Stéphanie Renaud

Vincent Corcé

Isabelle Cannie

Martine Ropert

Sylvie Lepage

Olivier Loréal

David Deniaud

François Gaboriau
email: francois.gaboriau@univ-rennes1.fr

Quilamine HQ1-44, an iron chelator vectorized toward tumor cells by the polyamine transport system, inhibits HCT116 tumor growth without adverse effect

Keywords: Iron chelator, iron, polyamine, polyamine transport system, tumor vectorization, cancer, colon adenocarcinoma

Tumor cell growth requires large iron quantities and the deprivation of this metal induced by synthetic metal chelators is therefore an attractive method for limiting the cancer cell proliferation. The antiproliferative effect of the Quilamine HQ1-44, a new iron chelator vectorized toward tumor cells by a polyamine chain, is related to its high selectivity for the Polyamine Transport System (PTS), allowing its preferential uptake by tumoral cells. The difference in PTS activation between healthy cells and tumor cells enables tumor cells to be targeted, whereas the strong dependence of these cells on iron ensures a secondary targeting. Here, we demonstrated in vitro that HQ1-44 inhibits DNA synthesis and cell proliferation of HCT116 cells by modulating the intracellular metabolism of both iron and polyamines.

Moreover, in vivo, in xenografted athymic nude mice, we found that HQ1-44 was as effective as cis-platin in reducing HCT116 tumor growth, without its side effects. Furthermore, as suggested by in vitro data, the depletion in exogenous or endogenous polyamines, known to activate the PTS, dramatically enhanced the antitumor efficiency of HQ1-44. These data support the need for further studies to assess the value of HQ1-44 as an adjuvant treatment in cancer.

1-Iron metabolism, a new target for anticancer therapy

Iron is essential in many biological functions and a potential target for anticancer therapy [START_REF] Richardson | Cancer cell iron metabolism and the development of potent iron chelators as anti-tumour agents[END_REF]. Initially developed for the treatment of iron overload, iron chelators have shown an antiproliferative action on tumor cells, thus demonstrating their potential value in the treatment of some cancers [START_REF] Richardson | Therapeutic potential of iron chelators in cancer therapy[END_REF]. Thus, the high efficiency and selectivity of new thiosemicarbazone chelators against various tumors have been reported in vitro and in vivo. [START_REF] Lane | Expanding horizons in iron chelation and the treatment of cancer: role of iron in the regulation of ER stress and the epithelial-mesenchymal transition[END_REF]. In fact, growing tumor cells require large quantities of iron, leading to an increase in the iron importing proteins and a decrease in the expression of iron exporting proteins [START_REF] Richardson | The molecular mechanisms of the metabolism and transport of iron in normal and neoplastic cells[END_REF][START_REF] Kwok | The iron metabolism of neoplastic cells: alterations that facilitate proliferation? Critical reviews in oncology[END_REF][START_REF] Brookes | Modulation of iron transport proteins in human colorectal carcinogenesis[END_REF]. In addition, iron exposure stimulates the proliferation of tumor cells in vitro and in vivo [START_REF] Torti | Iron and cancer: more ore to be mined[END_REF]. Therefore, decreasing the exchangeable intracellular iron level with synthetic iron chelators appears an attractive method to reduce the proliferation of cancer cells [START_REF] Merlot | Novel chelators for cancer treatment: where are we now? Antioxidants & redox signaling[END_REF]. Our previous results dealing with deferasirox (ICL670), an iron chelator currently used in the treatment of secondary iron overload disease, suggested that its antiproliferative activity results from its inhibitory effects on both iron and polyamine metabolisms [START_REF] Lescoat | Antiproliferative and apoptotic effects in rat and human hepatoma cell cultures of the orally active iron chelator ICL670 compared to CP20: a possible relationship with polyamine metabolism[END_REF]. However, these chelators were designed to treat patients exhibiting iron excess. Therefore, their use in patients without iron excess may lead to major side effects, thus limiting their use as antitumoral agents in patients not affected by iron overload. Thus, for potential iron chelators to be used in cancer therapy, they must be vectorized toward tumoral cells.

1.2-Polyamine, a vector for anticancer agents

In tumor cells, polyamine metabolism, including biosynthesis and uptake, is particularly amplified [START_REF] Thomas | Polyamine metabolism and cancer[END_REF][START_REF] Wallace | A perspective of polyamine metabolism[END_REF]. Targeting polyamine metabolism by using polyamine synthesis inhibitors or polyamine catabolism activators was demonstrated to prevent and reduce risk factors associated with the tumor development [START_REF] Babbar | Targeting polyamines and inflammation for cancer prevention. Recent results in cancer research Fortschritte der Krebsforschung Progres dans les recherches sur le cancer[END_REF]. The efficiency of the polyamine transport system (PTS) is also greatly increased in tumoral cells. This property is of potential interest for targeting antitumor agents [START_REF] Annereau | A fluorescent biomarker of the polyamine transport system to select patients with AML for F14512 treatment[END_REF][START_REF] Barret | F14512, a potent antitumor agent targeting topoisomerase II vectored into cancer cells via the polyamine transport system[END_REF][START_REF] Bergeron | Polyamine-iron chelator conjugate[END_REF][START_REF] Bergeron | Design, Synthesis, and Testing of Polyamine Vectored Iron Chelators Synthesis[END_REF][START_REF] Bergeron | Partitionvariant desferrithiocin analogues: organ targeting and increased iron clearance[END_REF][START_REF] Burns | Lipophilic lysine-spermine conjugates are potent polyamine transport inhibitors for use in combination with a polyamine biosynthesis inhibitor[END_REF][START_REF] Covassin | Synthesis of spermidine and norspermidine dimers as high affinity polyamine transport inhibitors[END_REF][START_REF] Dallavalle | Synthesis and cytotoxic activity of polyamine analogues of camptothecin[END_REF][START_REF] Delcros | Effect of spermine conjugation on the cytotoxicity and cellular transport of acridine[END_REF][START_REF] Delcros | Bis(7-amino-4azaheptyl)dimethylsilane: a new tetramine with polyamine-like features. Effects on cell growth[END_REF][START_REF] Esteves-Souza | Antitumoral activity of new polyamine-naphthoquinone conjugates[END_REF][START_REF] Gardner | Synthesis and Transfection Efficiencies of New Lipophilic Polyamines[END_REF][START_REF] Holley | Uptake and cytotoxicity of novel nitroimidazole-polyamine conjugates in Ehrlich ascites tumour cells[END_REF][START_REF] Tomasi | Targeting the polyamine transport system with benzazepine-and azepine-polyamine conjugates[END_REF][START_REF] Verschoyle | The comparative toxicity of chlorambucil and chlorambucil-spermidine conjugate to BALB/c mice[END_REF][START_REF] Zhuo | Synthesis and biological evaluation of boron-containing polyamines as potential agents for neutron capture therapy of brian tumors[END_REF].

Based on these observations, we have developed a new generation of iron chelators, the Quilamines, in which an 8-hydroxyquinoline chelating subunit is grafted onto polyamine vectors, in order to vectorize the iron chelator inside the cancerous cell with an overactive PTS [START_REF] Corce | Polyaminoquinoline iron chelators for vectorization of antiproliferative agents: design, synthesis, and validation[END_REF]. The difference in PTS activation A c c e p t e d M a n u s c r i p t 4 between healthy and tumor cells enables tumor cells to be targeted whereas the stronger dependence of these latter cells on iron ensures a secondary targeting.

We previously reported that the Quilamine HQ1-44 was highly taken up by the PTS and displayed an efficient antiproliferative activity [START_REF] Corce | Polyaminoquinoline iron chelators for vectorization of antiproliferative agents: design, synthesis, and validation[END_REF]. As expected during the design of Quilamines, the polyamine chain, involved in the metal coordination, reinforced the affinity for iron (III) and the ferric complex was shown to adopt preferentially a 1:2 [Fe/HQ1-44] stoichiometry at pH=7.4.

We extended the screening of the antiproliferative activity of HQ1-44 to various tumor cell lines and compared it to two non-tumoral cell lines [START_REF] Corce | Synthesis and Biological Properties of Quilamines II, New Iron Chelators with Antiproliferative Activities[END_REF]. The relationship between PTS activity and antiproliferative efficiency of HQ1-44 in these various cancer cell lines clearly demonstrated that the Quilamine HQ1-44 was the most efficiently recognized by the active PTS of human colon adenocarcinoma HCT116 cell lines.

Consequently, this Quilamine exhibited the higher antiproliferative efficiency on this cell line. In contrast, a low cytostatic effect was observed in normal cells. Colorectal cancer (CRC) is the second most common cause of death from cancer in France and the fourth most commonly diagnosed cancer in the world, so we choose to demonstrate the proof of the Quilamine concept on this kind of cancer.

In the present work, our aim was to investigate the impact of HQ1-44 on cell proliferation and the mechanisms involved as well as assessing its efficiency and tolerance in vivo as an antitumoral treatment in athymic nude mice xenografted with HCT116 cells, by comparison with cis-platin.

2-Materials and Methods

2.1-In vitro study

2.1.1-Chelator solutions

All reagents were obtained from Sigma-Aldrich (Saint Quentin Fallavier, France) at the highest available grade. Stock solutions of the Quilamine HQ1-44 (10 mM) were prepared in water. Cells were supplemented with iron by exposure to 20 µM exogenous iron-citrate complexes (1:10) for 72 h.

2.1.2-Cell studies

A c c e p t e d M a n u s c r i p t 5

The involvement of the PTS in the selective uptake of Quilamine HQ1-44 was investigated in the HCT116 cell line, derived from human colon carcinoma (ATCC number 91091005). We previously demonstrated the higher sensitivity of this cell line to Quilamines, compared to other carcinoma cells [START_REF] Corce | Synthesis and Biological Properties of Quilamines II, New Iron Chelators with Antiproliferative Activities[END_REF]. These cells were grown in McCoy's medium (Life Technologies, Invitrogenä, Saint Aubin, France), supplemented with 10% fetal calf serum (Eurobio, Les Ulis, France), 100 units/mL penicillin and 50 µg/mL streptomycin, at 37°C in 5% humidified CO 2 .

