is in France (Onelab LIP6) using 2 vCPU (2.6GHz, 4GB of RAM, runs Ubuntu 16.04.5 LTS in each instance. Our Hyperledger Fabric blockchain network is 1.4.1 version with RAFT consensus. The application for client as well as database (file server) are programmed in java, and we NodeJs for the gateway.

IV. CONCLUSION

In this paper we describe a broker based architecture for a publish/subscribe system where the broker role is played by Hyperledger Fabric. We instantiate our architecture for an online trading photo plateform and implemented a prototype. Our prototype is currently deployed on two private clouds running in France and Germany. A demonstration video can be found here: https://youtu.be/RFLmsSRmyMs.

Fig. 1 Fig. 1 .

 11 Fig. 1. Three phases architecture of Hyperledger Fabric.

Fig. 2 .

 2 Fig. 2. Architecture of our trading platform.

This usecase was proposed to us by Andromak start-up. http://www.andromak.com


Gaëlle Le Fer

Delphine Portes

Guillaume Goudounet

Jean-Michel Guigner

Elisabeth Garanger
email: garanger@enscbp.fr

Sébastien Lecommandoux
email: lecommandoux@enscbp.fr

Design and self-assembly of PBLG-b-ELP hybrid diblock copolymers based on synthetic and elastin-like polypeptides

Keywords: Elastin-like polypeptides, poly(γ-benzyl-L-glutamate), amphiphilic block copolymers, microfluidics, self-assembly

The precision synthesis and self-assembly of amphiphilic copolypeptides containing a recombinant elastin-like polypeptide (ELP) block used as macroinitiator for the ring opening polymerization (ROP) of γ-benzyl-L-glutamate (γ-BLG NCA) are herein presented. The molecular weight of the resulting PBLG-b-ELP block copolypeptides was precisely controlled without the use of complex initiators or demanding experimental setup. Diblock copolypeptides were obtained with an excellent control of the polymerization highlighted by the dispersity below 1.04. These amphiphilic hybrid synthetic/recombinant copolypeptides were self-assembled in water and nanoparticles obtained characterized by a combination of dynamic light scattering and electron microscopy. A variety of morphologies, namely polymersomes, inter-connected wormlike micelles and spherical micelles, were evidenced depending on the hydrophilic ratio of the diblocks as well as the self-assembly procedure.

Introduction

Over the past decades, several approaches have been developed for the elaboration of block copolymer systems based on proteins for various applications. For instance, therapeutic proteins can replace deficient or absent natural proteins, upregulate existing metabolic pathways, their conjugation with hydrophilic polymer being at the origin of important research area and biotechnology development. Upon them, poly(ethylene glycol) (PEG) is the most intensively used polymer for bioconjugation, allowing longer half-life in the bloodstream, and leading to less frequent dosing. 1 In addition, giant amphiphiles, copolymers resulting from the controlled conjugation of synthetic polymers to proteins 2 can be interestingly used for their self-assembly behavior. In this context, the perfect control of the chemical structure and bioconjugation mechanism is a prerequisite. In pioneered work in this area, Nolte et al. studied the self-assembly of precisely defined giant amphiphiles consisting of a single hydrophobic polystyrene tail covalently connected to a lipase [START_REF] Velonia | Lipase polystyrene giant amphiphiles[END_REF] and via cofactor reconstitution to horseradish peroxidase (HRP) or myoglobin. [START_REF] Boerakker | Giant amphiphiles by cofactor reconstitution[END_REF][START_REF] Boerakker | Aggregation behavior of giant amphiphiles prepared by cofactor reconstitution[END_REF] Velonia and co-workers showed the potential of hydrids BSA-polystyene to create bionanoreactors, [START_REF] Droumaguet | In Situ ATRP-Mediated Hierarchical Formation of Giant Amphiphile Bionanoreactors[END_REF] and the self-assembly behavior of tri-block giant amphiphilic BSAcopolymer synthesized by a combination of Atom Transfer Radical Polymerization (ATRP) and "click chemistry". [START_REF] Droumaguet | Formation of giant amphiphiles by post-functionalization of hydrophilic protein-polymer conjugates[END_REF] Thereafter, Bulmus et al. developed thermoresponsive BSA-poly(Nisopropylacrylamide) via the growth of the polymer chains from BSA-macroRAFT (Reversible Addition Fragmentation Chain Transfer) agent. [START_REF] Boyer | Well-defined protein-polymer conjugates via in situ RAFT polymerization[END_REF] More recently, vesicles were obtained from the self-assembly of BSA-poly(caprolactone) [START_REF] Liu | Self-Assembled Biodegradable Protein-Polymer Vesicle as a Tumor-Targeted Nanocarrier[END_REF] or HSA-poly(N-(2-hydroxypropyl) methacrylamide) [START_REF] Liu | In Situ Growth of Self-Assembled Protein-Polymer Nanovesicles for Enhanced Intracellular Protein Delivery[END_REF] and demonstrated great potential as drug-nanocarriers, taking advantage of the stealth character of BSA as the hydrophilic segment. In most recent example, Mann et al. [START_REF] Huang | Interfacial assembly of protein-polymer nano-conjugates into stimulus-responsive biomimetic protocells[END_REF] used such conjugates to built stimuli-responsive biomimetic protocells using BSA-PNIPAM based copolymers.

