Giancarlo Ranalli
email: ranalli@unimol.it.

Elisabetta Zanardini

Alessia Andreotti

Maria Perla Colombini

Cristina Corti

Pilar Bosch-Roig

Paola De Nuntiis

Giuseppe Lustrato

Paolo Mandrioli

Laura Rampazzi

Carlo Giantomassi

Donatella Zari

Hi-tech restoration by two steps biocleaning process of Triumph of Death fresco at the Camposanto Monumental Cemetery (Pisa, Italy)

Keywords: Hi-tech restoration, frescoes biocleaning, Pseudomonas stutzeri, optimization of fresco support, anticondensation system

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

The Camposanto Monumental Cemetery in Pisa during the II World War in 1944 was badly damaged by a bomb; considering the extremely precarious conditions, a large number of frescoes was rapidly detached with the 'strappo' technique using animal glue and gauze to tear off the fresco from the original walls and stored, once rolled, without any rigid support. The frescoes (a total surface area of about 1,500 m 2 ) dated back to the 14 th century and were painted by Bonamico Buffalmacco, Antonio Veneziano, Benozzo Gozzoli, Taddeo Gaddi, Francesco Traini, and Spinello Aretino. Later, in the 1960's, some of them were placed onto an asbestos cement support (eternit) in order to carry on with the restoration intervention and to finally put them back onto the original walls, but in some cases alteration processes such as swelling and detachment of the fresco paint film were observed. Therefore, in the 2000's the frescoes were once again detached from the Camposanto Monumental Cemetery walls and submitted to a new delicate restoration work which foresaw the initial removal of the adhering organic matter. Traditionally frescoes are restored both by traditional chemical and physical techniques, based on the removal of organic residual substances and salts with the use of ammonium carbonate solution and organic solvents, but these methods in some cases fail as in the case of Camposanto Monumental Cemetery frescoes (Ranalli et al. 2003a).

Analytical data on frescoes from the same site affirmed that some of the altered frescoes exhibited hydrophobic behavior related to weathering and protein polymerisation from past restoration works [START_REF] Ranalli | The use of microorganisms for the removal of nitrates and organic substances on artistic stoneworks[END_REF][START_REF] Ranalli | Bioremediation of cultural heritage: removal of sulphates, nitrates and organic substances[END_REF]Ranalli et al. 2003a;[START_REF] Ranalli | Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes[END_REF][START_REF] Lustrato | Fast biocleaning of mediaeval frescoes using viable bacterial cells[END_REF]) and therefore the removal of such compounds by using the traditional methods resulted ineffective. For these reasons, a crucial step in restoration is the chemical characterization in order to distinguish the original organic materials and those deriving from past restoration works and it is pivotal to define if the materials have undergone through transformation and weathering processes. The use of sensitive, selective and if possible, not destructive techniques is fundamental and this include techniques such as gas chromatography coupled with mass spectrometry (GC-MS) and pyrolysis/GC-MS [START_REF] Lluveras | A GC/MS analytical procedure for the characterization of glycerolipids, natural waxes, terpenoid resins, proteinaceous and polysaccharide materials in the same paint micro sample avoiding interferences from inorganic media[END_REF][START_REF] Rampazzi | The stucco technique of the Magistri Comacini: the case study of St. Maria dei Ghirli in Campione d'Italia (Como, Italy)[END_REF][START_REF] Rampazzi | Technology of Medieval mortars: an investigation into the use of organic additives[END_REF][START_REF] Orsini | Analyical pyrolysis of proteins in samples from artistic and archaeological objects[END_REF]. Previous studies highlighted that, when traditional methods resulted ineffective, the use of enzymes, surfactants and solubilizing agents showed a great potentiality [START_REF] Makes | Enzymatic consolidation of the portrait of Rudolf II as "Vertumnus" by Giuseppe Arcimboldo with a new multi-enzyme preparation isolated from Antarctic Krill (Euphasia superba)[END_REF]Bellucci and Cremonesi,1994;Bonomi,1994;[START_REF] Wolbers | Archetype. Cleaning painted surface: Aqueous Methods[END_REF][START_REF] Polo | Feasibility of removing surface deposits on stone using biological and chemical remediation methods[END_REF][START_REF] Troiano | Successful combination of chemical and biological treatments for the cleaning of stone artworks[END_REF] together with the application of a selected viable bacterial cells as "bio-restoration and bio-cleaning" agents, which can be an alternative and innovative technique to clean both frescoes and altered stoneworks [START_REF] Antonioli | Art-loving bugs: the resurrection of Spinello Aretino from Pisa' ' cemetery[END_REF][START_REF] Cappitelli | Bioconservation of the marble base of the Pietà Rondanini by Michelangelo Buonarroti[END_REF][START_REF] Cappitelli | Advantages of using microbial technology over traditional chemical technology in the removal of black crusts from stone surfaces of historical monuments[END_REF][START_REF] May | The BIOBRUSH project for bioremediation of Heritage stone[END_REF][START_REF] Alfano | The bioremoval of nitrate and sulfate alterations on artistic stonework: the case-study of Matera Cathedral after six years from the treatment[END_REF][START_REF] Gioventù | Comparing the bioremoval of black crusts on colored artistic lithotypes of the Cathedral of Florence with chemical and laser treatment[END_REF][START_REF] Bosch-Roig | New frontiers in the microbial biocleaning of artworks[END_REF]Bosch-Roig et al. 2013a;Bosch-Roiget et al. 2013b;Bosch Roig et al. 2014;[START_REF] Bosch-Roig | The safety of biocleaning technologies for cultural heritage[END_REF][START_REF] Mazzoni | Laponite micro-packs for the selective cleaning of multiple coherent deposits on wall paintings: The case study of Casina Farnese on the Palatine Hill (Rome-Italy)[END_REF][START_REF] Bosch-Roig | Connecting phenome to genome in Pseudomonas stutzeri 5190: An artwork biocleaning bacterium[END_REF].

As previously mentioned, using the "strappo" or "tear-off" technique a notable quantity of organic compounds such as animal glue and casein was distributed on both surfaces; on the front, as a fixative or consolidation agent and on the back of the fresco as a support adhesive (Ranalli et al. 2003a;[START_REF] Ranalli | Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes[END_REF]Bosch Roig et al. 2014). Therefore, at the Camposanto Monumental Cemetery, some fresco sections showed situations and conditions very complex and different.

Since 2000, the success of this technology have led the Technical Commission for Restoration (Pisa, Italy) to accept and approve the use of Pseudomonas stutzeri viable cells for biological applications. In fact, the first application of biocleaning technique on frescos at the Monumental Cemetery in Pisa was reported in pioneering studies, by Ranalli et al. 2005, where a protocol that put together the synergic combination of hydrolytic enzymatic action with microbial metabolism, was described for the fresco restoration. The case study featured the restoration of one of the frescoes in the Cemetery at Pisa, "The Conversion of Saint Ephysius and Battle" (Conversione di S. Efisio e Battaglia) by Spinello Aretino, which, as a result of the dramatic consequences of bombing during World War II had been removed from the walls using the "tear-off" technique, covering the surface with a strong cloth bound with generous layers of animal glue [START_REF] Antonioli | Art-loving bugs: the resurrection of Spinello Aretino from Pisa' ' cemetery[END_REF][START_REF] Ranalli | Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes[END_REF]. When, 10 years later, restorers had attempted to remove the cloth, they had found that the glue had polymerised following historical addition of formaldeyde to the surface, thus prohibiting effective use of a number of surfactants and solubilizing agents. At that time, preliminary tests with single, or with a mixture of commercial and purified enzymes showed no positive effects. The use of bacterial cells was, thus, the only way to restore the altered noble fresco. The biocleaning technique was performed by direct application of whole viable bacterial cells of Pseudomonas stutzeri, A29 strain, onto the artwork surface, for 10-12 hours. The biological pack removed a great amount of the glue, leaving only a little residue on a limited surface area which was then eliminated with the purified enzymes, collagenase and protease. Years later, another positive biocleaning was performed in only three 3 hours on a medieval fresco "Stories of the Holy Fathers" (Storie dei Santi Padri) altered by traces of animal glue [START_REF] Lustrato | Fast biocleaning of mediaeval frescoes using viable bacterial cells[END_REF]. Another important matter is related to the safety of the application of microorganisms on artwork (Bosch and Ranalli 2014). Microbiological controls after the biocleaning processes are also very important to guarantee that no bacterial cells are left on the cleaned surface at the end of the treatment. Different techniques have been used such as ATP surface analysis and contact plate microbial counts. (Bosch-Roig et al. 2013a).

In this work, we report the ex situ large-scale biotreatment of Triumph of Death fresco (5.6 x 15.0 m) painted by Buonamico Buffalmacco in the period 1336-41. The conservation state of this fresco showed evident problems due to the color loss caused by pulverization processes, but also due to recent restoration practices that in the course of time resulted inappropriate. The study aimed to the setting up of a "hi-tech restoration process" based on a two-steps biocleaning of the proteinaceous residues (mainly animal glue and casein) present both on the front and back of the fresco paintwork, including the removal of the old asbestos cement support. In addition, the adoption of a new support with specific properties in terms of resistance, insulation and mechanical characteristics for the relocation on the original walls and lastly, the design of a continuous monitoring system of environmental conditions (temperature and humidity) on the fresco surface and in the surroundings are also illustrated.

Materials and methods

Triumph of Death fresco description

The Triumph of Death, Buonamico Buffalmacco's fresco (1336-41), is the first of a series of three scenes for the Camposanto Monumental Cemetery and was painted on Dominican monk commission. The fresco, based on the theme of the end of the world, is divided in different scenes dominated by the horrid, grotesque, comic and serenity images.

