Estelle Deschamps

Annick Schaumann

Isabelle Schmitz-Afonso

Carlos Afonso

Emmanuelle Dé

Corinne Loutelier-Bourhis

Stephane Alexandre
email: stephane.alexandre@univ-rouen.fr

Stéphane Alexandre

Stéphane Alexandre

Membrane phospholipid composition of Pseudomonas aeruginosa grown in a cystic fibrosis mucus-mimicking medium

Keywords: Pseudomonas aeruginosa, CF sputum, membrane lipids, antibiotic resistance, culture medium, mass spectrometry CF, cystic fibrosis, MHB, Mueller Hilton broth, SCFM, synthetic cystic fibrosis medium, DOPC, 1,2-dioleoyl- tandem mass spectrometry, MIC, minimal inhibitory concentration, ESI-MS, electrospray ionisation-mass spectrometry, PAK, P. aeruginosa strain PAK, CFU, colony forming units, OD600, optical density at 600 nm, AFM, atomic force microscopy

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Introduction

P a g e 2 | 42

Pseudomonas aeruginosa is an opportunistic pathogen bacterium which is a major public health concern for its ability to survive on healthcare equipments [START_REF] Pachori | Emergence of antibiotic resistance Pseudomonas aeruginosa in intensive care unit; a critical review[END_REF]. This leads to nosocomial infections which are very difficult to eradicate. Due to its high pathogenicity for immunocompromised patients, such as cystic fibrosis (CF) patients [START_REF] Smith | Current and future therapies for Pseudomonas aeruginosa infection in patients with cystic fibrosis[END_REF][START_REF] Sadikot | Pathogen-host interactions in Pseudomonas aeruginosa pneumonia[END_REF], P. aeruginosa is one of the deadliest bacteria in hospitals. Moreover, P. aeruginosa can easily develop many mechanisms of resistance against antibiotics [START_REF] Bassetti | How to manage Pseudomonas aeruginosa infections[END_REF], leading to most antibiotics becoming useless. For these reasons, P. aeruginosa was set priority number one for the research and development of new antibiotics and treatments, by the world health organization (WHO) [START_REF] Tacconelli | research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis[END_REF].

In order to develop new antibiotics, new targets and strategies are required. One of the current new drug strategies involves targeting membrane phospholipids (e.g. cardiolipins [START_REF] Khoury | Targeting Bacterial Cardiolipin Enriched Microdomains: An Antimicrobial Strategy Used by Amphiphilic Aminoglycoside Antibiotics[END_REF]). Such new drugs could, by themselves, allow membrane lysis or could be used in synergy to increase the effectiveness of antibiotics [START_REF] Escriba | Membrane-lipid therapy: a new approach in molecular medicine[END_REF][START_REF] Epand | Molecular mechanisms of membrane targeting antibiotics[END_REF]. Previous analyses of the membrane lipid composition of P. aeruginosa were performed in culture media such as minimal glucose medium (MGM) [START_REF] Benamara | Impact of the biofilm mode of growth on the inner membrane phospholipid composition and lipid domains in Pseudomonas aeruginosa[END_REF][START_REF] Benamara | Characterization of membrane lipidome changes in Pseudomonas aeruginosa during biofilm growth on glass wool[END_REF], Muller-Hilton broth (MHB) [START_REF] Abbes | Identification by mass spectrometry of glucosaminylphosphatidylglycerol, a phosphatidylglycerol derivative, produced by Pseudomonas aeruginosa, Rapid communications in mass spectrometry[END_REF][START_REF] Deschamps | Determination of the collision cross sections of cardiolipins and phospholipids from Pseudomonas aeruginosa by traveling wave ion mobility spectrometry-mass spectrometry using a novel correction strategy[END_REF] or lysogeny broth (LB) [START_REF] Senechal | Phospholipid Content of Pseudomonas aeruginosa PAO1 Is Modulated by the Growth Phase Rather Than the Immobilization State[END_REF]. The membrane lipid composition mainly consisted of four lipid chemical classes: phosphatidylethanolamines (PE), phosphatidylcholines (PC), phosphatidylglycérols (PG) [START_REF] Senechal | Phospholipid Content of Pseudomonas aeruginosa PAO1 Is Modulated by the Growth Phase Rather Than the Immobilization State[END_REF] and cardiolipins (CL) [START_REF] Deschamps | Determination of the collision cross sections of cardiolipins and phospholipids from Pseudomonas aeruginosa by traveling wave ion mobility spectrometry-mass spectrometry using a novel correction strategy[END_REF]. Moreover, these phospholipids were described to be constituted of saturated, unsaturated or cyclopropylated, fatty acyl chains ranging from C14 to C18 [START_REF] Benamara | Characterization of membrane lipidome changes in Pseudomonas aeruginosa during biofilm growth on glass wool[END_REF].

Since P. aeruginosa is known to survive in diverse environments, by adapting its metabolism according to the available energy resources [START_REF] Oberhardt | Genome-scale metabolic network analysis of the opportunistic pathogen Pseudomonas aeruginosa PAO1[END_REF], an influence of the culture medium on the membrane lipids should be expected. Due to the high pathogenicity of P. aeruginosa in cases of CF lungs chronic infections, the CF lung sputum was chemically analysed with the purpose to reproduce it in laboratories [START_REF] Palmer | Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum[END_REF]. This sputum was characterized by specific amino acid concentrations, the presence of DNA, lipids and mucin and a high viscosity [START_REF] Turner | Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum[END_REF]. Thus, several media mimicking the CF sputum were described in the literature [START_REF] Palmer | Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum[END_REF][START_REF] Turner | Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum[END_REF][START_REF] Kirchner | Use of artificial sputum medium to test antibiotic efficacy against Pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung[END_REF]. The synthetic cystic fibrosis medium (SCFM) [START_REF] Palmer | Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum[END_REF], first described, contained amino acids, salts and sugars at adjusted concentrations, but lacked DNA, lipids and mucin. This medium was then improved, as SCFM2, by the addition of DNA, lipids and mucin [START_REF] Turner | Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum[END_REF].

P a g e 3 | 42

The Artificial Sputum medium (ASM) contained amino acids, mucin, DNA and egg yolk as a phospholipid nutrient [START_REF] Kirchner | Use of artificial sputum medium to test antibiotic efficacy against Pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung[END_REF]. However, most of these media are not compatible with membrane lipid extraction, as the high viscosity of such media [START_REF] Turner | Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum[END_REF][START_REF] Kirchner | Use of artificial sputum medium to test antibiotic efficacy against Pseudomonas aeruginosa in conditions more relevant to the cystic fibrosis lung[END_REF] does not allow enough bacterial growth for exploitable quantities of lipid material. For this specific reason, this work uses the commercial MHB as a reference medium and the SCFM one [START_REF] Palmer | Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum[END_REF]. By adding phosphatidylcholine to SCFM (SCFM-PC), we also analysed the impact of this choline nutrient source on the P. aeruginosa lipid composition. 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC) was chosen in order to simulate the presence of lipids from eukaryote cells, since DOPC is widely used for eukaryote cell modelling [START_REF] Zhao | Phase studies of model biomembranes: complex behavior of DSPC/DOPC/cholesterol[END_REF].

Herein, to our knowledge, this work reported, for the first time, the membrane phospholipid composition of P. aeruginosa grown in CF sputum-mimicking media -e.g. SCFM and SCFM-PC. The membrane lipids were identified and semi-quantified using liquid chromatography -mass spectrometry (LC-MS). Tandem mass spectrometry (MS/MS) experiments were performed to determine the fatty acyl composition of phospholipids. With the aim to link membrane lipid modifications and antibiotic resistance, the minimal inhibitory concentration (MIC) of four antibiotics was determined as well.

Material and methods

Chemicals

All SCFM components, chemicals required for the membrane lipid extraction and antibiotics were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), and phospholipids internal standards (PC 17:0/17:0, 0.5 μg; PE 17:0/17:0, 2.5 μg; PG 17:0/17:0, 1 μg; LPC 17:0, 1 μg; LPE 13:0, 2.5 μg; CL 14:0/14:0/14:0/14:0, 5 μg) were purchased from Avanti polar lipids (AL, USA). MHB media, brand Difco, was purchased from Fischer Scientific (UK). Milli Q grade water was obtained from an UltraClear TWF system (Evoqua water technologies, Germany).

P a g e 4 | 42

Methanol, chloroform, acetonitrile and isopropanol were purchased from Fischer Scientific (UK), ammonium formate from Sigma-Aldrich (Saint Quentin Fallavier, France), formic acid from Merck (Germany). Except methanol and chloroform which were HPLC grade, all solvents and buffers used were LC-MS grade.

SCFM and SCFM-PC media preparation

The SCFM medium was first described by Palmer et al. [START_REF] Palmer | Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum[END_REF]. In order to prepare this chemically defined medium more easily by hand, the number of individual stock solutions was reduced by mixing some of the individual stock solutions together (e.g. from 19

individual amino acids stock solutions to 6 individual or mixed amino acids stock solutions).

Moreover, part of the medium was autoclaved.

The stock solutions were prepared from powder, using either autoclaved Milli-Q grade water or NaOH solutions (concentrations will be detailed later) and stocked in the dark at 4°C. The bottles used for the stock solutions were autoclaved as well.

A first stock solution was prepared by mixing the following thirteen amino acids, whose final concentrations were: 360 mM alanine, 60 mM arginine -HCl, 40 mM cysteine -HCl, 300 mM glutamate -HCl, 240 mM glycine, 100 mM histidine -HCl, 420 mM lysine-HCl, 120 mM methionine, 140 mM ornithine -HCl, 340 mM proline, 280 mM serine, 200 mM threonine and 220 mM valine. A second stock solution was prepared by mixing isoleucine and phenylalanine at 110 mM and 50 mM, respectively. Leucine was stocked at 100 mM.

Aspartate was stocked at 103 mM in 0.5 M NaOH. Tyrosine was stocked at 100 mM in 1 M NaOH. Tryptophan was stocked at 130 mM in 0.2 M NaOH.

The following salts were mixed into a single stock solution at these concentrations: 23 mM NH4Cl, 153 mM KCl, 511 mM NaCl, 13 mM NaH2PO4, 13 mM Na2HPO4, 3 mM K2SO4, 4 mM NaNO3. Morpholinepropanesulfonic acid (MOPS) was stocked at 100 mM. The mixed salts and the MOPS stock solution were then autoclave sterilised for storage purpose.

P a g e 5 | 42

CaCl2 and MgCl2 were mixed as a stock solution at 877 mM and 303 mM respectively. FeSO4 was stocked at 4 mM. D-glucose was stocked at 1 M. Sodium L-lactacte was stocked at 10% w/w. CaCl2/MgCl2, Dglucose and Sodium L-lactacte stock solutions were filter sterilised through a 0.22-μm-pore-size filter. Since the FeSO4 stock solution could neither be autoclaved (precipitation) nor filter sterilised (filter blockage), the FeSO4 powder was diluted into an autoclave sterilised bottle using autoclave sterilised Milli-Q water. Fresh FeSO4 stock solution was systematically prepared to prevent precipitation during the storage. The full protocol for the preparation of the stock solutions for SCFM and SCFM-PC medium is given in the Supplementary Information S1.

