Thomas Sonntag

Mickael Rapp

Pascal Didier

Luc Lebeau

Françoise Pons

Anne Casset
email: casset@unistra.fr

Mucus-producing epithelial models for investigating the activity of gene delivery systems in the lung

Keywords: gene delivery, lipoplexes, airway mucus, cell models, rheology, multiple particle tracking Air-liquid interface, BSA: Bovine serum albumin, CCD: charge coupled device, CF: Cystic fibrosis, CLSM: Confocal laser scanning microscopy, Ct: carboxy terminal, cw laser: continuous wave laser, DLS: Dynamic light scattering, DOPC: 1,2-dioleoyl-sn-glycero-3-phospho-choline, EDMPC: 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, Genzyme lipid 67, HDM: House dust mite, HRP: Horseradish peroxidase, IgG Immunoglobulin G, L2k: Lipofectamine ® 2000, LLI: Liquid-liquid interface, MDa: Megadalton, MPT: Multiple particle tracking, MSD: Mean squared displacement, MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide, NTA: Nanoparticle tracking analysis, PBS: Phosphate-buffered saline, PdI: Polydispersity index, pDNA: plasmid DNA, r c : Radius of confinement, Rh-PE: 1,2-Dioleoyl-snglycero-3 3,3',5,5'- Wheat germ agglutinin

Inhaled transfection particles have to penetrate the mucus layer lining the airways to successfully deliver their therapeutic nucleic acid payload to target cells in the underlying epithelium. However, the in vitro models used for evaluating gene carrier efficiency often disregard this viscous defensive barrier. In this study, the two mucus-secreting cell lines NCI-H292 and Calu-3 were selected to develop a series of epithelial models displaying gradual mucus production. In NCI-H292 models, a gradual increase in the MUC5AC mucin was obtained after cell exposure to inducers. In Calu-3 models, MUC5AC production increased as a function of culture duration (3, 7, 14 days) at the air-liquid interface (ALI). Six DOPCderived cationic lipids were designed and their pDNA delivery activity was evaluated to validate these cellular models. The strongest impairment of the lipid delivery activity was observed in the Calu-3 14-d ALI model. The MUC5AC production in this model was the greatest and the mucus layer was 20 µm thick. The mucus exhibited a solid viscoelastic behaviour, and represented a major hindrance to lipoplex diffusion. The Calu-3 14-d ALI model will be highly useful for accurate evaluation of gene carriers intended for airway administration and characterization of their interactions with the mucus.

Introduction

Gene therapy relies on the successful delivery of therapeutic nucleic acids into target cells and holds great promise for curing congenital as well as acquired diseases. With respect to lung diseases, the pulmonary route is considered as a highly accessible and noninvasive administration route for therapeutics, including gene therapy products. It offers several advantages when compared to the systemic route, such as reduced drug side effects and limited interaction of drugs with serum proteins, and requires lower drug doses. On these bases, a large variety of gene carriers have been developed for lung gene therapy [START_REF] Griesenbach | Gene transfer to the lung: lessons learned from more than 2 decades of CF gene therapy[END_REF]. First to be developed, the viral gene vectors have rapidly shown some limitations such as toxicity, insufficient gene transfer in human airways requiring repeated administrations, and therapy-inactivating immunogenicity [START_REF] Griesenbach | Gene transfer to the lung: lessons learned from more than 2 decades of CF gene therapy[END_REF][START_REF] Kim | Barriers to inhaled gene therapy of obstructive lung diseases: A review[END_REF]. Non-viral gene carriers have been developed as an alternative, including polymer-and lipid-based vectors. Although they are generally less effective for intracellularly delivering nucleic acids, non-viral carriers are less toxic and less immunogenic than viral vectors, and their mass production is more feasible and much less costly. Among lipid-based gene vectors available to date, the Genzyme lipid 67 (GL67) is by far the most advanced in the clinic for the treatment of obstructive lung diseases by inhaled gene therapy. Nevertheless, its last clinical evaluation with repeated nebulization provided a significant but moderate improvement in lung function in patients with cystic fibrosis (CF) (Alton et al., 2015), suggesting that further work has still to be conducted to improve the bioavailability of gene carriers in the lung. Recently, a new family of biodegradable nucleic acid carriers derived from the 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), a major constituent of biological membranes, has been developed and transfection efficiency of these carriers was demonstrated in vitro and in vivo ( Pierrat et al., 2013;b Pierrat et al., 2016a). Upon airway administration, these cationic DOPC-detergent conjugates outperformed the best performing GL67-based formulation (GL67A), showing that these new delivery systems might be promising for lung gene therapy (Pierrat et al., 2016c). Nevertheless, in the particular case of administration into the lung, the transfection particles have to overcome the airway mucus, that constitutes a viscous defensive line which is overproduced in obstructive lung diseases.