The best preserved areas are those located in the higher part of the fresco (in particular the battle between angels and demons) and the two side scenes. As previously described, the fresco was already extremely damaged by pulverization processes and after the bomb in 1944 (1947-1960) it was detached from the walls and glued in a framework screwed onto asbestos cement eternity supports. The whole Triumph of Death fresco (about 85 m 2 ) was divided totally in 14 sections.

Microorganism, media and bacterial cells production

Pseudomonas stutzeri A29 strain (DISTAM-DISTAAM Strains Collections, University of Milan and University of Molise, Italy) was used for the biocleaning process. Two different growing conditions where used:

-bacterial cells suspension F for front treatment: the strain was incubated at 28°C for 24-36 hours on Standard Plate Count broth medium (CM463, Oxoid Ltd., Basingstone, England) containing pancreatic digest of casein, 5.0 g l -1 supplemented by animal glue in pearl (CTS srl, Altavilla Vicentina, Italy) at 0.5% w/v; -bacterial cells suspension B for back treatment: the strain was incubated at 28°C for 24-36 hours on Tryptone Soya broth medium containing pancreatic digest of casein, 17.0 g l -1 (CM0129, Oxoid Ltd., Basingstone, England).

Bacterial suspensions containing exponentially growing bacteria, approximately 10 8 CFU ml -1 , were obtained by inoculating 10 ml of an overnight broth-culture into 1,000 ml fresh broth medium (in flask of 3,000 ml total volume) and incubating it in a shaker (200 rpm) for 24 h at 28°C. The cells were centrifuged at 7,000 g for 10 min at 4°C, washed twice with phosphate-buffered saline, and re-suspended in sterile bi-distilled sterile water, pH 6.9. Final cell concentration was above 10 8 cells ml -1 , corresponding to an O.D.560 nm of about 1.6 and the cells were used immediately or stored at 4 °C during conservation and/or transport from the laboratory to the field application. [START_REF] Ranalli | Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes[END_REF]. A laboratory batch fermenter (20 l volume), fully computerized (mod. Biostat C, B. Braun, Biotech International, Germany), was used to produce a suitable amount of viable bacterial cell biomass to apply in the biocleaning treatment of the whole Triumph of Death fresco.

Present day 14 th -century fresco restoration

The present restoration procedure involves several steps performed on the front (F) and on the back (B) of the fresco: the biological and chemical cleaning treatments and the fixing phase are reported in detail in Table S1.

Artificially altered specimen biotreatments

In order to evaluate the effectiveness of the biocleaning process, artificial altered specimens, simulating the presence of animal glue and casein residues were prepared, separately. Adequate water solutions prepared using animal glue in pearl (CTS srl, Altavilla Vicentina, Italy) at 5-0 g l -1 and pancreatic digest of casein 17.0 g l -1 dissolved at 80°C for 15 min under agitation (100 rpm), was used. These solutions were applied by soft manual brush as a thin layer directly on the upper surface of gauze specimens (20 x 20 cm). Once the animal glue or casein formed a thin film, about 30 min, the artificially altered specimens were weighted using analytical balance (mod. Crystal, Gibertini, Elettronica, Novate Milanese, Italy) and submitted to the biocleaning tests using the same conditions for frescoes. The change in weight of organic materials (animal glue and casein residues, added separately) before vs after biocleaning process was calculated and related to a removal efficacy of the ancient frescoes.

Chemical and physical analyses GC-MS and PY/GC-MS analyses

Reagents, apparatus, analytical procedure based on selective extraction and hydrolysis assisted by microwave and GC-MS analysis are detailed in the literature [START_REF] Lluveras | A GC/MS analytical procedure for the characterization of glycerolipids, natural waxes, terpenoid resins, proteinaceous and polysaccharide materials in the same paint micro sample avoiding interferences from inorganic media[END_REF]. Twelve samples were collected by scalpel from the different sections of Triumph of Death fresco and a characterisation of the organic adhesive used both to detach the fresco from the wall and the adhesion onto the asbestos support, was performed by GC-MS and PY/GC-MS [START_REF] Orsini | Analyical pyrolysis of proteins in samples from artistic and archaeological objects[END_REF]) analytical procedures. Moreover, to identify the natural organic compounds, their decay products and the effectiveness over the time, the analytical procedures previously adopted by the same authors were used [START_REF] Ranalli | Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes[END_REF][START_REF] Lustrato | Fast biocleaning of mediaeval frescoes using viable bacterial cells[END_REF]. The thickness of the glue layers was measured in different areas of the fresco using a laser sensor micrometer, Thrubeam LX2-V mod. (Keyence Corp., Osaka, Japan).

Scanning Electron Microscopy analyses

The morphology of some micro-fragments taken from the fresco after the biocleaning and the treatment with the anionic resin was observed with a FEI/Philips XL30 ESEM [START_REF] Corti | Thermal analysis and archaeological chronology: the ancient mortars of the site of Baradello (Como, Italy)[END_REF][START_REF] Corti | Hellenistic Mortar and Plaster from Contrada Mella near Oppido Mamertina (Calabria, Italy)[END_REF]. Every sample was analyzed "as is" in low vacuum mode (1 torr) at 20kV, by using GSE and BSE detectors.

Monitoring of viable cell counts and ATP determination during and after the biocleaning At time 0 and 3 hours of the biocleaning step, the evaluation of the total viable cells has been performed. Small samples taken from the wet cotton layer (1.0 g), were analyzed using the plate counting technique in Standard Plate Count Agar medium (SPCA, Difco), with incubation at 28°C for 48 hours and ATP content (see below). In addition, residual bacterial cell presence both on the front and back of the biocleaned surfaces was monitored immediately after the washing steps with distilled sterile water, and at time 0, 30 and 180 days. For the fresco microbiological monitoring, sterile velvet cloths were pressed onto the cleaned fresco surface and then onto SPCA medium (Difco) plates. After the incubation at 28 °C for 48 hours the colony forming units (CFU) per 25 cm 2 were expressed.

At the same time, bacterial activity was measured by total ATP assay using sterile cotton swabs allowing a not destructive sampling of the surface of ancient frescoes. A Biocounter P1500 luminometer (Lumac B.V.) equipped with a photomultiplier tube set at 7,200 Relative Luminescence Unit (RLU) with 200 pg ATP in 100 µl -1 of Lumit buffer and Lumit-QM reagent, was used (Ranalli et al. 2003b;[START_REF] Ranalli | Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes[END_REF]Bosch-Roig et al. 2013a).

Choice and utilization and of a new support for the relocation of the recovered fresco sections

The previous support (glass-resin) used for other frescoes from the same site showed some limits and evidenced the need to optimize a new system with better physico-chemical characteristics and properties. The new fresco's support is composed by the restored fresco mounted on canvas, aluminum-centered honeycomb heating fabric of polyester and carbon fibers (Termotex srl, Vicenza; Studio Progetti, Galliera Veneta, PD, Italy), (Figure S1). Moreover, an additional metallic structure in aluminum grid (De Simone, Firenze) on the back of canvas can guarantee the fixing of large final panels on the original mural walls. More detailed information of the new support is reported in result section.

Statistical analysis

Data of ATP content and viable bacterial cell counts, in triplicate media were expressed as mean and standard deviation compared statistically by the Tukey's b test at the 5% level with SPSS 11.5 (SPSS for Windows, Version 11.5, USA).

Differences between values at P < 0.05 are considered not statistically significant.

Results

The "hi-tech" complex restoration and biocleaning of the 14 th century fresco Triumph of Death is summarized in Table S1 and here reported in detail.

Two-steps biocleaning treatment at the front and back of the fresco

The ex situ biorestoration treatments were performed by a two steps application (front and back) of the P. stutzeri strain A29 bacterial suspension on the altered fresco as indicated in Materials and Methods section (Figure 1). After 3 hours of biotreatment using bacterial suspension F (at the front of the fresco) and B (at the back of the fresco), the bacterial activity was so intense that the treated areas were largely biocleaned and no residues of proteinaceous materials were presence. The treatment was soft and delicate and did not show any structural damage (Figure 2).

Residues of glue from the front and casein from the back of the fresco, present as thin layer at the beginning of the treatments, were completely cleaned. At the back only where small spots of calcium caseinate were thicker (as 1-2 mm), some residues were still noticed, but no greater than around 0.4 mm. Anyway for the Restorers the residual traces of glue and casein were at very low level not visible to the naked eye and by use of a magnifying glass.

Even if a prolonged bacterial treatment (6 hours) was possible, we excluded this duration in order to avoid any risks to the fresco; a longer contact of the moist cotton with the fresco surfaces could lead indeed to the possibility of swelling and detachment of paint fragments.

