Bruno Paiva

Bertrand Santos

Mattia Garbay

Elsa Pasqua

Zoeisha Chevron

Christophe Chinoy

Katell Cullin

Sébastien Bathany

Joelle Lecommandoux

Hugo Amedee

Oliveira

Santos

Bertrand Garbay
email: bertrand.garbay@bordeaux-inp.fr

Mattia Pasqua

Elsa Chevron

Zoeisha S Chinoy

Christophe Cullin

Katell Bathany

Sébastien Lecommandoux

Joëlle Amédée

Hugo Oliveira

Elisabeth Garanger

Production, purification and characterization of an elastin-like polypeptide containing the Ile-Lys-Val-Ala-Val (IKVAV) peptide for tissue engineering applications

Keywords: Elastin-like polypeptides, recombinant expression, hydrogels, sensory neurons, tissueengineering

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

In any tissue-engineering application, the interaction of cells with the extracellular matrix is of major importance, as it supports the processes of cell adhesion, proliferation, migration and differentiation. In this sense, the development of biomaterials bearing guidance cues that can support these processes is fundamental in the establishment of novel, and more efficacious, tissue engineering applications. When considering for example bone tissueengineering applications, where the effective reconstruction of large bone segments remains a major unsolved problem, two major processes have shown to be critical: angiogenesis and neurogenesis [START_REF] Fukuda | Sema3A regulates bonemass accrual through sensory innervations[END_REF][START_REF] Grellier | author manuscript 24/46 osteogenic and endothelial lineages: implications for tissue engineering[END_REF][START_REF] Guerrero | Cell interactions between human progenitor-derived endothelial cells and human mesenchymal stem cells in a three-dimensional macroporous polysaccharide-based scaffold promote osteogenesis[END_REF][START_REF] Jones | Bone and brain: a review of neural, hormonal, and musculoskeletal connections[END_REF][START_REF] Lee | Novel extraneural role of neurite outgrowth inhibitor A: Modulation of osteoclastogenesis via positive feedback regulation of nuclear factor of activated T cell cytoplasmic 1[END_REF][START_REF] Silva | Dorsal root ganglion neurons regulate the transcriptional and translational programs of osteoblast differentiation in a microfluidic platform[END_REF]. As such, biomaterials development should consider the support of these processes. Additionally, when considering a clinical application, off the shelf, up-scalable and simpler biomaterial approaches may reduce regulatory, production and cost issues. Such an example relies on the use of elastin like polypeptides (ELPs). ELPs are recombinant peptide polymers, derived from the hydrophobic region of tropoelastin [START_REF] Urry | Phase-structure transitions of the elastin polypentapeptide-water system within the framework of composition-temperature studies[END_REF], that consist of multiple repeats of Val-Pro-Gly-Xaa-Gly pentapeptides, where the guest residue (Xaa) can be any amino acid except proline. ELPs are characterized by an inverse transition temperature (Tt) [START_REF] Li | The molecular basis for the inverse temperature transition of elastin[END_REF][START_REF] Urry | Free energy transduction in polypeptides and proteins based on inverse temperature transitions[END_REF]. Below the Tt, the free polymer chains of ELPs remain disordered, fully hydrated and thus soluble in aqueous solution. Above the Tt, the ELP chains fold hydrophobically to form insoluble aggregates. This phenomenon is fully reversible, influenced by the nature of the guest residue Xaa (e.g. polar, charged, hydrophobic) and by the ELP chain length [START_REF] Meyer | Quantification of the effects of chain length and concentration on the thermal behavior of elastin-like polypeptides[END_REF]. It also depends on the polypeptide concentration, as well as on the nature and concentration of co-solutes. Recombinant ELPs are especially attractive for tissue Journal of Biotechnology 298, 35-44 (2019) DOI: 10.1016/j.jbiotec.2019.04.010 author manuscript 5/46 engineering as they can include cell specific sequences, they can be engineered to approach the viscoelastic properties of native elastin upon cross-linking, and they can be produced in large scale. In addition, they are biocompatible, biodegradable and can be purified using their inherent transition temperature without the need for complex and expensive chromatographic approaches [START_REF] Changi | Biocompatibility and immunogenicity of elastinlike recombinamer biomaterials in mouse models[END_REF][START_REF] Nettles | Applications of elastin-like polypeptides in tissue engineering[END_REF][START_REF] Nicol | Cell adhesion and growth on synthetic elastomeric matrices containing Arg-Gly-Asp-Ser-3[END_REF][START_REF] Prieto | Biomimetic calcium phosphate mineralization with multifunctional elastin-like recombinamers[END_REF].

Several synthetic biomaterials were designed to incorporate signaling epitopes for cell-cell and cell-tissue interactions. A well-studied epitope is the short bioactive pentapeptide Ile-Lys-Val-Ala-Val (IKVAV), a motif derived from laminin, which has shown to promote neuron cell attachment, migration and neurite outgrowth [START_REF] Nomizu | Structure-activity study of a laminin α1 chain active peptide segment Ile-Lys-Val-Ala-Val (IKVAV)[END_REF][START_REF] Silva | Selective Differentiation of Neural Progenitor Cells by High -Epitope Density Nanofibers[END_REF]. This pentapeptide can also increase endothelial cell mobilization, capillary branching and revascularization [START_REF] Grant | Interaction of Endothelial-Cells With a Laminin-a Chain Peptide (Sikvav) Invitro and Induction of Angiogenic Behavior Invivo[END_REF][START_REF] Nakamura | Construction of multi-functional extracellular matrix proteins that promote tube formation of endothelial cells[END_REF]. This peptide was grafted to a number of different scaffolds such as aminated polymer surface [START_REF] Santiago | Peptide-surface modification of poly(caprolactone) with laminin-derived sequences for adipose-derived stem cell applications[END_REF], methacrylamide chitosan [START_REF] Yu | Peptide surface modification of methacrylamide chitosan for neural tissue engineering applications[END_REF], collagen [START_REF] Hosseinkhani | Engineering three-dimensional collagen-IKVAV matrix to mimic neural microenvironment[END_REF][START_REF] Li | IKVAV regulates ERK1/2 and Akt signalling pathways in BMMSC population growth and proliferation[END_REF], or to a small dipeptide such as PA [START_REF] Niece | Modification of gelation kinetics in bioactive peptide amphiphiles[END_REF][START_REF] Silva | Selective Differentiation of Neural Progenitor Cells by High -Epitope Density Nanofibers[END_REF]. It has been widely used in tissue engineering by immobilizing it onto hydrogel scaffolds [START_REF] Drury | Hydrogels for tissue engineering: scaffold design variables and applications[END_REF][START_REF] Shekaran | Nanoscale engineering of extracellular matrix-mimetic bioadhesive surfaces and implants for tissue engineering[END_REF][START_REF] Zhu | Bioactive Modification of Poly(ethylenglykol) Hydrogels for Tissue Engineering[END_REF].

Here, we have used the inherent capabilities of ELPs for purification and for the inclusion of cell-specific sequences to produce recombinant peptide polymers, containing the IKVAV to form a hydrogel capable of supporting neurotization. We describe the (i) production, purification, and characterization of IKVAV-containing ELPs, (ii) the use of this engineered polypeptide as a functional component for hydrogel production, and (iii) the application of this new synthetic extracellular matrix in tissue engineering. Enzymes required for recombinant DNA, including restriction endonucleases, OneTaq® hot start DNA polymerase, Antarctic phosphatase and Quick ligation™ kit (T4 DNA ligase) were obtained from New England Biolabs (NEB, Ipswich, MA, USA). The pET-44a(+) plasmid and the E. coli strain BLR(DE3) were obtained from Novagen Inc (Madison, WI, USA).

MATERIALS AND METHODS

Materials.

Synthesis of the ELP-IKVAV and ELP-VKAIV genes were achieved by Eurofin MWG Operon (Ebersberg, Germany). All custom oligonucleotides were synthesized by Eurogentec (Seraing, Belgium). Sequencing of the different constructs was performed by GATC biotech (Cologne, Germany). All chemicals were from Sigma-Aldrich (Saint-Louis, MO, USA).

Construction of expression vector for ELP genes.

Synthetic genes corresponding to the ELP-IKVAV and ELP-VKAIV sequences (Figure 1) provided in pUC19 plasmid, were extracted from the said plasmid by a double digestion with NdeI and BamHI, and the corresponding fragments were purified after agarose gel electrophoresis with the PCR clean-up, gel extraction kit (Macherey-Nagel GBMH, Düren, Germany). Purified fragments were ligated with the Quick ligation™ kit into similarly digested and dephosphorylated pET-44a(+). After transformation into E. coli BLR(DE3), transformants were selected by colony PCR with OneTaq® hot start DNA polymerase, and the primers pET-F (5'-TAATACGACTCACTATAGGG-3') and pET-R (5'-GTTATGCTAGTTATTGCTCAGCGG-3'). Plasmids from the positive clones were purified with the Plasmid DNA Purification kit (Macherey-Nagel, Düren, Germany) according to manufacturer's instructions, and verified by DNA sequencing.

Bioproduction of recombinant ELPs.

A single bacterial colony was selected and cultured overnight at 37°C in a rotary shaker at 200 rpm in 50 mL of rich LB medium (1% bacto tryptone, 0.5% NaCl, 1% yeast extract) containing 100 µg/mL ampicillin. Thereafter, this seed culture was inoculated into 150 mL of LB-rich medium containing 100 µg/mL ampicillin. Cells were cultivated at 37 °C in a 500 mL baffled shake flask in a rotary shaker at 150 rpm. When the optical density at 600 nm (OD 600nm ) reached a value close to 1.4, isopropyl β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 0.3 mM, and the cultivation temperature was decreased to 25 °C.

Purification of recombinant ELPs.

After 21 h of IPTG induction, the OD 600nm of the cultures reached a value close to 4. Cells were harvested by centrifugation at 3,000 g and 4 °C for 15 min, and the cell pellet was resuspended with 10 mL/g wet weight in PBS buffer (NaCl 137 mM, KCl 2.7 mM, Na 2 HPO 4 8 mM, KH 2 PO 4 2 mM, pH 7.4) supplemented with one tablet/10 mL of Complete Mini EDTA-free protease inhibitors (Roche, Basel, Switzerland). Thereafter, samples were incubated for 4 h at -80 °C, and thawed by incubation at 10 °C. Total cell lysis was performed by sonication for 10 min at 12 °C with several 4-second pulses at 125 W, 8 seconds apart. Then, the homogenates were centrifuged at 3,000 g and 4 °C for 15 min. For ELP-VKAIV, the supernatant was directly used for the polypeptide purification by four successive rounds of inverse transition cycling (ITC) [START_REF] Meyer | Purification of recombinant proteins by fusion with thermally-responsive polypeptides[END_REF]. Briefly, the ELP was precipitated at 37 °C in the presence of 1 M NaCl, and pelleted by centrifugation at 10,000 g and 25 °C for 10 min ("Hot Spin"). After elimination of the supernatant containing soluble proteins and re-suspension of the pellet in cold PBS buffer, the ELP was re-dissolved whereas insoluble proteins from E. coli were eliminated by centrifugation for 10 min at 10,000 g and 4 °C ("Cold Spin"). The supernatant containing the ELP was then processed through successive ITC cycles. NaCl was not added to the sample after the first round of ITC.