Airway mucus consists in a viscoelastic gel which contains a complex mixture of water (ca. 95 %), glycoproteins (mucins, ca. 2-5 %), non-mucin proteins, salts, lipids, DNA, cells, and cellular debris [START_REF] Bandi | Advanced materials for drug delivery across mucosal barriers[END_REF][START_REF] Bansil | Mucin structure, aggregation, physiological functions and biomedical applications[END_REF]. Thus, mucins are the major component of the mucus, and MUC5AC and MUC5B are the two main mucins of the human airway mucus. MUC5AC is secreted by goblet cells and MUC5B by submucosal glands [START_REF] Hovenberg | MUC5AC, but not MUC2, is a prominent mucin in respiratory secretions[END_REF][START_REF] Reid | Developmental expression of mucin genes in the human respiratory tract[END_REF][START_REF] Thornton | Structure and function of the polymeric mucins in airways mucus[END_REF]. Mucin proteins contain alternating regions of globular hydrophobic domains and hydrophilic O-glycan brushes. The globular hydrophobic domains are composed of cysteine residues and hydrophobic amino acids involved in intradomain disulfide bonds and hydrophobic interactions between mucin fibers. The O-glycan brushes have high sialic acid and sulfate contents and thus confer a strong net-negative surface charge to the biopolymer and impart stiffness of the mucin superstructure via electrostatic repulsions [START_REF] Leal | Physicochemical properties of mucus and their impact on transmucosal drug delivery[END_REF]. In addition, cysteine-rich domains in the carboxy-terminal (Ct) region of the mucin protein enable the formation of Ct-Ct mucin dimers and finally long multimers via disulfide bridges, leading to a cross-linked mucin 3D-array of molecular weight ranging from 0.5 to 20 MDa [START_REF] Carlstedt | Structure and macromolecular properties of cervical mucus glycoproteins[END_REF][START_REF] Leal | Physicochemical properties of mucus and their impact on transmucosal drug delivery[END_REF][START_REF] Thornton | From mucins to mucus: toward a more coherent understanding of this essential barrier[END_REF]. Hydrophilicity (O-glycans) and polymerization are the two key features of mucins leading to the formation of a hydrogel when in contact with water. Reversible interactions between naked and hydrophobic cysteineenriched domains that interrupt the heavily O-glycosylated regions of MUC5AC and MUC5B have been described a long time ago in the mucin network and are likely important in the viscoelastic properties of mucus on the microscale [START_REF] Demouveaux | Mucin CYS domain stiffens the mucus gel hindering bacteria and spermatozoa[END_REF][START_REF] Sheehan | Evidence for shared epitopes within the 'naked' protein domains of human mucus glycoproteins. A study performed by using polyclonal antibodies and electron microscopy[END_REF]. This grants mucus the ability to adsorb a wide range of molecules and particles [START_REF] Demouveaux | Gel-forming mucin interactome drives mucus viscoelasticity[END_REF]. Besides, mucus is continuously secreted (1.5-2 L, daily), forming a dynamic layer on epithelial cells that is in constant movement. The thickness of this mucus blanket is determined by the balance between the rate of mucin secretion and degradation.