2.1.3-Cell treatment

For experiments, cells were harvested with trypsin and seeded 24 h before the treatments in microplates (Becton Dickinson, Oxnard, CA, USA) at a density of 10,000 cells/cm 2 . In these conditions, cells reached confluency in 5-6 days. Chelator exposure was performed one day (D1) or one week (D8) after cell seeding in proliferating or confluent HCT116 cells, respectively. The effect of Quilamine on cell viability was also tested in the presence of DFMO (1 mM), a selective inhibitor of ODC, which induced a depletion of intracellular putrescine and spermidine and thus activated the PTS. In this case, preincubation, in the presence of the ODC inhibitor, was performed prior to the Quilamine treatment. In other experiments, 50 µM of spermidine was added to the culture medium to perform a competitive inhibition of Quilamine uptake through the PTS. Aminoguanidine (1 mM), an inhibitor of the serum amine oxidase, was added to the cell culture medium to prevent the oxidation of exogenous polyamine.

For the caspase 3/7 activity assay, Quilamine uptake quantification, polyamine concentration determination and iron status evaluation, cell lysates were obtained after 72 h of treatment. After supernatant collection for biochemical analysis, cells were washed twice with 1 mL of ice-cold phosphatebuffered saline solution (PBS). Cells were collected by scraping in 1 mL PBS, and then centrifuged for 5 min at 2,000 g. The cell pellet was sonicated for 30 min at 4°C in 500 µl of pure water. Protein content in cell extracts was measured according to a method adapted from Bradford (1976) (Bio-Rad Protein Assay, Bio-Rad, Ivry sur Seine, France) and absorbance was read at 595 nm.

2.1.4-Measurement of cytostatic and cytotoxic effects

Treatments was performed one day (D1) or one week (D8) after cell seeding in proliferating or confluent HCT116 cells, respectively. After 72 h of incubation at 37°C, cell supernatants were collected for A c c e p t e d M a n u s c r i p t 6 cytotoxicity evaluation (cytotoxicity detection kit -LDH, Roche, Penzberg, Germany). Results were reported as a percentage of extracellular LDH activity with respect to the control value.

Cell viability was determined by cell nuclei counting after Hoechst 33342 staining and the dose-effect curves were analyzed according to an established procedure [START_REF] Corce | Synthesis and Biological Properties of Quilamines II, New Iron Chelators with Antiproliferative Activities[END_REF]. The number of cell nuclei was reported as a percentage of the value obtained in control conditions. Due to their biphasic feature, the dose-response curves were fitted as the sum of two sigmoids (double 4-parameter fit) according to Rodbard [START_REF] Rodbard | Automated computer analysis for enzyme-multiplied immunological techniques[END_REF]. The various parameters, including the percentage and the IC 50 values of each sigmoid, were deduced from these fits. Three independent replicates were performed for each experiment, which was repeated three times.

2.1.5-Measurement of DNA synthesis

DNA synthesis was assessed by tritiated thymidine incorporation. Twenty-four hours before cell harvesting, [ 3 H] methyl-thymidine (Amersham, Uppsala, Sweden) was added to the culture medium at a final concentration of 0.5 µCi/mL. Cells were washed twice with PBS and lysed in 0.1 M NaOH. DNA precipitation was achieved with 30% trichloroacetic acid (TCA), followed by two washes with 10% and 5% TCA successively, and dissolution in formic acid. The radioactivity of 200 µl of this formic solution was counted in 5 mL of Instagel using a Packard Tricarb 2100TR scintillation counter b (Perkin Elmer, Waltham, MA, USA) and expressed as cpm/g of protein.

2.1.6-Analysis of the cell cycle

The cell cycle was assessed by flow cytometry. Cells were detached with trypsin and nuclei were stained with propidium iodide (Cycletest Plus Kit, Becton Dickinson, Le Pont de Clais, France). Data were acquired and analyzed with a FACSCalibur flow cytometer (Becton Dickinson) using Cell Quest software (Becton Dickinson).

2.1.7-Measurement of caspase 3/7 activity

Apoptosis was assessed through caspase 3/7 activity quantification, using the "AMC caspase-3/7 assay" kit (AnaSpec, Fremont, CA, USA), in accordance with the manufacturer's instructions. Caspase 3/7 activity measured by fluorescence at 440/460 nm was expressed as RFU/g of protein and reported as a percentage with respect to untreated control cells. Total RNA extracted using "SV total RNA isolation system" kit (Promega, Wisconsin, USA) was tested by spectrophotometrical dosage and electrophoretic separation on a 1.5% agarose gel. RNA was reversetranscribed to synthesize cDNA using Moloney Murine Leukemia Virus reverse-transcriptase (Promega, Wisconsin, USA). Gene expression was measured by real-time quantitative PCR performed in triplicate on an ABI Prism 7000 Sequence Detection System (Applied Biosystems, London, UK) using the qPCR Mastermix Plus for SYBR Green I kit and specific primer (Eurogentec, Seraing, Belgium) designed by Primer Express 1.0 software (Table 1). The relative quantification of mRNA levels was calculated after normalization using 18S RNA as an endogenous reference. The results were expressed as percentage compared to control culture.

2.1.9-Measurement of HQ1-44 uptake in HCT116 cells

HQ1-44 exposure at 1 µM was performed one day (D1) or one week (D8) after cell seeding in proliferating or confluent HCT116 cells, respectively. Quilamine uptake was also measured after activation of the PTS (1 mM DFMO, see below) or in the presence of 50 µM exogenous spermidine in the culture medium to perform a competitive inhibition of the HQ1-44 uptake by the PTS. Cell lysates were ultracentrifuged for 10 min at 15,000 g and HQ1-44 was quantified in the supernatant by LC/MSMS.

2.1.10-LC/MS quantification of HQ1-44 in HCT116 cells

Analyses were performed on a liquid chromatograph coupled with an ultra-high-resolution and accurate mass instrument (LC-HR-MS), Q ExactiveTM equipped with an Orbitrap mass analyzer (Thermo Scientific, San Jose, USA). An HESI source was used for the ionization of target compounds. Data acquisition, peak integration and calibration were performed using Xcalibur® 2.1 software (Thermo Scientific, San Jose, CA, USA).

LC separation was carried out using a gradient on a C18 Hypersil Gold column (100 mm x 2.1, 3 µm) from Thermo Scientific (San Jose, USA). The mobile phases used were 10 mM ammonium acetate buffer containing 0.1% (v/v) formic acid (solvent A) and methanol/water (95/5, v/v) (solvent B). The mobile phase was delivered at a flow rate of 300 µL/min using the following stepwise gradient elution program: initial conditions of 100% solvent A maintained for 3 minutes, run from 100% solvent B at 2 minutes, A c c e p t e d M a n u s c r i p t 8 maintained for 1 minute, run from 100% solvent A at 1 minute and conditions of 100% solvent maintained for 5 minutes for equilibration. All prepared samples were kept at 15°C in the autosampler until injection of 5 µL into the LC-HR-MS system (partial loop) in a thermostated column at 25°C. The MS conditions were as follows: HESI in positive mode, capillary temperature: 300°C; spray voltage: 4500 V; sheath and auxiliary gas (nitrogen) flow-rate: 30 and 15 (arbitrary units), respectively. Data were acquired in full scan mode. Full mass spectra were scanned in the mass range 120 to 700 m/z, the AGC Target was 1e 6 and the resolution was 140,000 (FWHM). The measured accurate m/z values of the protonated species was 317.2337. Quantitation was achieved by extracting the exact mass of the protonated species using a 10-ppm extraction window. Quilamine concentrations were then deduced from the calibration curves obtained from HCT116 cell lysate enrichments with various concentrations of HQ1-44, followed by their sonication and ultrafiltration.

Each HQ1-44 measurement was determined in triplicate and was corrected for protein content in the cell extracts.

2.2-In vivo study

2.2.1-Xenografted nude mice model studies

All animal experiments were approved by the Rennes Ethics Committee for Animal Experimentation and by the Ministry for Higher Education and Research. Mice were housed in individually ventilated cages in pathogen-free quarters in the animal handling facility of Rennes 1 University (ARCHE-Biosit). The 5week-old athymic female Swiss nude mice (Charles River, Saint Quentin Fallavier, France) were acclimated in a sanitary room for 1 week before the start of the experiments. Viable HCT116 colon cancer cells (3.0 ×10 6 in medium/100 μl) were injected subcutaneously into the right flank of the nude mice.

2.2.2-Comparison of antitumoral efficiency of HQ1-44 and cis-platin

Ten days after tumor cell inoculation, mice showing a tumor volume between 80-120 mm 3

2.2.3-Effect of activation of PTS on the antitumoral efficiency of HQ1-44

PTS activation was induced by a nutritional deficiency in polyamine (polyamine deficient chow, PDC) as previously described [START_REF] Quemener | Polyamine deprivation: a new tool in cancer treatment[END_REF] and/or by inhibition of the polyamine biosynthetic pathway with adifluoromethylornithine (DFMO, 3% (w/w) in the drinking water). This activation was associated or not with the Quilamine treatment.

Ten days after tumor cell inoculation, mice showing a tumor volume between 80-120 mm 3 were randomly divided into 6 treatment groups (9 mice per group). Mice received PDC supplemented or not with polyamine (21 mg.kg -1 putrescine.2HCl, 153 mg.kg -1 spermidine.3HCl, 48.7 mg.kg -1 spermine.4HCl) and/or DFMO 3% (w/w) in their drinking water. Quilamine-treated mice groups were treated daily by intraperitoneal injection of Quilamine HQ1-44 at 40 mg.kg -1 for 17 days. The mice were observed daily.

Mice weight and tumor growth were measured three times per week. Growth inhibition was calculated from the start of treatment by comparing the change in tumor volume for vehicle and treated groups.

Mice were sacrificed the day after the last injection. Blood was collected by intra-cardiac puncture after anesthesia. Liver, spleen, heart, kidneys and tumor were collected, weighed, and frozen at -80°C for biochemical and gene expression analyses and/or fixed in formaldehyde for histological analyses.