In the case of ELP-IKVAV, the 3,000 g pellet was re-suspended in 20 mL cold-PBS supplemented with β-mercaptoethanol at a final concentration of 0.68 M. After an overnight incubation at 4 °C on a rotary agitator, the homogenate was centrifuged at 3,000 g and 4 °C for 15 min, and the supernatant was subjected to three successive rounds of ITC purification as described above. Finally, the purified ELPs were dialyzed against ultrapure water at 4 °C in dialysis membranes with a 3.5-kDa cutoff (Slide-A-Lyer®Mini Dialysis Devices, Thermo Scientific, Rockford, IL, USA) for five hours. After a final overnight dialysis, samples were stored at -20°C. Protein content of the purified fraction was determined by spectrophotometry at 280 nm with a Nanodrop (NanoDrop-ND 1000). Due to their low Tt, the samples aggregated rapidly at room temperature. Therefore, they should first be diluted with ethanol (70% final concentration) and kept on ice until the measurement. Parameters for the Nanodrop were set at 5,500 for the extinction molar coefficient, and at 10,180 Da for the molecular mass.

Chemical and physico-chemical characterization of ELPs.

The molecular mass of each purified ELP was determined by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Analyses were performed on a MALDI-ToF-ToF (Ultraflex III, Bruker Daltonics, Bremen, Germany) equipped with a SmartBeam laser (Nd:YAG, 355nm). Samples at a final concentration of 0.5 mg/mL were mixed with the matrix sinapinic acid (Sigma Aldrich) at 10 mg/mL in acetonitrile/0.1% aqueous TFA (1/1, v/v). All MALDI-MS measurements were acquired in the linear positive mode and a mixture of standard proteins (Sigma-Aldrich, Saint-Louis, MO, USA) was used for external calibration covering the mass range from 8,500 Da to 17,000 Da. was defined as the temperature corresponding to the maximum of the first derivative of the turbidity versus temperature curve [START_REF] Christensen | Predicting transition temperatures of elastin-like polypeptide fusion proteins[END_REF].

Analysis of ELP-IKVAV aggregates

Amyloid-like fibril formation was monitored by thioflavin T (ThT) binding. Fluorescence emission of 20 µM ThT was measured in the presence of 150 µM ELP in PBS with a POLAR star Omega microplate reader (BMG Labtech, Champigny-sur-Marne, France). Excitation at 440-448 nm, and emission spectrum was recorded between 460 and 550 nm. Samples were preincubated at 4°C prior to the analysis to prevent gel formation in the wells.

Hydrogel production and rheology

PEG gels were composed of 4 arm thiol-ended PEG (SH-PEG, 20 kDa) (JenKem, USA), 4 arm acrylate PEG (Ac-PEG), at thiol to acrylate equimolar ratios. Photopolymerization was achieved using the photoinitiator Irgacure® 2959 (0.5% w/v) exposure to UV light (λ=305 nm) following a thiol-ene click Michael addition. Different mass ratios of ELPs and PEG were explored to attain optimal rigidity and appropriate sensory neuron attachment and neurite outgrowth. Briefly SH-PEG was substituted by ELPs at different mass rations.

Namely: (i) 50% (w/w) of Ac-PEG + 45% (w/w) of SH-PEG + 5 (w/w) of ELP, (ii) 50% (w/w) of Ac-PEG + 40% (w/w) of SH-PEG + 10% (w/w) of ELP and (iii) 50% (w/w) of Ac-PEG + 30% (w/w) of SH-PEG + 20% (w/w) of ELP.

The rheological properties of the hydrogels were evaluated upon 24 h of soaking in PBS by determining the frequency dependence of the storage (G') and loss moduli (G"). Frequency sweeps were performed at a constant strain (0.1) in the angular frequency range 0.1-100 rad s -1 . This characterization was performed for the composite hydrogels (containing ELPs) at a Biotechnology 298,35363738394041424344 final concentration of 15% (w/v) while varying the ELP concentration in the final hydrogel mass (i.e. 5%, 10% and 20% (w/w)). Measures were performed at 4 and 37 °C.

Journal of

Cell isolation, culture, and characterization

Primary sensory neurons were obtained from dorsal root ganglion from healthy 6-10 weekold Wistar rats as described elsewhere [START_REF] Malin | Production of dissociated sensory neuron cultures and considerations for their use in studying neuronal function and plasticity[END_REF]. Briefly, spinal columns was removed and opened from the caudal to the rostral in order to expose the dorsal root ganglions. They were individually harvested and digested with 1 mg/mL Collagenase Type IV (Gibco®) for 2 h at 37 °C. Subsequently, the digestion product was washed twice with culture medium supplemented with 2% (v/v) B-27 Serum-Free Supplement® (B-27, Gibco®), and 1% (v/v) Penicilin-Streptomycin, and mechanically dissociated using fire polished glass Pasteur pipettes (full diameter and ½ diameter). The cell suspension was finally washed three times with medium and resuspended in culture medium. The culture medium was supplemented with 1 µM cytarabine, to avoid the proliferation of the Schwann cells that were present in the primary culture.

Sensory neurons previously cultured on 100 µg/mL poly-D-lysine-and 10 µg/mL laminincoated coverslips for 7 days, were fixed in 1% (w/v) paraformaldehyde (PFA, Merck Millipore, Darmstadt, Germany) for 10 min at room temperature, washed once with PBS 0.1 M and permeabilized for 5 min with 0.1% (v/v) Triton® X-100 (EDM Millipore, Bilerica, MA, USA). A 1% (w/v) bovine serum albumin (BSA) solution was used for antigen blocking for 30 min at room temperature to minimize the non-specific binding. Goat anti-rat calcitonin gene-related peptide antibody (CGRP, 1:50; Abcam, Cambridge, UK) was used as antibody for 1 h at room temperature. Photomicrographies were taken using laser-scanning microscope (SPE, Leica Microsystems).

Neurite length evaluation

Sensory neurons were seeded on hydrogels at 20,000 cells per square cm and cultivated for 7 days. Subsequently, cells were fixed with 4% (w/v) paraformaldehyde at 4 °C for 30 min, permeabilized with Triton X-100 at 0.1% (v/v) for 30 min at 4 °C and blocked with BSA 1% (w/v) for 1 h. Primary antibody rabbit anti-rat beta III Tubulin (Abcam) was used at a dilution 1:500 overnight at 4 °C and second antibody goat anti-rabbit IgG conjugated with Alexa Fluor 568 (Molecular probes) at 1:200 dilution for 2 h at room temperature. Nuclei were labeled with DAPI (4', 6'-diamidino-2-phenylindole, FluoProbes) at 1 μg/mL for 30 min at room temperature. Sensory neurons morphology was visualized using a confocal laserscanning microscope (SPE, Leica Microsystems). The quantification of the neurite length was performed using Image J, using "Simple Neurite Tracer" tool. Neurites were measured tracing a path from the soma to the visible end and the length was converted in µm (n>4).

Subcutaneous hydrogel implantation

Balb/c mice (n=4 for each group) housed in the animal care facility of the 4A building of the University of Bordeaux (Bordeaux, France) were used for the in vivo evaluations. They were under conditions of circadian day-night rhythm, and they were fed ad libitum. Animal CO 2 chamber and samples with the surrounding tissue were collected and extensively rinsed with PBS 0.1 M (pH 7.4) and then fixed with 4% (w/v) PFA for 30 min at 4 °C. Sections of 10 µm thickness were obtained using a cryostat (Leica CM 1850 UV). Haematoxylin and eosin staining was performed under standard conditions, slides were then mounted using Clearmount medium (Invitrogen, USA).

Statistical analysis

Using the Graphpad Prism 5.0 Software, a D'Agostino and Pearson normality test was used in order to test if values obeyed to a Gaussian distribution. Statistically significant differences between several groups were analyzed by the non-parametric Kruskal-Wallis test, followed by a Dunns post-test. A p-value lower than 0.05 was considered to be statistically significant.

Results and discussion

Design of the ELPs

We have designed an ELP-based recombinant polymer with interspersed sequences of VPGVG (n = 8), VPGIG (n = 8) and VPGMG (n = 4). The gene sequence was selected according to E. coli codon usage while minimizing sequence repetition (Figure 1). We incorporated two sequences of the pentapeptide epitope IKVAV (or its scrambled version VKAIV) in the ELP backbone. Thus, ELP-IKVAV and ELP-VKAIV have identical MW (10,189 Da), theoretical pI (8.89), and grand average of hydropathicity (GRAVY, 1.236).

Tryptophan was included to monitor ELP during the final step of the purification process thanks to its UV absorption. Two cysteine residues were added close to the N-terminus and C-terminus of the ELP for subsequent cross-linking purposes and post-polymerization modifications [START_REF] Xu | Rheological Properties of Cysteine-Containing Elastin-Like Polypeptide Solutions and Hydrogels[END_REF][START_REF] Zhang | A Highly Elastic and Rapidly Crosslinkable Elastin-Like Polypeptide-Based Hydrogel for Biomedical Applications[END_REF]. Other post-production modifications could be performed at the methionine residues, as recently demonstrated [START_REF] Kramer | Quantitative Side-Chain Modifications of Methionine-Containing Elastin-Like Polypeptides as a Versatile Tool to Tune Their Properties[END_REF]. The GenBank accession numbers are KU743475 and KU743476, for ELP-IKVAV and ELP-VKAIV, respectively.

Production and purification of ELPs

In typical experiments, bacteria were cultivated at 37 °C in LB medium until OD 600nm reached a value around 1.4. IPTG was then added to a final concentration of 0.3 mM, and the cultivation temperature was decreased to 25 °C. In a first series of experiments, the timecourse accumulation of the two ELPs was monitored by SDS-PAGE. A band corresponding to the expected ELPs was detected 2 h after IPTG addition, and its amount increased up to 21 h of culture (data not shown). This duration of culture post-induction was thus selected for all the following experiments. After cell lysis, the homogenate was centrifuged at 3,000 g, and proteins from the corresponding pellet and supernatant were analyzed by gel electrophoresis.