Clearance of the mucus from the lower airways to the pharynx through the beating of cilia from epithelial cells enables a complete renewal of the mucus layer every 20 minutes in healthy subjects [START_REF] Di Gioia | Nanocomplexes for gene therapy of respiratory diseases: Targeting and overcoming the mucus barrier[END_REF]. This mucociliary clearance acts as a dynamic barrier to protect the respiratory tract from inhaled microorganisms, particles or drug molecules adsorbed or trapped in the mucus mesh structure [START_REF] Murgia | The role of mucus on drug transport and its potential to affect therapeutic outcomes[END_REF]. Therefore, airway mucus constitutes a physiological barrier due to i-its viscoelastic properties and mesh-spacing, resulting in size filtering mechanisms, ii-its composition and net-negative surface charge, resulting in interaction filtering mechanisms, and iii-its rapid turnover, resulting in very short time window for successful delivery of therapeutic particles, including nucleic acidcontaining particles, to epithelial cells.

In patients with obstructive lung diseases such as CF, chronic obstructive pulmonary disease or asthma, mucus hypersecretion and/or increase in mucus viscoelasticity are found, expanding the already complicated process of delivering therapeutic agents through the airways. In gene therapy strategies, rationally-designed approaches have been proposed to develop carriers capable of perfusing the altered mucus barrier and reaching the underlying target epithelial cells, taking into account that the thickness of the mucus layer covering the human airway epithelium from the trachea to the bronchi varies from 10 to 30 µm, and from 2 to 5 µm, respectively [START_REF] Kurbatova | Model of mucociliary clearance in cystic fibrosis lungs[END_REF][START_REF] Leal | Physicochemical properties of mucus and their impact on transmucosal drug delivery[END_REF]. However, cellular models usually used to evaluate gene transfer efficiency contain no or very low amount of mucus which may lead to significantly overrated performances of the investigated carriers [START_REF] Aissaoui | Efficient topical delivery of plasmid DNA to lung in vivo mediated by putative triggered, PEGylated pDNA nanoparticles[END_REF][START_REF] Mastorakos | Highly compacted biodegradable DNA nanoparticles capable of overcoming the mucus barrier for inhaled lung gene therapy[END_REF][START_REF] Osman | PEGylated enhanced cell penetrating peptide nanoparticles for lung gene therapy[END_REF][START_REF] Raik | Comparative study of diethylaminoethyl-chitosan and methylglycol-chitosan as potential non-viral vectors for gene therapy[END_REF][START_REF] Suk | N-acetylcysteine enhances cystic fibrosis sputum penetration and airway gene transfer by highly compacted DNA nanoparticles[END_REF]. Thus, cell culture models better mimicking in vivo physiological conditions are needed, and multiple parameters related to the intrinsic properties of the mucus layer in these models (e.g., mucus composition, viscoelastic properties, layer thickness…) should be considered for ultimately getting relevant data for a proper evaluation of gene carriers [START_REF] Ghanem | Optimizations of In Vitro Mucus and Cell Culture Models to Better Predict In Vivo Gene Transfer in Pathological Lung Respiratory Airways: Cystic Fibrosis as an Example[END_REF].