For 1 L of SCFM, the following volumes of stock solutions were added: 100 mL mixed salts, 25 mL MOPS, 805 mL Milli-Q water, 10 mL mixed amino acids (first solution), 10 mL isoleucine and phenylalanine (second solution), 16 mL leucine, 8 mL aspartate, 8 mL tyrosine, 0.1 mL tryptophan. The solution was then autoclave sterilised. After cooling to room temperature, the pH was adjusted to 6.5 using HCl 33% w/w. Finally, the following volumes of stock solutions were added: 2 mL CaCl2/MgCl2, 1 mL FeSO4, 3 mL D-glucose, 10.5 mL L-lactate.

The SCFM-PC medium preparation was based on the SCFM 2 developed by Turner et al. [START_REF] Turner | Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum[END_REF]. SCFM 2 is a modification of SCFM in which DOPC (PC 18:1 (9Z) / 18:1 (9Z)), DNA and mucin are added. However, in our study, we had to develop SCFM-PC in which only DOPC was added to SCFM. Indeed, when DNA and mucin are used, the extraction of membrane lipids cannot be performed due to the high viscosity of the medium (unpublished work). The SCFM-PC medium was prepared as the following: aliquots of DOPC stock solutions were prepared in order to make different volumes of SCFM-PC with a final DOPC concentration of 100 μg/mL, (aliquots of 10 mg, or 33 mg, to respectively prepare 100 mL, or 330 mL, of SCFM-PC). The stock solutions of DOPC (250 mg/mL) were prepared in chloroform and were stored under argon, in the dark and at -20 °C. The DOPC stock solution was slowly added to the fresh SCFM medium by making it run along the inside of the Erlenmeyer flask -in order to evaporate the chloroform as much as possible. Then, the solution was stirred until neither traces nor lipid droplets could be seen anymore. Finally, in order to make sure that no traces P a g e 6 | 42 of chloroform were left, the SCFM-PC medium was left to rest 30 min under a biosafety cabinet.

The full protocol for the preparation of SCFM and SCFM-PC medium are given in the Supplementary Information S1. To verify that the autoclave sterilisation had not degraded the amino acids (at the concentration of the medium and without adding sugars), SCFM medium was checked using electrospray ionisation-mass spectrometry (ESI-MS) before and after sterilisation (for MS parameters, see Section 2.6). No difference was observed suggesting that no degradation occurred. Growth curves of PAK into the autoclave sterilised or filtered sterilised media were performed as well and showed no impact of the sterilisation method on the growth of P. aeruginosa. MS spectra and growth curves are given in S2.

Bacterial strain, growth conditions and MICs determination

The P. aeruginosa strain PAK (PAK), used in this study, was obtained from Dr.

Lhousseine Touqui (Institut Pasteur, Paris, France). The P. aeruginosa PAK strain is known for its high virulence, a high expression of pili and flagella, glycosylations and pathogenicity islands [START_REF] Cain | Complete Genome Sequence of Pseudomonas aeruginosa Reference Strain PAK[END_REF] and is commonly used in medical studies. PAK was stocked in Muller-Hinton broth (MHB)/glycerol (70/30) at -80 °C. For growth kinetics, PAK was pre-cultured from this stock solution, overnight at 37 °C in MHB, SCFM or SCFM-PC. This pre-culture was used to inoculate 100 mL of MHB, SCFM or SCFM-PC (in a 250 mL Erlenmeyer flask) with 5.10 5 colony forming units (CFU)/mL. The culture was incubated at 37 °C for 24 h on a rotary shaker (140 rpm). Every hour for 8 h and at a final point of 24 h, OD 600 (optical density at 600 nm with Cary 100 Bio spectrophotometer, Varian) was monitored and 100 μL of culture were collected for CFU numeration. For the MICs determination of carbenicillin, gentamicin, ciprofloxacin, and colistin, the broth dilution method, in 96-well plates, was performed according to Wiegand et al. [START_REF] Wiegand | Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances[END_REF], using an inoculum at OD 600 = 0.001. All these experiments were performed in biological triplicates for MHB, SCFM and SCFM-PC. P a g e 7 | 42

Atomic force microscopy (AFM) imaging

All glass substrates (glass cover for haemocytometer, 20x26mm, 0.4 mm thick, Hirschmann) were cut in 4 parts, cleaned with ethanol and dried. Using a 6-well plates, one glass substrate was placed into each well and covered with 1 mL of MHB, SCFM or SCFM-PC.

A PAK preculture in either MHB, SCFM or SCFM-PC media (see Section 2.3) was used to inoculate each well at 10 7 CFU/mL. In order to have enough attached bacteria, but with as low as possible phenotype changes, the plate was incubated for 6 h at 37 °C, without any mixing, and protected from light. A bacterial monolayer and irreversible attachment of the biofilm could be achieved using this method. Then, the culture medium was removed using a Pasteur pipette. Each glass substrate was rinsed twice using water baths, carefully drained, then dried overnight.

AFM imaging was performed with a Nanoscope 8 Multimode microscope (Bruker Nano Surfaces, Santa Barbara, CA, USA) equipped with a 100 μm piezoelectric scanner.

Imaging was achieved in the air either using the contact mode (for bacteria length measurements) or using the PeakForce® mode (for image presentation). In the contact mode, the cantilevers used were characterized by a low spring constant of about 0.06 N/m and were equipped with classical pyramidal Silicon Nitride tip. All the measurements were performed with the feedback loop on. For the PeakForce® mode imaging, we used a soft Silicon Nitride cantilevers (spring constant of about 0.35 N/m) with sharp Silicon Nitride tip.

Images were obtained with a PeakForce Tapping frequency of 2kHz with the auto-amplitude on. Average force applied during scanning was 5 nN.

Membrane lipid extraction

The membrane lipid extraction of PAK was performed as described by Deschamps et al. [START_REF] Deschamps | Determination of the collision cross sections of cardiolipins and phospholipids from Pseudomonas aeruginosa by traveling wave ion mobility spectrometry-mass spectrometry using a novel correction strategy[END_REF]. Briefly, from a pre-culture overnight (see Section 2.3), PAK was grown for 7 h in P a g e 8 | 42 MHB, SCFM or SCFM-PC (5.10 5 CFU/mL for initial inoculum) and a pellet was collected by centrifugation.

Then, alginate lysis was performed using alginate lyase in PBS (0.15 mg/mL), followed by sonication, centrifugation and ultracentrifugation. Finally, lipids were extracted using a modified version of the Bligh and Dyer protocol [START_REF] Bligh | A RAPID METHOD OF TOTAL LIPID EXTRACTION AND PURIFICATION[END_REF]. In the present work, the liquid-liquid extraction was modified with the addition of internal standards as followed:

For a pellet of 20 mg, 500 μL of milli-Q water was added to resuspend the pellet and then vortex-mixed for 5 min. 1.2 mL of methanol was added and then vortex-mixed for 15 min, to degrade proteolipids. Then, the mixture was centrifuged (4000 ×g, 10 min, 22 °C) and the liquid phase recovered. After adding 0.6 mL of chloroform spiked with phospholipid standards (PC 17:0/17:0, 0.5 μg; PE 17:0/17:0, 2.5 μg; PG 17:0/17:0, 1 μg; CL 14:0/14:0/14:0/14:0, 5 μg), the solution was vortex mixed for 15 min and sonicated for 15 min. 0.6 mL of chloroform was added, then the solution was vortex mixed for another 15 min. 0.62 mL of 1 M ammonium acetate was added and the solution was vortex-mixed for 15 min. Finally, the mixture was left to rest for 30 min. One milliliter of the organic phase was recovered and 0.94 mL of chloroform was added to the aqueous phase for a second liquid-liquid extraction. After vortex mixing for 15 min, the mixture was left to rest again for 30 min. The aqueous phase was removed and organic phases were pooled together and evaporated under argon. Lipid extracts were conserved under argon at -20 °C. For other samples pellet masses, solvent volumes and standard masses were adjusted proportionally. This extraction was performed in biological triplicate.

Liquid chromatography -mass spectrometry (LC-MS) analysis

The sample preparation for LC-MS analyses, the MS and LC parameters -including LC gradient-were as described by Deschamps et al. [START_REF] Deschamps | Determination of the collision cross sections of cardiolipins and phospholipids from Pseudomonas aeruginosa by traveling wave ion mobility spectrometry-mass spectrometry using a novel correction strategy[END_REF]. Briefly, the dried lipids extracts were re-constituted into 50 μL of chloroform/methanol (2/1, v/v) and 1/5 diluted into isopropanol/ acetonitrile/water (4/3/1, v/v).

P a g e 9 | 42

The LC-MS experiments were performed using an UHPLC system (Dionex Ultimate 3000 RSLC, Thermo Scientific, San Jose, CA, USA) coupled to a hybrid quadrupole time-of-flight with an ion mobility cell instrument (Q-IMS-TOF, SYNAPT G2 HDMS, Waters MS Technologies, Manchester, UK) equipped with an electrospray ionisation source. LC -MS data were acquired in triplicate in positive and negative ion modes.

The column used was a 1.0 × 100 mm, Acquity UPLC CSH C18 1. [START_REF] Escriba | Membrane-lipid therapy: a new approach in molecular medicine[END_REF]

Results

Impact of the medium composition on the growth and MIC determination

The growth curves of PAK grown in MHB, SCFM or SCFM-PC are given in Fig. 1.

Measurements of OD 600 (Fig. 1.A) or CFU/ mL (Fig. 1.B) as a function of time showed a similar growth of PAK in the three media. This observation included no lag phase time, an exponential phase time of approximately 8 h and a doubling time of 43, 42 or 44 minutes in MHB, SCFM or SCFM-PC, respectively. As a matter of fact, using these growth curves, the growth time for the lipid extraction (7h) was chosen as it corresponds to the end of the exponential phase. Thus, exploitable quantities of bacteria could be collected without risking a lack of nutrients. AFM images (Fig. 2. A, B, C) show similar bacterial shapes for all media. For MHB and SCFM, the average lengths (2.8 µm ± 0.7 µm) of PAK were equivalent. However, when PAK was grown in SCFM-PC, the average length was a little smaller (2.6 µm ± 0.6 µm), but as a counterpart, the height was higher (Fig. The MIC values of four antibiotics (carbenicillin, gentamicin, ciprofloxacin, colistin) for PAK grown in MHB, SCFM or SCFM-PC are given in Table 1. Susceptibility (S) or resistance (R) to the antibiotic were given according to the norm Eucast SFM 2019. In fact, a two-fold MIC difference cannot be considered significant, since the experiment error is of one dilution (for the protocol, see 2.4). Consequently, the influence of the medium on the MIC values could significantly be noted for carbenicillin, gentamicin and colistin, but not for ciprofloxacin. The MIC of gentamicin and colistin were higher when grown in SCFM or SCFM-PC rather than into MHB. Moreover, a switch from susceptibility to resistance could be observed for colistin and gentamicin, depending on the medium of growth. Surprisingly, the MIC of carbenicillin was much smaller when PAK was grown in SCFM rather than in MHB, but was identical between MHB and SCFM-PC media.

Comparison of the membrane phospholipids composition

Semi-quantification

The membrane lipids of PAK were identified using a home-made retention time-m/z library, which was previously described for PAK grown in MHB [START_REF] Deschamps | Determination of the collision cross sections of cardiolipins and phospholipids from Pseudomonas aeruginosa by traveling wave ion mobility spectrometry-mass spectrometry using a novel correction strategy[END_REF]. Lipid annotation was P a g e 13 | 42 based on the nomenclature of Liebisch et al. [START_REF] Liebisch | Shorthand notation for lipid structures derived from mass spectrometry[END_REF]. Forty-two lipids were identified. Twenty phospholipids were identified using positive ionisation: 7 phosphatidylcholines (PC) and 13 cardiolipins (CL).