The result from a typical experiment is shown in Figure 2. For the two ELPs, a band migrating slightly lower than the 10 kDa protein marker was detected in the induced culture when compared to the non-induced one. This apparent MW is compatible with the one expected for the two ELPs, namely ELP-IKVAV and ELP-VKAIV. However, these two polypeptides behaved differently during the purification process. The ELP-IKVAV was predominantly retrieved in the 3,000 g pellets, suggesting that it mainly accumulated in the form of insoluble aggregates. To check that no substantial amount of ELP-IKVAV was present in the supernatant, we performed one round of ITC purification with this soluble fraction, and no significant amount of ELP-IKVAV was recovered in the final "cold spin" supernatant (Figure S1). On the contrary, substantial amounts of ELP-VKAIV were present in the 3,000 g supernatant, although some was present in the pellet. Indeed, densitometry analysis of this gel showed that 69% of ELP-VKAIV was in the soluble fraction. The fact that ELP-IKVAV and ELP-VKAIV exhibited different solubility was quite unexpected We then sought after a solubilization method for the ELP-IKVAV protein product. Several strategies were described to prevent the formation of insoluble aggregates during recombinant expression in E. coli. Their goal is usually to diminish the synthesis rate of the recombinant protein. Indeed, we did several experiments where the final concentration of IPTG was decreased from 0.5 mM to 0.3 mM, and then 0.1 mM, but this did not result in an increase in the ELP-IKVAV solubility. We also decreased the temperature of the culture after the induction by IPTG from 37°C to 30°C, 25°C or 20°C with no more success. We therefore sought for a solution to solubilize the polypeptide from the 3,000 g pellet obtained after cell lysis. Multiple buffers can be used to enhance protein solubility [START_REF] Bondos | Detection and prevention of protein aggregation before, during, and after purification[END_REF].

We chose to test the chaotrope urea (8 M final concentration), the detergent N-Lauroylsarcosine (14.6 mM final concentration) and the reducing agent β-mercaptoethanol (0.68 M final concentration). The latter was evaluated because both ELPs contained two cysteine residues that may trigger aggregation throughout the formation of intra-or intermolecular disulfide bridges. In all cases, fractions of the pellet 3,000 g were incubated overnight at 4 °C, centrifuged at 3,000 g, and the efficiency of solubilization was assessed by SDS-PAGE analysis of the supernatant and pellet fractions (Figure S2). This experiment showed that N-Lauroylsarcosine was inefficient at solubilizing ELP-IKVAV from the 3,000 g pellet. On the contrary, 90% of this polypeptide was recovered in the 3,000 g supernatant after treatment by 8 M urea. However, when ITC purification was performed with the corresponding soluble fraction, the majority of ELP-IKVAV did not precipitate during the hot spin step and was therefore lost in the supernatant. For some reason, urea somewhat prevented the coacervation that should occur when the ELP is placed at a temperature above its Tt. After one ITC round of purification, only trace amounts of the polypeptide could be S2). The profound effect of urea on the coacervation temperature of ELP has already been reported [START_REF] Miao | Structural determinants of cross-linking and hydrophobic domains for self-assembly of elastin-like polypeptides[END_REF]. Finally, the best results were obtained with β-mercaptoethanol. After an overnight incubation with this reducing agent, 58% of the ELP-IKVAV was solubilized from the initial 3,000 g pellet. More importantly, only trace amounts were lost in the hot spin supernatant or in the cold spin pellet during the ITC cycle (Figure S2). We thus decided to use β-mercaptoethanol to purify ELP-IKVAV in the following experiments. Indeed, starting with the supernatant post-βmercaptoethanol treatment (lane 3, Figure 3), we were able to purify to homogeneity the ELP-IKVAV by three successive rounds of ITC (Figure 3). To achieve good purity, four rounds of ITC were necessary for ELP-VKAIV because more proteins were present in the initial fraction (supernatant 3,000 g post-lysis, see lane 5, Figure 3). It should be noted that an additional band around 20 kDa MW was present in the two purified samples. This band corresponds in size to ELP dimers, which were quite resistant to the β-mercaptoethanol present in the sample loading buffer. Interestingly, SDS-PAGE performed in non-reducing conditions showed that numerous multimers (ranging from dimer to heptamer) were present in the two ELP purified fractions (Figure S3). The presence of multimers in the purified ELP-IKVAV sample was rather unexpected because this polypeptide was extracted from the 3,000 g insoluble pellet by using 0.68 M β-mercaptoethanol. This suggests that the formation of the intermolecular disulfide bridges occurred during the ITC cycles and/or during the dialysis step. For the two ELPs, the yield of the purified protein was 95 ± 25 mg/L (n=6). This yield was significantly higher than those previously obtained in our laboratory for hydrophobic ELPs [START_REF] Bataille | Recombinant production and purification of short hydrophobic Elastin-like polypeptides with low transition temperatures[END_REF][START_REF] Bataille | Expression and purification of short hydrophobic elastin-like polypeptides with maltose-binding protein as a solubility tag[END_REF].

Physico-chemical characterization of ELPs

The molecular mass of each purified ELP was determined by matrix-assisted laser desorption/ionization mass spectrometry (Figure S4). peptidase during the production in E. coli [START_REF] Ben-Bassat | Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure[END_REF]. Indeed, the N-terminal methionine is often cleaved off when the second residue in the primary sequence is small and uncharged [START_REF] Hirel | Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid[END_REF], which is the case for our ELPs with a cysteine residue following the initial methionine.

The thermal behavior of the two ELPs was studied by turbidimetry. A first series of experiments showed that the amount of multimers in the sample, which results from the formation of intermolecular disulfide bridges, dramatically affected the phase transition of the ELP. Indeed, Tts were measured for the same ELP-VKAIV sample which was either untreated, heated for 10 min at 95°C to increase aggregation [START_REF] Kaneko | Heat-Induced Formation of Intermolecular Disulfide Linkages between Thaumatin Molecules That Do Not Contain Cysteine Residues[END_REF], or treated with β-mercaptoethanol (Figure 4). For the three samples, we observed a sharp single transition as expected for ELP monoblocks. The relative amounts of the multimeric species present in each sample was evaluated by SDS-PAGE analysis (insert of Figure 4). The Tt measured, respectively at 18°C (untreated), 14°C (pre-heated) and 23°C (reduced), were inversely correlated to the proportion of multimers, the Tt decreasing when the amount of multimers increased. This result was expected because it has been previously demonstrated that the Tt of a given ELP is inversely related to its molecular mass [START_REF] Meyer | Quantification of the effects of chain length and concentration on the thermal behavior of elastin-like polypeptides[END_REF]. A similar conclusion could be drawn for ELP-IKVAV (Figure S5).

Taking into account the formation of intermolecular disulfide bridges, we chose to measure the Tts in the presence of 0.68 M β-mercaptoethanol to favor the presence of monomers because it was not possible to precisely control the state of multimerization between different samples. One of the hallmarks of the ELP is that the transition from the soluble form to the insoluble and aggregate state is reversible [START_REF] Urry | Phase-structure transitions of the elastin polypentapeptide-water system within the framework of composition-temperature studies[END_REF]. We therefore investigated the reversibility of the ELP-IKVAV and ELP-VKAIV solutions. To achieve this goal, the same solution of ELP was submitted to two consecutive runs of thermal denaturation (Figure 5). This experiment showed that the ELP-VKAIV behaved like a typical ELP, with a transition around 23 °C that was measured during the two successive experiments. On the contrary, the ELP-IKVAV showed a classical turbidity profile during the first run, but the aggregates that were formed above the Tt did not disintegrate when the temperature was lowered below 15 °C, and therefore the turbidity remained constant during the second experiment. This unexpected phenomenon did not result from the presence of intermolecular disulfide bridges because the experiment was performed in the presence of the reducing agent βmercaptoethanol. In addition, such irreversibility was not seen for the ELP-VKAIV, which contains also two cysteine residues, and therefore the same capacity for multimerization. The irreversibility of the transition was thus attributed to the presence of the IKVAV motifs in the ELP sequence. It should be noted that prolonged incubation of the solution of aggregated ELP-IKVAV at 4° C did not allow solubilization of the said ELP.

Insights into the insolubility of ELP-IKVAV

The difference in solubility between the two ELPs was difficult to understand. As previously stated, they have an identical amino acid composition and MW. Both contain two cysteine residues. The fact that β-mercaptoethanol enhanced the solubility of ELP-IKVAV during the purification process, suggests that the formation of intra-or inter-molecular disulfide bridges plays a role in the aggregation of the said polypeptide, but an additional mechanism is very likely to take place. Indeed, the same intra-or intermolecular disulfide bridges may be formed during the production of the ELP-VKAIV, but this ELP was mainly soluble (see Figure 2). In addition, turbidity measurements clearly showed that the aggregation of ELP- IKVAV was irreversible, whereas the transition of ELP-VKAIV was fully reversible. In fact, it has already been reported that a 12-mer peptide containing the IKVAV motif (called LAM-L) formed amyloid-like fibrils, whereas peptides containing a scrambled IKVAV motif did not [START_REF] Kleinman | Ile-Lys-Val-Ala-Val (IKVAV)containing laminin α1 chain peptides form amyloid-like fibrils[END_REF]. To test this hypothesis, samples of the two ELPs, each at a concentration of 150 µM, were incubated at 25 °C in the absence of β-mercaptoethanol. As expected, a gel rapidly formed in the two tubes as the temperature was above the Tt of the ELPs. However, after overnight incubation at 37 °C, a coacervate was still visible in the ELP-VKAIV tube whereas a precipitate accumulated at the bottom of the ELP-IKVAV tube. In this case, the gel structure collapsed and the ELP sedimented under the form of macroscopic aggregates (Figure S6). A similar phenomenon occurred when a similar sample was incubated for three days at 4°C.

To investigate if these aggregates contain amyloid-like structures, we used a specific test based on the binding of thioflavin T (ThT) [START_REF] Biancalana | Molecular mechanism of Thioflavin-T binding to amyloid fibrils[END_REF][START_REF] Khurana | Mechanism of thioflavin T binding to amyloid fibrils[END_REF]. We analyzed samples from ELP-IKVAV stored at -20 °C, or those stored at 4 °C for three days. A sample from ELP-VKAIV stored at 4 °C for three days was used as a control (Figure 6). A strong characteristic fluorescence signal at 482 nm was observed for the ELP-IKVAV sample, upon storage for three days at 4 °C. The signal was significantly lower for an ELP-VKAIV sample stored under the same conditions, or for an ELP-IKVAV sample stored at -20 °C and carefully defrost on ice just before the measurement. Using this test, we can conclude that the aggregates obtained after incubation of ELP-IKVAV at 4 °C possessed some characteristics of amyloid-like fibrils (i.e. rich in ß-strand secondary structures), and that these entities were not present in significant quantities when the same sample was stored at -20 °C or in the ELP-VKAIV sample. Since we aimed to produce hydrogels to contemplate neurotization and vascularization, we targeted a hydrogel with storage modulus (G') between 1 kPa and 1.5 kPa. Indeed, this range of substrate stiffness has shown to be optimal for dorsal root ganglia (DRG) neurons outgrowth [START_REF] Koch | Strength in the periphery: growth cone biomechanics and substrate rigidity response in peripheral and central nervous system neurons[END_REF]. Preliminary experiments showed that hydrogels obtained by crosslinking pure monomers of ELP-IKVAV or ELP-VKAIV do not meet this criterion.