In the past decade, the library of proteins was significantly extended, and recombinant proteinengineering techniques allowed the development of precision recombinant polymers with exquisite control over their primary structure. Among them, elastin-like polypeptides (ELPs) represent a unique class of recombinant proteins. Their main structure results in repeating sequences of [-Val-Pro-Gly-Xaa-Gly-] pentapeptides, the guest residue Xaa being any amino acid except proline. ELPs share structural characteristics with intrinsically disordered proteins naturally found in the body, such as tropoelastin, at the origin of their name. [START_REF] Urry | Phase-structure transitions of the elastin polypentapeptide-water system within the framework of composition-temperature studies[END_REF][START_REF] Urry | Physical chemistry of biological free energy transduction as demonstrated by elastic protein-based polymers[END_REF] The repeat sequences found in the biopolymer, as well as the monomer sequence an chain length, give each ELP a distinct structure, and influence the lower critical solution temperature (LCST), also referred to commonly as the T t . Recent contributions highlighted the tunable thermoresponsive properties of ELPs in water by chemoselective oxidation [START_REF] Petitdemange | Selective tuning of elastin-like polypeptide properties via methionine oxidation[END_REF] or alkylation [START_REF] Kramer | Quantitative Side-Chain Modifications of Methionine-Containing Elastin-Like Polypeptides as a Versatile Tool to Tune Their Properties[END_REF][START_REF] Petitdemange | Tuning Thermoresponsive Properties of Cationic Elastin-like Polypeptides by Varying Counterions and Side-Chains[END_REF] of all methionine residues, with the aim of providing polycationic derivatives in the later case or introducing new side-chain functionalities.

In this context, ELPs may find an original place as protein block in hybrid protein-polymer bioconjugates. Indeed, the elaboration of synthetic polymers-ELPs conjugates were reported, such as low molecular weight ELP-based side chain poly(methacrylate)s via ATRP [START_REF] Ayres | Elastin-based side-chain polymers synthesized by ATRP[END_REF][START_REF] Ayres | Stimulus responsive behavior of elastin-based side chain polymers[END_REF] and ELPgrafted poly(norbornene)s via ring-opening metathesis polymerization (ROMP) [START_REF] Roberts | Multifunctional thermally transitioning oligopeptides prepared by ring-opening metathesis polymerization[END_REF][START_REF] Conrad | Temperature-Responsive Polynorbornenes with Side Chains Based on an Elastin Peptide Sequence[END_REF] in presence of catalyst based on ruthenium. Moreover, ELPs obtained by chemical pathway (Boc-based liquid phase synthesis) were conjugated with polyamidoamine (PAMAM) dendrimers to gain potential stimuli-responsive drug-carrier. [START_REF] Kojima | Synthesis of Temperature-Dependent Elastin-Like Peptide-Modified Dendrimer for Drug Delivery[END_REF] More recently, Kiick and co-worker [START_REF] Luo | Noncovalent Modulation of the Inverse Temperature Transition and Self-Assembly of Elastin-b-Collagen-like Peptide Bioconjugates[END_REF] reported the self-assembly behavior in vesicles of elastin-b-collagen-like peptide bioconjugates obtained by copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC) "click" reaction.

Although peptide-polymer and protein-polymer conjugates were studied, conjugates of hybrid protein-synthetic polypeptide are surprisingly less explored whereas the polymerization of Ncarboxyanhydrides (NCAs) allows obtaining well-defined macromolecular structures, even from a macro-initiator. Indeed, block copolymer are usually prepared by using the first block with an amino end-group as a macroinitiator for the ring opening polymerization (ROP) of NCAs, this block being often PEG, [START_REF] Pu | Synthesis and Drug Release of Star-Shaped Poly (benzyl L-aspartate)-block-poly (ethylene glycol) Copolymers with POSS Cores[END_REF][START_REF] Shen | Conformation-specific self-assembly of thermo-responsive poly (ethylene glycol)-b-polypeptide diblock copolymer[END_REF][START_REF] Lee | Synthesis of poly (ethylene glycol)/polypeptide/poly (D, L-lactide) copolymers and their nanoparticles[END_REF][START_REF] Gao | Synthesis and thermoreversible gelation of coil-helical polyethylene-block-poly (γ-benzyl-L-glutamate) diblock copolymer[END_REF] even if some other example referred to poly[N- (2-hydroxypropyl) methacrylamide] (PHPMA) of high molecular weight (M n = 32,100 g.mol -1 ). [START_REF] Holley | pH-responsive HPMA-b-(l-Glu) copolymers synthesized via sequential aqueous RAFT and ringopening polymerizations[END_REF] The NCAs ROP can proceed via 2 mechanisms: the normal amine mechanism (NAM, induced by nucleophilic substitution) proposed by Waley and Watson 28 and the activated monomer mechanism (AMM, induced by deprotonation of the NCAs). 29 AMM yields polypeptides with high molar mass and broad dispersity. As a result, the NAM mechanism must be favored to obtain well-defined polypeptides. Most commonly, nucleophiles are used to initiates the polymerization, with primary amines being the most intensively investigated species. In addition to the versatility of synthesis and the intrinsic biodegradability of peptide bonds, the supramolecular organization of polypeptides provides an opportunity to produce hierarchical structures and can also be used to promote specific "bioactivity". [START_REF] Rodríguez-Hernández | Reversible inside-out micellization of pH-responsive and water-soluble vesicles based on polypeptide diblock copolymers[END_REF][START_REF] Holowka | Charged polypeptide vesicles with controllable diameter[END_REF][START_REF] Huang | Biologically active polymersomes from amphiphilic glycopeptides[END_REF] For instance, block copolymers combining polypeptide and oligosaccharide blocks have previously been employed to prepare glycoprotein biomimetic polymersomes. [START_REF] Upadhyay | Biomimetic doxorubicin loaded polymersomes from hyaluronan-block-poly (γ-benzyl glutamate) copolymers[END_REF][START_REF] Schatz | Polysaccharideblock-polypeptide Copolymer Vesicles: Towards Synthetic Viral Capsids[END_REF][START_REF] Upadhyay | The intracellular drug delivery and anti tumor activity of doxorubicin loaded poly (γ-benzyl l-glutamate)-b-hyaluronan polymersomes[END_REF] The goal of the present contribution is to combine the various advantages of ELPs and synthetic polypeptides to obtain a "hybrid" amphiphilic diblock recombinant/synthetic polypeptide capable of self-assembly in various biocompatible nano-structures. In this context, this article highlights the use of hydrophilic ELP as a macroinitiator for the ROP of γ-BLG-NCA. The diblock polypeptides were obtained with a high control of the polymerization mechanism, highlighted by the low dispersity of the resulting copolymers, below 1.04. The resulting amphiphilic hybrid diblocks were subsequently self-assembled, using a microfluidic chip system and more "classical" methods such as dialysis or direct dissolution. The goal of our study is to establish structure-property relationships between the weight fraction of each block and the size and morphology of the resulting nano-objects depending on the self-assembly process used.