The optimized biocleaning process is here reported in detail.

st -step biocleaning on the front of fresco paint layer for the removal of glue residues

The selected procedure involves the following cleaning steps: i soft cleaning step to remove dirt and dust from the surface using Japanese paper and water (40°C). The Japanesesilk paper, 9 g per m 2 , natural white un-buffered (Klug Conservation, Walter GmbH & Co. KG., Immenstatd D), was applied to protect the front of painting layer;

ii biocleaning step using P. stutzeri A29 viable cells previously adapted in animal glue. Bacterial cell suspension F (about 2.0 liters per m 2 of fresco) was distributed to the artwork surface covered by paper and the hydrophilic cotton layer (0.5 cm high, 40 cm wide, 6-10 m long) utilized as delivery system, was abundantly embedded with the viable cells and then applied to the selected area for 3 hours. Sterile water and cotton was the negative control [START_REF] Ranalli | Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes[END_REF]. Great care was taken to avoid fractures, creases on the thin silk paper;

iii final soft cleaning step on the front of the fresco surface by distilled water (3 soft manual sponge applications); iv final chemical cleaning step using an anion exchange resin (Ionex OH, CTS srl, Altavilla Vicentina) to remove the organic residues. 2 nd -step biocleaning on the back of the fresco for the removal of casein residues i mechanical removal from the asbestos support. Japanese -silk paper, 9 g per m 2 , natural white un-buffered (Klug Conservation, Walter GmbH & Co. KG., Immenstatd D) was applied in this case to protect the back of painting;

ii biocleaning of the casein residues using bacterial cell suspension B (see above) previously adapted for casein degradation. The cotton layer as delivery system was abundantly brushed with the living cells and then applied to the selected area for 3 hours;

iii final soft cleaning step on the back of the fresco surface by distilled water (3 soft manual sponge applications).

Chemical analyses and SEM observation

The analyses by GC-MS and PY/GC-MS techniques evidenced the presence of proteinaceous material, mainly animal glue, used in the past to glue the gauze onto the front of the fresco ("strappo" technique) and casein from the calcium caseinate used on the back of the painting. In a few samples in which the paint layer was most abundant respect to the overlaying patina, egg was also present, suggesting his use as organic binder of a few details of the fresco or in the retouching. Figure 3a shows the pyrogram of one of the samples, with the specific markers identified for each protein.

Benzeneacetonitrile and indole for casein, pyrrole for animal glue, and hexadecanenitrile, octadecanenitrile and cholesterol for egg (other peaks in the pyrogram are not labeled for simplicity, but they are typical rearrangement products of the pyrolysis of these proteinaceous materials). Finally, the GC-MS amino acidic profiles confirmed that all the proteins had undergone degradation. After 3 hours of biotreatment with bacterial cells suspension F and three gently manual washing steps, the proteinaceous materials were bio-dissolved and not more detectable since below the instrumental limit and sensitivity of the method adopted (see the absence of the main markers of proteinaceous material (Figure 3b). In a preliminary study, the biocleaning removal efficiency was calculated in weight respect to the control. On the basis of the specific enzymatic activities, the removal efficiency of P stutzeri A29 cells (2 liters per m 2 fresco, time 3 hours, on casein and collagen substrates) was 2.31 and 3.83 mg µl -1 h -1 for caseinolytic and collagenolytic, respectively [START_REF] Antonioli | Art-loving bugs: the resurrection of Spinello Aretino from Pisa' ' cemetery[END_REF]. In this work and in our real operative conditions, it means that the maximum amount removal of proteinaceous materials was 13.8 g and 21.6 g per m 2 , respectively for caseinolytic and collagenolytic activity.

The effectiveness was confirmed by the data obtained with the artificially specimen.

As already indicated in Materials and Methods section, at the end of the restoration, the front fresco surface was treated with anionic resin to remove traces of organic residues. Some representative micro-samples from the fresco were observed by SEM, at higher magnifications, and confirmed the absence of microorganisms after the treatment with the anionic resin and the large presence of spherical particles. In fact, as we observed from the low-magnification picture (Figure 4), spherical particles were detected with the diameter-around 10 μm; the same morphology has been previously reported in literature in SEM analyses of anionic resins similar to those used at the end of the restoration work [START_REF] Gupta | Suspension copolymerization of styrene and divinylbenzene: Formation of beads[END_REF][START_REF] Albuszis | Synthesis and in-depth characterization of reactive, uniform, crosslinked microparticles based on free radical copolymerization of 4vinylbenzyl azide[END_REF].

Monitoring of the bio-cleaning process

The monitoring is one of the most crucial aspect, both during and at the end of the treatment. To determine both the presence and the viability of bacterial cells plate count technique and ATP content determination by bioluminescence have been used. At the end of the biocleaning of the fresco surface, and after the final washing with sterile distilled water, these analyses were performed on the fresco surface to check the presence of residual bacterial cells. Once both front and back of the fresco surfaces were in fact completely dry, the presence of viable bacteria was checked immediately at time 0, 30 and 180 days following the biocleaning treatment in order to verify that no live bacteria remained on the surface of the artwork (Table 1).

The results of the plate count monitoring confirmed the absence of microbial cells detectable. These data were also confirmed by the total ATP content values, which were 0.015 -0.012 pg per 25 cm 2 immediately after biocleaning, both adopting bacterial suspension F or B (respectively for removal of animal glue or casein), 0.015-0.016, and 0.017-0.018 pg per 25 cm 2 at time 30 and 180 days. While P. stutzeri A29 bacterial cells used for the biocleaning treatment of altered animal glue (front) or casein residues (back fresco) showed an ATP average value over 21,000 pg ml -1 related to a viable cell value of 2*10 8 CFU ml -1 .

The average of colony forming units (CFU) and total ATP content as expression of microbial activity immediately after biocleaning (T0), at T30 and T180 days did not show significant differences (P < 0.05) both for the front and the back of the biocleaned artwork.

Microclimatic monitoring and new type of support

The microclimatic and environmental characterization study of Pisa's Camposanto highlighted in 2011 the need to provide a protection system against the formation of condensation of atmospheric humidity and the deposition of fine particles on the surface of the frescoes already restored and waiting for installation on the original walls. The solution adopted is based on the back heating of the frescoes in order to maintain the pictorial surface at temperature values slightly above the dew point temperature or the temperature at which of the water vapor must condense into liquid water. The new support, known as Compolam or Hexelite, is a panel of aluminum-centered honeycomb used extensively in aircraft industry because of their extreme rigidity, strength and lightweight. The sandwich panel has outer surfaces in fiberglass reinforced epoxy resin (Figure S1). Compolam is a complex material, which requires particular processes and therefore its production is limited in quantities and customized for unique use. The cost indicatively ranges between 80 and 120 €/m 2 . In addition, to complete the final relocation on the original wall, all parts of the fresco were fixed on a special aluminum structure with an additional cost ranging between 100-150 €/m 2 (De Simone, Firenze, IT). The protection of the frescoes occurs through heating of the painted surfaces that is achieved using heating fabric of polyester and carbon fibers adopted for military and civilian heated clothing. The low power supply voltage, the absence of metallic conductors and the reduced energy consumption, guarantee the long time reliability of this application. The heating sheets are based on a traditional manufacturing way, that is a longitudinal warp and a transversal weft, mainly in polyester yarns. At the ends of the fabric, or in the warp (lengthwise), tinned copper filaments are inserted to form two bands, about one cm wide each, which serve to feed the fabric. Transversely, or in the weave (in the sense of width), carbon filaments are inserted into continuous fibers that intersect the two copper bands at the ends of the warp. There are therefore many parallel strands, spaced apart by a few centimeters. The carbon fiber behaves like a current conductor characterized by a specific resistance such as to generate heat (due to Joule effect) when it is crossed by an electric current. Therefore, the carbon fibers inserted into the fabric behave as parallel resistors that take power from the two copper bands. The result was a fabric that, once powered by an appropriate electrical source, generated homogeneous heat over the entire surface, according to the desired temperature, the size of the fabric and the supply voltage. The fabric thus produced was then coupled on both sides with a thin polyurethane film which guaranteed an electrical insulation, an adequate protection of the fabric and a good mechanical stability. One of the two sides was then coupled with an expanded material and finally with a reflecting film to ensure good thermal insulation on the back side (passive) thus maximizing the heat transfer on the active face of the sheet which is placed in contact with the back fresco surface. In addition, to the conductive component, a high radiating component is obtained which also heats by infrared irradiation. So, the final support produced looks like a sandwich of several layers, of a uniform overall thickness of about 5 mm, robust but light, cut to size with a common scissor, able to develop specific powers up to a few hundred Watts / m 2 and temperatures up to one hundred Celsius degrees. In our specific case at Pisa, after relocation of the frescoes mounted on the new heated panels (Figure 5), the protocol adopted guaranteed to obtain a surface temperature of the frescoes at least 2°C higher than the air temperature, so as not to give the water vapor of the The charts (Figure 6 and Figure 7) show the frescoes surface temperature Ts and the dew point temperature Td. The vapor condensation conditions occur when the dots/yellow curve (Td) exceeds the solid/green curve (Ts). The unheated panel shows the condensation conditions during the same period. The variation in surface temperature between heating power-on and off is about 2.5°C.

Anti-condensation system

The water vapor condensation events are frequent in winter period (about ten) from November to February, when the moist air masses (above 90% RH) comes from the Tyrrhenian Sea, SW direction, and can last for a long time up to one week. During the microclimatic monitoring period (2007-2017) these phenomena mainly affected the frescoes of the south gallery, particularly the southeast corner, and northwest corner of the north gallery [START_REF] Mandrioli | Il monitoraggio assistito per la conservazione preventiva dei beni culturali: dal progetto all'applicazione[END_REF]

Discussion

The "hi-tech" complex biocleaning of the 14th-century fresco Triumph of Death reported in this paper is a new case of biorestoration in which the use of bacteria has been successfully applied to Cultural Heritage. The novelty in this study is the 2-steps biocleaning process using two different bacterial suspensions (F and B) of Pseudomonas stutzeri A29 viable cells; this biocleaning allowed the removal in 3 hours of both the animal glue and the residues of calcium caseinate, respectively from the front and the back of the fresco.