2.2.4-Blood analysis

Whole blood was collected in heparinized microtubes. After collection of the plasma for biochemical analyses (iron, transferrin, ferritin), 100 µL of red blood cells (RBC) was discarded for polyamine measurement according to a described method [START_REF] Gaboriau | Atmospheric pressure chemical ionization-mass spectrometry method to improve the determination of dansylated polyamines[END_REF] [START_REF] Seiler | Use of the dansyl reaction in biochemical analysis[END_REF]. The LC/MSMS analysis of the dansylated polyamines, adapted from a described method [START_REF] Ducros | Determination of dansylated polyamines in red blood cells by liquid chromatography-tandem mass spectrometry[END_REF][START_REF] Lescoat | Involvement of polyamines in iron(III) transport in human intestinal Caco-2 cell lines[END_REF], was performed with a Thermo Scientific TSQ Quantum Ultra (Thermo Fisher Scientific, Courtabeuf, France). Levels of intracellular iron were measured by a colorimetric method using OSR6286 kits (Olympus Diagnostics GmbH, Lismeehan, Ireland) on an Olympus AU 2700 chemistry autoanalyzer (Olympus Diagnostics GmbH, Hamburg, Germany).

2.2.5-Statistical analysis

Results from in vitro studies were replicated three times and expressed as means ± SD. Within the in vivo study, results from each animal group were expressed as means ± SD. Statistical analysis was performed using the non-parametric Mann-Whitney test. The significance level was set at 0.01.

3-RESULTS

3.1-HQ1-44-induced decrease in HCT116 cell growth involves the PTS

The dose-effect curves obtained after treatment of proliferating HCT116 cells (D1) with HQ1-44, could be deconvoluted into two components (Figure 1A). For Quilamine concentrations lower than 10 µM, the first component indicated the antiproliferative effect of the Quilamine (62% of component 1 with IC 50-1 = 1 µM). For higher concentrations, the second component reflected the cytotoxic effect (38% of component 2 with IC 50-2 = 165 µM), linked to membrane damage and a release of lactate dehydrogenase (LDH) into the supernatant (Figure 1B). In contrast, when confluent cells, eight days after cell seeding (D8), were treated with HQ1-44, the dose-effect curve was reduced to one component (IC 50 = 210 µM, Figure 1A), associated with LDH release into the supernatant (Figure 1B).

To explore the involvement of the PTS in HQ1-44 vectorization and consequently its antiproliferative effect, we used two approaches. Firstly, PTS activation was induced by a treatment with adifluoromethylornithine, DFMO (1 mM), an inhibitor of ornithine decarboxylase (ODC), leading to polyamine depletion and an increase in PTS expression. This activation improved the antiproliferative effect of HQ1-44 at D1, as shown by the increase in the first component (70%) and the decrease in the IC 50-1 value (0.5 µM) (Figure 1A). In contrast, co-incubation of the HCT116 cells in the presence of 50 µM spermidine, a natural polyamine, at D1 led to a competitive inhibition of HQ1-44 uptake by the PTS and thus an inhibition of its antiproliferative effect (60% of cytostatic component 1 with IC 50-1 = 3.5 µM).

A c c e p t e d M a n u s c r i p t 11 Secondly, the HQ1-44 uptake measured in cell lysates by LC/MSMS was six-fold higher in proliferating (D1) than in confluent (D8) HCT116 cells (Figure 1C), and the activation of the PTS by DFMO treatment in proliferating cells led to a two-fold increase in Quilamine uptake. In contrast, co-incubation of proliferating cells with exogenous spermidine (50 µM) competitively inhibited the Quilamine uptake by the PTS. In confluent cells, both DFMO and spermidine remained ineffective in activating or inhibiting, respectively, the Quilamine uptake.

3.2-HQ1-44 reduces DNA synthesis, stopping the cell cycle and inducing apoptosis

HCT116 cells were seeded at a cellular density allowing their analysis in the proliferative phase (10,000 cells/cm 2 ). In the untreated control cells, 3 H-thymidine incorporation, evaluating DNA synthesis, increased with time 24 h and 48 h after cell seeding and reached a plateau at 72 h (Figure 2A). After 72 h, cells reached confluency and DNA synthesis was slowed down. In the HCT116 proliferating cells treated with a concentration of HQ1-44 close to its IC 50-1 value (2 µM), DNA synthesis was weakly inhibited compared to the control after 48 h incubation and, to a lesser extent, at 72 h (45% of inhibition).

Analysis by flow cytometry of the percentage of cells in the various phases of the cell cycle in untreated control cells at different growth times after cell seeding (24, 48 and 72 h) showed a progressive reduction of cells in phases G2/M and S with time, whereas the number of cells in phase G0/G1 increased (Figure 2B). Cell culture treatment with HQ1-44, at a concentration close to its IC 50-1 value (2 µM), led to an arrest of the cell cycle after 72 h of treatment, resulting in an increase in the percentage of cells in phases G2/M (13.6% versus 5.0% in the control), S (31.9% versus 11.3% in the control) and a drop in those in phase G0/G1 (54.6% versus 83.6% in the control). No significant apoptosis was detected by the flow cytometry analysis at this HQ1-44 concentration.

Under the same conditions, these data were confirmed by in situ analyses of the size and shape of cell nuclei after DNA staining, showing the drop in the percentage of viable cells with a small nucleus (G0/G1 phase) and the relative increase in the percentage of cells with a 50% bigger nucleus, characteristic of cells in G2/M (Figure 3C). For concentrations lower than 5µM that are cytostatic but not cytotoxic, HQ1-44 was ineffective at inducing caspase 3/7 activity (Figure 2C), an early apoptosis marker, while for cytotoxic concentrations (e.g. up to 5 µM) HQ1-44 triggered a slight, but significant, increase in caspase 3/7 (+10%). The 20% increase in caspase 3/7 induced by 10 µM HQ1-44 remained lower than the effect of 50 µM hydrogen peroxide, used as a reference apoptosis inducer (+45%).

At sub-toxic HQ1-44 concentrations (<5 µM), an in situ analysis of nuclei after DNA staining showed no significant DNA fragmentation, which could reflect apoptosis (Figure 3A), thus supporting the caspase 3/7 activity data. In contrast, some fragmented nuclei were observed after HQ1-44 treatment with cytotoxic concentrations (10µM, Figure 3B).

3.3-HQ1-44 affects iron and polyamine metabolisms in HCT116 cells

Treatment with 20 µM iron-citrate induced a marked increase in the level of the L-ferritin protein subunit, and a decrease in the soluble transferrin receptor form in cell supernatants (Figure 4A). In contrast, treatment with the reference iron chelator, ICL670, at a concentration close to its IC 50 (8 µM, data not shown) caused an iron deficiency characterized by a reduction in the L-ferritin level and an increase in the level of the soluble transferrin receptor, also observed to a lesser extent at 2 µM (Figure 4A). At this concentration, HQ1-44 induced a larger reduction in the ferritin level and a greater increase in the level of soluble transferrin receptor 1 than ICL670, confirming a situation of iron deficiency.

Finally, an analysis of the TFRc mRNA, encoding the transferrin receptor 1 protein, showed a downregulation of the expression of this gene in iron overload conditions (20 µM iron-citrate) while an upregulation was observed in iron depletion conditions induced by both chelators, ICL670 and HQ1-44, at 2 µM (Figure 4B).

Polyamine metabolism was also affected by HQ1-44. The addition of 2 µM of HQ1-44 to the HCT116 cell culture medium induced a significant reduction in intracellular levels of putrescine and spermidine while the spermine level remained unchanged (Figure 4C). In the same way, a reduction in the level of acetylated spermine (ASpm) was found but with no effect on the acetylated spermidine level (data not shown). Under the same experimental conditions, ICL670 remained ineffective at modulating the polyamine level within HCT116 cells whereas it induced an inhibition of polyamine metabolism in rat and human hepatoma cells A c c e p t e d M a n u s c r i p t 13 at higher concentrations [START_REF] Lescoat | Antiproliferative and apoptotic effects in rat and human hepatoma cell cultures of the orally active iron chelator ICL670 compared to CP20: a possible relationship with polyamine metabolism[END_REF]. These data suggest that HQ1-44 may induce a disturbance both in the polyamine biosynthetic pathway, by inhibiting the synthesis of putrescine and spermidine, and in their retro-conversion, by inhibiting the level of acetylated spermidine (Fig. 4C). Analysis of the mRNA levels of genes involved in polyamine biosynthesis, including ornithine decarboxylase (ODC), antizyme (OAZ1), S-adenosylmethionine decarboxylase (SamDC) and polyamine oxidase (PAO), showed a significant downregulation of their expression (Figure 4D).

3.4-HQ1-44 inhibits tumor growth in vivo

To test the antiproliferative efficiency of HQ1-44 in vivo, we treated HCT116 xenografted athymic nude mice by i.p. injection with increasing doses of Quilamine (Figure 5, Table 2). After a treatment of 15 days, analysis of the tumor weights showed a non-significant reduction for doses of 6.6 and 20 mg/kg and a significant reduction for the dose of 40 mg/kg (-38%). Under the same conditions, cis-platin (4 mg/kg) induced a reduction of 29% of the tumor weight. As expected, the treatment with cis-platin also involved a marked reduction in the weight of the animal's body, liver and spleen (-17%, -17% and -44%, respectively) highlighting the toxicity of this compound. Histological analysis of the spleen (Figure 6)

showed that the 44% drop in spleen weight in animals treated with cis-platin was mainly due to a decrease in the red pulp of this organ (36% decrease with respect to the control), as suggested by its paler red color.

It is noteworthy that cis-platin administration also induced a behavioral change and reduced activity in mice, while no such changes were observed in Quilamine-treated mice.

By contrast, treatment with the efficient dose of HQ1-44 (40 mg/kg) did not affect the animal and organ weights, or behavior, thus suggesting a better tolerance of the Quilamine at this concentration.

Investigating the impact of HQ1-44 on different iron and polyamine parameters, we found that at doses of 6.6 and 20 mg/kg, the Quilamine did not significantly affect the plasmatic iron concentrations, whereas a significant decrease in iron level was observed for a dose of 40 mg/kg (-21%) (Table 2). By contrast, cisplatin had no effect on iron parameters. Finally, neither treatment (HQ1-44 and cis-platin) affected the intraerythrocytic concentration of polyamines.