Results and discussion

Synthesis of PBLG-b-ELP block copolypeptides

The most rapid and cost-effective process to prepare synthetic polypeptide chains with high degrees of polymerization and excellent control over the dispersity is the ROP of heterocycle αamino acid N-carboxyanhydrides (NCAs). [START_REF] Hadjichristidis | Synthesis of welldefined polypeptide-based materials via the ring-opening polymerization of α-amino acid Ncarboxyanhydrides[END_REF][START_REF] Cheng | Synthesis of polypeptides by ring-opening polymerization of α-amino acid N-carboxyanhydrides[END_REF][START_REF] Huang | Stimuli responsive synthetic polypeptides derived from Ncarboxyanhydride (NCA) polymerisation[END_REF] Poly(γ-benzyl-L-glutamate)-block-ELP (PBLG-b-ELP) copolypeptides studied in this work were therefore prepared in high efficacy and with high purity by ROP of γ-BLG NCA using the N-terminal group of the ELP as initiating group. A series of hybrid copolypeptides with different PBLG block lengths was synthesized in order to study the influence of this parameter, among others, on their self-assembly behavior.

The ELP used in this work was recombinantly produced in Escherichia coli using protein engineering techniques as described previously. [START_REF] Petitdemange | Selective tuning of elastin-like polypeptide properties via methionine oxidation[END_REF] This ELP contains a total of forty pentapeptide repeats according to the protein sequence MW[(VPGVG)(VPGMG)(VPGVG) 2 ] 10 corresponding to a molecular weight of 17,035 Da. From the ELP primary structure, the only functional group able to initiate the polymerization is indeed the primary amine present on the N-terminal group.

The 1 H NMR spectrum in CDCl 3 with 15 % of trifluoroacetic acid (TFA, to compare with spectrum after polymerization) of the ELP (Figure S1) was recorded and all peaks fully assigned for subsequent comparison with PBLG-b-ELP 1 H NMR spectra. The peak at 0.94 ppm attributed to the resonance of the CH 3 of the 70 valine residues (420 1 H total) was used for the calibration.