These results confirm the efficiency and degradation ability of the selected bacterial strain previously applied for recovering other valuable frescoes from the same site, as the Last Judgment and Hell [START_REF] Colombini | Gas chromatography/mass spectrometry for the characterization of organic materials in frescoes of the Monumental Cemetery of Pisa (Italy)[END_REF][START_REF] Ranalli | Biotechnology applied to cultural heritage: biorestoration of frescoes using viable bacterial cells and enzymes[END_REF]) and the Stories of the Holy Fathers [START_REF] Lustrato | Fast biocleaning of mediaeval frescoes using viable bacterial cells[END_REF]) by Bonamico Buffalmacco. The monitoring of the bacterial cell counts and cell viability during the bio-cleaning process and after the washing steps at time 0, 30 and 180 days shows that residual bacteria on both the surfaces (front and back) are not detectable after the biotreatment. Therefore, all the biocleaned frescoes monitored (five sections of Triumph of Death fresco) did not show any risk of biological re-contamination in the conservation period of about 8 months under controlled indoor conditions before their relocation onto the original wall at the Camposanto Monumental Cemetery (OpaPisa).

These data confirm that the adopted procedure does not leave residual cells on the fresco surfaces as we already showed in previous works and therefore it is a fast, safe, selective and free-risk methodology that can be utilised in Cultural

Heritage restoration field for the removal of different stone alterations (Ranalli et al. 2003a;[START_REF] Lustrato | Fast biocleaning of mediaeval frescoes using viable bacterial cells[END_REF]. In fact, bio-restoration through the use of not pathogenic selected bacteria shows no risk for human health and the environment, and represents a great potentiality for removing undesirable compounds from the surface of works of art also in complicated cases as the Triumph of Death fresco where two steps treatment (front and back) are required.

Therefore, in all these years, the "bio-restoration" technology has been continuously employed on about 1,500 m 2 of the medieval frescoes, where the critical conditions of several of them imposed further studies and changes/optimization in the working protocols.

Therefore, the Triumph of Death fresco biorestoration represents a novel and important case of a large scale application of viable bacterial cells and shows that this biotechnology can be extremely useful even when situations are more complex and related to the both side of the fresco (front and back).

With regards to the fresco support, the conservation scientists noticed undesired aspects following the utilization of the old glass-resin support. After the re-location to the original wall, a visible deformation of the glass resin has been observed with a high risk for the fresco surface caused by mechanical forces and absence of uniformity. In addition, phenomena of condensation on frescoes surfaces were frequently reported due to the fact that the walls of the galleries are facing the open-air interior courtyard. A further novelty in this work is represented by the special effort focused on the optimization of a new support with high technological properties and the planning of a continuous monitoring of the environmental temperature and humidity for the prevention and conservation of the recovered frescoes. The new support, known as Compolam or Hexelite, used for the first time, together with the design of an environmental monitoring system represent an innovative anti-condensation system for the solution for these problems and guarantee indeed a better preservation and conservation of these valuable frescoes.

In conclusion, the findings obtained in the Pisa Camposanto Monumental Cemetery have a significance and relevant impact in fresco restoration field and represent a real example of "active" preventive conservation where the biorestored and relocated frescoes, after 74 years since their detachment from the walls, are fixed on a new and innovative support and protected by condensation phenomena and deterioration processes.

This study is of a great importance since the Camposanto Monumental Cemetery will be fully re-opened in June the 17 th 2018 for its 750 th construction anniversary.

The remarkable results achieved in the large scale investigations on medieval frescos, using the proposed advanced biocleaning techniques, clearly show the success of this methodology for an extraordinary "resurrection" of degraded works of art, also in very complicated and delicate conditions such as the Triumph of Death fresco, defined for its dimension and artistic importance the 'Pisa's Sistina frescoes'. The biotechnology proposed is highly efficient, safe, non-invasive, risk-free and very competitive compared to the traditional cleaning methods. These findings can be of significant importance for further future new restoration activities and they are crucial for determining preservation strategies in this field.

Fixing by brush

The wet surfaces show altered animal glue residues.

Fixing is adopted when and where needed.

Here, after 3 hrs, animal glue residues are bioremoved.

With steam water, residual effluent appears of milk colour.

Step Here, after 3 hrs, caseinate residues are bioremoved.

By combining cleaning (solvent, bacteria and resin) results are satisfactory and colours appear bright.

Final adhesion onto new honeycomb panel: Alglass fiber and epoxy resin (Compolam)

A thermal blanket is interposed between the wall and the back-paint to avoid risks of condense on fresco surface.

Figure 1 -

 1 Figure 1 -Biocleaning 1-step on the front surface of "Triumph of Death" fresco altered by animal glue residues.

Figure 2 -

 2 Figure 2 -Particular of "Triumph of Death" fresco surface after the 1-step biocleaning.

Figure 3 -

 3 Figure 3 -Pyrogram of a sample from Triumph of Death front fresco surface with the specific markers identified for each protein (benzeneacetonitrile and indole for casein, pyrrole for animal glue, and hexadecanenitrile, octadecanenitrile and cholesterol for egg).

Figure 4 -

 4 Figure 4 -SEM image of a representative sample from the front fresco after the biocleaning and the anionic resin treatment (bar = 10 µm).

  2 and activity measured by ATP content (pg) per 25 cm 2 present on front and back of the of the Triumph of Death fresco surface, at time 0, 30 and 180 days after the 2-steps biocleaning. Standard Deviation (SD)were carried out by the Turkey test: mean values and standard deviation (SD) with the same superscript letter were not significantly different (P < 0.05).

  air a cold surface on which to condense. Through a constantly microclimatic monitoring, a new-casting model automatically provides the signals required to activate the heating of the frescoes and prevents the beginning of vapor condensation and the dust deposition on the painted surfaces.

Figure 5 -

 5 Figure 5 -Relocation of the frescoes mounted on the new heated panels onto the Camposanto Monumental Cemetery original walls.

Figure 6 -

 6 Figure 6 -Fresco on unheated panel. The vapor condensation conditions occur when the dew point temperature (dots) exceeds the fresco surface temperature (solid). Condensation events on 22 to 24 February 2017.

Figure 7 -

 7 Figure 7 -Fresco protected with heated panel. The vapor condensation conditions occur when the dew point temperature (dots) exceeds the fresco surface temperature (solid).

  by Al frame. Noble paint is covered by polyethylene film and fixed to new multilayer support Ammonium caseate was used in the old restauration for adhesion to asbestos support.

Table 1 -

 1 Total viable bacterial counts (CFU) per 25 cm

  as described previously (P. Mandrioli and P. De Nuntiis, presented at 23 th Fair of the Conservation, Technology of Cultural and Environmental Heritage, Ferrara, IT, 22-24 March 2017).

Table S1 -

 S1 Phases of the restoring intervention of the Triumph of Death fresco which included the two-steps biocleaning of altered animal glue and casein removal on front (F) and back (B), respectively, carried out by the application of P. stutzeri A29 cells.

	Fresco	Restoring phases Materials and Products		Methodology	Notes
	surface
	Step 1	Preliminary	Demineralised water, silk paper (japico	At room temperature,
		cleaning	612340 12 g/m 2 )			brush, sponge
		Fixing	Silk paper, Ammonium caseinate;	Large brush in wooded
			Lactic caseinate, ammonia;		handle (n°60)
	Front (F)	Biocleaning F	P. stutzeri A29 cells and growth on	Application by large
			SPCB medium + animal glue.		brush and cotton layers.
		Chemical	Anionic resin (Ionex OH, silk paper,	Cleaning by compress
		cleaning (1)	ethyil	alcohol,	nitro	solvent,	covered	by
			polyethylene film. Ratio 50:50 (v/v) of	polyethylene film to
			nitro solvent and alcohol.		reduce	evaporation
			10 % Paraloid B82 in blenched alcohol	solvents. Rinse by
		Localised fixing	94.				steam water.

ACKNOWLEDGEMENTS

The work was supported partially by the Opera Primaziale Pisana (OPA) Pisa, Ing. G. Bentivoglio and partially by the VAL I + d APOSTD/2013/024 project by Generalitat Valenciana, Spain. The Authors wish to thank the OPA Scientific Committee, under the direction of Prof. A. Paolucci, and the OPA Restoration Laboratories for the contribution of the conservation scientists, specifically S. Lupo and C. Pucci. The Authors would like to devote this manuscript to the memory of Donatella Zari. Finally, the Authors are grateful to S. Pool for editing the manuscript. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.

AUTHOR CONTRIBUTION

GR, EZ coordinated the project and participated in the conception of the work, in the analysis of results, and in the writing the draft of the manuscript, AA, MPC, PDN, PM, LR contributed to design the experiment, analysis of the data, and contributed in manuscript preparation and its critical revision, PB, GL contributed in the execution of part of the experimental work and in the acquisition of the data, CC contributed to the chemical analysis of samples, CG, DZ coordinated and supervised all the conservation activities. All authors reviewed the results and approved the final version of the manuscript.