In our quest for efficient carriers for gene delivery through the airways, we were pushed to design original biodegradable DOPC-derived cationic lipids based on results that were previously obtained with DOPC-detergent conjugates (Fig. 1) [START_REF] Pierrat | A cationic phospholipid-detergent conjugate as a new efficient carrier for siRNA delivery[END_REF]Pierrat et al., 2013a;Pierrat et al., 2013b;Pierrat et al., 2016a;Pierrat et al., 2016b;Pierrat et al., 2016c). However, in order to do a proper evaluation of the plasmid DNA (pDNA) delivery activity of these DOPC-based cationic lipids in the context of airway administration, we decided to improve the standard cell models commonly used in the field of lung gene therapy and develop more relevant ones with adjustable levels of mucus, with a particular emphasis on the characterization of the rheology and thickness of the mucus produced by these models and the capacity of the transfection particles to diffuse through it. To establish our cell models and evaluate the effect of the mucus layer on gene transfer activity of our new carriers, two main human pulmonary cell lines, the NCI-H292 and Calu-3 cells were used. These cells were cultured in plates and on inserts, and treated, or not, with mucus inducers or cultured at the air-liquid interface (ALI) to obtain 2D-or 3D-models producing increasing amount of mucus (Table 1). NCI-H292 cells are human pulmonary mucoepidermoid carcinoma cells known to constitutively express the epidermal growth factor receptor (EGF-R) which stimulation by its ligands results in MUC5AC production [START_REF] Takeyama | Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils[END_REF][START_REF] Takeyama | Epidermal growth factor system regulates mucin production in airways[END_REF]. Therefore, EGF and house dust mite (HDM) allergen extract were used to set up NCI-H292 2D-models with increased mucus production. Calu-3 cells are submucosal-gland carcinoma cells producing mucus when cultured in plates, and whose mucus production can be modulated by ALI culture on inserts [START_REF] Kreda | Coordinated release of nucleotides and mucin from human airway epithelial Calu-3 cells[END_REF][START_REF] Kreft | The characterization of the human cell line Calu-3 under different culture conditions and its use as an optimized in vitro model to investigate bronchial epithelial function[END_REF]. This cell line was thus used to set up 2D-and 3D-models (Table 1). In some experiments, 3D-models of NCI-H292, A549 or 16HBE were also used (Table 1). In the obtained cultures, mucus production was characterized by assessing MUC5AC levels in culture supernatants (2Dmodels) or in collected mucus samples (3D-models), and/or by visualizing mucus layer on the top of cultures by confocal microscopy (3D-models). These cellular models allowed us to investigate and finely characterize properties of mucus by rheology and transfection particle diffusion by multiple particle tracking (MPT). As well, gene transfer efficiency of our DOPCderived gene carriers was evaluated in these various in vitro models to probe the impact of mucus on their activity.

Table 1. 2D-and 3D-models used in this study. In 2D-models, NCI-H292 and Calu-3 cells were grown in plates and treated, or not, with EGF or mite allergen. In 3D-models, the different cell lines were cultured on inserts at the ALI for 3 (3-d ALI), 7 (7-d ALI) or 14 d (14-d ALI).

Model

Cell line Treatment/Culture duration

Materials and methods

Materials

Cell lines

The human epithelial adenocarcinoma cell line Calu-3 (HTB-55), the human epithelial mucoepidermoid carcinoma cell line NCI-H292 (CRL-1848), and the human epithelial carcinoma cell line A549 (CCL-185) were purchased from ATCC-LGC (Molsheim, France).

The SV40ori-transformed human bronchial epithelial cell line 16HBE14o-(16HBE) was a generous gift from Dr D. Gruenert (California Pacific Medical Center Research Institute, San

Francisco, CA, USA). [START_REF] Gorman | Efficient in vivo delivery of DNA to pulmonary cells using the novel lipid EDMPC[END_REF]), of EDOPC (MacDonald et al., 1999), and of the biolabile DOPC-based cationic lipids 1-6 investigated herein.

Reagents

Methods

Lipid synthesis

The detailed synthesis and characterization of the 6 cationic lipids investigated herein are provided as Supplementary Material.

Lipoplex preparation

Appropriate volumes according to the desired charge ratio (lipid ammonium/pDNA phosphate ratio, N/P) of a freshly prepared ethanolic solution of a cationic lipid (1-6) were deposited at the bottom of polyethylene tubes and dried under vacuum for 1 h using a SpeedVac concentrator. Then, 40 µL of a pCMV-Gluc solution (0.02 µg/µL prepared in 5%

(w/w) glucose) were added to each tube. The tubes were stirred by vortex for 20 s and allowed to stand at rt for 30 min before the resulting lipoplex preparations were used. The pDNA-L2k-based formulations were prepared as recommended by the manufacturer. Briefly, pDNA and L2k were diluted at the appropriate concentration in Opti-MEM ® medium. pDNA was then added to L2k (1:1, v/v) and the mixture was incubated for 5 min before use.