The ELP was subsequently used as macroinitiator for the ROP of γ-BLG NCA. Firstly, the polymerizations were initially performed in anhydrous DMF at 25 °C for 24 hours. Pure products were isolated by precipitation in diethyl ether. The degree of polymerization (DP) of each compound was determined by 1 H NMR in CDCl 3 containing 15% of TFA. The DP was calculated by comparing the integral of the peak at 5.10 ppm corresponding to the resonance of the methylene group in α of the benzyl ring (2 1 H per BLG unit) to the integral of the peak at 0.94 ppm used for calibration (vide supra, 420 1 H). Four PBLG-b-ELP copolypeptides were obtained with DP ranging from 21 to 110. Because the experimental DP in each case was far below the targeted DP (Table S1, e.g. a DP 21 was obtained when DP 52 was targeted), we concluded that the polymerizations stopped before total consumption of the monomer. The increased polydispersity index Đ of PBLG-b-ELP copolypeptides with DP above 61 (Table S1) attributed to the presence of a shoulder in the size exclusion chromatography (SEC) profiles (Figure 3a) also suggested the uncontrolled character of the polymerization under these conditions. Indeed, it was previously reported that side reactions, such as chain-end terminations and the formation of formyl end-groups can occur at 25 °C during the polymerization of NCAs. [START_REF] Habraken | How controlled and versatile is N-carboxy anhydride (NCA) polymerization at 0 C? Effect of temperature on homo-, block-and graft (co) polymerization[END_REF] More precisely, regarding the synthesis of PBLG, Hanby and co-workers [START_REF] Hanby | Synthetic polypeptides. Part II. Polyglutamic acid[END_REF] described in the 50's, the polypeptide chain termination, where the amine chain-end reacts with the carbonyl of the last unit side chain to form a stable 5-atoms heterocycle (i.e., pyrrolidone). When occurring during polymerization, this reaction leads to the termination of the growing chain and is responsible for the poor control of the polymerization. [START_REF] Hadjichristidis | Synthesis of welldefined polypeptide-based materials via the ring-opening polymerization of α-amino acid Ncarboxyanhydrides[END_REF][START_REF] Habraken | How controlled and versatile is N-carboxy anhydride (NCA) polymerization at 0 C? Effect of temperature on homo-, block-and graft (co) polymerization[END_REF] To avoid this problem that is specific to the ROP of γ-BLG NCA, the polymerization reactions were performed in anhydrous DMF at low temperature (5 °C) (Scheme 1). Indeed, it was previously demonstrated that low reaction temperatures reduce the frequency of this premature termination since this reaction requires a higher activation energy. [START_REF] Habraken | How controlled and versatile is N-carboxy anhydride (NCA) polymerization at 0 C? Effect of temperature on homo-, block-and graft (co) polymerization[END_REF][START_REF] Vayaboury | Living Polymerization of α-Amino Acid N-Carboxyanhydrides (NCA) upon Decreasing the Reaction Temperature[END_REF][START_REF] Habraken | Optimization of Ncarboxyanhydride (NCA) polymerization by variation of reaction temperature and pressure[END_REF][START_REF] Lu | Recent advances in amino acid N-carboxyanhydrides and synthetic polypeptides: chemistry, self-assembly and biological applications[END_REF] Under these conditions, all N-terminal groups of ELP chains initiated the ROP as evidenced by the excellent yields. (Table 1) Complete conversions were achieved according to the excellent agreement between theoretical and experimental DPs as assessed by 1 H NMR and targeted DPs (Figure 1). Four PBLG-b-ELP copolypeptides with DP ranging from 25 to 180 were obtained, corresponding to PBLG blocks between 5,500 and 39,500 g.mol -1 molecular weights and diblock PBLG-b-ELP copolypeptides between 22,500 and 56,500 g.mol -1 molecular weights, respectively. All hybrid copolypeptides were characterized by SEC in DMF (0.8 mL.min -1 ) at 50 °C in the presence of LiBr (1 g.L -1 ) with RI detector and polystyrene used as standard. Using this setup, the apparent molar mass of the ELP was 18,000 g.mol -1 , while diblock copolypeptides' apparent molar masses were comprised between 33,300 and 56,200 g.mol -1 . Values estimated by SEC were found in relatively good adequacy with the values obtained by 1 H NMR. (Table 1) The SEC chromatograms evidenced monomodal distributions of polypeptide chains with narrow dispersities (1.02 -1.03) indicating the well-controlled character of the polymerization. (Table 1 and Figure 2b) Consequently the presence of the carboxylic group at the C-terminal end of the ELP did not appear to be disadvantageous in the polymerization. Aoi and co-workers actually showed that the presence of a nucleophilic carboxylate provides control over the polymerization of sarcosine-NCA. [START_REF] Nakamura | Controlled Synthesis of a Chitosan-Based Graft Copolymer Having Polysarcosine Side Chains Using the NCA Method with a Carboxylic Acid Additive[END_REF] To summarize the synthetic part of this work, the ROP of γ-NCA BLG initiated by a high molecular weight recombinant ELP was conducted at 5 °C to prevent chain termination reactions during the polymerization. A total of four, well-defined, hybrid diblock copolymers associating a synthetic hydrophobic polypeptide block and a biocompatible elastin-like polypeptide block were obtained. These differ by the length of the hydrophobic PBLG block and therefore feature different hydrophilic weight fractions (f) -PBLG 25 -b-ELP (f = 76 %), PBLG 45 -b-ELP (f = 63 %), PBLG 95 -b-ELP (f = 45 %) and PBLG 180 -b-ELP (f = 30 %) -a critical parameter that influences amphiphilic copolypeptide self-assembly. [START_REF] Chécot | Structure of polypeptide-based diblock copolymers in solution: Stimuliresponsive vesicles and micelles[END_REF][START_REF] Peyret | Synthetic glycopolypeptides: synthesis and self-assembly of poly (γ-benzyl-Lglutamate)-glycosylated dendron hybrids[END_REF][START_REF] Bonduelle | Synthesis and self-assembly of "tree-like" amphiphilic glycopolypeptides[END_REF]

Self-assembly of PBLG-b-ELP block copolypeptides

The self-assembly in water of the different PBLG-b-ELP copolypeptides was performed using different methodologies, more precisely dialysis or microfluidics and direct solubilization in the case of PBLG 25 -b-ELP with the highest hydrophilic weight fraction (f = 76 %). These methods were selected among others to obtain stable nanoparticles of controlled size and morphologies under kinetic or thermodynamic control. Temperature of the solutions during the self-assembly process and the dialysis was carefully maintained at 20°C to overcome the impact of this parameter. The self-assembly studies were performed at the concentration of 4 g.L -1 , namely the highest polypeptide concentration avoiding the formation of macroscopic aggregates in the dialysis tubing or in the microfluidic chip. Concentrations lower than 2 g.L -1 resulted in the formation of similar nanostructures that were however difficult to characterize because of the high dilution after dialysis. The resulting self-assemblies were characterized by transmission electron microscopy (TEM) (Figures 3 andS2a) and dynamic light scattering at 90° (DLS) (Figures S2b andS3).

The block copolypeptide with the shortest hydrophobic PBLG block, and therefore the highest hydrophilic weight fraction, (f = 76 %), was first self-assembled by direct solubilization in ultrapure water at 1 mg.mL -1 at room temperature. DLS measurements showed a monomodal size distribution, with a hydrodynamic radius (R h ) of 39 nm and relatively low polydispersity index (PDI) of 0.13 (Figure S2a) that is consistent with the formation of spherical nanoparticles. TEM micrographs revealed the presence of a highly homogeneous dispersion of spherical nanoparticles (Figure S2b) with a rather strong intrinsic contrast certainly due to the high electron density of the benzyl group of the hydrophobic PBLG block. It is worth mentioning that PBLG 25 -b-ELP has the largest hydrophilic weight fraction of the whole series of copolypeptides; it is therefore supposed to have the highest critical aggregation concentration (CAC). The CAC of PBLG 25 -b-10 ELP was determined at 0.22 mg.mL -1 . Self-assembly of all copolypeptides was therefore studied at concentrations above the CAC.