Twenty-two were identified using negative ion mode: 13 phosphatidylethanolamines (PE) and 9 phosphatidylglycerols (PG). Please note that PE and CL could be observed in both positive and negative ion modes. However, using our analytical conditions (especially the LC mobile phase with buffers), a higher signal was observed in positive ionisation mode for CL and in negative ionisation mode for PE.

When PAK grown in SCFM or SCFM-PC, no new phospholipids were detected.

Therefore, a semi-quantitative approach was used in order to highlight the influence of the medium. Please note that, while PC 34:0, PE 34:0 and PG 34:0 were detected as traces in the membrane of PAK, the relative area of these phospholipids could not be determined.

Actually, using our chromatographic conditions, these phospholipids could not be separated from the standards (i.e. PC 17:0/17:0, PE 17:0/17:0, PG 17:0/17:0), therefore, the PC 34:0, PE 34:0 and PG 34:0 produced by PAK were excluded from the study. Caution must be taken regarding the high relative area of PC 36:2 extracted from PAK grown in SCFM-PC. Indeed, this may be due to i) direct incorporation of DOPC inside the bacteria membrane -without any modifications, ii) contamination of our samples with the culture medium DOPC or iii) both of i) and ii). However, it is clear from the over-production of PE 36:2, PG 36:2 and phospholipids containing oleic acid, that DOPC is at least recycled by the bacteria to produce phospholipids. Then, the presence of DOPC directly incorporated into the bacteria membrane would be a very likely hypothesis.

Fatty acid composition

In order to evaluate the influence of the growth medium on the fatty acid composition, MS/MS experiments were performed, on the most intense phospholipids, in biological triplicates and for each medium. Fragment identification was performed using phospholipid fragments described in the literature [START_REF] Hsu | Electrospray Ionization with Low-energy Collisionally Activated Dissociation Tandem Mass Spectrometry of Glycerophospholipids: Mechanisms of Fragmentation and Structural Characterization[END_REF][START_REF] Colsch | Mechanisms governing the fragmentation of glycerophospholipids containing choline and ethanolamine polar head groups[END_REF]. S3 gives all the fatty acid fragments of interest in negative ion mode -carboxylate anions R 1 COO ̶ -for PC/PE/PG 32:1, 34:1 and 36:2. Please note that for SCFM medium, PC species could not be detected in negative ion mode, since they were only produced as traces in this medium (see 3.2.1) and PC are known to not ionize well in negative ion mode, even with the [M+HCO 2 ] -adduct.

P a g e 15 | 42

As previously described by Hsu et al. [START_REF] Hsu | Electrospray Ionization with Low-energy Collisionally Activated Dissociation Tandem Mass Spectrometry of Glycerophospholipids: Mechanisms of Fragmentation and Structural Characterization[END_REF], the loss of the fatty acid in the sn-2 position is favoured. Hence, in order to compare the proportion of the two sn-1/2 position isomers (e.g. PG 16:0/16:1 and PG 16:1/16:0) between the three media, the following ratio was calculated ratio 1=

Intensity fatty acid sn-

Discussion

To our knowledge, this is the first study to analyse the membrane lipid composition of P. aeruginosa in CF sputum-like media (e.g. SCFM and SCFM-PC) and compare it with a classical medium. The three following media were used: the commercial MHB -as a P a g e 18 | 42 reference medium, SCFM [START_REF] Palmer | Nutritional cues control Pseudomonas aeruginosa multicellular behavior in cystic fibrosis sputum[END_REF] and a modified version of SCFM by the addition of a phosphatidylcholine (SCFM-PC).

When studying the influence of the culture medium on the production of molecules by bacteria, checking that the bacterial growth is not affected by the culture medium is crucial. Here, growth curves (see 3.1) showed that the growth of PAK was identical when grown in MHB, SCFM or SCFM-PC. As detailed in the protocol of the membrane lipid extraction (see 2.5), the culture was stopped at the same time for every medium (end of the exponential phase, see 3.1). Therefore, the following discussion could be made assuming that the membrane lipid composition modifications were not linked to a difference in the bacterium growth phase. This should be noted as a recent work by Le Sénéchal et al. [START_REF] Senechal | Phospholipid Content of Pseudomonas aeruginosa PAO1 Is Modulated by the Growth Phase Rather Than the Immobilization State[END_REF] showed that the phospholipid content of P. aeruginosa PAO1 was closely related to the bacterial growth.

Before going further into the discussion, one should note that a plausible criticism of our methodology is that P. aeruginosa was grown in planktonic conditions instead of biofilm conditions, which should better reproduce the CF sputum growth conditions. However, since laboratory biofilm are grown on abiotic sterile surfaces, the adherence of the biofilm is likely to differ from the one in CF sputum, leading to possible lipid modifications. Moreover, many studies on clinical P. aeruginosa were performed in shaken flasks since even the sampling of the bacterium would be enough to alter its metabolic phenotype [START_REF] Van Gulik | Fast sampling for quantitative microbial metabolomics[END_REF]. Despite these limitations, we believe that SCFM-PC is a good compromise medium for rigorous analysis of the membrane lipids of P. aeruginosa.

The main focus of this study was to investigate the influence of the medium on the phospholipids composition of the membrane. Using LC-MS in qualitative manner, as a first approach, we could not observe any influence of the medium on the type of identified phospholipids.

Therefore, a semi-quantification approach (comparison of relative intensities, using internal standards) was used (see 3.2.1). The most remarkable result was that most phospholipids were less produced into SCFM, compared to MHB. Knowing that lipids could be found in CFsputum [START_REF] Son | In vivo evidence of Pseudomonas aeruginosa nutrient acquisition and pathogenesis in the lungs of cystic fibrosis patients[END_REF], SCFM-PC was made, by adding DOPC to SCFM, in order to avoid the lack of PC P a g e 19 | 42 species. When PAK was grown in SCFM-PC, we could see the increase of the phospholipids with the longest fatty acyl chains (see Fig. 3.C). This increase might counterbalance the shortage of other lipids. However, for SCFM, we haven't identified any new phospholipids nor increase of other phospholipids. Ornithine-lipids, which were described to be produced by P. aeruginosa when grown in phosphate limiting conditions [START_REF] Lewenza | The olsA gene mediates the synthesis of an ornithine lipid in Pseudomonas aeruginosa during growth under phosphate-limiting conditions, but is not involved in antimicrobial peptide susceptibility[END_REF], were not detected. Two hypothesis can be assumed regarding the decrease of phospholipids of PAK grown in SCFM: i) novel phospholipids species were produced, but were neither extracted using our extraction conditions (see 2.6), nor detected using our analytical conditions (see 2.7), ii) the lipid-protein ratio, which is supposed to be around 1/1 (w/w) for bacteria [START_REF] Barák | The role of lipid domains in bacterial cell processes[END_REF], was modified.

The hypothesis that PAK grown in SCFM could be smaller than grown in MHB -leading to an overall lighter weight of phospholipids-was also considered. However, as shown by the AFM images in Fig. 2. (see 3.1), no significant influence of the medium on size nor on the shape of PAK was observed, consequently this hypothesis was rejected.

MS/MS experiments were performed, as well, in order to evaluate the influence of the growth medium on the fatty acid composition of phospholipids (see 3.2.2). The most remarkable observation was that the fatty acid C18:1 was strikingly more abundant in phospholipids produced by PAK grown in SCFM-PC than SCFM or MHB. This resulted into an inversion of the most abundant isomer for PC 32:1 and PG 34:1 (from MHB or SCFM to SCFM-PC). Indeed, for PC 32:1, both PC 14:0/18:1 and PC 16:0/16:1 were detected into MHB and SCFM-PC, but for SCFM-PC the major isomer was PC 14:0/18:1, whereas for MHB, the major isomer was PC 16:0/16:1. Also, for PG 34:1, the major isomer was PG 16:0/18:1 in the case of SCFM-PC, whereas for MHB or SCFM, the major isomer was PG 18:1/16:0. Moreover, isomers containing C 18:1 in sn-2 position were favoured when PAK was grown in SCFM-PC.

Regarding PG 32:1, PC 34:1 and PE 34:1, the respective 16:1/16:0, 16:0/18:1 and 16:0/18:1 isomers were significantly more produced in SCFM-PC than in SCFM or in MHB.

The most likely explanation for these observations would be that P. aeruginosa would take DOPC from the SCFM-PC, and modify it, in order to produce its own phospholipids, as previously described for polyunsaturated fatty acids [START_REF] Baker | Pseudomonas aeruginosa responds to exogenous polyunsaturated fatty acids (PUFAs) by modifying phospholipid composition, membrane permeability, and phenotypes associated with virulence[END_REF]. As a matter of fact, this hypothesis can also be supported by the overproduction of PE 36:2 and PG 36:2 (see Fig. 3.2.1) in SCFM-PC. Indeed, in most bacteria, PC are synthesised from PE, while PG are synthesised using another pathway [START_REF] Sohlenkamp | Bacterial membrane lipids: diversity in structures and pathways[END_REF]. However, in the case of P. aeruginosa, it was demonstrated that PC are synthesised from a phosphatidic acid (PA) and a choline [START_REF] Wilderman | Pseudomonas aeruginosa synthesizes phosphatidylcholine by use of the phosphatidylcholine synthase pathway[END_REF].

Therefore, PC, PE and PG are synthesised from three different pathways which uses PA as a common precursor. Thus, PA 36:2, recycled from DOPC, is likely used to synthesise PE 36:2 and PG 36:2. Consequently, these modifications would include: i) change of the polar head group and ii) replacement of the sn-1 fatty acid. Since adding a phospholipid to the growth medium had an influence on the membrane phospholipids of P. aeruginosa, we should note that caution must be taken when choosing this phospholipid, in regards to the aim of the study.

Finally, as described in 3.4.1, the other less abundant isomer of PE 36:2 (i.e. PE 19:1/17:1) and PG 36:2 (i.e. PG 20:1/16:1) were produced for MHB and SCFM, but only in traces for SCFM-PC. Taking into account these observations and the inversion of the major isomer previously described, one should note that caution must be taken when analysing the fatty acid composition of phospholipids. Our results show that for an identical fatty acyl chain length, the fatty acid composition might not be the same for different polar heads (e.g. PC 32:1, PE 32:1 and PG 32:1).

In a second approach, the MIC values of four antibiotics were determined (see 3.1) with the aim to link membrane lipids modifications and antibiotic resistance. Indeed, the antibiotic susceptibility of P. aeruginosa is related to its ability to guarantee membrane homeostasis -maintenance of the membrane physical properties [START_REF] Zhang | Membrane lipid homeostasis in bacteria[END_REF]. This homeostasis can happen by modifying phospholipids in different ways. The stability of the membrane and, accordingly, the decrease of the permeability, can be enhanced by increasing the fatty acyl chain length [START_REF] Goldfine | Membrane Lipid Biogenesis[END_REF]. Conversely, the fluidity and permeability can be increased by introducing a double bond or a cyclopropyl (more stable to acidic conditions than a double bond), or a terminally branched methyl on the fatty acyl chain [START_REF] Romantsov | Contributions of Membrane Lipids to Bacterial Cell Homeostasis upon Osmotic Challenge[END_REF].