3.5-Polyamine deprivation increases the inhibitory effect of HQ1-44 on tumor growth

A c c e p t e d M a n u s c r i p t 14

As the in vitro data suggested an additive impact of DFMO with Quilamine, we investigated whether PTS activation might potentialize the Quilamine impact in our mouse model. Thus, polyamine deprivation was carried out by combining a blockade of endogenous sources, inhibition of intracellular polyamine biosynthesis with DFMO [START_REF] Heston | Differential effect of a-difluorométhylornithine on the in vivo uptake of 14 C-labeled polyamines and méthylglyoxal bis(guanylhydrazone) by a rat prostatederived tumor[END_REF] and/or a reduction in exogenous sources by nutritional deficiency in polyamine with a polyamine deficient chow (PDC) [START_REF] Quemener | Polyamine deprivation: a new tool in cancer treatment[END_REF][START_REF] Quemener | Polyamine deprivation enhances antitumoral efficacy of chemotherapy[END_REF]. As shown in Table 3, DFMO treatment alone, associated to a nutritional deficiency in polyamine (PDC) or not (PA), inhibits tumor growth, leading to a 33% and 35% tumor growth inhibition for PA and PDC animal groups, respectively. Our results showed that polyamine deprivation was efficient in reducing tumor development. The monitoring of tumor size under the different treatments is summarized in Figure 7. After 15 days of treatment, tumour reduction induced by HQ1-44 (-30%) was amplified in the group receiving 3% DFMO in their drinking water (-56%). Polyamine deprivation in the group of animals receiving the PDC, in the absence of any other treatment, had no impact on tumor growth while the PDC diet increased the tumor growth inhibitory effect of the combined treatment with HQ1-44 and DFMO (-65% of tumor growth inhibition). The various treatments did not significantly modify the animal and organ weights, except for DFMO associated with HQ1-44 treatment, which induced a significant reduction in the spleen weights (Table 3). The decrease in spleen weight in animals receiving this combined treatment is low (-16% with respect to the control, Table 3) compared to the spleen toxicity of the treatment with 4 mg/kg cis-platin (-44%, Table 2). Contrary to the treatment by this alkylating agent, the histological analysis of the spleen after the combined treatment with PDC, DFMO and HQ1-44 did not show any significant decrease in the red pulp of this organ.

A significant decrease in circulating iron in serum was observed in the group of animals receving the Quilamine in conjunction with the polyamine-free diet (Table 4). No change in the ferritin and transferrin levels was detected in these groups while an unexpected decrease in the serum ferritin level was observed in the group of untreated animals receving the polyamine-free diet (PDC). The various treatments did not modify the polyamine levels in RBC (Table 4).

4-Discussion

A c c e p t e d M a n u s c r i p t 15 Our objective was to investigate whether the vectorized iron chelator HQ1-44 might be of value in the treatment of cancer. Our data demonstrate in vivo that HQ1-44 is effective, by limiting tumor growth of HCT116 cells in xenografted nude mice. The antitumor efficiency of the Quilamine HQ1-44 is similar to that of the reference alkylating antitumor agent, cis-platin, and is not associated with toxicity, unlike the reference treatment. In addition, both in vivo and in vitro, the results obtained are helpful in understanding the mechanisms involved, suggesting an impact of HQ1-44 on the cell cycle through an alteration of both iron and polyamine metabolisms.

The decrease in the intracellular ferritin protein level and the increase in soluble transferrin receptor concentration in culture medium in the presence of HQ1-44 clearly show that HQ1-44 induces a reduction in the intracellular iron pool in tumor cells, which in turn regulates the expression of these two genes through the Iron Responsive Element/Iron Regulatory Protein (IRE/IRP) system. These data are in accordance with those we previously reported, demonstrating the iron-chelating activity of Quilamines, including HQ1-44. In HQ1-44, the iron-chelating activity of the chelating motif is reinforced by the polyamine backbone, with both the 8-HQ moiety and the polyamine part participating in the iron coordination [START_REF] Corce | Polyaminoquinoline iron chelators for vectorization of antiproliferative agents: design, synthesis, and validation[END_REF]. In addition, a role of the amino groups of polyamines in the chelation of metallic cations and in the stability of iron chelates has previously been reported [START_REF] Løvaas | Antioxidative and metal-chelating effects of polyamines[END_REF][START_REF] Palmer | Polyamine complexes with seven-membered chelate rings: Complexe formation of 3-azaheptane-1,7-diamine, 4-azaoctane-1,8-diamine (spermidine), and 4,9-diazadodecane-1,12-diamine (spermine) with copper(II) and hydrogen ions in aqueous solution[END_REF]. Such a high capacity for chelating iron is likely to participate in the strong antiproliferative activity, as demonstrated in a previous study, showing that the higher antiproliferative activity of the bis-8-hydroxyquinoline benzylamine chelator S1 in hepatocytes, compared to the iron chelator o-Trensox, was closely connected to the high iron-chelating capacity of this tetradentate ligand [START_REF] Lescoat | Antiproliferative and iron chelating efficiency of the new bis-8-hydroxyquinoline benzylamine chelator S1 in hepatocyte cultures[END_REF]. For low concentrations of chelating agents (2 µM), the higher iron-chelating capacity of Quilamine HQ1-44, compared to deferasirox, may explain the stronger impact on L-ferritin and transferrin receptor levels, through the IRE/IRP system.

It is noteworthy that in vivo data showing that the serum iron level is only slightly affected with the highest HQ1-44 dose, without a significant change in both systemic ferritin and soluble transferrin receptor levels, support the concept that HQ1-44 is an iron chelator targeting tumoral cells. As reported above, such a concept has been proposed to target other molecules. Our in vivo data, with a low level of side effects, reinforce those obtained in vitro showing the higher sensitivity of proliferating HCT116 cells compared to A c c e p t e d M a n u s c r i p t 16 confluent cells, as well as those previously reported, showing that cells not expressing the PTS are less sensitive to HQ1-44 [START_REF] Corce | Polyaminoquinoline iron chelators for vectorization of antiproliferative agents: design, synthesis, and validation[END_REF]. In addition, the HQ1-44 targeting can be reinforced through polyamine metabolism manipulation using polyamine depletion tools by providing animals with a polyaminedeficient diet and DFMO, an inhibitor of polyamine biosynthesis, in their drinking water. The polyamine depletion induced by these experimental conditions are currently believed to activate the PTS in tumor cells, as a feedback regulation to maintain the higher polyamine levels required for tumor growth [START_REF] Moulinoux | Circulating polyamines as biological markers for cancer[END_REF].

In addition to its impact on intracellular iron metabolism, HQ1-44 could also exert its antiproliferative activity by modulating polyamine metabolism. Indeed, polyamines are involved in the control of cell cycle progression and cell proliferation [START_REF] Wallace | A perspective of polyamine metabolism[END_REF]. Thus, the ubiquitous polyamine molecules modulate the G1/S transition phase [START_REF] Wallace | A perspective of polyamine metabolism[END_REF]. Our results show that the Quilamine HQ1-44, like deferasirox at higher concentrations, disturbs the metabolism of natural polyamines by inhibiting their biosynthetic pathway. HQ1-44 reduces the gene expression of the metabolism of polyamines, such as those of ornithine decarboxylase (ODC), antizyme (OAZ1) and S-adenosylmethionine decarboxylase (SamDC). This leads to a significant decrease in putrescine, spermidine and a slight, non-significant, reduction in spermine.

Such findings are characteristic of a biosynthetic pathway inhibition, such as that of ODC by DFMO. HQ1-44 also modulates the retro-conversion oxidative pathway of polyamines, causing inhibition of the production of N1-acetyl spermine and increased levels of N1-acetyl spermidine. The contribution of the inhibitory effect of chelating iron, such as by Quilamine HQ1-44, to the metabolism of polyamines and their impact on cell proliferation is under study.

In conclusion, HQ1-44 Quilamine is effective in inhibiting HCT116 in vitro and in vivo in xenografted mice. This effect is related to the iron chelator impact of the iron chelator motif, which is reinforced by the polyamine backbone. An inhibitory impact of HQ1-44 on polyamine metabolism may also play a role by affecting cell cycle progression. The polyamine backbone of the molecule enables tumor cells to be targeted and limits the toxicity and side effects of the molecule. Taken together, these results suggest that Quilamines are a way of exploring and adding to the cancer therapeutic panoply in association with other treatments.

A c c e p t e d M a n u s c r i p t 7 2. 1 . 8 -

 718 Measurement of gene expression

  were divided randomly into different treatment groups (6 mice per group) and were treated daily by intraperitoneal injection of Quilamine HQ1-44 at different doses (0, 6.6, 20 and 40 mg.kg -1 ) or cis-platin (4 mg.kg -1 ) for 17 days. The mice were observed daily. Mice weight and tumor volume were measured three times per A c c e p t e d M a n u s c r i p t 9 week with a digital caliper. Tumor volumes were calculated according to the formula for a spheroid: tumor volume = 0.5 x length x width 2 . Growth inhibition was calculated from the start of treatment by comparing the change in tumor volume for vehicle and treated groups.

  A c c e p t e d M a n u s c r i p t 10 Ferritin and transferrin were quantified in plasma by ELISA using a mouse-specific assay kit in accordance with the manufacturer's recommendations (Mouse Ferritin Elisa Kit, Abnova; Mouse Transferrin Elisa Kit, Genway).

  as means ± SD from three independent measurements. Statistical analysis was performed using the non-parametric Mann-Whitney test (* p≤0.05, ** p≤0.01).

Table 4 :

 4 Effect of polyamine depletion on the antitumor efficiency of HQ1-44 in HCT116 xenografted immunocompromised nude mice. Effect on blood levels of iron and polyamine parameters. Results from each animal group were expressed as means ± SD from three independent measurements. Statistical analysis was performed using the non-parametric Mann-Whitney test (* p≤0.05, ** p≤0.01).