Self-assembly of PBLG 25 -b-ELP (f = 76 %) was triggered either by dialysis or microfluidics. leading to micellar morphology, irrespective of the technique used (Figure 3). Due to the use of DMSO as organic solvent to solubilize the diblock copolypeptides, the intrinsic α-helix conformation of the PBLG block was lost in favor of the coil conformation. [START_REF] Bradbury | Nmr studies of the helix-coil transition of polypeptides in non-protonating solvent mixtures[END_REF] The α-helix conformation is however most likely reformed during the self-assembly process, as evidenced by circular dichroism (data not shown), ensuring the stability of the final structures. [START_REF] Holowka | Charged polypeptide vesicles with controllable diameter[END_REF] The suspension obtained by dialysis was heterogeneous as evidenced by the TEM micrograph (Figure 3a) as well as the DLS profile (Figure S3a) showing two distinct populations of particles: one most likely corresponding to individual nanoparticles with a R h of 27 nm and one around 100 nm in diameter most likely corresponding to aggregates. Self-assembly from an organic solution in DMSO in which the copolypeptide is well solubilized thus leads to the formation of smaller spherical micelles than a direct solubilization of the copolypeptide in water. The microfluidic technique proved more efficient in producing nanoparticles of homogeneous size at a ratio of 70:30 DMSO/H 2 O. The DLS intensity profile indeed showed a monomodal size distribution centered on R h = 15 nm with a narrow polydispersity (PDI = 0.14) (Figure S3b).

The hybrid block copolypeptide with a 63 % hydrophilic weight fraction, namely PBLG 45 -b-ELP, self-assembled similarly and formed spherical nanoparticles both by dialysis and microfluidics (Figure 3). The microfluidic technique allowed the formation of nanoparticle of homogeneous size at R h = 17 nm (PDI = 0.08), while dialysis resulted in a more heterogeneous dispersion (R h = The micellar morphology observed with PBLG 25 -b-ELP and PBLG 45 -b-ELP is consistent with previous studies performed on amphiphilic diblock copolymers based on PBLG. In particular, poly(ethylene oxide)-b-PBLG [START_REF] Jeong | Cellular recognition of paclitaxel-loaded polymeric nanoparticles composed of poly (γ-benzyl Lglutamate) and poly (ethylene glycol) diblock copolymer endcapped with galactose moiety[END_REF] (from 21 % to 85 % f values) as well as polysarcosine-b-PBLG [START_REF] Heller | Directed Interactions of Block Copolypept (o) ides with Mannose-binding Receptors: PeptoMicelles Targeted to Cells of the Innate Immune System[END_REF] (f = 90 %) block copolymers self-assembled into spherical nanoparticles by dialysis of an organic solution against water or solvent displacement method. We also evidenced that, for these two copolypeptides with short hydrophobic PBLG chains and f above 60 %, microfluidics provided homogeneous solvents mixing resulting in monodisperse and smaller micelles as compared to the dialysis method.

The self-assembly technique showed however to have a greater impact in the case of larger hydrophobic PBLG block. Dialysis against water of PBLG 95 -b-ELP (f = 45%) led to the formation of polydisperse inter-connected worm-like micelles as shown by the TEM micrographs. (Figure 3, panel e) This observation explained the large distribution obtained by DLS with R h around 130 nm and high polydispersity (PDI = 0.32). (Figure S3, panel e).

Because the water content plays a very important role in the self-assembly, decreasing the solvent quality for the PBLG block firstly solubilized in DMSO and changing the chain mobility, in this part, we describe the formation of the copolymer self-assemblies depending on the DMSO/H 2 O ratio precisely controlled by the microfluidic device.

Using a total flow rate of 100 μL.min -1 and DMSO/H 2 O ratio of 70:30, PBLG 95 -b-ELP formed inter-connected worm-like micelles, similar to that obtained by dialysis, as shown on Figure 3f. Indeed, the DMSO content being rather large after microfluidic, the system is still dynamic and able to re-organize during the dialysis step, a situation similar to that resulting from the slow solvent mixing process. This observation is consistent with previous results reported for different amphiphilic copolymer systems within a similar range of hydrophilic fraction. [START_REF] Discher | Polymer vesicles[END_REF][START_REF] Lee | Crystallization-driven thermoreversible gelation of coil-crystalline cyclic and linear diblock copolypeptoids[END_REF][START_REF] Kakkar | Amphiphilic PEO-b-PBLG Diblock and PBLGb-PEO-b-PBLG Triblock Copolymer Based Nanoparticles: Doxorubicin Loading and In Vitro Evaluation[END_REF] When reducing the relative amount of DMSO in H 2 O (30:70) and thus increasing the amount of selective solvent, slightly distorded micelles were observed on Figure 3g. We assumed these structures to be metastable morphologies kinetically trapped during the experimental time scale, due to the faster diffusion of water in DMSO and high hydrophobicity and rigidity of the PBLG segment. This observation confirmed previous findings that nanoparticle morphology can be controlled not solely by the thermodynamic rules, related to the macromolecular parameters (the degree of repulsion between the blocks, the lengths of the block and consequently the hydrophilic weight fraction) but also by inducing self-assembly with different techniques [START_REF] Huang | Biologically active polymersomes from amphiphilic glycopeptides[END_REF] or by varying the ratio between the organic solvent and the selective solvent allowing the self-assembly via kinetic control. [START_REF] Zhang | Thermodynamic vs kinetic aspects in the formation and morphological transitions of crew-cut aggregates produced by self-assembly of polystyrene-bpoly (acrylic acid) block copolymers in dilute solution[END_REF][START_REF] Cui | Block copolymer assembly via kinetic control[END_REF] Indeed, if the water content is too high, reducing drastically the solubility and chain mobility, the kinetic process of polymer self-assembly and structural rearrangement of the aggregates can be too slow to establish a dynamic equilibrium.