The ratio of negatively charged phospholipids (CL and PG) and neutral (PE) or positively charged phospholipids (PC) can also be modified in order to make the membrane less sensitive to pH or to the medium ion composition [START_REF] Lewis | Surface charge markedly attenuates the nonlamellar phase-forming propensities of lipid bilayer membranes: calorimetric and (31)P-nuclear magnetic resonance studies of mixtures of cationic, anionic, and zwitterionic lipids[END_REF]. The replacement of PE by PC was P a g e 21 | 42 also described to enhance the membrane stability due to the ability of PC to form bilayers more easily than PE [START_REF] Goldfine | Membrane Lipid Biogenesis[END_REF].

As described in 3.4.1., PAK was found to produce less PC species when grown in SCFM than into MHB. MIC values of carbenicillin were also seemingly smaller when PAK was grown in SCFM (MIC SCFM = 16 µg/mL) than in MHB (MIC MHB = 64-128 µg/mL). Carbenicillin mechanism of action consists in inhibiting the synthesis of the bacterial cell-wall peptidoglycan. Also, since carbenicillin is an hydrophobic antibiotic (logP = 1.13 [36]), we may assume that this antibiotic can cross the lipid membrane instead of going through the porins. Hence, we can make the hypothesis that PAK, when grown in SCFM, was more susceptible to carbenicillin because the membrane cell was less stable due to the lack of PC species. Then, when DOPC was added to the SCFM medium, the presence of PC in the membrane restore a susceptibility to carbenicillin similar to the one in MHB (MIC MHB = MIC SCFM-PC ).

Regarding the other antibiotics tested, the influence of the growth medium on the MIC values was observed for colistin and gentamicin, but not for ciprofloxacin (see 3.1). It is notable that, MIC values of gentamicin, colistin and ciprofloxacin in SCFM were in agreement with those from Pusic et al. [START_REF] Pusic | Harnessing Metabolic Regulation to Increase Hfq-Dependent Antibiotic Susceptibility in Pseudomonas aeruginosa[END_REF] SCFM medium). We also observed an increase of MIC values from a rich medium (MHB) to the chemically defined SCFM (see Table 1). This observation was also described by Pusic et al. (from LB to SCFM), and for colistin between MHB and ASM, by Diaz Iglesias et al. [START_REF] Diaz Iglesias | Activity of Antibiotics against Pseudomonas aeruginosa in an In Vitro Model of Biofilms in the Context of Cystic Fibrosis: Influence of the Culture Medium[END_REF]. In SCFM-PC, the MIC values of colistin and gentamicin were equal to the ones in SCFM. Therefore, the link between the membrane stability (decrease of the membrane stability in SCFM but restored in SCFM-PC) and the MIC, described for carbenicillin, might not be suitable for other antibiotics. The membrane permeability changes, induced by the growth medium, may partly explain P. aeruginosa antibiotic susceptibility modifications. Actually, it is rather a multi-factor issue since the nutrients present in the growth medium can induce particular resistance mechanisms of P. aeruginosa [START_REF] Son | In vivo evidence of Pseudomonas aeruginosa nutrient acquisition and pathogenesis in the lungs of cystic fibrosis patients[END_REF].

P a g e 22 | 42

5.

Conclusion

In this study, we reported the membrane lipid composition of P. aeruginosa grown into CF sputum-mimicking media (SCFM and SCFM-PC). We showed that the growth medium had an influence on the phospholipid quantities. We observed that PC species were produced in trace level in SCFM. Implementing DOPC to SCFM (SCFM-PC medium), as a choline nutrient, not only had an influence on the quantities of PC species, but also on the length of the fatty acids parts of the phospholipids and phospholipid isomerism. Such modifications may affect the membrane permeability. Actually, MIC variations of the hydrophobic carbenicillin antibiotic support this hypothesis. These results suggest that the membrane lipid composition should be taken into account for antibiotic susceptibility testing. Since we showed that the growth medium can influence the membrane lipid composition, caution must be taken when choosing a medium, especially for clinical studies.

SCFM-PC preparation

In order to add the aliquot of DOPC to SCFM (SCFM not older than 3 weeks):

/!\ Use an erlenmeyer or a bottle at least 3 times bigger than the volume of required SCFM-PC.

Under a biosafety cabinet:

1 Rince Paster pipette several times with chloroform, 2 Add DOPC slowly 20μL per 20μL, making it run along the inside of the Erlenmeyer flask -in order to evaporate the chloroform, /!\ Lipid traces and lipid droplets will be visible on the inside of the flask, and in the bottom of the flask, respectively.

3 Gently manually shake to "rinse" the lipids traces, 4 Shake until lipids droplets can't be seen anymore: the medium will appear cloudy.

Fig. 1 .

 1 Fig. 1. Growth curves of PAK strain in MHB, SCFM and SCFM-PC. black circles for MHB, red squares for SCFM, blue diamonds for SCFM-PC. A. optical density (OD) plotted against time. B. Colony Forming Unit (CFU)/ mL plotted against time. Error bars: standard deviation. P a g e 11 | 42

  2.C), showing that PAK seemed to spread less on the surface than in the other media. Interestingly, as contrary to the other two media, colonies of PAK grown in SCFM-PC showed bacteria were not glued to each other, probably due to the presence of DOPC in the growth medium. P a g e 12 | 42

Fig. 1 .

 1 Fig.1. AFM images of PAK young biofilms grown on glass substrates. Images are shown as topographic images overlaid with a local contrast image (Kernel size: 2; blending depth: 2; weight: 1). Classical filters as flattening and 3-points levelling were applied to the topographic image using the Gwyddion software (http://www.gwyddion.net). A. PAK grown in MHB; B. PAK grown in SCFM; C. PAK grown in SCFM-PC.

Fig. 3 .

 3 Fig. 3.A., 3.B. and 3.C. show relative areas of some depictive examples of the membrane phospholipids extracted from PAK grown in MHB, SCFM or SCFM-PC media. The relative areas for all the identified phospholipids are given in S2 (Fig S2.4 to S2.7).

Fig. 2 .

 2 Fig. 2. Relative areas of phospholipids extracted from PAK. R elative area= PAK PL area PLstandard area . Black bars: PAK grown in MHB, red bars: PAK grown in SCFM, blue bars: PAK grown in SCFM-PC. Error bars: standard deviation (7≤n≤9 for PE, PC and PG, 3≤n≤6 for CL). P-values are reported -between MHB and SCFM, SCFM and SCFM-PC and MHB and SCFM-PC -as: * for p<0.05, ** for p<0.01, *** for p<0.001, ns for non-significant. A. PC less or equally produced in SCFM-PC than MHB and mostly not produced in SCFM; B. phospholipids more produced in MHB than SCFM or SCFM-PC; C. phospholipids more produced in SCFM-PC than SCFM or MHB. Depending on the medium of growth, three trends of relative areas could be observed. As shown in Fig. 3.A., apart from PC 34:1 and PC 36:2 (Fig. 3.C.), PC were significantly less produced for PAK grown in SCFM rather than MHB or SCFM-PC. As shown in Fig. 3.B. and S2 (Fig S2.4 to S2.7), most phospholipids (23) were more produced in MHB than SCFM or SCFM-PC. As shown in Fig. 3.C., phospholipids containing oleic acid (C18:1) were greatly overproduced in SCFM-PC, especially for PE 34:1, PC 34:1, PG 34:1 and PE 36:2, PC 36:2, PG 36:2.

Fig. 4 MSMS

 4 Fig. 4 MSMS Spectra of [PG 34:1-H] -and determination of the ratio 1.Ratio 1 allows to compare the proportion of the two sn-1/2 position isomers ( ratio 1= Intensity fatty acid sn-1 Intensity fatty acid sn-2 ) between the three media. Bracket values: ion intensities. A. PAK grown in MHB; B. PAK grown in SCFM; C. PAK grown in SCFM-PC.

  3.C. in part P a g e 20 | 42

Fig. S2. 1 .

 1 Fig. S2.1. Mass spectra, in ion negative mode, of SCFM media collected during preparation, before (A) or after (B) autoclave sterilisation. The ions detected whose m/z are specified correspond to the deprotonated molecules [M-H] -of the amino acids.

Fig. S2. 2 .Fig S2. 3 .Fig S2. 4 .Fig S2. 5 .Fig S2. 6 .Fig S2. 7 .

 234567 Fig. S2.2. Optical Density (A) or Colony Forming Unit (B) plotted against growth time for PAK grown into SCFM autoclave sterilised (red squares) or filtered sterilised (green triangles). Experiments were made in biological triplicates. Standard deviations are represented by error bars.

  Data processing, automatic identification and integration of peaks were performed using UNIFI 1.8.2.169 (Waters, Manchester, UK). Relative areas were calculated by dividing the area of the identified phospholipids by the area of the standard from the corresponding

	MS/MS experiments were performed in negative ion mode using a collision energy ramp
	from 25 eV to 45 eV. For PE (i.e. PE 32:1, PE 34:1, PE 36:2) and PG species (i.e. PG 32:1, PG
	34:1, PG 36:2), the ion selected in the quadrupole was the [M-H] -, whereas for PC species
	(i.e. PC 32:1, PC 34:1, PC 36:2), the ion selected was the [M+HCO 2 ] -. MS/MS spectra were
	processed using MassLynx 4.1 (Waters).
		μm column (Waters,
	Manchester, UK) equipped with a 0.2 μm prefilter. Mobile phase A consisted of
	acetonitrile/aqueous 10 mM ammonium formate (60/40) + 0.1% formic acid and mobile
	phase B consisted of isopropanol/acetonitrile/aqueous 10 mM ammonium formate (88/8/4)
	+ 0.1% formic acid. Data acquisition was performed using MassLynx 4.1 (Waters,
	Manchester, UK).
	class of phospholipids (e.g. relative area=	Area of PE 34 :1 Area of PE 17 :0/17 :0
	the repeatability of the measurements. Anova tests and Turkey mean comparisons were
	performed using OriginPro 2016 (64-bit) b9.3.226.
	MS/MS experiments were performed in biological triplicate, using the parameters
	previously described [12].
		P a g e 10 | 42

). Areas were automatically calculated, from the extracted ion chromatograms, using the monoisotopic peak of [M+H] + or [M-H] -ions, for positive or negative ionisation respectively. The monoisotopic peak was used, instead of the sum of all isotopes, since not all isotopes could be observed for phospholipids at small concentrations (e.g. CL 67:3). We also selected only protonated or deprotonated species, instead of the sum of all detected adducts (i.e. [M+Na] + , [M+NH 4 ] + , [M+CH 3 COO] -) for similar reasons. The standard deviation and relative standard deviation of the relative area (the number of replicates (n) in which the phospholipids were identified were; 7≤n≤9 for PE, PC and PG, 3≤n≤6 for CL) were calculated to plot error bars and to check

Table 1 .

 1 Minimal

	medium/	carbenicillin	gentamicin	ciprofloxacin	colistin
	antibiotic		(MIC in µg/mL)
	MHB	64-128	0.5 (S)	0.125 (S)	0.125-0.25 (S)
	SCFM	16	4-8 (R)	0.125 (S)	2-4 (R)
	SCFM-PC	64-128	4-8 (R)	0.25 (S)	2 (R)

inhibitory concentrations (MIC) for PAK strain. (S) for susceptibility of PAK to the antibiotic, (R) for resistance. Both (S) and (R) are given according to the norm Eucast SFM 2019 -apart for carbenicillin for which reference values was not given in the norm.