Hydrogel production

Therefore, we chose to use poly(ethylene glycol) (PEG) as a crosslinking agent to enhance the rheological properties. PEG is currently FDA-approved for several medical applications and presents several characteristics that favor its use in biomedical applications, such as low toxicity, low protein adsorption, and low immunogenicity. Albeit PEG is slowly biodegradable, it has been shown that PEGs with a molar mass below 20 kDa are easily excreted in urine (Pasut and Veronese, 2007). Moreover, it has already been shown that the ELP-PEG hydrogels have substantially improved optical transparency compared with pure ELP hydrogels, which facilitate in vitro analyses [START_REF] Wang | Hybrid elastin-like polypeptide-polyethylene glycol (ELP-PEG) hydrogels with improved transparency and independent control of matrix mechanics and cell ligand density[END_REF].

As means to achieve the production of PEG-ELP hydrogels, we used equimolar ratios of thiol to acrylate groups at different final polymer concentrations. Rheological analyses were performed to assess the storage (G') and loss (G'') moduli. Preliminary experiments showed that PEG gels made of 15% polymer with equimolar ratios of thiol to acrylate groups had a (G') value of 1.4 kPa. Then, further analyses were conducted using the 15% (w/v) final concentration hydrogel composition where we substituted the thiol PEG by different mass percentages of ELP-IKVAV, namely 5%, 10% and 20% (w/w). Composite hydrogels showed distinct behavior depending on the measurement temperature and on the percentage of ELP composition (Figure 7). At 4 °C, below the ELPs transition temperature, the composite gels showed a reduction of the elastic modulus (G'), more relevant for the higher ELP compositions. Conversely, the transition from 4 to 37 °C, resulted in an increase of the storage modulus of + 8%, + 67% and +78 %, for the 5, 10 and 20% (w/w) ELP hydrogels, respectively. Similar results were obtained with the ELP-VKAIV hydrogel (Figure S7). This behavior can be explained by the inherent transition temperature of the polymers, ELP chains being soluble and fully extended below the Tt value. In response to strain, force is transmitted largely through these extended chains and chemical crosslinks. In addition, the polypeptide chains do not have much contact with each other (Trabbic-Carlson, Setton, & Chilkoti, 2003).

At higher temperature, ELP chains are insoluble and contracted, decreasing water retention and increasing their intermolecular interactions, resulting in a tighter hydrogel network. As expected, these intermolecular interactions are proportional to the ELP concentration [START_REF] Trabbic-Carlson | Swelling and Mechanical Behaviors of Chemically Cross-Linked Hydrogels of Elastin-like Polypeptides Swelling and Mechanical Behaviors of Chemically Cross-Linked Hydrogels of Elastin-like Polypeptides[END_REF].

Evaluation of neurite growth on the hydrogels

Sensory neurons primary cultures were characterized by confocal microscopy through CGRP and β III tubulin immunostaining. Cells expressed CGRP in the cell body and neurite, and β III tubulin labelling was used to monitor neurite formation [START_REF] Silva | Dorsal root ganglion neurons regulate the transcriptional and translational programs of osteoblast differentiation in a microfluidic platform[END_REF]. In order to evaluate the capacity of the IKVAV-containing hydrogels to support neurite growth, we evaluated the behaviour of sensory neurons after 7 days of culture. As shown in Figure 8A, initial sensory neurons adhesion was observed for all the conditions studied. In scrambled peptide formulations, sensory neurons formed larger cellular aggregates with neurite wrapped around the cell bodies. Sensory neurons showed more dispersed cellular distribution and longer neurite when grown on IKVAV-containing hydrogels. Image analysis for average neurite length confirmed that hydrogels containing the highest IKVAV concentration (20% w/w) had longer neurite (168 ± 75 µm) relative to the same scrambled concentration (45 ± 19 µm, p < 0.001) (Figure 8B). The average neurite length for the 20% (w/w) IKVAV containing gels is similar to those previously published for adult rat sensory neurons grown for 7 days in the absence of nerve growth factor (NGF). isolated from adult rats, grown on collagen hydrogels for 7 days [START_REF] Kim | Effects of proinflammatory cytokines on axonal outgrowth from adult rat lumbar dorsal root ganglia using a novel threedimensional culture system[END_REF]. They showed that the neurite length reached 229 ± 102 µm in the absence of NGF. In our study, we deliberately chose to evaluate the sensory neurons growth in the absence of supraphysiological concentrations of NGF, as means to analyze the particular impact of the hydrogel structure and to reflect the in vivo scenario, where adult cells from peripheral tissues can secrete neurotrophic factors that support neurotisation [START_REF] Madduri | Schwann cell delivery of neurotrophic factors for peripheral nerve regeneration[END_REF][START_REF] Wilkins | Human bone marrow-derived mesenchymal stem cells secrete brain-derived neurotrophic factor which promotes neuronal survival in vitro[END_REF].

In vivo evaluation of cytotoxicity

Based on the results obtained in vitro, 50 µL of hydrogels containing 20% (w/w) of ELP-IKVAV, or the scrambled sequence, were implanted subcutaneously in Balb/c mice (n=4 for each group). After 4 weeks, the samples were collected with the surrounding tissue and analyzed histologically (Figure 9). For both compositions, the implanted material was integrated in the surrounding tissue with a thin layer of fibrous tissue formed around the implanted material. These observations, coupled with the absence of any major inflammatory response, suggest a suitable biocompatibility for our material in vivo. This supports previous reports showing that ELP-based hydrogels do not trigger important cytotoxicity or inflammatory response after their in vivo implantation [START_REF] Ibáñez-Fonseca | Biocompatibility of two model elastin-like recombinamer-based hydrogels formed through physical or chemical cross-linking for various applications in tissue engineering and regenerative medicine[END_REF][START_REF] Nettles | In situ crosslinking elastin-like polypeptide gels for application to articular cartilage repair in a goat osteochondral defect model[END_REF][START_REF] Testera | Biocompatible elastin-like click gels: design, synthesis and characterization[END_REF][START_REF] Zhang | A Highly Elastic and Rapidly Crosslinkable Elastin-Like Polypeptide-Based Hydrogel for Biomedical Applications[END_REF].

Conclusion

We designed, produced, purified and characterized a recombinant ELP containing the IKVAV adhesion peptide, and formulated it with PEG to obtain biofunctional hydrogels. This approach allowed the modulation of both the mechanical properties and the density of the biologically relevant adhesion sequence, relevant for the optimal cell response. These hydrogels supported sensory neurons growth, triggered neurite lengthening, and showed good biocompatibility after in vivo implantation. These features make the IKVAV-containing ELP hydrogels a promising engineered construct to be used as extracellular matrix in biomedical applications with a special need for neurotization.

  The transition temperatures of ELPs at two different concentrations in PBS solution were determined by measuring the turbidity at 350 nm between 10 and 40 or 50 °C, at 1 °C min -1 scan rate. Data were collected on a Cary 100 Bio UV-visible spectrophotometer equipped with a multi-cell thermoelectric temperature controller from Varian (Palo Alto, CA). The Tt Journal of Biotechnology 298, 35-44 (2019) DOI: 10.1016/j.jbiotec.2019.04.010 author manuscript 9/46

  ) DOI: 10.1016/j.jbiotec.2019.04.010 author manuscript 10/46

  primary antibody overnight at 4 °C and Alexa Fluor® 488-conjugated chicken anti-goat secondary antibody (1:250; Molecular Probes®, Life Technologies) was used as secondary Journal of Biotechnology 298, 35-44 (2019) DOI: 10.1016/j.jbiotec.2019.04.010 author manuscript 11/46

  experimentation protocol was approved by the local Ethic Committee (protocol nº APAFIS #4375-2016030408537165 v4). 15% (w/v) hydrogels containing IKVAV, or the scrambled adhesion sequence, were produced in 96 wells in a volume of 50 µL and were implanted subcutaneously at the mediodorsal position in sterile conditions. After 28 days of implantation, mice were euthanized in a Journal of Biotechnology 298, 35-44 (2019) DOI: 10.1016/j.jbiotec.2019.04.010 author manuscript 12/46

  Journal of Biotechnology 298, 35-44 (2019) DOI: 10.1016/j.jbiotec.2019.04.010 author manuscript 14/46 because they possess identical physico-chemical characteristics (MW, pI, GRAVY index, cysteine content).

  The average masses measured by Journal of Biotechnology 298, 35-44 (2019) DOI: 10.1016/j.jbiotec.2019.04.010 author manuscript 16/46 MALDI-MS were 10,055.2 Da and 10,054.6 Da for ELP-IKVAV and ELP-VKAIV, respectively. With the theoretical molecular weight being 10,189.4 Da, the likely hypothesis to explain the mass difference between the experimental and theoretical values (~134 Da) is that the N-terminal Met (131.2 Da) was excised from the polypeptides by methionine amino

  Indeed, Kim and collaborators studied the effect of several growth factors on sensory neurons Journal of Biotechnology 298, 35-44 (2019) DOI: 10.1016/j.jbiotec.2019.04.010 author manuscript 21/46

  Journal of Biotechnology 298, 35-44 (2019) DOI: 10.1016/j.jbiotec.2019.04.010 author manuscript 22/46

Figure 1 :

 1 Figure 1: Schematic representation of the nucleic acid and protein sequences of ELP-

  Journal of Biotechnology 298, 35-44 (2019) DOI: 10.1016/j.jbiotec.2019.04.010 author manuscript

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  Journal of Biotechnology 298, 35-44 (2019) DOI: 10.1016/j.jbiotec.2019.04.010 author manuscript

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Journal of Biotechnology 298, 35-44 (2019) DOI: 10.1016/j.jbiotec.2019.04.010 author manuscript

Acknowledgements

The authors would like to thank the Fédération de Recherche Technologies pour la Santé (TecSan), Bordeaux Consortium for Regenerative Medicine (BxCRM), Université de Bordeaux, and Conseil Régional de Nouvelle Aquitaine.