We finally studied the last copolypeptide of the series, namely PBLG 180 -b-ELP, having the lowest hydrophilic weight fraction (f = 30 %). In this case, the process proved critical on the selfassembled morphologies observed. Dialysis against water of a solution of PBLG 180 -b-ELP led to relatively polydisperse (PDI = 0.28) spherical particles of R h = 59 nm (Figures 3h andS3g).

Spherical micelles were also reported as stable self-assembled structures of PBLG-b-PEG diblock copolymers with similar hydrophilic fractions and via a similar dialysis method. [START_REF] Jeong | Cellular recognition of paclitaxel-loaded polymeric nanoparticles composed of poly (γ-benzyl Lglutamate) and poly (ethylene glycol) diblock copolymer endcapped with galactose moiety[END_REF] The use of microfluidics (DMSO:H 2 O ratio of 70:30) however allowed the formation of vesicles as evidenced by TEM and cryo-TEM (Figure 3i). DLS measurements provided an average R h = 76 nm and PDI = 0.19 (Figure S3h). The size of vesicles as measured on TEM micrographs (n = 150) appeared slighty smaller with an average radius of 61 nm, this difference being attributed to the hydration sphere of the ELP segment in solution. These results mainly highlight the major advantage of the microfluidic device in controlling the formation of monodisperse nanoparticles in a metastable morphology and in highly reproducible manner. With microfluidics, the proportion of selective solvent (i.e., water) in the organic phase containing the soluble copolypeptides is directly related to the solvent mixing rate and thus the flow rate of the streams directly controls the formation of spherical or worm-like micelles or polymersomes. [START_REF] Bleul | Techniques to control polymersome size[END_REF] When the kinetic of self-assembly is slowed down by a highly selective solvent (here the water) and no free self-assembly is possible, the formation of polymersomes need significantly more time than the formation of spherical micelles. The vesicular structures obtained can be explained by the slower and homogenous mixing of DMSO and water in the micro-mixer, allowing the selforganization of the rigid and hydrophobic PBLG block to form the thin membrane. Thereafter, the self-assembly of this series of giant amphiphiles in water was studied using several methods: dialysis, microfluidics and direct solubilization only for PBLG 25 -b-ELP (f = 76 %). This study allows to explore the thermodynamic and kinetic conditions the most suitable for the system to obtain stable nanoparticles of controlled size and shape.

Conclusion

Depending on the diblock copolypeptide composition and the self-assembly protocol, the morphology of the structures formed could be controlled, ranging from micelles, worm like micelles to polymersomes and the results highlighted the advantages of using a microfluidic device to obtain more monodisperse structures of nano-objects of interest in a reproducible manner.

Overall, this contribution provides the first example of nano-assemblies of hybrid diblock based on recombinant ELPs and synthetic polypeptides. Due to their intrinsic biocompatibility, these new nanomaterials can potentially serve as platforms for the development of original nanovectors.

Experimental section

Materials

All chemicals were purchased from Sigma-Aldrich and used as received. γ-benzyl-L-glutamate N-carboxyanhydride (γ-BLG NCA) was purchased from Isochem.

NMR spectroscopy

Avance I NMR spectrometer operating at 400 MHz, and equipped with a Bruker multinuclear zgradient direct probe head capable of producing gradients in the z direction with 53.5 G.cm -1 strength. 1 H NMR spectra were recorded with a D1 of 2 sec and 64 scans.

Size exclusion chromatography

The molar masses and the dispersities of poly(γ-benzyl-L-glutamate)-block-ELP and the ELP precursor were determined by size exclusion chromatography (SEC) on a PL GPC50 integrated system from Agilent equipped with two KD-804 Shodex gel columns (300 x 8 mm) (exclusion limits from 4000 Da to 200 000 Da). The detector used is a refractive index detector (Jasco 1530-RI), dimethylformamide (DMF) has been used as eluent (0.8 mL.min -1 ) at 50 °C in the presence of LiBr (1 g.L -1 ).

Dynamic Light Scattering

To determine the hydrodynamic radius (R h ) and the polydispersity index (PDI, a dimensionless measure of the broadness of the size distribution), single-angle light scattering analysis was achieved with an ALV laser goniometer, with a 22 mW linearly polarized laser (632.8 nm HeNe)

and an ALV-5000/EPP multiple tau digital correlator (125 ns initial sampling time). All the measurements were performed at a constant temperature of 25°C and with an angle of 90°. The solutions were placed in 10 mm diameter glass cells. Data were acquired with ALV correlator control software, and the counting time was fixed for each sample at 10 s. The concentration of the solutions was 1 mg.mL -1 . The autocorrelation functions (g 1 (t)) were analyzed in terms of relaxation time distribution (τ) (equation 1). For the suspension in a DMSO/water mixture (before dialysis) the hydrodynamic radius and the polydispersity index (PDI) were determined by DLS with a Zetasizer Nano ZS from Malvern

Instruments operating with a He-Ne laser source (wavelength 633 nm, scattering angle 90°). The correlation functions were analyzed using the cumulants method.

Transmission Electron Microscopy (TEM) micrographs of polymer nanoparticles (NPs) were recorded on a Hitachi H7650 microscope working at 80 kV equipped with a GATAN Orius 10.5

Megapixel camera (Bordeaux Imaging Center, Bordeaux, France).A drop of NPs dispersion was deposited on an Agar Scientific Formvar/Carbon film grid (200 mesh) without staining and the excess was removed after 1 min.