SUPPORTING INFORMATION


Jeroen Louwsma

Alain Carvalho

Jean-François Lutz

Stéphane Joly

Delphine Chan-Seng
email: delphine.chan-seng@ics-cnrs.unistra.fr

Adsorption of phenylalanine-rich sequence-defined oligomers onto Kevlar

Keywords: Adsorption, phenylalanine, Kevlar fibers, interfacial agent, sequence-defined oligomer

come

INTRODUCTION

As recently highlighted, the future of polymer science requires the development of new generations of high-performance polymers as well as the rejuvenation of classical polymer materials. [START_REF] Abd-El-Aziz | The next 100 years of polymer science[END_REF] Fiber-reinforced thermoplastic composite materials (FRTCM) are widely used in a large number of applications including civil engineering, [START_REF] Bakis | Fiber-reinforced polymer composites for construction-State-of-the-art review[END_REF][START_REF] Uddin | Developments in fiber-reinforced polymer (FRP) composites for civil engineering[END_REF] aircraft, [START_REF] Soutis | Fibre reinforced composites in aircraft construction[END_REF] and automotive [START_REF] Beardmore | The potential for composites in structural automotive applications[END_REF][START_REF] Holbery | Natural-fiber-reinforced polymer composites in automotive applications[END_REF] industry. The growing interest for FRTCM, commonly using glass [START_REF] Sathishkumar | Glass fiber-reinforced polymer composites -a review[END_REF] and carbon [START_REF] Chung | 2 -Introduction to carbon composites[END_REF] fibers, is driven by the substitution of metal components to develop materials having similar or higher performances compared to traditional materials like steel and aluminum. The main advantages of FRTCM are their higher strength-to-weight ratio used to decrease the weight of vehicles and thus fuel consumption and CO 2 production, their higher resistance to corrosion, and the ability to tune the performances according to their composition and design. Para-aramid fibers provides lightweight, strength superior to steel, and high impact and abrasion resistance. While its tensile strength (3.6 GPa) and tensile modulus (131 GPa) are similar or superior to those of glass fibers (3.4 and 72 GPa respectively), but inferior to those of carbon fibers (4.1 and 242 GPa respectively), [START_REF] Erden | 3 -Fiber reinforced composites[END_REF] its density is lower (1.4 g cm -3 as compared to 2.5 and 1.8 g cm -3 for glass and carbon fibers respectively) electing Kevlar fibers as a competitor to carbon fibers and showing superiority to glass fibers in terms of strength-toweight ratio.

FRTCM involves fibers as reinforcement constituents embedded in a polymer matrix such as polypropylene that hold the reinforcement constituents together. The proper control of the interface between the fibers and matrix and thus the mechanism of interfacial adhesion are beneficial to reach FRTCM with improved structural integrity and mechanical properties. [START_REF] Kim | Engineered interfaces in fiber reinforced composites[END_REF] Various approaches focused on the surface treatment of fibers have been developed to optimize the adhesion at the fiber-matrix interface through either chemical etching to induce fiber surface roughening or chemical modification to introduce functional groups at the fiber surface. In the case of Kevlar fibers, chemical etching has been conducted using different strategies such as bromination, [START_REF] Lee-Sullivan | Effects of bromination and hydrolysis treatments on the morphology and tensile properties of Kevlar-29 fibres[END_REF] chlorosulfonation, [START_REF] Lin | The effect of chemical treatment on reinforcement/matrix interaction in Kevlar-fiber/bismaleimide composites[END_REF][START_REF] Wu | Kevlar fiber-epoxy adhesion and its effect on composite mechanical and fracture properties by plasma and chemical treatment[END_REF] and plasma treatment, [START_REF] Wu | Kevlar fiber-epoxy adhesion and its effect on composite mechanical and fracture properties by plasma and chemical treatment[END_REF][START_REF] Su | The effect of oxygen-plasma treatment on Kevlar fibers and the properties of Kevlar fibers/bismaleimide composites[END_REF][START_REF] Mathur | Modification of mechanical properties of Kevlar fibre by polymer infiltration[END_REF] while amine, [START_REF] Wu | Chemical modification of Kevlar fiber surfaces and of model diamides[END_REF] alkoxysilane, [START_REF] Ai | Effect of grafting alkoxysilane on the surface properties of Kevlar fiber[END_REF] alkyl, [START_REF] Takayanagi | A new chemical method of surface-treatment of Kevlar fiber for composites with epoxy resin[END_REF] and phosphoric acid [START_REF] Li | Interface correlation and toughness matching of phosphoric acid functionalized Kevlar fiber and epoxy matrix for filament winding composites[END_REF] groups have been inserted at the surface of Kevlar fibers.

In the field of polymer blends, a common approach is the compatibilization by addition. Various types of additives have been investigated including copolymers [START_REF] Pospiech | Influencing the interface in polymer blends by compatibilization with block copolymers[END_REF] and Janus particles. [START_REF] Cardinaels | Chapter 8 -Compatibilization of polymer blends by Janus particles[END_REF] These materials are tailored to have sections compatible to those of the polymers composing the blend through specific interactions such as hydrogen bond and dipole-dipole interactions. [START_REF] Utracki | Compatibilization of polymer blends[END_REF] The compatibilizer should place itself at the interface of the incompatible polymers modifying the molecular structure of the interface and reducing the interfacial tension for a better stabilization of the polymer blend morphology and interfacial adhesion. Examples of this approach applied to Kevlar fiber-reinforced thermoplastic composite materials are sparser focusing generally on the use of maleic anhydride-grafted polyolefins, a reactive compatibilizer, and combined in some cases to surface-modified Kevlar fibers, leading to improved tensile strength and modulus of the composite materials. [START_REF] Saikrasun | Kevlar reinforcement of polyolefin-based thermoplastic elastomer[END_REF][START_REF] Ahmad | Effects of fiber composition and graft-copoly(ethylene/maleic anhydride) on thermoplastic natural rubber composites reinforced by aramid fiber[END_REF][START_REF] Maity | Direct fluorination of Twaron fiber and preparation of PP/Twaron fiber composites using MA-g-PP as a compatibilizer[END_REF][START_REF] Fu | Combined effect of interfacial strength and fiber orientation on mechanical performance of short Kevlar fiber reinforced olefin block copolymer[END_REF] It has been shown in recent years that sequence-defined oligomers open up interesting new avenues in materials science; [START_REF] Hartmann | Polymers for control freaks: Sequence-defined poly(amidoamine)s and their biomedical applications[END_REF][START_REF] Badi | Microstructure control: An underestimated parameter in recent polymer design[END_REF][START_REF] Trinh | Synthesis of monodisperse sequence-defined polymers using protecting-group-free iterative strategies[END_REF][START_REF] Solleder | Recent progress in the design of monodisperse, sequence-defined macromolecules[END_REF] in particular for blends and composites compatibilization. [START_REF] Perry | 100th Anniversary of macromolecular science viewpoint: Opportunities in the physics of sequence-defined polymers[END_REF] The group of Simmons has recently demonstrated by molecular dynamics simulations that the use of copolymers with specific sequences as interfacial agents in polymer blends can better reduce interfacial energy than block or random copolymers. [START_REF] Meenakshisundaram | Designing sequence-specific copolymer compatibilizers using a molecular-dynamics-simulation-based genetic algorithm[END_REF] Furthermore, the group of Börner has identified peptide-polymer conjugates with specific peptide sequences able to act as interfacial agents for poly(ethylene oxide)/magnesium fluoride nanoparticle composite materials. [START_REF] Samsoninkova | Peptide-polymer conjugates for bioinspired compatibilization of internal composite interfaces: via specific interactions toward stiffer and tougher materials[END_REF][START_REF] Hanßke | Via precise interface engineering towards bioinspired composites with improved 3D printing processability and mechanical properties[END_REF] As the sequence of the repeat units constituting the interfacial agent has been identified as beneficial in the improvement of the interfacial adhesion in FRTCM, sequence-defined interfacial agents for Kevlar fiber-reinforced polyolefin composite materials have been designed considering as repeat units phenylalanine to interact with Kevlar fibers through π-π interaction and hydrogen bond, and alkyl chains to be compatible with the polyolefin (i.e. poly(propylene-co-ethylene)) matrix using an iterative synthesis on a solid support. [START_REF] Chan-Seng | Synthesis of macromolecules containing phenylalanine and aliphatic building blocks[END_REF] Herein, a library of sequence-defined oligomers (Figure 1) were prepared and deposited on Kevlar fibers by dip coating technique to investigate the effect of the number of successive phenylalanine units and the length of the alkyl chains on the coating integrity and quantity adsorbed on the fibers. A comparative study was conducted with model molecules in the attempt to understand which parameters drive the adhesion between Kevlar fibers and the polyolefin matrix.

RESULTS AND DISCUSSION

Synthesis and thermal characterization of phenylalanine-based oligomers

The oligomer HOOC-(Fh) 3 Fh-N 3 , where F stands for the phenylalanine unit, h the hexyl chain and  for the triazole group, was prepared by performing four repeating cycles by successive addition of Fmoc-L-Phe-OH (followed by Fmoc removal), 6-azidohexanoic acid, and propargyl amine on a 2-chlorotrityl chloride resin as previously reported. [START_REF] Chan-Seng | Synthesis of macromolecules containing phenylalanine and aliphatic building blocks[END_REF] The library of oligomers was first extended to evaluate the influence of the presence of four successive phenylalanine units per repeating cycles affording HOOC-(F 4 h) 3 F 4 h-N 3 . Due to difficulties in synthesis, the 2-chlorotrityl chloride resin was replaced by Tentagel®-S-PHB prefunctionalized with Fmoc-Phe-OH with a low loading in phenylalanine (0. oligomers obtained with incomplete reaction steps) and PEG (i.e. PEG leakage due to acidic hydrolysis of ester linkage between PEG and polystyrene composing the Tentagel resin). The observation of the molecular peak of the oligomers was ardeous due to the difficulty to ionize them. Due to their limited solubilities in common solvents and requiring the addition of a relatively large amount of acids (i.e. at least 5 vol%), their purification by standard techniques, e.g. high-performance liquid chromatography, could not be achieved and the oligomers were used without further purification.