Figure 1 :

 1 Figure 1: Relationship between PTS efficiency and the antiproliferative effect of HQ1-44 in HCT116 cell

Figure 2 :

 2 Figure 2: Effect of HQ1-44 on DNA synthesis, cell cycle and apoptosis in proliferating HCT116 cultured

Figure 3 :

 3 Figure 3: Effect of HQ1-44 on cell nuclei of HCT116 cells (shape and size). The impact of HQ1-44 was

Figure 4 :

 4 Figure 4: Effect of a 72 h treatment of proliferating HCT116 cells with HQ1-44 (2 µM), ICL670 (2 µM)

A c c e p t e d M a n u s c r i p t 22 Figure 5 :

 225 Figure 5: Antitumor effect of HQ1-44 compared to cis-platin in HCT116 xenografted athymic nude mice.

Figure 6 :

 6 Figure 6: Histological analysis of spleen. Hematoxyline eosine saffron (HES) staining from HCT116-

Figure 7 :

 7 Figure 7: Effect of polyamine depletion induced by DFMO and/or by a polyamine-deficient diet (PDC) on

  Figure1

Figure

  Figure

Figure

  Figure

Table 3 Table A

 3A

			Iron Parameter			Polyamines
		Iron	Ferritin	Transferrin	Putrescine	Spermine	Spermidine
		(µM)	(µg/l)	(g/l)	(µM)	(µM)	(µM)
	PA	38,30±4,95	1187±328	2,46±0,93	0,24±0,14 7,73±3,30 0,48±0,27
	PDC	38,37±5,65	769±222**	1,79±0,46	0,18±0,14 6,24±1,06 0,35±0,12
	M 932±146 2,48±1,14 33,97±13,88 33,97±13,88 34,25±8,44 1,80±0,66 37,00±9,50 920±158 2,19±0,52 26,73±6,11** 1118±535 2,05±0,34 38,00±13,88 1165±324 1,65±0,66 a n u s c r i p t 0,22±0,17 11,04±4,8 0,73±0,45 0,14±0,05 8,8±2,58 0,64±0,28 0,24±0,17 5,78±1,53 0,30±0,10 0,19±0,11 8,58±4,00 0,69±0,51 0,18±0,14 7,04±1,60 0,53±0,3 PDC+HQ1-44+DFMO 28,94±6,62** 1098±146 PA+DFMO PDC+DFMO PA+HQ1-44 PDC+HQ1-44 PA+HQ1-44+DFMO 1,73±1,12 0,18±0,11 8,64±3,47 0,58±0,25
		c c e p t e d

Table 4

 4

5-Acknowledgments

This work was supported by the development funds Ouest-Valorisation and FEDER Europe (Brittany region). The authors are grateful to the Conseil Régional Pays de la Loire, to the French Ministry of Education, to the Ligue Nationale contre le Cancer (LNCC, Ille et Vilaine/Loire Atlantique), to the Association pour la Recherche sur le Cancer (ARC) and to the Centre National de la Recherche Scientifique (CNRS) for financial support. The authors thank P. Loyer (Inserm UMR991, Rennes, France) and I. Morel (CHU Pontchaillou, Rennes, France) for scientific assistance in the flow cytometry and LC/MSMS analyses, respectively. In vivo experiments were carried out in the animal handling facility of the university, the ARCHE platform of the Structure Fédérative de Recherche BIOSIT in Rennes (BiogenOuest and Cancéropôle Grand Ouest network). The histological analysis were carried out in the histopathological platform H2P2 of the Structure Fédérative de Recherche BIOSIT in Rennes (BiogenOuest and Cancéropôle Grand Ouest network).

6-References

Rev: 5'-TCGAGGCCCTGTAATTGGAA-3' Fw : 5'-CCCAAGATCCAACTACGAGCTTT-3'

TfR1 :

Rev : 5'-GCTTTCCCTTTCCTTGCATATTCT-3' Fw : 5'-GTGGTACCCAAATAAGGATAATCTGT-3' L-ferritine : Rev : 5'-AGGGCCCCTTGCAAATTAA-3' Fw : 5'-GCCTATTGGCTGGAGGGAG-3'

PAO:

Rev: 5'-GGTAGGCTGGGACCGTCATT-3' Fw : 5'-AAGGTGGCCCTGGTTACCA-3'

OAZ1 :

Rev: 5'-AATTCCGCCGAAAAAGAGAAG-3' Fw ; 5'-CTCCTAGGCCCTCTGGGTAGTT-3'

SAMDC :

Rev : 5'-TCGTCTTCCGCAGTAAGACCTAT-3' Fw : 5'-CTTTGCAGTAACACCATCTTTGATG-3'

ODC :

Rev : 5'-CGGTACAGCCGCTTCCTACA-3' Fw : 5'-CGGTGCCACGCTCAGAAG-3'


Kevin Hardonnière

Laurence Huc

Normand Podechard

Morgane Fernier

Xavier Tekpli

Isabelle Gallais

Odile Sergent

Dominique Lagadic-Gossmann

come

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> B #? # # $ , -. / >:-. ? G ( ( (# ( $ / 9 $ ( (( # #

5


V Shubina

L Gaillet

S Ababou-Girard

V Gaudefroy

T Chaussadent

F Farças

T Meylheuc

C Dagbert

J Creus

The influence of biosurfactant adsorption on the physicochemical behaviour of carbon steel surfaces using contact angle measurements and X-ray photoelectron spectroscopy

We investigated modifications to carbon steel surfaces due to the adsorption of a biosurfactant derived from Pseudomonas fluorescens bacteria cells using contact angle measurements (CAM) and X-ray photoelectron spectroscopy (XPS). After conditioning carbon steel in solutions with three different concentrations of biosurfactant molecules: 0.05, 0.3 and 1 g.L -1 , the average thickness of the biosurfactant layer on the carbon steel specimens was 7.9±0.3, 12.1±0.5 and 16.4±0.7 Å, respectively. The biosurfactants changed the composition of both the Fe 2+ and Fe 3+ mixed-oxide layer and the outer layer, mostly composed of Fe 3+ associated with magnetite. Contact angle measurements indicate decreased hydrophobic properties after the carbon steel was modified by biosurfactant. It was shown that the carbon steel surface free energy depends on the biosurfactant concentration, due to an acquisition of strong electron-donating properties.

Many bacteria are able to produce amphipathic substances (also called "biosurfactants"), which are potentially interesting compounds for many fields of study, due to having both hydrophilic and hydrophobic moieties in the same molecule. This internal "opposition" allows them to exhibit surface active properties and favour aggregation at phase boundaries or interfaces. Their hydrophobic parts can be saturated/unsaturated hydrocarbon chains or fatty acids; while their hydrophilic parts are mainly composed of an acid, peptide or mono-, di-, or polysaccharides [START_REF] Fracchia | Biosurfactants and bioemulsifiers biomedical and related applications -Present status and future potentials[END_REF].

Interest in biosurfactants has been increasing in recent years, as they have advantages, compared with chemical surfactants, such as environmental compatibility, lower toxicity, higher biodegradability [START_REF] Mulligan | Environmental applications for biosurfactants[END_REF][START_REF] Banat | Microbial biosurfactants production, applications and future potential[END_REF]. They have already numerous applications in different industrials fields: pharmaceutical and cosmetic industries, enhanced oil recovery and bioremediation [START_REF] Desai | Microbial production of surfactants and their commercial potential[END_REF]. Nevertheless, their large-scale application are limited by the high cost of production and by limited understanding of their interactions with different surfaces and with environment [START_REF] Desai | Microbial production of surfactants and their commercial potential[END_REF].

Different kinds of bacteria synthesise a wide range of compounds such as glycolipids (Pseudomonas aeruginosa, Rhodococcus erythropolis [START_REF] Ciapina | Biosurfactant production by Rhodococcus erythropolis grown on glycerol as sole carbon source[END_REF][START_REF] Guerra-Santos | Pseudomonas aeruginosa biosurfactant production in continuous culture with glucose as carbon source[END_REF]) phospholipids (Corynebacterium lepus [START_REF] Cooper | Production of surface-active lipids by Corynebacterium lepus[END_REF]), fatty acids (Nocardia erythropolis [START_REF] Macdonald | Surface-active lipids from Nocardia erythropolis grown on hydrocarbons[END_REF]), polymeric surfactants (Acinetobacter calcoaceticus [START_REF] Zhao | Biosurfactants from Acinetobacter calcoaceticus BU03 enhance the solubility and biodegradation of phenanthrene[END_REF]) and lipopeptides (Bacillus subtilis, Pseudomonas fluorescens, Serratia marcescens [START_REF] Cooper | Enhanced Production of Surfactin from Bacillus subtilis by Continuous Product Removal and Metal Cation Additions[END_REF][START_REF] Nielsen | Production of cyclic lipopeptides by Pseudomonas fluorescens strains in bulk soil and in the sugar beet rhizosphere[END_REF][START_REF] Matsuyama | A novel extracellular cyclic lipopeptide which promotes flagellumdependent and -independent spreading growth of Serratia marcescens[END_REF]).

Lipopeptides class of biosurfactants are of interest because of their wide range of biological activities like antibiotic, antifungal, antiviral and antitumour [START_REF] Mandal | Lipopeptides in microbial infection control: Scope and reality for industry[END_REF][START_REF] Rosenberg | High-and low-molecular-mass microbial surfactants[END_REF]. Lipopeptides are stable at high temperatures, over a large range of pH and at high salt concentrations [START_REF] Rosenberg | High-and low-molecular-mass microbial surfactants[END_REF].

Meylheuc et al. [START_REF] Meylheuc | Adsorption of biosurfactant on solid surfaces and consequences regarding the bioadhesion of Listeria monocytogenes LO28[END_REF] ascertained that lipopeptide biosurfactants derived from Pseudomonas fluorescens improve antibacterial properties and repulse the A c c e p t e d M a n u s c r i p t 4 adhesion of pathogenic Listeria monocytogenes. This kind of biosurfactant barrier coating on the stainless steel surface was studied using CAM, AFM, XPS and PM-IRRAS [START_REF] Meylheuc | Adsorption on stainless steel surfaces of biosurfactants produced by gram-negative and gram-positive bacteria: Consequence on the bioadhesive behavior of Listeria monocytogenes[END_REF]. As a matter of fact, AFM observations enabled to say that probably the biosurfactant layer covered the surface with patches. Dagbert et al. [START_REF] Dagbert | Corrosion behaviour of AISI 304 stainless steel in presence of a biosurfactant produced by Pseudomonas fluorescens[END_REF][START_REF] Dagbert | Pit formation on stainless steel surfaces pre-treated with biosurfactants produced by Pseudomonas fluorescens[END_REF] found a difference in the corrosion behaviour of a stainless steel surface when modified by biosurfactant obtained from gram-negative bacterium Pseudomonas fluorescens in 0.15 M NaCl.