Cryo-Transmission Electron Microscopy (cryo-TEM) micrographs were obtained as following. A drop of suspension was deposited on "quantifoil"® (Quantifoil Micro Tools GmbH, Germany) carbon membrane. The excess of liquid on the membrane was absorbed with a filter paper and the membrane was quench-frozen quickly in liquid ethane to form a thin vitreous ice film including NPs in the holes of the grid. Once placed in a Gatan 626 cryo-holder cooled with liquid nitrogen, the samples were transferred in the microscope and observed at low temperature (-180 °C). Cryo-TEM images were recorded on ultrascan 2k CCD camera (Gatan, USA), using a LaB6 JEOL 2100 (JEOL, Japan) cryo microscope operating at 200kV with a JEOL low dose system (Minimum Dose System, MDS) to protect the thin ice film from any irradiation before imaging and reduce the irradiation during the image capture.

The critical aggregation concentration (CAC) was determined by fluorimetric method: Nile red (0.94 mg, 3.0 μmol) was dissolved in 9 mL of CH 2 Cl 2 and 0.1 mL of this solution was added to a series of vials. The CH 2 Cl 2 was removed under a stream of nitrogen. A series of concentrations of the nano-objects suspension ranging from to 2 mg.L -1 to 1000 mg.L -1 was prepared by serial twofold dilutions. The suspensions were added to the vials containing Nile red and were allowed to equilibrate with stirring overnight. The fluorescence spectra were obtained on a Varian Cary Eclipse fluorescence spectrometer from Agilent Technologies. An excitation wavelength of 550 nm was used for Nile red and the emission spectra were recorded from 565 to 700 nm. The emission intensity at maximum of excitation was recorded for each concentration.

Design of encoding gene, bioproduction, purification and isolation of recombinant ELP-M-40

ELPs with high molecular weight have been efficiently produced in a large scale using Escherichia Coli as expression system. The ELP-M-40 was obtained as previously reported. [START_REF] Petitdemange | Selective tuning of elastin-like polypeptide properties via methionine oxidation[END_REF] The amino acid sequence is: MW[(VPGVG)(VPGMG)(VPGVG) 2 ] 10 so the molecular weight is precisely 17,035 g.mol -1 . The 1 H NMR spectrum in CDCl 3 is shown in the supporting information (Figure S1).

Synthesis of block copolymers poly(γ-benzyl-L-glutamate)-block-ELP

Diblock copolypeptides poly(γ-benzyl-L-glutamate)-block-ELP were synthesized by ring opening polymerization (ROP) of γ-BLG NCA using ELP as macroinitiator. Various

[monomers]/[initiator] molar ratios were used to obtain copolypeptides with different hydrophilic weight fractions f. For example, to prepare PBLG 25 -b-ELP, γ-BLG NCA (9.42×10 -2 g, 3.58×10 -1 mmol) was weighed in a glovebox under pure argon, introduced in a flame-dried schlenk, and dissolved in 2 mL of anhydrous DMF. The solution was stirred for 10 min, and the ELP (2.44×10 -2 g, 1.43×10 -3 mmol, M = 17,035.4 Da) was added with a nitrogen purged syringe from a solution in anhydrous DMF (1×10 -2 g.mL -1 , injected volume: 24.4 mL). The mixture was stirred for one week at 5°C (or at 25°C) precipitated in diethyl ether, and dried under vacuum to afford a white powder. Yield: 3.12×10 -1 g (97 %). A copolypeptide with a molecular mass of 22.5×10 3 g.mol -1 (determined by 1 H NMR) corresponding to the targeted degree of polymerization of 25 was obtained.

Study of self-assembly behavior of poly(γ-benzyl-L-glutamate)-block-ELP

The diblock amphiphilic copolypeptides PBLG-b-ELP were self-assembled using various methods to compare the structure and the size of the nano-objects obtained depending on the process used.

Direct solubilization

The first method, the direct solubilization in water was used only for the copolypeptide with the highest hydrophilic weight fraction f, PBLG 25 -b-ELP (f = 79 %). Indeed, it was not possible to self-assemble under these conditions the others copolymers due to their lower f values without formation of macroscopic aggregates. Shortly, the copolypeptide was added in ultrapure water at a concentration of 1 mg.mL -1 and the mixture was stirred overnight at room temperature.

Dialysis

The second method used, the dialysis, consists in a solution of copolymers at 4 g.L -1 in a common solvent of both blocks (here DMSO) dialyzed against a non-solvent of the hydrophobic block of PBLG (here deionized water). The membrane used has a cut off of 15 KDa and the water was renewed 4 times in 24 h. During this process, water diffused into the DMSO phase, leading to aggregation of the hydrophobic chains and driving the self-assembly process of the amphiphilic block copolypeptides.

Microfluidics

The last method consists in using a microfluidics system from Dolomite ® . A solution of copolypeptides at 4 g.L -1 in DMSO and deionized water were mixed in a chip containing twelve steps of mixing. Two pumps under pression of nitrogen brought the solutions with a flow rate controlled by the "Mitos Flow Control Center" software from Dolomite ® (Scheme S.1). This technique allowed controlling perfectly the speed of mixing depending on the ratio DMSO/water and the total flow rate. For our study, the total flow rate is 100 µL.min -1 , the flow rates of the solution of copolypeptides in DMSO and the water are 30 µL.min -1 and 70 µL.