Table 2

 2

	34:1 and PG 34:1. The major isomer was identical in MHB and SCFM media. However, when
	compared to MHB and SCFM, the SCFM-PC medium shows different isomers for two
	phospholipids: PC 32:1 and PG 34:1. In SCFM-PC, the major isomer was PC 14:0/18:1 for PC
	32:1 (PC 16:0/16:1 in MHB), and PG 16:0/18:1 for PG 34:1 (PG 18:1/16:0 in MHB or SCFM).
	Regarding sn-1/2 position isomers, for PG 32:1, PC 34:1/ PE 34:1/ PG 34:1, the respective
	P a g e 17 | 42

shows the major isomers identified for PC 32:1, PE 32:1, PG 32:1, PC 34:1, PE

Table 2 . Fatty acid composition of the major isomer for six phospholipids, depending on the medium of growth.

 2

		PC 32:1	PE 32:1	PG 32:1	PC 34:1	PE 34:1	PG 34:1
	MHB	16:0/16:1 16:0/16:1 16:0/16:1 18:1/16:0 18:1/16:0 18:1/16:0
	SCFM	nd	16:0/16:1 16:0/16:1	nd	18:1/16:0 18:1/16:0
	SCFM-PC	14:0/18:1 16:0/16:1 16:0/16:1* 18:1/16:0* 18:1/16:0* 16:0/18:1
	other isomers were observed for PE 36:2 and PG 36:2. For PE 36:2, PE 19:1/17:1 was
	detected, in trace level, in MHB (ratio 2 = 0.07 ± 0.02), SCFM (ratio 2 = 0.05 ± 0.02) and
	SCFM-PC (ratio 2 = 0.01 ± 0.01). For PG 36:2, PG 20:1/16:1 was observed in MHB (ratio 2 =
	0.55 ± 0.09), SCFM (ratio 2 = 0.52 ± 0.19) and in trace level in SCFM-PC (ratio 2 = 0.04 ± 0.01).

nd: not detected; * for PG 32:1, PC 34:1 and PE 34:1, the respective 16:1/16:0, 16:0/18:1 and 16:0/18:1 isomers were significantly more produced in SCFM-PC than in SCFM or in MHB.

Regarding PC/PE/PG 36:2 (see S3), the major isomers were PC/PE/PG 18:1/18:1 for all media. However, whereas no other isomer could be observed for PC 36:2 in any media,

Isoleucine and phenylalanine stock solution to dilute into 100 mL of Mili-Q water amino acid m (g)

	Using Mili-Q
	Stock solutions preparation for the SCFM medium
	Part I: amino acids
	Solutions can be kept in the dark at 4 °C for less than two months.
	Amino acids stock solution to dilute into 100 mL of Mili-Q water
	amino acid	m (g)
	Alanine	1.60
	Arginine -HCl Cysteine -HCl	0.63 0.32
	Glutamate -HCl Glycine	2.75 0.90
	Histidine -HCl Lysine -HCl	0.96 3.84
	Methionine Ornithine -HCl	0.90 1.18
	Proline Serine	1.96 1.47
	Threonine Valine	1.19 1.29
	Note: a few minutes of mixing at 37°C can help the dissolution of the amino acids.
	Isoleucine Phenylalanine	1.44 0.83
	Leucine stock solution to dilute into 200 mL of Mili-Q water
	amino acid	m (g)
	Leucine	2.62
		P a g e 27 | 42

water, prepare the following NaOH solutions for the last 3 amino acids

  Solutions can be kept in the dark at 4 °C for less than two months.

		Part II: salts and others
	solutions
	Mixed	C (mol/L) 1 0.5	V (L) 0.10 0.10	m (g) 2.00 4.00
		0.2	0.10	0.80
	Aspartate stock solution to dilute into 50 mL of NaOH 0.5 M
		amino acid	m (g)
		Aspartate	0.69
	Tyrosine stock solution to dilute into 50 mL of NaOH 1 M
		amino acid	m (g)
		Tyrosine		0.91
	Tryptophan stock solution to dilute into 50 mL of NaOH 0.2 M
		amino acid	m (g)
		Tryptophan	1.33
				P a g e 28 | 42

salts stock solution to dilute into 500 mL of Mili-Q water

  This solution can be autoclave sterilised for storage purpose.

	Salt	m (g)
	NH 4 Cl	0.61
	KCl	5.70
	NaCl	14.92
	NaH 2 PO 4	0.78
	Na 2 HPO 4	0.72
	K 2 SO 4	0.24
	NaNO 3	0.15
	Morpholinepropanesulfonic acid (

MOPS) to dilute into 250 mL of Mili-Q water

  This solution can be autoclave sterilised for storage purpose.

	Buffer	m (g)
	MOPS	5.23

CaCl 2 / MgCl 2 stock solution to dilute into 25 mL of Mili-Q water Filter sterilise through a 0.22-µm-pore-size filter.

	Salt	m (g)
	CaCl 2	2.43
	MgCl 2	0.72
	FeSO4

stock solution to dilute into an autoclave sterilised bottle using 100 mL of autoclave sterilised Mili-Q water Regularly check for precipitation; prepare fresh stock solution in case of precipitation.

	D-
	Salt	m (g)
	FeSO4	0.10
		P a g e 29 | 42

glucose stock solution to dilute to 50 mL using Mili-Q water Filter sterilise through a 0.22-µm-pore-size filter.

  !\1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) should be Avanti polar Lipids quality.Make DOPC aliquots of 10mg or 33mg (depends on the required volume of SCFM-PC required; in SCFM-PC, DOPC is at a final concentration of 100μg/mL), in chloroform, at approximately 250 mg/mL. Stock these aliquots in chloroform, under argon, at -20°C, in the dark.

	SCFM preparation Before SCFM-PC preparation
	This medium needs to be prepared in sterile conditions.
		Sugar	m (g)
	Stock solution	D-glucose V for 1 L of SCFM 9.01	V for 330 mL of SCFM
	60 % (w/w) Sodium L-Lactate to dilute to 10 % (w/w) using Mili-Q water Mixed salts 100 mL 33 mL MOPS 25 mL 8.25 mL
	Filter sterilise through a 0.22-µm-pore-size filter. Mili-Q water 805 mL Mixed amino acids 10 mL	265 mL 3.3 mL
	Isoleucine + Phenylalanine Leucine	10 mL 16 mL	3.3 mL 5.28 mL
	Aspartate Tyrosine		8 mL 8 mL	2.64 mL 2.64 mL
	Tryptophan		0.1 mL	33 µL
		Autoclave sterilise
	Wait for the medium to cool to room temperature
	Adjust pH to 6.5 using HCl 33 % w/w (approx. 600 µL for 330 mL)
	CaCl 2 / MgCl 2		2 mL	0.66 mL
	FeSO 4		1 mL	0.33 mL
	D-glucose Sodium L-lactate		3 mL 10.5 mL	0.99 mL 3.5 mL
				P a g e 30 | 42 P a g e 31 | 42

/

Funding information

The authors gratefully acknowledge European Regional Development Fund (ERDF, no. HN0001343), Labex SynOrg (ANR-11-LABX-0029) and Région Normandie for their financial support. P a g e 23 | 42

Conflict of interest

The authors declare that they have neither conflict of interest nor competing financial interest.

Supplementary Information S1: SCFM and SCFM-PC preparation


Aurélie Paganin-Gioanni

Marie-Pierre Rols

Justin Teissié

Muriel Golzio
email: muriel.golzio@ipbs.fr

Cyclin B1 knockdown mediated by clinically approved pulsed electric fields siRNA delivery induces tumor regression in murine melanoma

Keywords: electroporation, electro-geno-therapy, electro-chemo-therapy, RNAi, tumors, fluorescence imaging, cyclin B1

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION.

RNA interference (RNAi) is a promising therapeutic approach to induce specific gene silencing within cells. This strategy is developed against a variety of diseases, and is being evaluated in pre-clinical and clinical trials [START_REF] Devincenzo | Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV)[END_REF][START_REF] Davis | Evidence of RNAi in humans from systemically administered siRNA via targeted nanoparticles[END_REF][START_REF] Crunkhorn | Trial watch: pioneering RNAi therapy shows antitumour activity in humans[END_REF][START_REF] Barata | RNA-targeted therapeutics in cancer clinical trials: Current status and future directions[END_REF][START_REF] Kaczmarek | Advances in the delivery of RNA therapeutics: from concept to clinical reality[END_REF]. The small interfering RNA (siRNA) is a synthetic molecule, which is particularly attractive due to its lower ability to generate anti-viral responses, in comparison to viral vectors, which may be used to induce RNAi. Nevertheless, the siRNA's ability to exert its full potential as an anti-cancer agent cannot be attained, unless better methodologies for its targeted intracellular delivery to cells and tissues are developed [START_REF] De Fougerolles | Interfering with disease: a progress report on siRNA-based therapeutics[END_REF][START_REF] Ledford | Drug giants turn their backs on RNA interference[END_REF][START_REF] Zhou | Aptamer-targeted cell-specific RNA interference[END_REF][START_REF] Pecot | RNA interference in the clinic: challenges and future directions[END_REF]. Small interfering RNAs are very sensitive to nucleases degradation and can rarely cross cancer cell membranes, to reach the cytoplasm, where their target (mRNA) and the enzymatic machinery are present (RISC) [START_REF] Elbashir | Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells[END_REF][START_REF] Liu | Argonaute2 is the catalytic engine of mammalian RNAi[END_REF][START_REF] Behlke | Progress towards in vivo use of siRNAs[END_REF]. A delivery strategy is thus necessary to overcome the cellular barriers, promoting cellular uptake and offering protection to the RNAi molecules. RNAi delivery system can be categorized into viral and non-viral vectors, according to their nature, and these delivery systems were extensively reviewed [START_REF] Couto | Viral vector-mediated RNA interference[END_REF][START_REF] Zhang | In vivo gene delivery by nonviral vectors: overcoming hurdles?[END_REF][START_REF] Kanasty | Delivery materials for siRNA therapeutics[END_REF][START_REF] Boisguerin | Delivery of therapeutic oligonucleotides with cell penetrating peptides[END_REF]. While poor safety profile, high production cost and the risk of immunogenicity limit the applications of viral vector mediated RNAi as a laboratory tool, most of the non-viral vectors take advantages of endocytic mechanisms.

Following cell entry, the RNAi molecules are entrapped in early endosomes. The late endosomes then fuse with lysosomes, which contain hydrolases that degrade the RNAi molecules. In order to exert a biological effect, the RNAi molecules must escape from endosomes at early stage.

Increasing number of reports showed that nanoparticles (NPs) including lipid-based-NPs and polymeric NPs, are now capable of protecting siRNA from nuclease degradation, can be functionalized with targeting molecules and facilitate cellular uptake. Nevertheless, in the case of hepatocytes, only a tiny fraction of the injected dose arrives safely to the cytosol of target cells, as the NPs have first to cross the blood vessels walls and escape the endosomal and recycling pathways, once they entered the cells [START_REF] Dominska | Breaking down the barriers: siRNA delivery and endosome escape[END_REF]. The intracellular trafficking of NPs may also generate some undesirable effects, such as activation of the innate immune system and toll-like receptor stimulated inflammatory response [START_REF] Jackson | Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application[END_REF][START_REF] Scherman | Genetic pharmacology: progresses in siRNA delivery and therapeutic applications[END_REF][START_REF] Sun | Enhancing the Therapeutic Delivery of Oligonucleotides by Chemical Modification and Nanoparticle Encapsulation[END_REF][START_REF] Pandi | Dendrimer as a new potential carrier for topical delivery of siRNA: A comparative study of dendriplex vs. lipoplex for delivery of TNF-alpha siRNA[END_REF][START_REF] Perez | Lipid-based siRNA Nanodelivery Systems: A Learning Process for Improving Transfer from Concepts to Clinical Applications[END_REF][START_REF] Zhang | Lipidation of polyethylenimine-based polyplex increases serum stability of bioengineered RNAi agents and offers more consistent tumoral gene knockdown in vivo[END_REF].