The corrosion behaviour of carbon steel was also studied in simulated concretepore solutions contaminated by chlorides (0.5 M NaCl) with and without adding lipopeptides from Ps. fluorescens [START_REF] Shubina | Utilisation des biosurfactants comme « biorevêtement » anticorrosion sur armatures béton armé[END_REF][START_REF] Shubina | Biosurfactant coatings as innovative inhibitors against corrosion of steel rebars[END_REF][START_REF] Shubina | Biomolecules as a sustainable protection against corrosion of reinforced carbon steel in concrete[END_REF]. Polarization curves and electrochemical impedance spectroscopy demonstrated that these biosurfactants can be regarded as good inhibitors of steel corrosion in the presence of chloride ions. Despite these extensive studies [START_REF] Meylheuc | Adsorption of biosurfactant on solid surfaces and consequences regarding the bioadhesion of Listeria monocytogenes LO28[END_REF][START_REF] Meylheuc | Adsorption on stainless steel surfaces of biosurfactants produced by gram-negative and gram-positive bacteria: Consequence on the bioadhesive behavior of Listeria monocytogenes[END_REF][START_REF] Dagbert | Corrosion behaviour of AISI 304 stainless steel in presence of a biosurfactant produced by Pseudomonas fluorescens[END_REF][START_REF] Dagbert | Pit formation on stainless steel surfaces pre-treated with biosurfactants produced by Pseudomonas fluorescens[END_REF][START_REF] Shubina | Utilisation des biosurfactants comme « biorevêtement » anticorrosion sur armatures béton armé[END_REF][START_REF] Shubina | Biosurfactant coatings as innovative inhibitors against corrosion of steel rebars[END_REF][START_REF] Shubina | Biomolecules as a sustainable protection against corrosion of reinforced carbon steel in concrete[END_REF], many aspects of layer composition and thickness are still poorly understood. The interesting electrochemical results encouraged the authors to further investigate the process occurring between the carbon-steel surface and the biomolecules used in previous studies [START_REF] Shubina | Biomolecules as a sustainable protection against corrosion of reinforced carbon steel in concrete[END_REF].

Therefore, in the present work contact angle measurements (CAM) and XPS analysis were conducted, to make a more precise assessment of the surface modification of low carbon steel by biosurfactants. Three concentrations of biomolecules, corresponding to three different states of molecules in the solution, were used in this study, among them the concentration of 1 g.L -1 that was reported as a promising one in [START_REF] Shubina | Biomolecules as a sustainable protection against corrosion of reinforced carbon steel in concrete[END_REF]. In addition, we attempted to estimate the biosurfactant layer thickness from XPS data. Complementary FTIR analysis was used to confirm the possible lipopeptide nature of P. fluorescens biosurfactants provided earlier in the literature (viscosin, surfactin, viscosinamide [START_REF] De Faria | Production and structural characterization of surfactin (C14/Leu7) produced by Bacillus subtilis isolate LSFM-05 grown on raw glycerol from the biodiesel industry[END_REF]).

MATERIALS AND METHODS

A c c e p t e d M a n u s c r i p t

2.1Steel substrates and preparation

The carbon steel for this study was kindly provided by PLBArmatures (Allians-Snaam, Carquefou, France). The chemical composition of the carbon steel is presented in Table 1. Figure 1 shows the ferritic-pearlitic microstructure deduced from Nital etching of the low carbon steel. We observe that the pearlite grain density is very low compared to the ferritic grain density. days' incubation, the KBA surfaces were scraped, then the cells were suspended in 150 mL of sterile deionized water and shaken strongly for 3 min (Vortex, Bioblock, France). The supernatant containing the biosurfactant was separated from the cells by centrifuging for 30 min at 18,000 g. The supernatant was carefully recovered then sterilized (121°C, 20 min) and stored at 4°C.

Fourier transform infrared spectroscopy analysis

Fourier transform infrared spectroscopy (FTIR) is based on how the test substrate absorbs infrared radiation. The result of FTIR analysis is a spectrum that is like a fingerprint of a sample with absorption peaks that correspond to the vibration frequencies between the bonds of the atoms making up the material.

The FTIR spectra in transmission mode were obtained by analysing an evaporated biosurfactant sample on a potassium bromide (KBr) pellet. The resulting pellet was analysed by transmission with a Nicolet 380 spectrometer between 4000 cm -1 and 400 cm -1 with a scan number of 32 and a resolution of 2 cm -1 .

Conditioning of carbon steel surfaces

Before conditioning, the surfaces were cleaned with deionized water at 20°C.

Conditioned surfaces were obtained by immersion of carbon steel coupons in four solutions with biomolecules concentrations of 1 g.L -1 (BS-1), 0.3 g.L -1 (BS-A c c e p t e d M a n u s c r i p t 0.3), 0.05 g.L -1 (BS-0.05) and 0 g.L -1 (Blank sample) for 20 h at 20°C (the necessary time was determined in [START_REF] Meylheuc | Adsorption of biosurfactant on solid surfaces and consequences regarding the bioadhesion of Listeria monocytogenes LO28[END_REF][START_REF] Meylheuc | Adsorption on stainless steel surfaces of biosurfactants produced by gram-negative and gram-positive bacteria: Consequence on the bioadhesive behavior of Listeria monocytogenes[END_REF]). Non-adsorbed compounds were then removed from the carbon steel surface by being gently soaked in deionized water and dried on laminar air flow. A carbon steel sample not subjected to any treatment (Blank sample) was also studied.

Surface tension and contact angle measurements

The aqueous BS solutions were prepared using double distilled water (Millipore Q, 12.6 m 72.28 mN.m -1 ) by surface tension measurements before the solutions containing was measured by the Drop Shape Analyser DSA100 (KRUSS, Gmbh) according to the pendant drop method. The sterile syringes (NE 43, KRUSS, Gmbh) were used for each new measurement. The standard deviation depending on the surfactant concentration ranges from ± 0.10 to ± 0.65 mN.m -1 . The test chamber temperature was controlled using two thermostatic water baths. All the experiments were carried out at 20°C. For each concentration, more than 15 successive measurements were carried out.

Contact angle measurements for four different liquids (distilled water, diiodomethane, glycerol and ethylene glycol) were carried out at room temperature. The sessile drop method for the contact angle determination was used through deposition of liquid drops on solid surfaces with a micrometric syringe and controlled by means of a camera. The contact angles were determined from the droplet image using the tangent method. At least contact angles of 5 droplets were measured for each solvent on three independently conditioned surfaces. The final values were averaged on at least two different locations on the surface. Curve fitting of high resolution XPS spectra and Semi-Quantification Analysis was performed using CasaXPS data software.

XPS measurements

RESULTS AND DISCUSSION

3.1Characterization of isolated BS

FTIR analysis

The FTIR spectrum illustrated in Figure 2 is a signature of the BS.

A c c e p t e d M a n u s c r i p t

Figure 2. FTIR spectrum of the dried BS

The main FTIR absorption peaks attributions reported in Table 2 highlight the presence of amide bands. They are in good accordance with the composition of peptide compounds, which represent the hydrophobic part of biosurfactants. The presence of polysaccharides is due to the use of the non-purified biosurfactant (polysaccharides are substances secreted by bacteria to organize them into biofilms) [START_REF] De Faria | Production and structural characterization of surfactin (C14/Leu7) produced by Bacillus subtilis isolate LSFM-05 grown on raw glycerol from the biodiesel industry[END_REF]. The carbon was also stored as lipids: CH 2 stretching in the 2850-2950 cm -1 region and C=O stretching at 1743 cm -1 .

These absorptions confirm the lipopeptide nature of the biosurfactant [START_REF] Mohamed | Application of FT-IR Spectroscopy for Rapid and Simultaneous Quality Determination of Some Fruit Products[END_REF][START_REF] Ferré | Conformational studies of the cyclic l, d-lipopeptide surfactin by Fourier transform infrared spectroscopy[END_REF] and correlate positively with the XPS analysis.

Table 2. Attribution of the main BS FTIR absorption peaks [22-24].

Wavenumber (cm

Surface tension isotherm

The biosurfactant surface tension plot is presented in The surface tension of 30.01 mN.m -1 is in good accordance with the data of previous studies on lipopeptides reported in literature [START_REF] Singh | Synthesis of stable cadmium sulfide nanoparticles using surfactin produced by Bacillus amyloliquifaciens strain KSU-109[END_REF]. The surface tension curve can also provide information on the critical micellar concentration point (CMC). Above the CMC point, surfactant molecules have a tendency to selforganize into micelles or aggregates in the solution and surface tension values do not vary significantly. The literature data about a CMC point for lipopeptides was mainly obtained from surface tension values changing as a function of their concentrations and is in the range 1×10 -4 g.L -1 -2 ×10 -2 g.L -1 [START_REF] Soledade | Structure, chemistry, and biological activity of pseudophomins A and B, new cyclic lipodepsipeptides isolated from the biocontrol bacterium Pseudomonas fluorescens[END_REF]. However, our CMC value was higher than those reported. This difference may be due either to

28].

After exploring the biosurfactants surface tension, three points (indicated in The plateau of this zone in Figure 3 indicates that no further change in surface tension was produced.

A sample conditioned in sterile deionized water without addition of biosurfactant molecules was used as Blank with further designation BS-0.

Contact angle measurements (CAM) and surface free energy calculations

The results of contact angle measurements are shown in Table A-1 (Appendix).

For sample BS-0 the measurements were not carried out due to zones of localized corrosion (pitting) on the surface, which may have affected the spreadability of drops and distort the measurements. Owens/Wendt theory (also named the "harmonic method") was used to determine the surface free energy for our case. This approach considered the surface energy of a solid as a two-component model: a dispersive component (y=mx+b), (Eq.1).

Thus, the contact angle ( ) was measured using different liquids with known values of surface tension for the liquids and their polar and dispersive components (Table A-2 of Appendix). The surface free energy components of carbon steel samples can be obtained using (Eq. 1). The parameters listed in Table 3 were calculated from experimental data for liquid contact angles on unconditioned (Blank) or conditioned carbon steel (BS-0.05, BS-0.3, BS-1). was also established due to masking a surface. This is another proof of good adsorption of biosurfactant molecules.