The preparation of composite materials requires working at a temperature above the melting temperature of the polymer matrix. As poly(propylene-co-ethylene) was aimed as polymer matrix and its melting temperature is 137 °C, the working temperature to process the composite material was expected to be at least 167 °C. Thus, the molecules to be used as interfacial agents should not thermally degrade at or below this temperature. The thermal stability of the oligomers synthesized was studied by thermogravimetric analysis. HOOC-(Fh) 3 Fh-N 3 was previously investigated showing at 167 °C a cumulative weight loss of 7 wt% attributed to the loss of the terminal groups, [START_REF] Chan-Seng | Synthesis of macromolecules containing phenylalanine and aliphatic building blocks[END_REF] showed an initial degradation temperature between 214 and 230 °C (ESI, Figure S6). The inobservance of earlier degradation for these oligomers could be attributed to their higher molecular weight and thus a contribution of the terminal groups corresponding to 2 wt% that may be difficult to detect.

Adsorption of interfacial agents on Kevlar fibers

Dip coating is a simple and widely used technique to deposit a substance dissolved in a solution onto a substrate as a film at its surface. [START_REF] Tang | Dip-coating for fibrous materials: Mechanism, methods and applications[END_REF] The deposition of the synthesized interfacial agents onto Kevlar fibers was considered through dip coating individual Kevlar fibers in 95/5 acetonitrile (ACN)/trifluoroacetic acid (TFA) solutions at a concentration of 1 mM of interfacial agent. After being air-dried overnight, the fibers were analyzed by SEM (Figures 23). For all the interfacial agents considered, the coating integrity on the fibers was low showing heterogeneous deposition as depicted for HOOC-(Fh) 3 Fh-N 3 in Figure 2. The adsorption of the oligomer on Kevlar was attempted to be further characterized by energy dispersive X-ray spectroscopy (EDS). However, the close chemical composition of the oligomers and Kevlar fibers (ESI, Table S1) and the effect of the nature of these samples (curved surface, heterogeneity of the coating, etc.) on the accuracy to determine the composition limited the possibility to discriminate precisely the oligomers from Kevlar fibers. For these reasons, the adsorption of all the oligomers on Kevlar fibers was thus conducted qualitatively and quantitatively by SEM and gravimetrical analysis respectively.

To estimate the contribution of the triazole moiety through hydrogen bond and dipole-dipole interactions, [START_REF] Valverde | Synthesis of a biologically active triazole-containing analogue of Cystatin A through successive peptidomimetic alkyne-azide ligations[END_REF] HOOC-(Fh) 4 -N 3 was considered. No significant difference in the coating formation was observed between HOOC-(Fh) 3 Fh-N 3 and HOOC-(Fh) 4 -N 3 indicating no significant influence of the triazole group on their adsorption on Kevlar fibers. Furthermore, as phenylalanine is expected to adsorb on Kevlar fibers thanks to potential π-π interactions and hydrogen bond, the relevance of the choice of phenylalanine as anchoring unit to promote the adsorption of the oligomers on Kevlar fibers was evaluated. The contribution of these interactions was investigated using oligostyrene (PS, possessing solely π-π interactions, solubilized in toluene instead of 95/5 ACN/TFA), tetraglycine (HOOC-G 4 -NH 2 , involving hydrogen bond), and tetraphenylalanine (HOOC-F 4 -NH 2 , combining π-π interactions and hydrogen bond). The fibers dipped in the solution of PS showed patches of coating on the fibers. The dewetting phenomenon observed (even more pronounced after six months after the deposition on the fiber, ESI, Figure S7) could be attributed to low interactions between PS and the fibers. The presence of HOOC-G 4 -NH 2 deposited on Kevlar fibers was difficult to detect. The distinction between the bare Kevlar fibers and the fibers coated with HOOC-G 4 -NH 2 was complexified by the inhomogeneity observed at the surface of bare Kevlar fibers (ESI, Figure S8) that may be due to some remaining finish on the fibers besides the treatment performed by the supplier. If HOOC-G 4 -NH 2 was present on the fiber, the coating seemed to be thin and relatively smooth. The fibers treated with HOOC-F 4 -NH 2 exhibited the presence of a coating that seemed to be more prominent as compared to HOOC-G 4 -NH 2 and more homogeneous than when PS was used. These experiments emphasize the need of the combined contribution of π-π and hydrogen bond interactions provided by phenylalanine residues for the adsorption of interfacial agents on Kevlar fibers. The structure of the oligomers synthesized was further tuned by first increasing the number successive phenylalanine units on the oligomer. HOOC-(F 4 h) 3 F 4 h-N 3 (i.e. oligomers with four successive phenylalanine units per repeating cycle) was deposited on Kevlar fibers by dip coating showing a more prominent presence on the fibers emphasized by the cracks observed on the coating that was relatively homogeneous (Figure 3). The influence of the length of the alkyl chains was then investigated using oligomers with spacing units comprising three, four, five, and ten methylenes (HOOC-(

F 4 b) 3 F 4 b-N 3 , HOOC-(F 4 v) 3 F 4 v-N 3 , HOOC-(F 4 h) 3 F 4 h-N 3 , HOOC-(F 4 u) 3 F 4 u-N 3 respectively
). Among these oligomers, the fibers coated with HOOC-(F 4 v) 3 F 4 v-N 3 exhibited the roughest and thickest coating at the surface of Kevlar fibers (Figure 3). The roughness of the coating may be advantageous when embedding the fibers in the polymer matrix. The interfacial adhesion between the fiber and the polymer matrix may be enhanced acting as nucleating agents for the crystallization of the polymer matrix in the close proximity of the fiber. [START_REF] Batista | Correlation between degree of crystallinity, morphology and mechanical properties of PPS/carbon fiber laminates[END_REF] The fibers treated with HOOC-(F 4 v) 3 F 4 v-N 3 were characterized again by SEM after six months to evaluate their stability: some fibers remained visually unchanged, while others exhibited some delamination (ESI, Figure S9).

Quantification of interfacial agents deposited onto Kevlar fibers

The previously prepared fibers were not suitable for gravimetric quantification due to the weight of one Kevlar fiber (i.e. approximatively 9 µg per fiber having a length of 5 cm). Kevlar fiber pellets were thus prepared by placing approximatively 75 mg of fibers (i.e. corresponding to an estimated number of 8000 fibers) in a stainless-steel crucible and compressing them using a hydraulic press. The pellets obtained had a diameter of 13.37 ± 0.13 mm and a height of 1.15 ± 0.03 mm (ESI, Figure S10 and Table S2). These of oligomer adsorbed on the fibers was determined by gravimetry (Table 1). The results for the pellets treated with the oligomers were relatively consistent. The average value of the number of moles of oligomers adsorbed per gram of fibers was similar for these oligomers ranging from 3.84 to 5.21 µmol g -1 without any identified trend associated to the length of the spacing unit. However, it seemed that the rougher surface of HOOC-(F 4 v) 3 F 4 v-N 3 might be associated to higher adsorption density in the oligomer.

Preliminary investigation of the composite material

Preliminary investigations on fiber-reinforced composite materials were conducted with bare Kevlar fibers and fibers treated with HOOC-(F 4 v) 3 F 4 v-N 3 . After embedding the fibers in a matrix of poly(propylene-coethylene) upon thermal treatment at 180 °C, the composite materials were investigated by fracture test.

The fragments were analyzed by EDS selecting for each sample at least one spot on the matrix and one on the fiber (Figure 5). The results seem to suggest an enrichment of the presence in matrix on the fiber when the interfacial agent was used.

CONCLUSION

Oligomers based on phenylalanine have been designed as potential candidates as interfacial agents for

Kevlar fiber-reinforced polyolefin composite materials. The evaluation of their adsorption on Kevlar fibers has been evaluated visually by SEM on individual fibers and gravimetrically on pellets of fibers. While the use of a larger number of successive phenylalanine residues on the oligomers increased the adsorption of the oligomer on Kevlar fibers, the length of the aliphatic chains did not seem to have a notable influence.

This work is the first step in determining the potential of these oligomers by evaluating their adsorption, however it will be also essential to further study their interaction with the polymer matrix to validate their potential as interfacial agents for fiber-reinforced composite materials.

EXPERIMENTAL

Materials

Sodium azide (NaN 3 , 99 %), 6-bromohexanoic acid (>98%), 5-bromovaleric acid (97%), 4-bromobutyric acid (97%), piperidine (99%), phenol (>99%), copper(I) bromide (CuBr, 98%) and All the syntheses on solid support were performed in solid phase extraction (SPE) tubes (60 mL polypropylene tubes with polyethylene frits, 20 μm porosity, SUPELCO® purchased from Sigma-Aldrich)

and shaken using a modified IKA KS 130 basic shaker.

4-Azidobutyric acid [START_REF] Sacchetti | Microwave-assisted synthesis and click chemistry as simple and efficient strategy for RGD functionalized hydrogels[END_REF] (b), 5-azidovaleric acid [START_REF] Voliani | Cancer phototherapy in living cells by multiphoton release of doxorubicin from gold nanospheres[END_REF] (v), and 6-azidohexanoic acid [START_REF] Chan-Seng | Primary structure control of oligomers based on natural and synthetic building blocks[END_REF] (h) were synthesized as previously reported.

Tetraglycine (HOOC-G 4 -NH 2 , >99.9%) and tetraphenylalanine (HOOC-F 4 -NH 2 , >97%) were purchased from Bachem. Polystyrene standard (PS) having an average number-molecular weight (M n ) of 2100 g mol -1 (Đ = 1.05) was purchased from PSS-Polymer. HOOC-(Fh) 4 -NH 2 (>90%) was purchased from Laboratoire Altergen. HOOC-(Fh) 3 Fh-N 3 was synthesized as previously reported. [START_REF] Chan-Seng | Synthesis of macromolecules containing phenylalanine and aliphatic building blocks[END_REF] Kevlar® short fiber aramid (SFA A) fibers, having an average length of 50 mm and diameter of 12 µm and pre-treated by the supplier to remove the finish present at their surface, were received as a sample from DuPont de Nemours S.A.