So, the challenge in RNAi use is to develop local delivery methods to transfer RNAs directly into targeted cells and to avoid its degradation in extracellular medium and intracellular compartments. Pulsed electric field (PEF) application is a non-viral method, which can be successfully used to transfer therapeutic molecules into cancer cells [START_REF] Testori | Electrochemotherapy for cutaneous and subcutaneous tumor lesions: a novel therapeutic approach[END_REF][START_REF] Agerholm-Larsen | Preclinical validation of electrochemotherapy as an effective treatment for brain tumors[END_REF][START_REF] Curatolo | Electrochemotherapy in the treatment of Kaposi sarcoma cutaneous lesions: a two-center prospective phase II trial[END_REF][START_REF] Chabot | Targeted electro-delivery of oligonucleotides for RNA interference: siRNA and antimiR[END_REF]. This method was first based on the local application of microsecond electrics pulses (100 microseconds) at high voltage (1000-1300 V/cm), which permeabilizes the cell membrane and allows the transfer of small molecules (< 5kDa). Bleomycin or cisplatin may thus enter the cell by diffusion as long as the cell membrane is permeabilized [START_REF] Mir | Electrochemotherapy, a new antitumor treatment: first clinical trial[END_REF][START_REF] Mir | Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses[END_REF][START_REF] Mir | Effective treatment of cutaneous and subcutaneous malignant tumours by electrochemotherapy[END_REF][START_REF] Gothelf | Electrochemotherapy: results of cancer treatment using enhanced delivery of bleomycin by electroporation[END_REF]. Since the 90's, thousands of patients have been treated by electrochemotherapy (ECT) all over the world [START_REF] Belehradek | Electrochemotherapy, a new antitumor treatment. First clinical phase I-II trial[END_REF][START_REF] Domenge | Antitumor electrochemotherapy: new advances in the clinical protocol[END_REF][START_REF] Heller | Treatment of cutaneous and subcutaneous tumors with electrochemotherapy using intralesional bleomycin[END_REF][START_REF] Gehl | Efficient palliation of haemorrhaging malignant melanoma skin metastases by electrochemotherapy[END_REF][START_REF] Rodriguez-Cuevas | Electrochemotherapy in primary and metastatic skin tumors: phase II trial using intralesional bleomycin[END_REF][START_REF] Testori | Electrochemotherapy for cutaneous and subcutaneous tumor lesions: a novel therapeutic approach[END_REF][START_REF] Agerholm-Larsen | Preclinical validation of electrochemotherapy as an effective treatment for brain tumors[END_REF][START_REF] Hampton | Electric pulses help with chemotherapy, may open new paths for other agents[END_REF][START_REF] Curatolo | Electrochemotherapy in the treatment of Kaposi sarcoma cutaneous lesions: a two-center prospective phase II trial[END_REF]. PEF is very promising for the targeted transfer of nucleic acids in cells or tissues, however, this method was not efficient enough for the transfer of macromolecules like the plasmids DNA (pDNA) in cancer cell [START_REF] Mir | High-efficiency gene transfer into skeletal muscle mediated by electric pulses[END_REF][START_REF] Satkauskas | Mechanisms of in vivo DNA electrotransfer: respective contributions of cell electropermeabilization and DNA electrophoresis[END_REF]. With the development of more sophisticated electric field generators, millisecond electric pulses (5 -100 msec) at low voltage (50 -800 V/cm) have been shown to improve the transfer of macromolecules like pDNA and siRNA into cells and tissues [START_REF] Golzio | In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery[END_REF][START_REF] Cemazar | Cancer electrogene therapy with interleukin-12[END_REF]. The transfer of nucleic acid has been called electrogenotherapy (EGT). Because of nucleic acids are negatively charged, electrophoretic forces are essential for their transfer into cells during PEF. Therefore, the use of millisecond durations (EGT parameters) is needed for better pDNA uptake [START_REF] Golzio | Direct visualization at the single-cell level of electrically mediated gene delivery[END_REF]. The exact mechanism leading to enhanced intracellular nucleic acid delivery remains unclear, but at least for pDNA in vitro, it might involve the induction of local physical interactions between the nucleic acid and the cell membrane, followed by a slow intracellular release into the cytoplasm after PEF [START_REF] Golzio | Direct visualization at the single-cell level of electrically mediated gene delivery[END_REF][START_REF] Berezhna | siRNA in human cells selectively localizes to target RNA sites[END_REF]. A different behavior was observed with siRNA using millisecond PEF.

No complex between siRNA and the cell surface is formed; siRNAs go through the membrane during the PEF application and stop their penetration after PEF application, as no diffusion was observed. Naked siRNA have a rapid and direct access into the cytoplasm of permeabilized cells [START_REF] Paganin-Gioanni | Direct visualization at the single-cell level of siRNA electrotransfer into cancer cells[END_REF]. This is also observed for LNA/DNA oligo nucleotides [START_REF] Chabot | LNA-based oligonucleotide electrotransfer for miRNA inhibition[END_REF]), but chemically-modified oligonucleotide increased stability negatively correlates with its efficacy despite efficient electrotransfer [START_REF] Pelofy | Chemically modified oligonucleotide-increased stability negatively correlates with its efficacy despite efficient electrotransfer[END_REF]. EP has been used successfully for in vivo siRNA electrotransfer in a wide variety of tissues such as skin [START_REF] Zhang | Enhanced delivery of naked DNA to the skin by noninvasive in vivo electroporation[END_REF], muscle [START_REF] Kishida | Sequence-specific gene silencing in murine muscle induced by electroporation-mediated transfer of short interfering RNA[END_REF][START_REF] Golzio | Inhibition of gene expression in mice muscle by in vivo electrically mediated siRNA delivery[END_REF], joint tissue [START_REF] Inoue | Electro-transfer of small interfering RNA ameliorated arthritis in rats[END_REF], eyes [START_REF] Matsuda | Electroporation and RNA interference in the rodent retina in vivo and in vitro[END_REF], brain [START_REF] Akaneya | RNAi-induced gene silencing by local electroporation in targeting brain region[END_REF]) and kidney [START_REF] Takabatake | Exploring RNA interference as a therapeutic strategy for renal disease[END_REF]. The mechanism of uptake following PEF is clearly specific, and depends, among other, upon the physico-chemical properties of the electrotransferred molecule [START_REF] Chabot | Delivery of RNAi-Based Oligonucleotides by Electropermeabilization[END_REF]. We can also hypothesize that different PEF durations can also act differently on the cell membrane and on charged molecules. The effects of these durations need to be further evaluated.

In this study, we used B16-F10 mouse melanoma cell line stably expressing the enhanced green fluorescent protein (EGFP), to evaluate in vivo the efficiency of specific siRNAs delivery by a clinically approved protocol using microsecond PEF duration. Subsequently, the transfer efficiency was compared between microsecond and millisecond PEF duration protocols. Different siRNAs were electrotransferred into melanoma cells: negative control, anti-GFP and anti-cyclin B1 siRNAs. Anti-GFP siRNA was used as a positive control and its efficiency was followed by whole body fluorescence imaging. Anti-cyclinB1 siRNA was selected as an endogeneous target. Knockdown of cyclin B1 inhibits tumor proliferation in vitro as well as in vivo. Indeed, cyclin B1 is the regulatory subunit of cyclin-dependent kinase 1 (Cdk1) and essential for the transition from G2 phase to mitosis. Cyclin B1 is found to be overexpressed in many cancer cell lines. Inhibition of cyclin B1 decreases tumor growth [START_REF] Androic | Targeting cyclin B1 inhibits proliferation and sensitizes breast cancer cells to taxol[END_REF][START_REF] Crombez | Targeting cyclin B1 through peptide-based delivery of siRNA prevents tumour growth[END_REF][START_REF] Kedinger | Sticky siRNAs targeting survivin and cyclin B1 exert an antitumoral effect on melanoma subcutaneous xenografts and lung metastases[END_REF].

Moreover, targeting cyclin B1 sensitizes breast cancer cells to taxol, suggesting that specific cyclin B1 targeting is an attractive strategy for the combination with conventionally used agents in cancer therapy [START_REF] Yuan | Stable gene silencing of cyclin B1 in tumor cells increases susceptibility to taxol and leads to growth arrest in vivo[END_REF][START_REF] Androic | Targeting cyclin B1 inhibits proliferation and sensitizes breast cancer cells to taxol[END_REF]. We showed that the clinically approved protocol using microsecond pulse duration allowed efficient transfer of siRNA delivery in solid tumors in vivo, as shown by the tumor growth delay.

Repeated treatments improved the tumor growth delay. When compared to millisecond PEF protocol, similar tumor growth delay was obtained despite a better delivery using one single treatment. Our results show that existing clinical protocol based on PEF can be used efficiently for targeted tumor delivery of unmodified siRNA, targeting a therapeutic gene.

Moreover, this protocol can be repeated several times. Our approach provides new opportunities into siRNA delivery field, as it can be combined with other anti-cancer strategies.

MATERIALS AND METHODS.

siRNA sequences. egfp siRNA duplexes were designed according to Caplen and co-workers and labeled with Alexa Fluor 546 at the 3' position of the sense strand [START_REF] Caplen | Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems[END_REF]) (Qiagen Xeragon, Germantown). The sequence was given in [START_REF] Paganin-Gioanni | Tracking in vitro and in vivo siRNA electrotransfer in tumor cells[END_REF]. The labeling of the siRNA does not significantly modify the global negative charge of the native siRNA. The siRNA anti cyclin B1 was a pool of three target-specific 21 nt siRNAs (sc-29285A: sense: 5' AAG UCA GCG AAG AGC UAC ATT, antisense: UGU AGC UCU UCG CUG ACU UTT; sc-29285B: sense: AAC CUC UGU AGU GAA UAU GTT, antisense: CAU AUU CAC UAC AGA GGU UTT; sc-29285C: sense: GAG CAG UCA GUU AGA CCA ATT, antisense: UUG GUC UAA CUG ACU GCU CTT) (Santa Cruz Biotechnology). The negative control siRNA showed no significant sequence similarity to the mouse gene sequences (sense: 5' UUC UCC GAA CGU GUC ACG UdT dT, antisense: 5' ACG UGA CAC GUU CG GAGA AdT dT) (Qiagen Xeragon).

Cells. B16-F10 murine melanoma cells expressing EGFP were obtained after retroviral transduction and maintained in culture according to a previously described protocol [START_REF] Golzio | In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery[END_REF]). Electrotransfer apparatus. Electropulsation (EP) was operated by using a CNRS cell electropulsator (Jouan), which delivered square-wave electric pulses. An oscilloscope (Enertec) monitored pulse shape. A polarity inverter built in the laboratory allowed the triggering of pulses with alternating (bipolar) polarities.