A comparison between the surface energy values in

CAM has allowed us to elucidate the adsorption of biosurfactant molecules as a function of concentration on the macro scale. It was shown that the higher the concentration (up to 1 g.L -1 ), the higher carbon steel surface free energy will be, due to a surface modification. However, when the solution is near (BS-0.3) or above the CMC (BS-1), no significant increase of surface energy polar component was noticed. To confirm and study this in greater depth, a more sensitive physicochemical method, XPS analysis, was used.

XPS analysis

A c c e p t e d M a n u s c r i p t XPS analysis has been of key importance in the field of corrosion science for organic films of inhibitors [START_REF] Swift | Surface analysis of corrosion inhibitor films by XPS and ToF-SIMS[END_REF] in different corrosion media. In this paper, XPS analysis was used to investigate the composition of the biosurfactant layer adsorbed on the carbon steel surface, and to evaluate the layer thickness for different BS concentrations: BS-0.03, BS-0.5 and BS-1.

Survey scan analysis

We first recorded the survey scans of all the samples. Carbon steel surfaces of Blank, BS-0 and BS-1 samples are presented in Another feature for all biosurfactant-conditioned surfaces was the presence of a nitrogen peak. It can be attributed to a biosurfactant fingerprint on the carbon steel surface.

For the BS-0 sample the surface is seen to be highly oxidized (corrosion products on the surface), and the substantial increase and slight shift of Fe 2p peak can be seen.

We then studied the high resolution spectra for C 1s, O 1s, N 1s and Fe 2p to get a better understanding and more information about adsorbed biosurfactant molecules and the corrosion protection mechanism.

Analysis of C 1s spectra

The deconvoluted C 1s core level spectra for BS-0 (Blank) and BS-1 samples are presented in Figure 6 (a,b).

A c c e p t e d M a n u s c r i p t For the Blank sample, the largest contribution is attributed to the first peak of a carbon single bond with the other carbon or hydrogen located at approximately 285.0 eV. The second peak at 286.4 eV is attributed to a carbon making a single bond with oxygen (C-O). This binding energy corresponds also to a carbon making a single bond with nitrogen (C-N), but we considered that that contribution was weak (see the N 1s spectrum in Figure 8). The last component at 288.2 eV is a small contribution and suggests creation of double carbon-oxygen bonds (O=C). This peak can be explained by a presence of a thin hydroxide layer on the carbon steel surface, i.e. OH-Fe. This decomposition could be completed by the fourth component, at higher binding energy, to reconstitute the experimental spectrum. However, we decided only to play on the ratio of G/L for the last component. The full width at half maximum (FWHM) value of The second and third components at 286.6 eV and 288.2 eV increase significantly and can be ascribed to involving new bonds between carbon and nitrogen or oxygen atoms in C-N and C-OH bonds. The peak at 288.2 eV is sometimes attributed to compounds such as ester, acetate, amide, carbonyl and carboxylate compounds, which are in good accordance with the biosurfactant's structure. The complementary peak that appears at 289.7 eV can be ascribed to the creation of the double carbon bonds to oxygen and nitrogen.

Analysis of O 1s spectra

The O 1s core level spectra for all samples clearly show two components. The asymmetry of spectra at higher binding energies expects two and three component-shoulders for the Blank and biosurfactants samples, respectively at (Figure 7 (a,b)).

By taking as example Blank and BS-1 samples, the peaks were fitted as follows:

-The first peak at 529.8 eV/529.9 eV is attributed to O 2-, which represents the lattice oxygen

Analysis of N 1s spectra

For the Blank sample, the nitrogen signal is low (Figure 8 The N 1s spectra envelopes for all the biosurfactant-conditioned samples were fitted using two components with FWHM = 1.7 eV and G/L = 30, as shown in Assuming that (i) the thickness of the oxide film on the Blank sample (which was not subjected to any treatment) will be used as a reference point, and that (ii) we obtained a uniform layer of biosurfactants on the steel surface (Figure 11), a semi-quantitative approach can be used. Accordingly, the biosurfactant layer thickness can be calculated from the intensity ratios, as: The calculated average biosurfactant layer thickness for BS-0.05, BS-0.3 and BS-1 are 7.9±0.3, 12.1±0.5 and 16.4±0.7 Å, respectively. From these estimations, we can consider that the adsorption of biosurfactant molecules takes place for all the conditioning samples (i.e. BS-X). In addition, the results suggest that the average thickness of biosurfactant layer slightly increases with increasing BS concentration. These average layer thicknesses cannot be accurate values, because many parameters have not been taken into account. However, it can give us an idea about the organization of layers. It is logical that the values 12.1±0.5 and 16.4±0.7 Å are close to each other, because the biosurfactant solution concentrations 0.3 g.L -1 and 1 g.L -1 are close to threshold point of the other hand, we can see that insufficient biomolecules concentration (0.05 g.L -1 ) could create a layer with about 7.9±0.3 Å thick.

To estimate the biosurfactant layer on the surface in another way, we supposed that for the low biosurfactant concentration, the molecules are tilted and lie closer to the surface. When the biosurfactant concentration increases, the molecules of biosurfactant "stand up" on the surface. Assuming that N 1s signal indicates the presence of the biosurfactant (i) and that (ii) the biosurfactant molecules are distributed uniformly on the surface, we attempted to take into account the N 1s core level to find layer density. Using the relation of I N1s BS (intensity signal of N 1s on biosurfactant conditioned surface (see BS-X the carbon steel surface, and comparing with reference (HOPG, N s =4×10 15 atomes/cm 2 ), the signal of N 1s core level sample is equivalent to 5×10 14 , 3×10 14 and 1×10 14 atoms/cm 2 for BS-1, BS-0.3 and BS-0.05 samples respectively. These quantitative results suggest that the density of the biosurfactant layer on the surface increases with increasing biosurfactant concentration. It must be underlined once again, that the heterogeneity of the layer has not been taken into account; therefore this information is valid only in the case of a homogenous ad-layer. This result is consistent with that of iron: when biosurfactant concentration increases, the biosurfactant layer thickness increases and the number of molecules on the surface increases. These molecules then will have to "stand up" (more compact layer) on the surface instead of "lie down" at low concentrations.

The more compact layer was also specified using contact angle measurement results. The significant increase in surface energy due to an increased polar component is due to creation of a compact biosurfactant layer by anchoring -NH 2 functional groups on the metal surface. Consequently, the lipid part of biosurfactant molecules is turned outwards from the surface. These lipids, which are presumably hydrophobic, have an important property, reported by Boggs [START_REF] Boggs | Effect of lipid structural modifications on their intermolecular hydrogen bonding interactions and membrane functions[END_REF][START_REF] Boggs | Lipid intermolecular hydrogen bonding: influence on structural organization and membrane function[END_REF].That is their ability to create a network of intermolecular hydrogen bonds to increase the packing density in the biosurfactant layer, and increase the polar component of the surface energy.

CONCLUSIONS

Modification of carbon steel using three concentrations: 0.05, 0.3 and 1 g.L -1 of biosurfactants derived from Pseudomonas fluorescens was studied using CAM and XPS measurements. From this study, we can draw the following conclusions: -The N 1s core level was specified as a signature of biosurfactant adsorption. The N 1s core level signal is equivalent to 5×10 14 , 3×10 14 and 1×10 14 atoms/cm 2 for samples BS-1, BS-0.3 and BS-0.05 respectively.

-Assuming a homogenous ad-layer, the thickness of the biosurfactant layer was found to be approximately 7.9±0.3, 12.1±0.5 and 16.4±0.7 Å, for 0.05, 0.3 and 1 g.L -1 of biomolecules, respectively.

In this work, the conditioning of steel coupons in deionized water leads to the formation of a uniform film of adsorbed molecules on the steel surface. The properties of this film are strongly dependent on the concentration of the BS.

The thickness of the adsorbed films slightly increases with the BS concentration up to a threshold value corresponding to a specific arrangement of molecules on the surface that could be also related to the modification of the composition of the iron oxide film in presence of BS molecules. This finding can explain our first positive results concerning corrosion prevention by using biomolecules but further research using electrochemical methods must be done to confirm these results. In addition, the ellipsometry technique can be applied to support our experimental results of adsorbed biosurfactant thickness values deduced from XPS analysis.

APPENDIX

A c c e p t e d M a n u s c r i p t

Figure 1 .

 1 Figure 1. Carbon steel microstructure: the light ferrite region and the dark pearlite regions.The samples were disk cut from rebars of two different diameters: 0.6 cm (for XPS) and 2 cm (for CAM). The samples were embedded in epoxy resin with an exposed surface area of 0.28 cm 2 and 3.14 cm 2 , respectively. The specimens were mechanically polished with SiC emery paper down to grade 1200, then with 6 and three times in distilled water, and finally dried under an air flow.

A c c e p t e d M a n u s c r i p t 6 2. 2

 62 Biosurfactant productionThe biosurfactant was kindly provided by Thierry Meylheuc and was prepared as described previously in reference[START_REF] Meylheuc | Adsorption of biosurfactant on solid surfaces and consequences regarding the bioadhesion of Listeria monocytogenes LO28[END_REF]. Briefly, isolated bacteria were kept frozen at -80°C in cryotubes before production. Petri dishes of King B Agar (KBA, Merck) were used for bacteria growth. Each KBA plate (about 80 plates to 150 mL of BS) was inoculated with cryotube suspension for dense coverage of the agar surface. Inoculated plates were then incubated at 20-22°C. After 4

A c c e p t e d M a n u s c r i p t 8 XPS

 8 photoemission (obviously due to an insulating film on the surface) which therefore requires the use of the neutralizer during all experiments. To check the reproducibility of the results, survey spectra and high resolution spectra for Fe 2p, C 1s, O 2s and N1s were recorded on two different locations on each specimen. Moreover, two independently prepared specimens for each concentration of interest were studied. Binding energies were determined by reference to the C 1s component (recorded from HOPG sample) set at 285.0 eV.

Figure 3 .

 3 It is clear that in the presence of biosurfactant, the water's surface tension dropped linearly with increasing biosurfactant concentration until the biosurfactant critical micelle concentration (CMC) of approximately 0.6 g.L -1 was reached. The shape of this surface tension isotherm depends on two factors: (i) density and (ii) orientation of biomolecules at the interface layer.

A c c e p t e d M a n u s c r i p tFigure 3 .