  VPGVG)(VPGMG)(VPGVG) 2 ] 10 and named ELP-M-40 was efficiently used as macroinitiator for the ROP of the γ-benzyl-L-glutamate N-carboxyanhydride. The controlled ammonium-mediated ROP mechanism allowed us to afford library of define hybrid recombinant/synthetic diblock polypeptides with varying hydrophilic weight fractions (f): PBLG 25 -b-ELP (f = 76 %), PBLG 45 -b-ELP (f = 63 %), PBLG 95 -b-ELP (f = 45 %) and PBLG 180 -b-ELP (f = 30 %) with a dispersity below 1.04.

g 1 (

 1 t) = ∫ 𝐴𝐴 (𝜏𝜏) exp ( -𝑡𝑡 𝜏𝜏 )𝑑𝑑𝑑𝑑 equation 1 Hydrodynamic radius (R h ) was determined from the Stokes-Einstein relation (equation 2). is diffusion coefficient, η s ids the viscosity of the solvent, T is absolute temperature and k B the Boltzmann constant.

  min -1 respectively (ratio DMSO:H 2 O of 70:30), and also 70 µL.min -1 and 30 µL.min -1 for the PBLG 95 -b-ELP (f = 45%) (ratio DMSO:H 2 O of 30:70). Thereafter, the DMSO was removed by dialysis (in a membrane bag with a cut off of 15 KDa). Except for the suspension of PBLG 180 -b-ELP for which the characterizations were carried out before.

Scheme 1 .

 1 Scheme 1. Synthesis of hybrid diblock copolymers poly(γ-benzyl-L-glutamate)-block-ELP by

Figure 1 . 1 H

 11 Figure 1. 1 H NMR spectrum of poly(γ-benzyl-L-glutamate) 180 -block-ELP in CDCl 3 (15 % TFA).

Figure 2 .

 2 Figure 2. Size exclusion chromatography traces of ELP and hybrid diblock copolypeptides

Figure 3 .

 3 Figure 3. TEM micrographs of nano-objects obtained from PBLG n -b-ELP depending on the self-

Table 1 .

 1 Molecular characteristics of ELP and of the different hybrid diblock copolypeptides PBLG-b-ELP obtained by ROP of γ-BLG NCA at 5 °C. f: hydrophilic weight fraction determined from the M n calculated by1 H NMR **SEC in DMF (0.8 mL.min -1 ) at 50 °C in the presence of LiBr (1 g.L -1 ) with RI detector and polystyrene used as standard.

	Copolypeptide	Expected DP (PBLG)	DP PBLG ( 1 H NMR)	M n 1 H NMR (g.mol -1 )	f (%)*	M n SEC** (g.mol -1 )	Ð SEC**	Yield (%)
	ELP	-	-	-	-	18,000	1.03	-
	PBLG 25 -b-ELP	25	25	22,500	76	33,300	1.02	97
	PBLG 45 -b-ELP	52	45	26,900	63	40,100	1.02	85
	PBLG 95 -b-ELP	117	95	37,800	45	49,800	1.03	88
	PBLG 180 -b-ELP	181	180	56,500	30	56,200	1.03	93

*

Table 2 .

 2 Characteristics of nanometer-sized particles obtained from the self-assembly of amphiphilic PBLG-b-ELP hybrid copolypeptides.

	Copolypeptide	f (%) 1 H NMR	Self-assembly process	R h * PDI	Structure of nano-objects**
			Dialysis	27 nm 0.32	Micelles + aggregates
	PBLG 25 -b-ELP	76	Microfluidics	15 nm 0.14	Micelles
			Direct solubilization	39 nm 0.13	Spheres
	PBLG 45 -b-ELP	63	Dialysis Microfluidics	30 nm 0.25 17 nm 0.08	Micelles Micelles
			Dialysis	133 nm 0.32	Worm-like micelles
	PBLG 95 -b-ELP	45	Microfluidics	128 nm 0.29	Worm-like micelles
			Microfluidics***	42 nm 0.47	Spheres
	PBLG 180 -b-ELP	30	Dialysis Microfluidics	63 nm 0.28 76 nm 0.19	Spheres Vesicles
	*Determined by DLS at 90° at 1 mg.mL -1
	**Observed by TEM
	***Ratio DMSO:H 2 O of 30:70

H NMR experiments were performed in CDCl 3 (with 15 % of TFA) at

°C on a Bruker

Acknowledgments

The authors thank Amélie Vax for the SEC analyzes and Julien Rosselgong for general discussions. The Transmission Electron Microscopy (TEM) was performed at the Bordeaux Imaging Center, a service unit of the CNRS-INSERM and Bordeaux University, member of the national infrastructure France BioImaging; authors are grateful to Sabrina Lacomme and Mélina Petrel for the training.

Synthesis mechanism hypothesis

Because the purification of ELP involves the use of sodium chloride, we can reasonably assume the polymerization to be initiated by the N-terminal ammonium group of the ELP with the chloride Cl -as counter-anion. Consequently, the block copolypeptides poly(γ-benzyl-Lglutamate)-block-ELP were synthesized by ROP of the γ-BLG NCA initiated by the primary ammonium end group of the ELP.

It was previously postulated by Schlaad et al. that the ammonium-mediated ROP mechanism may lead to a controlled propagation comparable to nitroxyde-mediated radical or living cationic polymerizations involving an equilibrium between dormant (ammonium) and active (amine) chain ends 1 provided that the counter-anion, here the nucleophilic chloride anion, is quite mobile in the medium. 1,2 The ammonium salt in addition is suspected to suppress the activated monomer mechanism (AMM) due to protonation of the NCA anions. 1 Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.

Interestingly, in the present case of the polymerization of γ-BLG NCA, we suspect the ELP macroinitiator to afford sufficient polarity to the medium to allow an excellent control of ammonium-mediated ROP with the chloride counterion. We additionally presume the formation of hydrogen bonds between the polypeptide chain amide groups and the γ-BLG NCA, stabilizing the latter and limiting the AMM mechanism, and consequently favoring the normal NAM amine mechanism.