Murine tumor model. Female

In vivo electropulsation. The protocol was previously described in [START_REF] Golzio | Targeted gene silencing into solid tumors with electrically mediated siRNA delivery[END_REF][START_REF] Golzio | Targeted gene silencing into solid tumors with electrically mediated siRNA delivery[END_REF])). In summary, fifty microliters of saline solution (i.e. PBS containing 40 units of the RNAse inhibitor RNAsin) (TebuBio) and 5 µg of EGFP siRNA, negative siRNA or anti cyclin B1 siRNAs were slowly injected into the tumor. In the control conditions, the volume of siRNA was replaced by the siRNA suspension buffer containing RNAsin to keep the injection conditions similar and are denominated "PBS" in figures. Thirty seconds after injection, electropulsation was applied. Square-wave pulses were delivered as described previously [START_REF] Golzio | In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery[END_REF]). The electric field strength (600 V/cm for EGT condition and 1300 V/cm for ECT condition), pulse duration (5 msec for EGT condition and 100 µsec for ECT condition) and frequency of pulses (1 Hz) were all preset. The electrodes were of the parallel-plate type, 1 cm long, 1 cm thick, and with 4, 5 or 6 mm of distance between the plates, depending on the tumor size (in order to obtain the desired electric filed strength, the voltage was adjusted following the equation V = E x d, where E is the electric field strength and d is the distance between the electrodes). At day 2 and 4, the treatment was repeated. First we delivered a train of four pulses, and subsequently we delivered an additional train of four pulses with reverse polarity.

Non-invasive fluorescence macroscopy imaging in live animals. GFP expression in the tumor cells was detected directly through the skin by a fluorescence macroscope (Macrofluo, Leica) equipped with a 12-bit camera binning 1x1 (Coolsnap HQ, Roper) and with the acquisition software Metavue. The observations were made under a 2% isoflurane volatile anesthesia. eGFP was observed with the Leica filter set n°11513880, L5 (excitation filter 480nm ±40nm, dichroic mirror 505 long-pass, emission filter 527nm ±30nm). All the acquisitions were made with a lens X1, with a zoom 1, with an exposure time of 1000 msec.

The EGFP fluorescence from the tumor was measured quantitatively during one week as described in [START_REF] Golzio | In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery[END_REF]). Briefly, images were processed using ImageJ software (National Institutes of Health). The mean fluorescence intensity (MFI) of the same ROI was quantified for each image in the scan series.

Quantitative RT-PCR. RNA extraction for quantitative real time PCR RNA was carried on by using RNeasy Protect mini kit (Qiagen). Immediately after harvesting the tumor, the tissues were put in RNAlater stabilization reagent and stored at 4°C for at least 24 h. For RNA extraction, stabilized tissues were disrupted using a needle of 22-G and total RNA was purified according to Qiagen's protocol. Possible traces of contaminating genomic DNA were eliminated using the DNase RNase free kit (Qiagen). The quality of the RNA was controlled by agarose gel electrophoresis. cDNAs were produced from 5 µg of total RNA by a reverse transcription at 55°C for 50 min using random hexamer primers followed by treatment with RNase H using the Superscript III First strand (InVitrogen).

Quantitative real-time PCR analyses were performed on 50 ng of cDNAs and 300 nM of each primers into the SYBR Green PCR Master Mix (Applied Biosystems) using an Abi Prism 7000 Sequence Detector systems (Applied Biosystems). For each sample, measurements of gene expression were carried out in triplicate in 96-well reaction plates with the current conditions of PCR (50°C 2 min then 40 cycles: 95°C during 10 min then 60°C during 1 min).

geNorm44 VBA applet for Microsoft Excel was used to determine the most stable reference mRNA transcript using a set of six PCR primer couples targeting different mouse housekeeping genes. Cyclin B1 expression values were normalized by a factor calculated by geNorm from the three most stable reference genes: β-glucuronidase, tyrosine 3monooxygenase (YWHAZ) and β-actin-2. All PCR primers used in this study were purchased form Sigma and were validated for quality of the amplification by controlling the dissociation curves of the products and amplification efficiencies. Primers used for Cyclin B1 amplification were Cyclin B1-Forward 5' -CAG AGT TCT GAA CTT CAG CCT G -3' and Cyclin B1-Reverse 5' -ATG GTG AAC TGT GGC CTC ACA AA -3' that were previously described by Wasner and coworkers [START_REF] Wasner | Cyclin B1 transcription is enhanced by the p300 coactivator and regulated during the cell cycle by a CHR-dependent repression mechanism[END_REF]. Other primers were designed using Primer Express software (Applied biosystems). Sequences of PCR primers for the selected housekeeping reference genes are the following: β-glucuronidase: Forward 5' -ACTTTA TTG GCT GGG TGT GG -3' and Reverse 5' -AAT GGG CAC TGT TGA TCC TC -3'; YWHAZ: Forward 5' -CGT GGA GGG TCG TCT CAA GT -3' and Reverse 5' -CTC TCT GTA TTC TCG AGC CAT CTG -3'; β-actin-2: Forward 5' -AGC CAT GTA CGT AGC CAT CCA -3' and Reverse 5' -TCT CCG GAG TCC ATC ACA ATG -3'.

Statistical analysis.

For in vivo experiments, the number of mouse per condition was 4 to 6.

RESULTS.

Efficient siRNA delivery in vivo by a clinically approved PEF protocol.

In order to evaluate the efficiency of the clinically approved protocol (ECT parameters) for siRNA in vivo delivery and silencing into cancer cells, siRNA anti-EGFP was transferred into B16F10-EGFP tumors of 5 to 7 mm diameter. About 30 seconds after slow intra-tumor injection, eight pulses of 100 µsec at a frequency of 1 Hz were delivered at 1300 V/cm and compared with our previous experiments performed using pulses of 5 msec delivered at 600 V/cm. The fluorescence of the subcutaneous tumors was followed and quantified by direct fluorescence imaging of the animal (Fig. 1A). As expected, EGFP fluorescence levels were not modified in the different control groups (Fig. 1B). Indeed, electric pulses alone and unrelated siRNA electrotransfer (negative siRNA) did not modify the percentages of EGFP fluorescence intensities at 72h when compared to non-treated tumors as already shown in our previous study using an EGT protocol [START_REF] Golzio | In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery[END_REF]). When anti-EGFP siRNA was electrotransferred (siRNA+EGT and siRNA+ECT), significant decrease in EGFP fluorescence was observed during the 3 days following the electrical treatment (Fig. 1B). The decrease was maximal at day 3 for both protocols. EGFP fluorescence decreased down to 40% and 30% of the initial fluorescence for ECT and EGT protocols, respectively. When compared with our previous results, similar behavior was observed [START_REF] Golzio | Targeted gene silencing into solid tumors with electrically mediated siRNA delivery[END_REF].

Cyclin B1 inhibition in murine melanoma by siRNA electrotransfer.

In order to confirm the efficiency of the ECT parameters for the delivery of siRNAs in cancer cells, a pool of three anti-cyclin B1 siRNAs were electrotransferred into subcutaneous tumors B16F10-EGFP and tumor growth was monitored during the following days (Fig. 2). The growth of tumors was not modified in the different control groups (Fig. 2A). Indeed, the ECT parameters alone, siRNA control (neg siRNA) or anti-cyclin B1 siRNAs injection alone did not affect tumors growth. When anti-cyclin B1 siRNAs were electrotransferred, a significant slowdown of the tumor growth was observed within 24 h (P Value = 0,005). The tumor growth delay was significant until the second day (P = 0,012).

In order to obtain a more pronounced effect, anti-cyclin B1 siRNAs electrotransfer was repeated three times every other day (Fig. 2B). As expected, the growth of tumors was not significantly modified in the different control groups. Indeed, PBS injections followed by ECT protocol, injections of 5 µg of anti-cyclin B1 siRNAs or injections of 5 µg of unrelated siRNA followed by ECT protocol did not modify tumor growth (Fig. 2B). When repeated injections of 5 µg of anti-cyclin B1 siRNAs were followed by ECT parameters, a significant slowdown of the tumor growth was observed during 6 days as shown in Fig2C. Once the treatment stopped, the tumor growth returned to an exponential regime, meaning that the effect of the siRNA was not maintained after 2 days.

To demonstrate that the slowdown of tumor growth was specifically due to anti-cyclin B1 siRNAs electrotransfer, we performed quantitative real-time PCR to evaluate the cyclin B1 mRNA content within the treated tumors (Fig. 2D). siRNA electrotransfer has been already

shown efficient for the specific gene silencing in EGT condition, but the delivery of other siRNAs using ECT condition could potentially induce some off-target effect or unspecific knock-down of an untargeted gene involved in the cell cycle. At day 2, mice were sacrificed and tumors were processed for RNA extraction. Quantitative RT-PCR using GeNorm analysis revealed a 30% reduction of the cyclin B1 mRNA content only when anti-cyclin B1 siRNAs was electrotransferred into tumors (Fig. 2D). Indeed, with all other control treatments, no significant differences in cyclin B1 mRNA levels were observed: after anti-cyclin B1 siRNAs injection, PBS injection followed by ECT parameters, unrelated siRNA injection followed by ECT parameters or no treatment. These data demonstrated that tumor growth delay observed after electrotransfer of the anti-cyclin B1 siRNA truly originated from a siRNA-mediated degradation of cyclin B1 mRNA. for each group at day 5. (D) Cyclin B1 mRNA levels after siRNA delivery into murine melanoma. The steady-state transcript levels of the cyclin B1 gene were measured by quantitative RT-PCR analysis of total RNA extracted from tumors 2 days after treatments. Cyclin B1 mRNA levels were calculated in comparison to untreated tumors. Three tumors were analyzed in each case. Histograms represent the mean of normalized cyclin B1 mRNA ± standard deviation. Two-sided Student t-tests were followed for the anti-cyclin B1 siRNA electrotransfer. * P ** P*** P 0.001.

siRNA electrotransfer using millisecond PEF duration for cyclin B1 inhibition

In order to compare the clinically approved protocol (ECT protocol) versus the nucleic acid delivery protocol (EGT protocol) done in our previous study [START_REF] Golzio | In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery[END_REF], same experiments than in Fig. 2 were performed. Only the PEF parameters were changed.

The growth of tumors was not modified in the different control groups (Fig. 3A). Indeed, the EGT protocol alone, siRNA control (neg siRNA) or anti-cyclin B1 siRNAs injection alone (0.25 mg/kg) did not modify tumors growth. When anti-cyclin B1 siRNAs were electrotransferred, a significant slowdown of the tumor growth was observed within 24 h (P Value = 0.005). The tumor growth delay was significant until the third day (P = 0.046). This observation showed that a single EGT protocol treatment led to a more sustained delay in tumor growth than one single ECT protocol treatment.

When treatments were repeated three times every other day (Fig. 3B), the growth of tumors was not significantly modified in the different control groups. Indeed, PBS injections followed by EGT protocol, injections of 5 µg of anti-cyclin B1 siRNAs or injections of 5 µg of unrelated siRNA followed by EGT protocol did not modify tumor growth (Fig. 3B). When injections of 5 µg of anti-cyclin B1 siRNAs were followed by the EGT parameters, a significant slowdown of the tumor growth was observed during 6 days. The slowdown of the tumor growth lasted until the eighth day for the both treatments (Figs. 2B and3B).

Our results clearly indicate that the clinically approved protocol parameters is well suited for siRNA delivery, when repeated treatments are needed.

DISCUSSION.

The main obstacle to the use of siRNAs for in vivo cancer therapy remains the need of an efficient and specific intracellular delivery method, which would avoid off-target effects or immune response [START_REF] Rossi | Helping RNAi deliver[END_REF][START_REF] Dykxhoorn | The silent treatment: siRNAs as small molecule drugs[END_REF][START_REF] Xie | Harnessing in vivo siRNA delivery for drug discovery and therapeutic development[END_REF].