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After culture of the ELP clones, cells were recovered by centrifugation and lysed by sonication. After centrifugation at 3,000 g, the insoluble pellet containing the ELP-IKVAV was mixed with a solution of PBS containing β-mercaptoethanol (0.68 M final concentration). After an overnight incubation on a rotator at 4° C, the homogenates were centrifuged at 3,000 g for 10 min at 4° C. Supernatants were used for ITC purification. For ELP-VKAIV, ITC purification was performed directly on the 3,000 g supernatant obtained after cell lysis. 1: pellet 3,000 g post-lysis; 2: pellet 3,000 g post-incubation with βmercaptoethanol; 3: supernatant 3,000 g post-incubation with β-mercaptoethanol; 4 purified ELP after three rounds of ITC; 5 supernatant 3 000 g post-lysis; 6: purified ELP-VKAIV after four rounds of ITC; M: Precision Plus Protein Standards. 8 µL of each protein sample were loaded in the lanes. Total volume of the fractions was 10 mL for samples 1, 2, 3 and 5 After culture of the ELP-IKVAV clone, cells were recovered by centrifugation and lysed by sonication. After centrifugation at 3,000 g, the supernatant was used for one round of ITC purification. ELP was precipitated from the 3,000 g supernatant at 37 °C in the presence of 1 M NaCl, and pelleted by centrifugation at 10,000 g and 25 °C for 10 min ("Hot Spin"). After re-suspension of the pellet in cold PBS buffer, the ELP was re-dissolved whereas insoluble proteins from E. coli were eliminated by centrifugation for 10 min at 10,000 g and 4 °C ("Cold Spin"). S: supernatant; P: pellet; M: Precision Plus Protein Standards. InstantBlue TM was used to stain the gel. solution of PBS containing β-mercaptoethanol (0.68 M final concentration). After overnight incubation on a rotator at 4°C, the homogenates were centrifuged at 3,000 g for 10 min at 4° C. Supernatants were used for ITC purification (one round of purification). P: pellet 3,000 g after incubation with NLS, urea or β-mercaptoethanol; S: supernatant 3,000 g after incubation with NLS, urea or β-mercaptoethanol; SHS: supernatant of the "Hot Spin" step; PCS: pellet of the "Cold Spin" step; SCS: supernatant of the "Cold Spin" step; M: Precision Plus Protein Standards (corresponding MW are indicated on the figure). InstantBlue TM was used to stain the gel. Total volumes of the fractions were 1 mL for P and S, 300 µL for SHS, PCS and SCS. 10 µL of each fraction were loaded onto the gel. The solution was incubated overnight at 37 °C.

Figure S7. Rheological properties of ELP-VKAIV hydrogels

The rheological properties of the hydrogels were evaluated after 24 h of soaking in PBS by determining the frequency dependence of the storage (G') and loss moduli (G"). Frequency


Guillaume Sudre
email: guillaume.sudre@espci.fr

Yvette Tran
email: yvette.tran@espci.fr

Costantino Creton
email: costantino.creton@espci.fr

Dominique Hourdet
email: dominique.hourdet@espci.fr

pH/Temperature control of interpolymer complexation between poly(acrylic acid) and weak polybases in aqueous solutions

Keywords:

The formation of inter polymer complexes (IPC) between poly(acrylic acid) (PAA) and poly(acrylamide) (PAM), poly(N,N-dimethylacrylamide) (PDMA), and statistical copolymers of acrylamide (AM) and N,N-dimethylacrylamide (DMA) has been studied as a function of pH, salt concentration and temperature (0-70 °C). The cloud points of dilute solutions were measured by turbidimetry and phase diagrams were determined as a function of temperature and pH in pure water and as a function of pH and salt concentration at room temperature. For each temperature and salt concentration a critical pH (pH crit ) below which IPC are observed was defined. In the case of PAA/PAM, pH crit continuously decreased with increasing temperature, from pH 3.5 at 0 °C to pH 1.9 at 60 °C (UCST-type). In the case of PAA/PDMA, pH crit increased with temperature. The LCST-type behavior of the hydrogenbonding complex formed between PAA and PDMA was attributed to the dimethyl substitution of amide groups that puts in hydrophobic interactions at high temperature. PAA and statistical copolymers P(AM-co-DMA) showed an intermediate behavior between PAA/PAM and PAA/PDMA with a continuous shift from UCST-type to LCST-type with increasing amount of DMA. This behavior can be attributed to changes in configurational entropy due to the IPC formation and (for PDMA) to the release of water molecules initially confined in hydrophobic hydration cages around DMA units. While at low salt concentration, the stability of PAA/PAM and PAA/PDMA complexes only slightly increases with the screening of ionized acrylic units, there is a sharp increase of pH crit at high salt concentration in relation with the weakening of the solvent quality. In this regime, the complex formation of PAA/PDMA is greatly enhanced compared to PAA/PAM due to the interference of hydrophobic interactions.

Introduction

Responsive macromolecular devices, able to adapt their properties to environmental stimuli, form today a very challenging class of materials for high tech applications in microfluidics, mechanical transducers, reversible adhesion, tissue engineering or drug delivery. [START_REF]Responsive Gels: Volume Transitions I & II[END_REF][2][3][4][5][6] For a given stimulus, these materials can display responsive bulk properties, with modification of their volume (by swelling), permeability, solubility, etc., as well as responsive surfaces properties that are of fundamental importance in scientific areas such as wetting, lubrication and adhesion.

Many of these responsive materials are water-soluble or water-swellable polymers and their responsivity is related to the presence of intermolecular interactions and/or to the formation of interpolymer complexes (IPC). In essence a modification of molecular interactions due to an external stimulus (temperature, pH, electric field, light…) triggers a modification in solubility or conformation of the polymers or triggers the formation of an IPC, which in turn results in a macroscopic change in properties. Although in some cases the correspondence between the molecular scale interactions and the macroscopic effect is obvious, in some others such as adhesion between two surfaces, the connection is much less well understood. In this case molecular interactions occur at an interface, while a change in adherence of an object on a surface is a coupling between interface and bulk property.

In order to investigate systematically that molecular/macro connection it is essential to study both molecular interactions and macroscopic effects. In this paper we report a systematic study on the effect of pH and temperature on molecular interactions in a water-soluble polymer system which can also be readily used for macroscopic adhesion tests. Since the objective is to develop a multiresponsive model system by coupling pH and temperature, the key features that have to be taken into account in the selection of polymers are typically ionizable groups (weak acidic or basic moieties), hydrogen bonding and hydrophobic interactions.

Setting hydrogen bonding as the main driving force, we selected a weak polyacid (polyacrylic acid (PAA)) as the responsive polymer. PAA is well known to form hydrogen-bonding complexes with a large number of non-ionic polybases, such as poly(ethylene oxide) (PEO), poly(acrylamide) (PAM) and its derivatives, poly(N-vinyl-2-pyrrolidone) (PVP), etc. [START_REF] Khutoryanskiy | In Hydrogen-bonded interpolymer complexes. Formation, structure and applications[END_REF] The formation of IPC is generally studied with standard methods [START_REF] Bekturov | [END_REF] such as gravimetry, 9 potentiometry, 9,10 turbidimetry, [11][12][13][14] viscosimetry, 10,[14][15][16][17] fluorescence study after the labeling of one of the polymers 17,18 and light scattering 13 or more specific ones like small angle neutron scattering 19 and infrared 13 or NMR spectroscopies. 20 In aqueous solution, the formation of interpolymer complexes (IPC) between PAA and a proton acceptor polymer has been shown to occur only below a critical value of pH (pH crit ), 11 where the degree of ionization was low enough. It is generally assumed 11 that the value of the critical pH is an index of the complexing ability of the polymer pair: the higher the critical pH of complexation, the stronger the complex. In other words, the lower the protonation of PAA for complexation, the higher the affinity between the two polymers. The absence of complexation between isolated monomers and the relatively low measured values of critical pH of IPC led to the idea that complex formation must involve cooperative effects, such as "non-interrupted linear sequences of bonds", described by a ladder structure 9,10,12,18 and at the origin of the so-called "zipper effect". 21 For the specific case of PAA, the interaction between the two polymers is almost stoichiometric but the polybase/polyacid ratio can change according to the chemical structure and steric hindrance of the polybase interacting with PAA with acid:base molar ratios of 2:3 or 1:1 proposed for PAA/PAM 22 up to 3:2 for PAA/PNIPAM (poly(N-isopropylacrylamide))

or PAA/PDEAM (poly(N,N-diethylacrylamide)). 10,22 While in organic solvents, the strength of IPC complexes formed between PAA and proton accepting polymers mainly depends from the difference between interaction forces polymer/polymer and polymer/solvent, the situation becomes more subtle in aqueous media as hydrophobic interactions and the dissociation state of PAA must also be taken into consideration. Hydrophobic interactions which often interfere in the complexation mechanism have been reported to favor the complex formation (increase of pH crit ) and to modify the temperature dependence of its stability. [START_REF] Khutoryanskiy | In Hydrogen-bonded interpolymer complexes. Formation, structure and applications[END_REF] For instance, while the formation of IPC between PAA and PAM is characterized by negative values of standard enthalpy and entropy that underline the formation of a hydrogen-bonded complex characterized by lower configurational entropy, the complex formation between PAA and PNIPAM is characterized by thermodynamic values of opposite sign. In that case, the gain of entropy is attributed to the release of water molecules initially confined in hydrophobic hydration cages formed around isopropyl groups of PNIPAM. 23 As the complex formation between polymer pairs in aqueous solution involves material parameters, such as molar mass of the polymers, relative concentration and composition and environmental parameters such as pH, temperature or ionic strength, the formation of IPC has been investigated under many different angles. However to the best of our knowledge, and in spite of numerous studies devoted specifically to PAA complexes, we did not find a description of the whole "temperature-pH" dependence of the complex stability. As a matter of fact, most of the studies generally focus on a single stimulus, i.e. the temperature dependence of IPC at a given pH 12,13 or conversely its pH dependence at a given temperature, generally room temperature.

The current study was therefore mainly focused on IPC formed in aqueous solution between PAA and two H-bonding acceptor polymers, PAM and PDMA. Working at the same relative concentration for all polymers, we established comprehensive "pH-temperature" phase diagrams for these two complexes between 0 and 70 °C and below pH 4 by using turbidimetry measurements.

Experimental part

Chemicals

Acrylamide (AM, 99%, Sigma), N,N-dimethylacrylamide (DMA, 99%, Aldrich), ammonium peroxodisulfate (APS, 99,5% Aldrich), sodium metabisulfite (SMB, 97%, Acros), N,N,N',N'tetramethylethylenediamine (TEMED, 99.5% Sigma-Aldrich), 4,4'-azobis(4-cyanovaleric acid) (ACVA, 75%, Aldrich), ammonium chloride (99.5% Aldrich) and 3-mercaptopropionic acid (≥ 99% Fluka) were used as received. All organic solvents were analytical grade and water was purified with a Millipore system combining an inverse osmosis membrane (Milli RO) and ion exchange resins (Milli Q).