Aceso® Lumicene® MR10MM0 (random copolymer of propylene and ethylene) was obtained from Total.

Characterization

Nuclear magnetic resonance (NMR) spectra were recorded in DMSO-d 6 on a Bruker Avance I 400 MHz spectrometer equipped with an Ultrashield TM magnet.

Mass measurements were carried out on an Autoflex TM MALDI-TOF mass spectrometer (Bruker Daltonics GmbH, Bremen, Germany). This instrument was used at a maximum accelerating potential of 20 kV in positive mode and was operated in mode reflector at 19 kV. The delay extraction was fixed at 80 ns and the frequency of the laser (nitrogen 337 nm) was set at 5 Hz. The acquisition mass range was set to 400-4000 m/z with a matrix suppression deflection (cut off) set to 500 m/z. The equipment was externally calibrated with a standard peptide calibration mixture that contained seven peptides (Bruker Peptide Calibration Standard #206196, Bruker Daltonics GmbH, Bremen, Germany) covering the 1000-3200 m/z range. Each raw spectrum was opened with flexAnalysis 2.4 build 11 (Bruker Daltonics GmbH, Bremen, Germany) software and processed using the following parameters: signal-to-noise threshold of 1, Savitzky-Golay algorithm for smoothing, median algorithm for baseline subtraction, and SNAP algorithm for monoisotopic peak detection and labelling in reflectron mode. In all cases, resolution was higher than 9000. Sample preparation was performed with the dried droplet method using a mixture of 0.5 µL of sample with 0.5 µL of matrix solution dry at room temperature. The matrix solution was prepared from a saturated solution of α-cyano-4-hydroxycinnamic acid in water/acetonitrile 50/50 diluted three times in water/acetonitrile/trifluoroacetic acid 50/49.9/0.1. The sample solution was prepared by dissolving ~5 mg of oligomer in 1 mL of a solution of concentrated HCl in tetrahydrofuran (1/9).

Thermogravimetric analyses (TGA) were performed on a Mettler-Toledo TGA2 thermogravimeter at a heating scan rate of 50 °C min -1 using alumina crucibles using an atmosphere of N 2 (gas flow rate: 100 mL min -1 ). The TGA curves were recorded from 50 °C to 850 °C. The temperature of the onset of degradation (T d , onset) was determined as the crossing point of the tangents of the first weight loss observed on each TGA curve.

Fourier transform infrared (FTIR) spectra were recorded on a Bruker Vertex 70 spectrometer using the attenuated total reflectance (ATR) technique.

Energy dispersive X-ray spectroscopy (EDS) measurements were performed on a JEOL JSM6490LV microscope (max resolution 3 nm) equipped with an Oxford instruments AZTEC (lateral resolution 200 nm) EDS module at 5 keV with a spot size of 62 and measuring time of 60 s. The average atomic composition and standard deviation were determined on nine measurements.

For quantification experiments, all weights were recorded on a vibration-stabilized Mettler-Toledo AX 205 analytical balance located in an air-conditioned room.

Synthesis of 11-azidoundecanoic acid (u)

The synthesis of 11-azidoundecanoic acid was adapted from the protocol used to prepare 6-azidohexanoic acid. [START_REF] Chan-Seng | Primary structure control of oligomers based on natural and synthetic building blocks[END_REF] Briefly, 11-bromoundecanoic acid (28 g, 106 mmol) and sodium azide (17 g, 264 mmol) were added to a solution of 100 mL of acetone (100 mL) and 200 mL of reverse-osmosed water in a round bottom flask. The solution was stirred in an oil bath thermostated at 60 °C for 5 h. The reaction mixture was then cooled in an ice bath to 0 °C and acidified with concentrated HCl until pH 2 was reached. The mixture was then extracted with ethyl acetate three times. The organic layers were combined, washed with brine, dried over anhydrous sodium sulphate, concentrated by rotary evaporation and dried overnight under vacuum. The final product was obtained as an off-white solid (23 g, 96 %). Caution: Organic azides are potentially explosive substances. Routine precautions were taken to minimize the effect of possible explosions at all stages in the preparation and handling of any ω-azidoalkanoic acids.

These compounds are stored in the fridge.

Synthesis of oligomers HOOC-(Fx 4 ) 3 Fx 4 -N 3

The oligomers were prepared using the Fmoc-Phe-Tentagel® S PHB resin by adding iteratively the different building blocks using the protocol described below illustrated for oligomers with a hexyl linker (h).

i) Resin deprotection. 3 g of Fmoc-Phe-Tentagel® S PHB resin (0.72 mmol of functional groups) was weighted in a SPE tube, swollen in 10 mL of CH 2 Cl 2 for 10 min and washed three times with 10 mL of CH 2 Cl 2 . The Fmoc-protecting groups were removed by agitation for 3 min with 10 mL of a 25% piperidine solution in DMF followed by filtration and agitation with a fresh cleavage solution for 20 min. The resin was then washed six times with 10 mL of DMF and six times with 10 mL of CH 2 Cl 2 .

ii) Addition of 6-azidohexanoic acid. 13 C interfacial agent or model molecule in 2 mL 95/5 ACN/TFA (toluene in the case of PS). Subsequently the solution was emptied into a Petri dish (diameter = 3.5 cm, height = 1 cm) and fiber pellets were dipped into solution to fully submerge for 5 min, turning the pellet midway through. After treatment, coated fibers were left to dry on the tweezers overnight, after which the weight difference was recorded. All weights recorded were a result of an average of 2-3 separate instances of the same sample being weighted. In the case of a large standard deviation, more than triplicate experiments were performed. All weighted differences have been corrected for with a constant mass of solvent (<m s > = 0.41 mg) that was determined dip coating pellets in 2 mL of the solvent used for dip coating. Three sets of pellets were treated with 95/5 ACN/TFA and two sets with toluene that were weighed before (m pellet ) and after 16-18 h of soaking in the solution (m f ). The measurements were performed in triplicate to obtain average values for mass differences.

SEM measurements

For the individual fibers, the fibers placed on the sample holder were held in with carbon tape (Agar Scientific). Scanning electron microscopy (SEM) experiments were performed on a FE-SEM Hitachi SU8010 with a voltage of 1 kV.

For the fiber pellets, the pellet was placed on a carbon substrate and treated with platinum sputter coating (Emitech K575 sputter coater was used with the following settings: platinum as metal, pressure of 10 -3 mbar, current of 65 mA, and sputter distance of 3 cm). Scanning electron microscopy (SEM) experiments were performed on a JEOL JSM6490LV microscope (max resolution 3 nm) under a pressure of ~10 -5 mbar with a voltage of 15 kV with a spot size of 55.

Fracture test

Untreated or treated aramid fibers were sandwiched between two layers of poly(propylene-co-ethylene) using eight to ten granules for each layer in a ceramic crucible (bottom diameter of 13 mm). The crucible was placed in a thermostated oven at 180 °C for 15 min to melt the polymer, after which the crucible was removed and allowed to cool to room temperature for 15 min. The composite was then removed from the crucible by gently pulling. The composite was then plunged into liquid nitrogen for 10 min after which it was fractured using a hammer and pick. Fragments of the composite were placed on several carbon substrates. Subsequently, the substrate was treated with platinum sputter coating. SEM/EDS analysis followed with SEM images taken at 15 keV with a spot size of 55 and EDS spectra taken at 5 keV with a spot size of 62 and measuring time of 35 s using X-Max X-ray module.

Figure 1 .

 1 Figure 1. Library of phenylalanine-based oligomers synthesized and model molecules considered in this study as compatibilizers of Kevlar-reinforced composite polyolefin composite materials. The functional groups involved in hydrogen bond and π-π interactions are indicated in red and blue respectively.

  24 mmol per gram of resin), and the solvent used for the addition of Fmoc-Phe-OH and 6-azidohexanoic acid by a mixture of solvent consisting in dichloromethane, N,N-dimethylformamide, and 1-methyl-2-pyrrolidinone in equivolumes. Similarly, HOOC-(F 4 b) 3 F 4 b-N 3 , HOOC-(F 4 v) 3 F 4 v-N 3 , and HOOC-(F 4 u) 3 F 4 u-N 3 were prepared to study the effect of the length of the alkyl chain by replacing 6-azidohexanoic acid (h) by 4-azidobutyric acid (b), 5-azidovaleric acid (v), and 11-azidoundecanoic acid (u) respectively. The oligomers were characterized by NMR spectroscopy and MALDI-ToF mass spectrometry (ESI, Figure S2-S5). Both characterization techniques indicated the presence of oligomers with deleted sequences (i.e.

  while the other oligomers (i.e. HOOC-(F 4 b) 3 F 4 b-N 3 , HOOC-(F 4 v) 3 F 4 v-N 3 , HOOC-(F 4 h) 3 F 4 h-N 3 , and HOOC-(F 4 u) 3 F 4 u-N 3 )

Figure 2 .

 2 Figure 2. SEM images (scale bar = 10 µm) of one single Kevlar fiber treated by dip coating (1 µmol mL -1 of interfacial agent in 95/5 ACN/TFA) with oligomer HOOC-(Fh) 3 Fh-N 3 in comparison to treatment conducted with HOOC-(Fh) 4 -NH 2 , PS, HOOC-G 4 -NH 2 , and HOOC-F 4 -NH 2 .