Indeed, naked siRNA is relatively unstable in blood and is rapidly cleared from the body by nucleases degradation [START_REF] Whitehead | Knocking down barriers: advances in siRNA delivery[END_REF]). The systemic delivery method seems feasible to transfer siRNA into cancer cells, but the resulting low efficiency silencing of the gene may occur due to a poor siRNA cellular uptake. In addition, the latter can also induce unintended effects with siRNA interactions or absorption by bystander (untargeted) cells [START_REF] Jackson | Recognizing and avoiding siRNA off-target effects for target identification and therapeutic application[END_REF][START_REF] Samuel-Abraham | Staying on message: design principles for controlling nonspecific responses to siRNA[END_REF][START_REF] Walton | Designing highly active siRNAs for therapeutic applications[END_REF][START_REF] Scherman | Genetic pharmacology: progresses in siRNA delivery and therapeutic applications[END_REF].

EGT and ECT parameters are physical techniques that allow the local delivery of therapeutic molecules into cells without carrier molecules. They have been previously used by different groups including ours, to locally deliver siRNAs in various tissues [START_REF] Zhang | Enhanced delivery of naked DNA to the skin by noninvasive in vivo electroporation[END_REF][START_REF] Golzio | Inhibition of gene expression in mice muscle by in vivo electrically mediated siRNA delivery[END_REF][START_REF] Inoue | Electro-transfer of small interfering RNA ameliorated arthritis in rats[END_REF][START_REF] Takabatake | Exploring RNA interference as a therapeutic strategy for renal disease[END_REF][START_REF] Golzio | In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery[END_REF]. EGT protocol consists in the application of millisecond electric pulses, which are required to load macromolecules into cells [START_REF] Rols | In vivo electrically mediated protein and gene transfer in murine melanoma[END_REF][START_REF] Cemazar | Electrotransfer of therapeutic molecules into tissues[END_REF][START_REF] Golzio | In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery[END_REF]). We have previously described the electrotransfer mechanism of the siRNA into cancer cells, in which siRNA molecules have a rapid and direct access to the cytosol of the electropermeabilized cells, allowing a direct transfer of the siRNA into cells compartment, where siRNA's enzymatic machinery (RISC) and its target (mRNA) are present. Cytoplasmic delivery of synthetic siRNAs by electroporation into adherent peripheral blood mononuclear cells did not induce either inflammatory cytokines or interferons [START_REF] Sioud | RNA interference below the immune radar[END_REF].

The present study shows that using a clinically approved protocol (ECT) is a promising method to transfer molecules including siRNAs into cancer cells. This protocol consists in the application of short electric pulses (µsec) at high electric field intensities (100-1300 V/cm) to transiently permeabilize the cells and allow the diffusion of small molecules like cisplatin or the bleomycin [START_REF] Mir | Electrochemotherapy potentiation of antitumour effect of bleomycin by local electric pulses[END_REF][START_REF] Heller | Treatment of cutaneous nodules using electrochemotherapy[END_REF][START_REF] Mir | Effective treatment of cutaneous and subcutaneous malignant tumours by electrochemotherapy[END_REF]. In this study, we have tested the efficiency of this delivery method into murine melanoma for the transfer of siRNAs, targeting either a reporter gene (anti-GFP) or an endogenous gene (anti-cyclin B1).

In vivo, ECT parameters alone, intra-tumor injection of siRNA alone or unrelated siRNA electrotransfer did not affect egfp expression (Fig. 1). On the contrary, when tumors were electropermeabilized after injection of anti-EGFP siRNA, a 50% decrease of the EGFP fluorescence EGFP was observed after 72 h (Fig. 1B). This observation is in agreement with our previous in vitro and in vivo observations obtained with EGT parameters [START_REF] Golzio | In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery[END_REF]).

Electrotransfer of anti-cyclin B1 siRNA by ECT or EGT protocols induced a slowdown of tumor growth (Figs. 2A and 3A). This observation showed the efficacy of both electric treatments (ECT and EGT) to target an endogenous gene involved in the cell cycle. Moreover, this phenomenon can be maintained by repeating the treatment (Fig. 2B, 2C and 3B). After three treatments, the treated tumor growth was delayed, compared to other control groups. We confirmed that the cyclin B1 mRNA level was decreased after siRNA electrotransfer, indicating that RNAi gene silencing mechanism was involved (Fig. 2D and3C). The anti-EGFP siRNA electrotransfer effect was transient; it was maximal at 24-48 h and returned to its initial value at day 7. The silencing was transient because the intracellular concentration of siRNA was diluted during the cellular division [START_REF] Chiu | RNAi in human cells: basic structural and functional features of small interfering RNA[END_REF][START_REF] Paroo | Challenges for RNAi in vivo[END_REF].

A delay of 24-48 h was observed, mainly because of the 24 h lifetime of the EGFP [START_REF] Corish | Attenuation of green fluorescent protein half-life in mammalian cells[END_REF]. B16F10 is a very rapid growing tumor model. Anti-cyclin B1 siRNA effect induced only a slowdown of the tumor growth by blocking the cell cycle division, but did not kill the tumor cells (did not completely inhibit cell division).

Our results show that ECT and EGT protocols were an efficient method for the local delivery of siRNA into cancer cells. The repeated siRNA administration is feasible and efficient using PEF protocols. In addition, we also show that the application of the electric field alone does not induce a tumor growth delay. Electropermeabilization induces a transient permeabilization of the plasma membrane and a rapid electrophoretic transfer of siRNAs into the cytoplasm.

Our conclusions show that an existing clinical procedure could be used for siRNA delivery that targets endogenous gene involved in tumor progression [START_REF] Ferrari | Frontiers in cancer nanomedicine: directing mass transport through biological barriers[END_REF][START_REF] Kaczmarek | Advances in the delivery of RNA therapeutics: from concept to clinical reality[END_REF]. Knockdown of cyclin B1 inhibits cell proliferation in vitro as well as in vivo. Moreover, targeting cyclin B1 sensitizes breast cancer cells to taxol, suggesting that specific cyclin B1 targeting is an attractive strategy for the combination with conventionally used agents in gynecological cancer therapy [START_REF] Yuan | Stable gene silencing of cyclin B1 in tumor cells increases susceptibility to taxol and leads to growth arrest in vivo[END_REF]. As far as electroporation of siRNA is concerned, only solid and visible tumors can be treated.

Nevertheless, with the development of immunotherapy strategies based on cytokine expression [START_REF] Pasquet | Pre-clinical investigation of the synergy effect of interleukin-12 gene-electro-transfer during partially irreversible electropermeabilization against melanoma[END_REF]) and other strategies targeting T-cells (with the downregulation of PD-1 alone or simultaneously with CTLA-4) [START_REF] Simon | The siRNA-mediated downregulation of PD-1 alone or simultaneously with CTLA-4 shows enhanced in vitro CAR-T-cell functionality for further clinical development towards the potential use in immunotherapy of melanoma[END_REF], we are convinced that siRNA delivery by electroporation, using a clinically approved protocol, can open the way to additional therapeutic approaches against disseminated melanoma.

  C57Bl/6 mice were obtained from Rene Janvier and were subjected to an adaptation period of at least 10 days before experiments. They were maintained at constant room temperature with a 12 h light cycle in a conventional animal facility. The mice were 10-14 weeks old at the beginning of the experiments; weighing 20-25 g. Tumors were implanted subcutaneously in the right flank of the mice by inoculation of 10 6 B16F10-EGFP cells in PBS and grown to a size of 5-7 mm in diameter. Procedures were performed with approved protocols from the local ethical committee N°20110329/129, in accordance with the French CNRS and EU commission regulations for laboratory animals' care. Tumor growth was followed by measuring two orthogonal tumor diameters (a and b) with a vernier caliper. Tumor volumes were calculated by the formula V = π × a × b × b/6; (a) being the longest diameter. The arithmetic mean of tumor volumes and standard error of the mean (SE) were calculated for each experimental group.

  Statistical analyzes were performed by using unpaired two-sided Student t-tests in Microsoft Excel software (version 2010). * P ** P*** P 0.001. The error bars represent the standard deviation.

Figure 1 .

 1 Figure 1. In vivo anti-EGFP siRNA electrotransfer in B16F10-EGFP tumors. B16F10-EGFP tumors were non-treated (NT) or injected with anti-EGFP siRNA (siRNA) or with PBS (EGT+PBS ant ECT+PBS). Anti-EGFP siRNA was electrotransferred into tumors by EGT (EGT+siRNA) or ECT (ECT+siRNA) protocols. (A) Digital images were used to visualize fluorescence of B16F10-EGFP tumors as a function of time after EGT and ECT protocols with PBS as a control and with anti-EGFP siRNA. The scale bar corresponds to 5 mm. (B) For each animal, the relative tumor mean fluorescence was determined as the ratio between the fluorescence intensity at day 3 and the fluorescence intensity measured before treatment (day 0 referred as 100% value). The number of mice was: NT, n=6; siRNA, n= 6; EGT+PBS, n=4; ECT+PBS, n=4; EGT+siRNA, n=5; ECT+siRNA, n=5. Two-sided Student ttests were performed. ** P*** P 0.001.

Figure 2 .

 2 Figure 2. SiRNAs anti cyclin B1 electrotransfer into tumor cells by ECT parameters. For each animal, the tumor growth was quantified on a relative scale using as a 100% reference value the tumor size just before treatment (day 0). (A) B16F10 tumor growth was followed after no treatment (NT), after one injection of PBS followed by ECT protocol, after one injection of 5 µg of the siRNA anti cyclin B1 (siRNA 5 µg x1) alone. The siRNA anti cyclin B1 was electrotransferred into tumors by ECT parameters (siRNA 5 µg + ECT). (B) Mice were treated as previously described at Day 0, day 2 and Day 4. The number of mice was: NT, n=6; siRNA 5µg x1, n= 5; PBS+ECT x1, n=4; siRNA 5µg+ ECTx1, n=5; siRNA 5µg x3, n= 6; PBS+ECT x3, n=5; Neg siRNA+ECT x3, n=5; siRNA 5µg+ ECT x3, n=5. For statistical analysis, a two-way ANOVA analysis with the Bonferroni post hoc test was performed. * P ** P*** P 0.001 show statistically differences between ECT parameters and electro-transferred siRNA. (C) Representative pictures of mice after 3 treatments

Figure 3 .

 3 Figure 3. SiRNAs anti-cyclin B1 electrotransfer into tumor cells by EGT parameters. For each

Acknowlegdements. We thank Pr Bettina Couderc for the generation of the B16F10-EGFP and Dr Marie-Jeanne Pilaire for the RT-PCR assistance. This work has been supported by the Région Midi Pyrénées, ITAV programs, ANR programs Biodendridots, Cemirbio and the Seventh Framework European Program (FP7) OncomiR [grant number 201102]. We thank the "Toulouse Réseau Imagerie" and Anexplo core IPBS facilities (Genotoul, Toulouse, France). Research was conducted in the scope of the EBAM European Associated Laboratory (LEA) and resulted from the networking efforts of the COST Action TD1104 (http://www.electroporation.net).

CRediT author statement

Aurélie

Paganin-Gioanni: Methodology, Visualization, Investigation, Data curation, formal analysis, Writing-Original draft preparation. Marie-Pierre Rols: Conceptualization, Resources, Funding Acquisition. Justin Teissié: Conceptualization, Supervision, Resources, Funding Acquisition, Writing-Reviewing and Editing. Muriel Golzio: Conceptualization, Investigation, Supervision, Validation, Writing-Reviewing and Editing.