Polymers

Poly(acrylic acid) (PAA, M w = 50 kg.mol -1 from Polysciences) was used as received. PAM, PDMA and their copolymers were prepared by radical polymerization following two different procedures. For PDMA and its copolymers, we modified the method previously developed by Bokias et al. 24 to control the molar mass of vinyl monomers like N-isopropylacrylamide and acrylic acid. Targeting polymers with molar masses of about 30 kg mol -1 , we set the total monomer concentration at 1 mol L -1 , using the red-ox initiator APS/SMB with equal concentrations of 10 -2 mol L -1 . However, since the homopolymerization of AM with persulfate initiators is known to give high molar masses and high polydispersities, 25 we specifically used a chain transfer agent. 26 The main details of the syntheses can be summarized as follows.

PDMA and P(AM-co-DMA). Ammonium chloride (0.5 mmol) and the proper quantities of monomers (50 mmol in total) were dissolved in water (50 mL) and the pH of the mixture was adjusted around 5-6 with hydrochloric acid before deoxygenation with nitrogen bubbling (1 h). A 1 mL solution of SMB (0.5 mmol) and a 1 mL solution of APS (0.5 mmol) were prepared, deoxygenated, and then added to the monomer solution under nitrogen atmosphere.

The reaction was allowed to proceed at room temperature during 24 h. The final solution was dialyzed against milli-Q water using a membrane with a MWCO equal to 6 to 8 kg mol -1 and the polymer was finally recovered by freeze-drying.

PAM. 3-mercaptopropionic acid (0.18 mmol) and acrylamide (180 mmol) were initially dissolved in 1 L of water at 60 °C under nitrogen bubbling. ACVA (1.8 mmol) was separately dissolved in water (18 mL) after ionization of carboxylic units by drop-wise addition of concentrated sodium hydroxide solution. After deoxygenation the initiator solution was added into the monomer solution. The reaction was left to proceed during 1 h at 60 °C followed by 1.5 h at 50 °C. The reaction medium was then concentrated under reduced pressure, precipitated in ethanol, filtered and washed several times before drying under vacuum.

The polymers were characterized by 1 H NMR in D 2 O (Bruker, 400 MHz) and size exclusion chromatography using a Viscotek SEC system equipped with three Shodex OH Pack columns equilibrated at 25 °C in a 0.5 M solution of sodium nitrate. The absolute molar masses of the samples were determined by the three detectors in line (refractometer, viscometer and light scattering), coupled with a calibration based on poly(ethylene oxide) standards. The composition and macromolecular characteristics of P(AM-co-DMA) copolymers are given in Table 1.

We can notice that the synthesis initiated by persulfate gives rise to molar masses M w in the range of 30 to 40 kg mol -1 , as expected from the literature. 24 In the case of radical copolymerization of AM and DMA, McCormick et al. 27 have shown that DMA tends to react a little bit faster than AM on a growing chain, but the reaction constants remain very close to one another. Consequently, we will consider that the distribution of AM and DMA in the polymer chain is almost random. Although polymerized in the presence of thiol, the molar mass of homo-PAM is clearly higher but the polydispersity remains rather low for this type of radical polymerization. AM (mol%) [START_REF]Responsive Gels: Volume Transitions I & II[END_REF] DMA (mol%) [START_REF]Responsive Gels: Volume Transitions I & II[END_REF] Yield (%) M w (kg mol -1 ) ( (1) the composition was determined by 1 H NMR.

(2) the molar mass M w and polydispersity index (Ð) were determined by SEC.

Preparation of ternary mixtures

Initial solutions containing 5 wt% of polymer in pure water were prepared at least 24 h before the analysis. Then, 0.5 mL of PAA solution (proton-donor polymer) was mixed with 0.5 mL of the solution containing the proton-acceptor polymer and the resulting mixture was diluted 10 times by adding water and adjusting the pH with a small amount of concentrated NaOH or HCl solutions. For experiments exploring the influence of the ionic strength, a given amount of solid NaCl was slowly added to the previous solution and dissolved under stirring. If needed, the pH was adjusted during this procedure to avoid the formation of macroaggregates. In the following, all the ternary systems have been studied at the same weight composition: PAA (0.25 wt%) / Polymer 2 (0.25 wt%) / water (99.5 wt%).

Analysis of IPC formation

Complex coacervation in ternary systems (polymer-1 / polymer-2 / solvent) gives rise to the formation of polymer aggregates that significantly impact the physical properties of the mixture. When this phase separation process remains under control of environmental conditions, the initiation of the phase transition can be readily followed by various techniques like rheology, potentiometry, light scattering (turbidimetry) or differential scanning calorimetry if enthalpy absorption or release is associated with the phase transition. [START_REF] Khutoryanskiy | In Hydrogen-bonded interpolymer complexes. Formation, structure and applications[END_REF] In the framework of the present study, the complex formation was studied by turbidimetry that highlights the growth of large aggregates with typical size greater than the wavelength of the light. The determination of the cloud point, that defines the transition threshold, can be made by eye but a more systematic analysis was preferred by using a UV-visible light spectrometer (Hewlett Packard 8453) with wavelengths ranging from 200 to 1200 nm. The quartz cell (1 cm thickness) containing the ternary solution was maintained under stirring in the chamber of the spectrometer and the temperature was accurately controlled with a Peltier system. The temperature range was typically scanned from 7.5 °C to 70 °C with a heating rate of about 0.7 °C/min. As shown in Figure 1a and 1b, the transmittance of the ternary system under investigation increases with temperature, as the solubility of the complex increases, but the transmittance strongly depends on the wavelength. By comparing spectrometric data with those obtained by naked eye, the best agreement (within a degree) was obtained by setting the transition threshold for a transmittance value of 0.90 at 480 nm. These conditions, that have been systematically applied in this work, are typically in the spectrometric range (400-600 nm) generally considered for turbidimetry. [START_REF] Khutoryanskiy | In Hydrogen-bonded interpolymer complexes. Formation, structure and applications[END_REF]12

Results and discussion

PAA/PAM phase diagram

Figure 2 shows the value of the transition points for the PAA/PAM IPC as a function of pH and temperature. At room temperature, PAA and PAM start to self-assemble reversibly below pH 3. However, when the temperature is changed, the pH transition zone is not sharp and it is possible to shift the association process with temperature by exploring the pH on a larger scale, typically between 1.8 and 3.4. In this range of pH, the IPC formation/dissociation can be finely tuned between 0 and 60 °C. As shown in Figure 2, there is a small hysteresis 7.5 °C between the transition temperatures determined during heating and cooling that could be attributed to the slow dynamics of dissociation and solubilization of the two polymers with respect to the heating rate. Below pH 3.4, the complex formed between PAA and PAM clearly exhibits a UCST-type behavior (Upper Critical Solution Temperature) that mainly originates from hydrogen bonding between the two polymers. As shown by various authors like Staikos et al. 23 and Deng et al., 21 the thermodynamics of formation of hydrogen-bonding complexes between PAA and PAM is an enthalpy-driven process (ΔH 0 < 0) with a loss of configurational entropy (ΔS 0 < 0). In this case hydrogen-bonding is the main factor stabilizing the IPC and its strength is weakened by increasing the temperature and/or the pH. In the absence of other additives, the thermodynamic properties of the ternary mixture PAA/PAM/water also depends on the molar mass of the polymers, their molar fraction with respect to the complex stoichiometry and their concentration. In this work, the study of IPCs was mainly carried out at low polymer concentration (0.25 wt%) in order to focus on the thermodynamic reversibility of the complexation while avoiding a dramatic impact of kinetics, especially for the dissolution of large aggregates. Nevertheless, we have explored more specifically for the PAA/PAM/water mixture the concentration dependence of the critical pH for complex formation (pH crit ); i.e. the pH where the IPC starts to form. As shown in Figure 3, the critical pH slightly increases of 0.15 units over one concentration decade; the two polymers are being added at the same weight ratio (1/1) and molar ratio (1/1) in the case of PAA/PAM. As observed, the formation of IPC is favored with increasing polymer concentration since the degree of ionization of PAA is expected to decrease in such conditions. These results are in agreement with those reported by Mun et al., 11 although they found that the critical pH of the transition increased by 0.3 unit over one concentration decade but carried out their experiments with polymers of higher molar masses.

PAA/PDMA phase diagram

A similar study was carried out with the ternary system PAA/PDMA/water and Figure 4 shows the cloud points as a function of pH and temperature. At room temperature the formation of interpolymer complexes occurs at pH below 3.5 for PAA/PDMA mixtures prepared at the same relative weight concentration (0.25 wt% in water). This tendency for "PAA/PDMA" to form IPC at higher pH than PAA/PAM typically highlights the stronger complexation ability of PDMA towards PAA as already reported. [12][13][14] Moreover, contrary to PAA/PAM, the IPC formation of PAA/PDMA is favored at high temperature and there is a narrow pH range, typically between 3.45 to 3.70, where ternary mixtures turn from clear at room temperature into turbid at high temperatures. All these features support the idea that in addition to hydrogen-bonding, the formation of PAA/PDMA complexes in aqueous solutions is stabilized by hydrophobic interactions. Such a coupling between hydrogen bonds and hydrophobic interactions has been previously reported for various polymer pairs where one of the polymer exhibits some hydrophobic character. In these conditions, the entropic penalty due to the formation of hydrophobic cages around low polar groups of polymers is generally considered responsible for dehydration and complexation processes observed by increasing temperature (ΔS 0 > 0); the associated enthalpy is generally positive (ΔH 0 > 0). 23,28 This is the case for instance for poly(methacrylic acid) (PMAA) which forms stronger complexes than PAA, at higher critical pH values. 29 For instance, Ikawa et al. 30 and Kabanov et al. 31 have shown that the minimal critical segment length, which corresponds to the number of monomer units needed for the formation of complexes, increases with temperature for PAA/PEO and decreases for PMAA/PEO due to the increased hydrophobicity of PMAA compared to that of PAA. The formation of IPC between PAA and proton-acceptor polymers has been widely studied and it is well established that the introduction of hydrophobic comonomers into the proton-acceptor macromolecule enhances the complexation ability and the critical pH value. 32 The same holds for polymer pairs involving PAA and LCST polymers. In this case, IPC are formed at higher critical pH, compared to analogous polymers without LCST, and the complex itself can exhibit a LCST behavior. 10,23,33 Although PDMA is water-soluble in the usual temperature range (0-100°C), it has an intermediate chemical structure between PAM, which is fully soluble in water, and poly(N,Ndiethylacrylamide) (PDEA) that displays a LCST behavior at about 30°C. 10 Consequently, we can reasonably assume that PDMA is able to develop hydrophobic interactions through its methyl groups and theoretical predictions of a LCST phase transition for PDMA in water above 200 °C34 support this idea. Although a LCST-type complex has been identified in the case of PAA/PDMA mixtures and grafted copolymers (PAA-graft-PDMA) by Aoki and coworkers, 12,13 the details of the temperature dependence of the complex formation as a function of pH has never been studied. As shown in Figure 4, the pH range where the complex formation between PAA and PDMA can be tuned with temperature is very narrow (less than 0.3 pH units over 60 °C) compared to PAA/PAM (more than 1.5 units over 60 °C).