Figure 3 .

 3 Figure 3. SEM images (scale bar = 10 µm) of one single Kevlar fiber treated by dip coating (1 µmol mL -1 of interfacial agent in 95/5 ACN/TFA) with oligomers having spacing units of various lengths: HOOC-(F 4 b) 3 F 4 b-N 3 , HOOC-(F 4 v) 3 F 4 v-N 3 , HOOC-(F 4 h) 3 F 4 h-N 3 , and HOOC-(F 4 u) 3 F 4 u-N 3 .

3 HOOCFigure 4

 34 Figure 4 using HOOC-(F 4 u) 3 F 4 u-N 3. The pellets exhibited loose and flattened areas with a smooth coverage of the interfacial agent deposited at the surface of the Kevlar fibers.

Figure 4 .

 4 Figure 4. SEM images of fiber pellets that have been submerged in a solution of HOOC-(F 4 u) 3 F 4 u-N 3 (1 µmol mL -1 of interfacial agent in 95/5 ACN/TFA) highlighting a) loose and b) flattened areas. The white bars represent 100 µm. White arrows are highlighting three of the Kevlar fibers.

Figure 5 .

 5 Figure 5. SEM images and EDS data of Kevlar-reinforced composite material with a poly(propylene-co-ethylene) matrix a) without adding an interfacial agent and b) upon treatment of the fiber with HOOC-(F 4 v•) 3 F 4 v-N 3 . The white rectangles are highlighting the spot analyzed by EDS, for which the experimental values are reported in the table.

Table 1 :

 1 Quantification of the amount of oligomer deposited on Kevlar fiber pellets after immersion in a solution of oligomer at a concentration of 1 mM in 95/5 ACN/TFA for 5 min and air-drying overnight.

	oligomer	m oligomer a (mg)	m pellet b (mg)	m f c (mg)	adsorption density d (µmol g -1 ) individual pellet average
	HOOC-(F 4 b•) 3 F 4 b-N 3	5.99	74.52	75.81	3.96	4.46 ± 0.71
		6.03	74.48	76.06	5.27
		6.02	74.47	75.80	4.15
	HOOC-(F 4 v•) 3 F 4 v-N 3	6.14	74.25	75.81	5.10	5.21 ± 0.89
		6.09	74.45	76.25	6.15
		6.08	74.59	75.99	4.37
	HOOC-(F 4 h•) 3 F 4 h-N 3	6.24	74.07	75.36	3.84	3.84 ±0.34
		6.20	74.36	75.73	4.18
		6.18	74.56	75.78	3.51
	HOOC-(F 4 u•) 3 F 4 u-N 3	6.80	74.68	76.26	4.64	4.84 ± 0.21
		6.76	74.50	76.18	5.05
		6.81	74.26	75.88	4.83

a mass of oligomer dissolved in 2 mL of 95/5 ACN/TFA, b weight of the Kevlar fiber pellet before treatment, c weight of the Kevlar fiber pellet after immersion and drying, d number of moles of oligomer adsorbed per gram of Kevlar fibers (adsorption density = (m f -m pellet -0.41 mg)/(M oligomer .m pellet ))

  Cl 2 /DMF/NMP(10 mL) were added to the vessel and agitated for 1 h. After filtration, the resin was washed six times with 10 mL of DMF and six times with 10 mL of CH 2 Cl 2 . The completion of this step was monitored using the Kaiser test[START_REF] Kaiser | Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides[END_REF] that should be negative (yellow solution), and a measurement by FT-IR spectroscopy that should show an absorbance band at 2100 cm -1 characteristic of the asymmetric stretching vibration of azide groups. If the tests indicate an incomplete reaction, this step was repeated. The solution was agitated under argon for 1 h. After filtration, the resin was washed six times with 10 mL of DMF and six times with 10 mL of CH 2 Cl 2 . The completion of this step was monitored by FTIR spectroscopy that should show the disappearance of the absorbance band at 2100 cm -1 characteristic of the asymmetric stretching vibration of azide groups. If the test indicates an incomplete reaction, this step Cl 2 /DMF/NMP (10 mL) were added to the peptide vessel, and agitated for 1 h. After filtration, the resin was washed six times with 10 mL of DMF. The completion of this step was monitored using the Kaiser test that should be negative (yellow solution). If the test indicates an incomplete reaction, this step was repeated. The Fmoc-protecting group was removed by agitation with 10 mL of a 25% piperidine in DMF solution for 3 min followed by filtration and agitation with a fresh solution of piperidine for 20 min. The resin was then washed six times with 10 mL of DMF and six times with 10 mL of CH 2 Cl 2 . v) Cleavage from the resin and isolation of the phenylalanine-based macromolecule. The resin was washed twelve times with 10 mL of CH 2 Cl 2 before transferring the resin to a round-bottom flask and adding the cleavage solution (10 mL of 95/2.5/2.5 TFA/TIPS/H 2 O) to the flask. The resin was agitated with the cleavage solution for 2 h and collected in a clean round-bottom flask. These tasks were performed three times. The resin was then washed six times with 10 mL of CH 2 Cl 2 . The filtrates were combined, stirred with

	6-Azidohexanoic acid (0.35 g, 2.19 mmol), HBTU (0.83 g, 2.18 mmol), HOBt (0.29 g, 2.17 mmol), DIPEA (0.70 mL, 4.05 mmol) and a 1/1/1 mixture of CH 2 iii) Copper-assisted alkyne-azide cycloaddition with propargylamine. CuBr (27 mg, 0.19 mmol) was added to the vessel, which was then degassed through three vacuum/argon cycles. Anhydrous CH 2 Cl 2 (10 mL) were added to the vessel, followed by PMDETA (0.07 mL, 0.34 mmol) and propargylamine (0.14 mL, 2.23 iv) Addition of Fmoc-L-Phe-OH. Fmoc-L-Phe-OH (0.84 g, 2.19 mmol), HBTU (0.83 g, 2.18 mmol), HOBt (0.29 g, 2.17 mmol), DIPEA (0.70 mL, 4.05 mmol) and a 1/1/1 mixture of CH 2 CupriSorb TM overnight to remove residual copper, concentrated into a viscous solution by rotary evaporation and precipitated in diethyl ether to remove TFA and TIPS followed by precipitation in reverse-osmosed water. Aggregates of oligomers were crushed to obtain a finely dispersed powder, which was washed six times with 10 mL of reversed-osmosed water. The product was then frozen in liquid nitrogen and subjected to six vacuum-argon cycles (these tasks were performed three times) before drying it under vacuum overnight at room temperature. HOOC-(F 4 b•)F 4 b-N 3 . 427 mg (31 % yield). 1 H NMR: δ (DMSO-d 6 , 2.50 ppm) 12.77 (bs, 1H), 8.26-8.44 (m, 5H), 7.92-8.26 (m, 14H), 7.46-7.54 (m, 3H), 6.99-7.33 (m, 80H), 4.41-4.62 (m, 16H), 3.99-4.41 (m, 12H), 3.50 (PEG), 2.54-3.12 (m, 32H), 1.91-2.05 (m, 8H), 1.75-1.89 (m, 8H). 13 C NMR: δ (DMSO-d 6 , 39.52 ppm) 172.63, 171.09, 171.02, 170.86, 170.78, 170.68, 170.59, 170.47, 144.39, 137.97, 137.58, 137.49, 137.44, 137.33, 129.20, 129.08, 128.19, 128.05, 127.98, 127.92, 126.44, 126.29, 126.21, 126.15, 126.11, 122.50, 69.78 (PEG), 53.83, 53.64, 53.50, 53.46, 49.93, 48.60, 37.85, 37.63, 37.50, 37.41, 36.75, 34.29, 31.83, 24.39. MALDI MS (m/z): [M+H] + calculated from C169H182N31O21 2981.41, found 2982.16. mmol). was repeated. HOOC-(F 4 v•)F 4 v-N 3 . 450 mg (29 % yield). 1 H NMR: δ (DMSO-d 6 , 2.50 ppm) 12.77 (bs, 1H), 8.27-8.46 (m,

4H), 7.87-8.27 (m, 15H), 7.50-7.57 (m, 3H), 6.97-7.37 (m, 80H), 4.37-4.66 (m, 16H), 4.08-4.37 (m, 12H), 3.50 (PEG), 3.17 (t, 2H), 2.56-3.11 (m, 32H), 1.89-2.05 (m, 8H), 1.40-1.60 (m, 8H), 1.14-1.40 (m, 8H).

ACKNOWLEDGMENTS

The authors are thankful to J.-M. Strub for the MALDI-ToF measurements and C. Saettel for TGA measurements. The authors acknowledge the polymer characterization facilities at the Institut Charles Sadron, the electron microscopy facilities at the Institut Charles Sadron, and the Cronenbourg NMR core facilities. The research leading to these results has received funding from the H2020 program of the European Union (project Euro-Sequences, H2020-MSCA-ITN-2014, grant agreement no. 642083). The doctoral position of JL was supported by the project Euro-Sequences. The CNRS and PSA Groupe are also acknowledged for financial support.

DATA AVAILABILITY

The raw/processed data required to reproduce these findings cannot be shared at this time due to technical or time limitations.

CREDIT AUTHORSHIP CONTRIBUTION STATEMENT

Jeroen Louwsma: methodology, investigation, writing; Alain Carvalho: methodology, investigation; Jean-François Lutz: funding acquisition, conceptualization; Stéphane Joly: funding acquisition, conceptualization, supervision; Delphine Chan-Seng: conceptualization, supervision, visualization, writing.

APPENDIX A. Supplementary material

Supplementary data to this article can be found online at .