 3 Figure 3. Surface tension measurements ( solutions versus logarithm of BS concentration (C).

  A c c e p t e d M a n u s c r i p t the use of non-purified biosurfactant or to the fact that a wide variety of cyclic lipopeptides (CLs) are produced by bacteria. It was reported that the BSs produced by Pseudomonas fluorescens can be a mixture of 4-5 homologues [27-

Figure 3 )

 3 Figure3), corresponding to the three different configurations of biomolecules in aqueous solution were selected:

  A c c e p t e d M a n u s c r i p tAs can be seen from Table A-1 (Appendix), the most remarkable changes were the increase of spreadability of water droplets, i.e. the decrease of contact angle values of water from 76.7° (Blank) to 28.9°, 0° and 0°, with BS-0.05, BS-0.3 and BS-1, respectively. Because of complete wetting of the surface for two samples (BS-0.3 and BS-1) we had difficulty in ascertaining the contact angles, so a value of 0° was used. Moreover, regarding the change in water contact angle over time, presented in Figure 4 (curve b), for Blank and conditioned surfaces, we can assume a substantial increase of surface wetting properties. In other words, this data suggested that carbon steel surfaces had been modified by surface-active biomolecules, this effect having been already evidenced on stainless steel surface in [15]. Characteristically, these facts indicate of favourable adsorption of the test liquid on the surface [29].A c c e p t e d M a n u s c r i p t

Figure 4 .

 4 Figure 4. Evaluation of water contact angles over time on Blank (a) and conditioned (BS-1) samples (b). The modification due to the adsorption of biomolecules is accompanied by a change of carbon steel surface tension. The surface tension of conditioned samples was calculated from a set of liquid/solid contact angles, developed by bringing four liquids (diiodomethane, ethylene glycol, glycerol and water) in contact with surfaces [30].

(

  van der Waals and other non-specific interactions with applied liquid) and a polar component (dipole-dipole, dipole-induced dipole, hydrogen bonding and other site-specific interactions). The combination of the equations of Good and Young produces the Owens/Wendt theory:

A c c e p t e d M a n u

  s c r i p t acidic components gave in sum the polar component of surface energy [34]. The basic characteristic resulted from the presence of chemical groups such as COO - or HSO 3 -on the conditioning surfaces [35]. Otherwise, strong surface hydrophilicity can be due to a well-organized surfactant layer, terminated by COOH or COO -groups and not a mixture of them, as was investigated by Vallée et al. [36]. After the conditioning in different biosurfactant concentrations (0.05, 0.3 and 1 g.L -1 ), the corrosion inhibition of carbon steel was observed, compared to sample BS-0 (conditioned in distilled water without addition of BS). Because BSs contain nitrogen, they can be considered to be hard bases and so they have good adsorption properties for metal surfaces [37]. Moreover, the FTIR analysis demonstrated a cyclic BS structure, which is often better for adsorption than an acyclic structure, as a ring leaves the nitrogen lone pair more exposed for donation, according Lewis acid-base theory [30]. In that way, chemical corrosion inhibitors based on amine compounds improve their corrosion inhibition properties. In addition, a decrease of dispersive component from 39.21 to 19.36 mN.m -1

Figure 5 .

 5 Collected XPS survey spectra provide information about the presence of C 1s at 298.0 eV, N 1s at 400.0 eV, O 1s at 530.9 eV and all characteristic peaks of iron, with the main peak situated between 710.0 and 730.0 eV with doublet structure Fe 2p 3/2 and Fe 2p 1/2 . For all the spectra, we observed that the signals of Fe, O and C are intensified compared to the other elements. Traces of other elements (Ca 2p, P 2p) were found in small quantities for biosurfactant-conditioned samples, (acceptable values, caused by the presence of phosphate salts used for bacteria growth for biosurfactants production), and will not be discussed in this paper.

A c c e p t e d M a n u s c r i p tFigure 5 .

 5 Figure 5. XPS survey spectra of carbon steel of Blank (blue line), BS-0 (red line) and BS-1 (green line) samples.

Figure 6 .

 6 Figure 6. C 1s high resolution XPS spectra obtained for Blank sample (a) and BS-1 samples (b).

A c c e p t e d M a n u s c r i p t 21 different

 21 carbon peak components was considered to be 1.56 eV. Compared to the FWHM of 0.8 eV for C 1s recorded from the HOPG (Highly Oriented Pirolitic Graphite) reference sample, the width increase demonstrated complex and heterogeneous sample surfaces. After the conditioning at different biosurfactants concentrations (BS-1 sample is presented in 6 (b)), the total carbon increases (+20%), and we observed the same three components with significant contribution. Indeed, the first peak at 285.0 eV is slightly decreased ( nds [40].

[ 40 ].

 40 It could though be attributed to oxygen atoms bonded with iron in the Fe 2 O 3 and Fe 3 O 4 [41, 42]. The two fold decrease in integrated intensity from Blank to biosurfactant samples is due to the creation of the biosurfactant layer on the carbon steel (as was deduced from survey scans data for the Fe 2p signal).

A c c e p t e d M a n u s c r i p t 22 -Figure 7 .

 227 Figure 7. O 1s high resolution XPS spectra obtained for Blank (a) and BS-1 samples (b).

Figure 8 .

 8 Figure 8. N 1s high resolution XPS spectra obtained for Blank (a) and BS-1 samples (b).

Figure 8 ( 5

 85 Figure 8 (b). The first peak at 400.2 eV has the highest contribution and is mainly attributed to nitrogen atoms characteristic of non-protonated nitrogen, N-CH. This component (FWHM [47]; or a nitrogen atom coordinated with the steel surface (N-Fe) [39]. The second N 1s component at 402.4eV indicates a new bond involving nitrogen groups attributed to positively charged nitrogen [45]. The presence of protonated nitrogen can be explained by hydrogen bonding between one hydrogen atom

A c c e p t e d M a n u s c r i p tFigure 9 .

 9 Figure 9. An example of curve fitting for a Fe 2p high resolution spectrum for the Blank sample. The Fe 2p spectra of five specimens (i.e. Blank, BS-0, BS-0.05, BS-0.3 and BS-1) are compared in Figure 10 and can be used to qualitatively compare the film thickness of adsorbed biosurfactant. It is clear that conditioning in biosurfactant solutions reduces the intensities of iron peaks. Since iron peaks are recorded under the same experimental parameters, the intensity of Fe 2p peaks can be used as an indicator to estimate the thickness of the biosurfactants layer.

Figure 10 .

 10 Figure 10. Evolution of intensity (cps) for the Fe 2p high resolution XPS spectra obtained from Blank, BS-0, BS-0.05, BS-0.3, BS-1 carbon steel specimens.

Figure 11 .

 11 Figure 11. Model of carbon steel surface with biosurfactant layer.

  is the thickness of the biosurfactant layer, I Fe blank and I Fe BS-X are the integrated intensities of the Fe 2p 3/2 iron component for Blank and BS-X (X= 0.05, 0.3, 1) samples, respectively, obtained from Fe 2p spectra and BS X Fe is a IMFP (Inelastic Mean Free Path) for Fe 2p, taken here as 24 Å [49].

A c c e p t e d M a n u s c r i p t 32 -

 32 Adsorption of the biosurfactant increases the polar component of the surface energy due to a compact layer being created as -NH 2 functional groups are anchored on a metal surface; -The significant surface changes were investigated from XPS study of C 1s, O 1s, N 1s and Fe 2p peaks. The biosurfactant adsorption changes the stoichiometry of the surface's outer layer; the intensity of the component associated with magnetite increases.

Table 1 . Chemical composition of carbon steel (wt. % of main elements)

 1

	C	S	P	N	Cu	Fe
	0.24	0.055	0.055	0.014	0.85	balance

Table 3 . Surface free energy components by Owens/Wendt theory. Surface free energy components, (mN/m) Sample

 3

		D	P
		S	S	S
	Blank	39.21±1.50	5.90±1.20	45.11±2.70
	BS-0.05	24.01±0.80	31.26±1.30	55.27±2.10
	BS-0.3	20.25±0.90	43.56±2.00	63.81±2.

90 BS-1 19.36±0.80 43.56±2.00 62.92±2.80 The

total surface energy and components for the Blank sample are in good accordance with literature data [31, 32].

Table 3

 3

	shows that the

properties represent principally an increase in a basic component, which with

Table A -3 of

 A

Table A -1. Contact angle measurements of carbon steel samples after immersion at different biomolecule concentrations.

 A

			Contact angles, °Samples
	diiodomethane ethylene glycol glycerol	water
	Blank	43.3 ± 2.9	51.3 ± 1.2	33.8 ± 2.5 76.7 ± 1.2
	BS-0.05	58.4 ± 1.8	12.4 ± 1.0	36.3 ± 1.3 28.9 ± 0.45
	BS-0.3	61.8 ± 2.1	11.8 ± 0.8	38.5 ± 2.2	0*
	BS-1	60.7 ± 1.6	10.7 ± 0.9	36.7 ± 1.8	0*
	* estimated angle

Table A -2. Surface tension and components for testing liquids (at 20 °C)

 A

	Liquid	L ,mN/m	D L ,mN/m	P L ,mN/m
	Water (W)	72.8	21.8	51.0
	Diiodomethane (D)	50.8	50.8	0
	Ethylene glycol (E)	48.0	29.0	19.0
	Glycerol (G)	64.0	34.0	30.0

Table A -3. XPS elements concentration (at.%) of the surface films for Blank, BS-0.05, BS-0.3 and BS-1 samples.

 A

		Fe, at.%	O, at.%	C, at%	N, at.%
	Blank	19.1 ± 2.5	21.1 ± 2.5	59.8 ± 1.5	0.12 ± 0.1
	BS-0.05	3.5 ± 0.8	48.6 ± 1.5	42.8 ± 1.4	2.9 ± 0.5
	BS-0.3	1.1 ± 0.5	33.0 ± 1.8	57.1 ± 2.3	5.3 ± 0.7
	BS-1	0.5 ± 0.5	32.6 ± 1.7	57.3 ± 1.5	9.7 ± 1.5

ACKNOWLEDGMENTS

The authors are thankful to Sophie Ricordel, Michel Grasset and Jonathan Hamon for technical assistance and to Thomas Le Noir for advice on preparing this manuscript.