Interestingly the two systems behave similarly close to 0 °C where the two ternary systems phase separate below the same critical pH around 3.5. We can also notice that the temperature dependence of the complex PAA/PDMA is initially very weak below 20 °C and becomes larger above 20 °C and up to 60 °C. Nevertheless, as both entropy and enthalpy of complex formation may vary with the temperature, as it was shown with various polymer pairs like PAA/PEO 35 and PAA/PVP 36 , it is difficult to specifically address the temperature behavior of the complex to hydrogen bonding, hydrophobic interactions or a coupled effect. Moreover, on this specific aspect, we can mention that while most of the background originates from experiments performed on a large number of systems, there is no unifying theory taking into account distinctly the contribution of hydrogen bonds and hydrophobic forces. While UCST is readily understood in terms of intermolecular forces, interpretation of LCST generally takes into account 1) differences in thermal expansion of solvent and solute or 2) order/disorder transitions as encountered in systems capable of forming hydrogen bonds, with or without hydrophobic contribution. 37 In the latter case, the hydrophobic contribution could be taken into account through the introduction of a strong correlation between neighboring water molecules along the polymer chain (cooperative hydration). 38

Influence of ionic strength

The stability of hydrogen-bonding complexes can be modified by adding molecules that will interfere with the interactions. While the addition of urea, a well-known competitor for hydrogen bonding, clearly destabilizes the complex formation at low pH and increases the solubility of each polymer, the effect of the addition of inorganic salts could be more complex to interpret since three competing mechanisms are involved, namely:

1) an increase in the level of dissociation of the carboxylic groups of the PAA which is unfavorable for hydrogen-bonding, 2) a screening of the electrostatic repulsions between ionized residues which favors the hydrogen-bonded complex formation, 3) a decrease of the thermodynamic quality of water which strengthens interactions between polymer chains.

The influence of different concentrations of sodium chloride upon complex formation of PAA with PAM and PDMA is reported in Figure 5. PAA/PAM (○) and PAA/PDMA (•). The one-phase system is at high pH.

Starting with the PAA/PAM complex we can notice that the addition of sodium chloride increases the critical pH and favors the formation and stability of the complex. At low salt concentration, typically below 1 mol/L, the critical pH increases only very slightly (about 0.2 units between 10 -2 and 1 mol/L) and this can be ascribed to the screening of electrostatic interactions that prevail upon dissociation of carboxylic acids. This behavior is in good agreement with the literature where IPC having a relatively low critical pH (pH crit between 2.45 and 2.88) are known to increase their stability and pH crit upon the addition of inorganic salts, while stronger complexing systems (with pH crit in the range 3.66-4.85) show the opposite trend. [START_REF] Khutoryanskiy | In Hydrogen-bonded interpolymer complexes. Formation, structure and applications[END_REF]39 At high salt concentration (above 1 mol/L), the PAA/PAM complex displays a significant increase of the critical pH due to the weaker hydration of polymer chains (decrease of solvent quality).

In the case of PAA/PDMA, the salt dependence of the critical pH is positive at low salt concentration but much weaker than the one obtained for PAA/PAM. This result is in good agreement with the critical pH value of PAA/PDMA that is higher than PAA/PAM and we can reasonably conclude that screening and ionization effect almost compensate each other.

Nevertheless, the situation becomes different at higher salt concentration where a strong increase of the critical pH is observed at about 0.1 mol/L; i.e. one decade below the two other complex. This feature can be put in line with the "hydrophobic" character of PDMA that is responsible for the LCST behavior of the PAA/PDMA complex. This hydrophobicity is expected to provide a much higher responsivity towards added molecules that will compete in the hydration process. This well-known "salting out" effect, which impacts the solubility of LCST polymers like PEO, PNIPAM and others, clearly depends from the nature of salt but becomes very significant typically for salt concentrations above 0.1 mol/L. 40,41

Complex formation between PAA and P(AM-co-DMA) copolymers

As the complex formation in aqueous solution between PAA/PAM and between PAA/PDMA display an opposite behavior with temperature, we investigate how random copolymers of AM and DMA behave in the presence of PAA. Such problem has been initially partly investigated in the literature by authors like Wang et al. 18 For a given temperature, the critical pH for complexation continuously increases with DMA content. However the impact of DMA increases significantly with temperature with a common intercept at about 0°C where all the complexes behave similarly with a critical pH at about 3.5. Interestingly we can notice on Figure 6 that the temperature dependence of the cloud point changes progressively from a UCST-type for PAA/PAM, dominated by hydrogen bonds, to a LCST-type for PAA/PDMA where hydrophobic interactions are strongly coupled to hydrogen bonds. Moreover by tuning the level of hydrophobic interactions with the DMA composition, one can control the temperature dependence of the complex formation and find stable complexes that do not respond at all to the temperature. From a thermodynamic point of view, it is possible to correlate the temperature dependence of the complex stability with the entropy change of the complex formation, which is expected to be negative for PAA/PAM and positive for PAA/PDMA; then, ΔS 0 goes through 0 for a given copolymer composition. This is almost the case for the complex formed between PAA and CopAM 34 that can be tuned with temperature only in an extremely narrow range of pH (about 0.1 unit over 60 °C).

As already shown with the complex formation of (co)poly(vinyl ether) with PAA, the incorporation of hydrophobic fragments into macromolecules enhances the hydrophobic stabilization of IPC in aqueous solutions which leads to an increase of the pH-stability. 32 While PDMA is generally considered as a hydrophilic polymer, the complex formation with PAA reveals that its hydrophobic character is strong enough to readily modify and overturn the temperature dependence of hydrogen bonding complexes formed between PAA and P(AM-co-DMA). Such tuning should be reflected in the level of interactions between surfaces and provide a guidance on which systems are likely to provide the broadest range of tunability.

Conclusion

The formation of hydrogen bonded IPC between PAA on the one hand and homopolymers of PDMA, PAM and statistical copolymers of AM and DMA on the other hand has been studied as a function of pH, salt concentration and temperature. In the case of PAA/PAM IPCs, the hydrogen-bonding complex takes place around 0°C at pH 3.5 and the critical pH for the complex formation continuously decreases with increasing temperature, up to pH 1.9 at 60 °C. This behavior, already described in the literature, is typically expected when the IPC formation is driven by hydrogen bonding in water, in which case both the enthalpy and the entropy of the complex formation are negative. Although PDMA is very soluble in water and chemically close to PAM, we have shown that methyl groups in DMA units were responsible for the opposite LCST behavior of the IPC formed with PAA. While the role of hydrophobic interactions on IPC stability in aqueous media has been well described using LCST polymers like PNIPAM or polyethers, this work clearly evidences that even weak hydrophobic contribution can strongly impact the complex formation in water and its stability. The original contribution of this work also comes from the determination of comprehensive "pHtemperature" phase diagrams of hydrogen-bonding complexes formed between PAA and statistical copolymers of AM and DMA which provide a good understanding of the impact of hydrophobic forces in hydrogen bonding complexes. Although negligible at temperature close to 0 °C, the contribution of hydrophobic interactions increases with temperature and at 60 °C the critical pH for complex formation can be shifted by about two pH units by changing the monomer composition of P(AM-co-DMA). Similarly, working at a fixed pH, the complex stability can be controlled with temperature as statistical copolymers display an intermediate behavior between PAM and PDMA with a continuous evolution from UCST-type to LCST-type with increasing amount of DMA. For a molar composition close to 1AM/2DMA the complex formed with PAA has no temperature dependence. This can be explained if we assume that the loss of configurational entropy related to the IPC formation compensates the gain of entropy coming from the release of water molecules initially confined in hydrophobic hydration cages around DMA units.

Comparing PAM and PDMA complexes formed with PAA, the influence of salt is mainly significant at high salt concentration (above 0.1 mol/L) where the decrease of the solvent quality becomes noticeable, especially for PAA/PDMA which involves hydrophobic interactions. From a general point of view, this work illustrates the potentiality of IPC to design responsive devices with well controlled interactions. Based on hydrogen-bonding complexes between PAA and P(AM-co-DMA) we have defined a large "pH-temperature" playground where responsive interactions can be used to trigger responsive adhesion between macromolecular surfaces prepared with the same polymers. Such work is currently undergoing in our laboratory and will be reported elsewhere.

Figure 1 .

 1 Figure 1. Temperature dependence of the transmission spectra of a ternary mixture PAA (0.25 wt%) / CopAM 69 (0.25 wt%) / water (99.5 wt%) at pH 2.93. (a) The spectra were measured every 2.5 °C between 7.5 °C and 70 °C (from right to left). (b) The temperature dependence of the transmission was determined every 50 nm from 350 nm to 800 nm (left to right).

Figure 2 .

 2 Figure2. "pH-temperature" phase diagram of the ternary system PAA/PAM/water. Cloud points were determined during heating (Δ) or cooling ( ). The one-phase system is at high pH, in the clear domain.

Figure 3 .

 3 Figure 3. Critical pH of the IPC formation (PAA/PAM) at room temperature as a function of polymer concentration in water (PAA or PAM). The one-phase system is at high pH

Figure 4 .

 4 Figure 4. "pH-temperature" phase diagram of the ternary system PAA/PDMA/water. Cloud points were determined while heating () or cooling (). The one-phase system is at high pH, in the clear domain.

Figure 5 .

 5 Figure 5. Salt dependence of the critical pH for hydrogen-bonding complex formation at 25 °C. All the ternary systems were studied in aqueous solutions at the same polymer concentration (0.25 wt% for each polymer):

Figure 6 .

 6 Figure 6. "pH-temperature" phase diagram of ternary systems PAA/P(AM-co-DMA)/water. The one-phase system is at high pH.

Table 1 .

 1 Composition and macromolecular characteristics of P(AM-co-DMA) copolymers.

	2)	Ð (2)

Acknowledgements

We thank Alicia Forstneric, Linh Bon Nguyen, Stéphanie Genain, Claire Goldmann and Clément Bottois for their experimental contribution to the realization of the phase diagrams.

We also gratefully acknowledge the ANR Blanc Programme: project ADHGEL for its financial support.


J L Autran

D Munteanu

S Moindjie

T Saad Saoud

S Sauze

G Gasiot

P Roche

ASTEP (2005-2015): Ten Years of Soft Error and Atmospheric Radiation Characterizations on the Plateau de Bure

This paper surveys ten years of experimentation conducted on the Altitude SEE (Single Event Effects) Test European Platform (ASTEP), a permanent mountain laboratory opened in 2005 on the Plateau de Bure (Devoluy, France) at the altitude of 2,552 m and primarily dedicated to the characterization of soft errors in electronic circuits subjected to terrestrial cosmic rays. The paper retraces the foundations of the project and gives an extensive overview of the different past, current and future experiments conducted on ASTEP in the fields of SER (Soft Error Rate) realtime testing and natural radiation monitoring and metrology.

Introduction

Since soft errors induced by terrestrial natural radiation have been considered as one of the most important primary limits for modern electronics reliability at ground level, determining the soft error rate (SER) of a given circuit is a major issue for IC manufacturers and users [START_REF] Autran | Soft Errors: from particles to circuits[END_REF]. In complement to the popular accelerated SER tests using intense particle sources or beams, realtime (RT) measurements represent a very accurate experimental solution to determine the SER from the monitoring of a population of devices subjected to the true natural radiation environment and operating under nominal conditions [START_REF] Autran | Introductory Invited Paper -Real-time soft-error rate measurements: A review[END_REF]. Conducting real-time tests is therefore not trivial because such experiments cannot be launched without a solid logistic, a dedicated test location and a long-term technical support. In order to benefit from a permanent facility precisely dedicated to RT testing, we have developed since 2005 a test platform at mountain altitude, the Altitude Test Singleevent European Platform (ASTEP). Located in south French Alps at 2552 m of elevation, ASTEP has been continuously used from this date to investigate the impact of atmospheric radiation (terrestrial cosmic rays primarily composed of atmospheric neutrons) on the SER of circuits manufactured in different technological nodes and various CMOS bulk technologies. ns This paper retraces the foundations of the project (Section 2) and gives an extensive overview of the different experiments conducted on ASTEP in the fields of SER (Soft Error Rate) real-time testing (Section 3) and natural radiation monitoring and metrology (Section 4). New additional data concerning real-time experiments on advanced SRAM technologies will be presented at the conference and included in the final paper, as well as details about the forthcoming experiments on CMOS SOI technologies and scientific perspectives for the future of ASTEP.

A brief history of ASTEP

The project to create an altitude laboratory dedicated to long-term experiments in the domain of soft error characterization in devices, circuits and systems was launched in 2001 by STMicroelectronics and JB R&D. The initial idea was to build a permanent facility with a dual academic and industrial research vocation, opened to the radiation effect community and to conduct, in the same location, both soft error rate (SER) measurements and terrestrial cosmic ray metrology. The project was formally named "Altitude SEE (Single Event Effects) Test European Platform" (ASTEP) in 2003; it has been supported by L2MP-CNRS laboratory (now IM2NP) in Marseille since this date. The same year, the site of the Plateau de Bure (Devoluy, France) and the host support of the institute for millimeter radio astronomy (IRAM) were definitively chosen. A host convention was signed in 2004 between CNRS and IRAM to formalize the installation of the ASTEP platform on the Plateau de Bure. In 2005, the ancient building "POM2" of the IRAM Observatory was fully rehabilitated by the City of Saint-Etienne en Dévoluy in order to host the scientific instruments of the platform (Fig. 1). The first equipments were installed on site in September 2005. The permanent network connection (wireless between the Plateau de Bure and the Devoluy valley) between ASTEP and IM2NP in Marseille was also successfully tested. In parallel, the first real-time experimental bench dedicated to the characterization of STMicroelectronics 130 nm SRAM memories was designed and integrated. The of the Xilinx's Rosetta experiment [START_REF] Lesea | The Rosetta Experiment: Atmospheric Soft Error Rate Testing in Differing Technology FPGAs[END_REF] (see Fig. [START_REF] Autran | Real-Time Soft-Error Testing of 40nm SRAMs[END_REF]. Additional results concerning thermal neutron sensitivity of these 40 nm SRAMs were presented at the 2012 Nuclear and Space Effects (NSREC) Conference [START_REF] Autran | Soft-Error Rate Induced by Thermal and Low Energy Neutrons in 40 nm SRAMs[END_REF]. In 2013, the first results concerning the characterization of flash memories subjected to natural radiation were presented at IRPS in Monterey (USA) [START_REF] Autran | Soft-Error Rate Induced by Thermal and Low Energy Neutrons in 40 nm SRAMs[END_REF]. The 40 nm experiment was stopped and transported to Marseille for sea level measurements. Finally, in 2014, a fourth generation of real-time setup embedding more than 4 Gbits of ST 28nm SRAMs manufactured in FD-SOI technology was designed and integrated. This setup will be installed on ASTEP in June 2015.

Atmospheric neutron and muon flux monitoring

Since 2008 and 2011, respectively, ASTEP is equipped with a neutron and a muon monitors. The two original instruments, designed and constructed by IM2NP for ASTEP, are shown in Fig. 3. Note that a clone copy of each instrument is available at IM2NP in Marseille on the new established TERRAMU platform [11]. Both monitors have been used to determine the acceleration factor of the real-time experiments with respect to sealevel conditions. Annual, seasonal and daily variations of the particle flux can thus be evidenced, as a function of atmospheric pressure, solar cycle variations and solar event (Forbush decreases). These points will be carefully detailed in the final paper and presentation.

Real-time SER experiments

Table 1 gives the current status of the different realtime experiments conducted on ASTEP, at LSM (Underground Laboratory of Modane) and at IM2NP in Marseille during the last decade. Two experiments have been recently moved from ASTEP (40 nm, see Fig. 4) and from LSM (65 nm) to Marseille. The final paper will report for the first time these additional sealevel measurements and will discuss the adequacy of these results with respect to the estimated contribution of the internal chip radioactivity. A status about the test of two flash technologies will be also reported.

Figure 1 .

 1 Figure 1. Evolution of the ASTEP building since 2004. The new cupola constructed in 2008 hosts, since this date, the Plateau de Bure Neutron Monitor (PdBNM). The configuration of the ASTEP main experimental room changed several times since 2005, as a function of hosted experiments. Since 2012, the Plateau de Bure Muon Monitor (PdBM2) is definitively installed in this room.

  2). One month later, the ST 130 nm SRAM setup was installed on ASTEP and RT measurements started on April 29, 2005. The first experimental measurements were presented at the European Conference on Radiation and its Effects on Components and Systems (RADECS 2006) in September 2006 in Athens [4]. In 2007, the rehabilitation works of the first floor of the ASTEP building (ancient cupola of the POM2 building) were launched during the summer period. Works were therefore interrupted by the snow and bad weather conditions in October. The construction of a second real-time test setup dedicated to ST 65 nm SRAM memories was launched. In January 2008, the 65 nm setup was installed on ASTEP. Rehabilitation works resumed at the end of May 2008. The Plateau de Bure Neutron Monitor (PdBNM) was installed on the first floor of the ASTEP building on July 23, 2008 (see Figs. 1 and 2). The instrument has been fully operational since this date. The first results concerning 65 nm SRAMs were presented at RADECS 2008 in Jyväskylä (Finland), in September 2008 [5]. In 2009, high temperature tests (85°C) were conducted on the 65 nm SRAMs to investigate Single Event Latchup (SEL) and micro-latchup mechanisms. In 2010, the ASTEP platform and collaboration program were presented during an invited talk at ESREF 2010 [6]. In 2011, a third real-time setup embedding more than 7 Gbits of ST 40 nm SRAMs was designed and integrated. The setup was installed on ASTEP in March (see Fig. 2). The Plateau de Bure Neutron Monitor joined the Neutron Monitor Database international network [7]. The Plateau de Bure Muon Monitor (PdBM2), developed at IM2NP-CNRS laboratory in Marseille, was installed on the ASTEP platform during July/August 2011. A new type of waferlevel characterization based on more than 50 Gbit of 90 nm NOR flash memories subjected to natural radiation was also launched. The first results concerning the 40 nm SRAM experiment were presented at the International Reliability Physics Symposium (IRPS 2012) in Anaheim (USA)

Figure 2 .

 2 Figure 2. Panel of SER real-time experiments conducted on the ASTEP platform since 2005.

Figure 3 .

 3 Photomul%plier+

Figure 4 .

 4 Figure 4. Definitive results for the 40 nm single-port SRAMs characterized in altitude on the ASTEP platform Top: Cumulative number of bit flips versus test duration. Test has been conducted under nominal conditions: V DD = 1.1V, room temperature, standard checkerboard test pattern. Bottom: Bit flip SER (FIT/MBit) versus test duration calculated from above data. The 90% confidence interval is also indicated (hatched area).

Table 1

 1 Summary of RTSER experiments conducted on the ASTEP platform and at LSM during the period 2006-2015.

	Platform	Circuit & Technology	Test temperature	Initial Mbit under test	Start date	Stop date	Effective hours of test Cumulated	Main Reference
	ASTEP			3,664	03/31/2006 11/26/2006	5,200	[4]
		130 nm SRAM	RT
	LSM			3,472	10/16/2007 10/15/2010	24,747	[6]
			RT	3,216	01/21/2008 05/07/2009	11,278	[5]
	ASTEP
			85 °C	2,884	06/26/2009 01/24/2013	28,482	[2]
		65 nm SRAM
	LSM		RT		04/11/2008 01/25/2015	57,058	[5]
				3,226
	Marseille		RT		03/03/2015	Ongoing	~ 2,500	This work
	ASTEP		RT		11/03/2011 06/03/2014	28,495	[8]
		40 nm SRAM		7,168
	Marseille		RT		10/16/2014	Ongoing	~5,000	This work
		90 nm Flash						[10]
	ASTEP		RT	~50,000	07/2011	Ongoing	> 32,000
		(wafer-level)
	ASTEP	55 nm Flash	RT	~40,000	11/30/2012	Ongoing	> 20,000	This work
		(wafer-level)

Acknowledgments

The European Regional Development Fund (ERDF) program, the Regional Council of Provence-Alpes Côte d'Azur and the General Council of Hautes Alpes initially supported the creation of the ASTEP platform. The different real-time experiments conducted on ASTEP have been supported through the MEDEA+ Project #2A704 ROBIN, the CATRENE Project # CA303 OPTIMISE (OPTImisation of MItigations for Soft, firm and hard Errors) and the DGA project EVEREST. The French Ministry of Economy, Finances and Industry has supported these programs under research conventions #052930215, #062930210, #092930487 and #132906128.