Measurement of size and zeta potential of the lipoplexes

O O O O O P O N O O O O O O P O N O O O P O N O O O O TfO Cl O O O O R 1 O R 2 Cl EDMPC EDOPC 1: R 1 = i-C 3 H 7 O ; R 2 = H 2: R 1 = n-C 4 H 9 O ; R 2 = H 3: R 1 = n-C 8 H 17 O ; R 2 = H 4: R 1 = n-C 12 H 25 O ; R 2 = H 5: R 1 = n-C 12 H 25 O ; R 2 = CH 3 6: R 1 = n-C 12 H 25 OC(O)C 2 H 4 ; R 2 = H
The average diameter of lipoplexes was measured by dynamic light scattering (DLS) using a Zetasizer ® nano-ZS instrument (Malvern Instruments, Palaiseau, France) at 25 °C with a scattering angle of 173°. Freshly prepared lipoplexes were diluted to the tenth in a 5% glucose solution before measurement. The Z-averaged sizes and polydispersity indexes (PdI) were obtained by cumulant analysis of the autocorrelation function. Each analysis, giving an average of 3 measurements, was carried out on 3 independent preparations. Simultaneously, the size distribution and the concentration of lipoplex preparations were determined by nanoparticle tracking analysis (NTA) using a Zetaview ® instrument (Particle Metrix GmbH, Inning, Germany). The samples were diluted to reach the optimal range of 50 to 200 particles per frame and measurements were made at rt. For each sample, videos were acquired for 8 s at the frame rate of 3.75 s -1 , as recommended by the manufacturer with regard to the size range of the particles. Detected tracks were analyzed using the Zetaview ® software and translated into particle size distribution profile from which the mean diameter of the particles was expressed. Zeta potential (z-potential) of the lipoplexes was determined at 25 °C on samples diluted to the tenth in a 5% glucose solution, using the Zetasizer ® nano-ZS instrument. Measurements were performed on 3 independent preparations and analyzed with the Zetasizer ® software, using the electrophoretic mobility based on the Smoluchowski approximation. Data were expressed as mean value ± standard deviation (SD).

Cell culture

A549, Calu-3 and 16HBE cells were grown in a DMEM/F12 medium containing 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 5 mM HEPES and 2 mM L-Glutamine. NCI-H292 cells were grown in a RPMI-1640 medium supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 10 mM HEPES, 1 mM sodium pyruvate and 2 mM L-Glutamine. All cultures were carried out in culture flasks using a humidified incubator at 37 °C under a 5% CO 2 atmosphere. At 80-100% confluency, cells were released from the flasks with trypsin-EDTA (0.05%, w/w, in PBS) and transferred into new flasks, to establish subcultures, or seeded in 96-well culture plates (2D-models) or on polyethylene terephthalate inserts with 0.4 µm pore size (3D-models), for experiments. Calu-3, NCI-H292, 16HBE and A549 cells were used from passage 26 to 38, 10 to 37, 54 to 62, and 84 to 101, respectively.

For experiments, Calu-3 and NCI-H292 cells were seeded in 96-well plates at a density of 1.0 × 10 5 and 3.0 × 10 4 cells/well, respectively, in 100 µL of culture medium. Calu-3 cells were seeded on 24-well inserts at a density of 1.5 × 10 5 cells/insert in 400 µL of culture medium, or on 6-well inserts at a density of 2.1 × 10 6 cells/insert in 2.5 mL of culture medium. NCI-H292, 16HBE and A549 cells were seeded on 24-well inserts at a density of 1.0 × 10 5 , 1.1 × 10 5 and 6 × 10 4 cells/insert, respectively, in 400 µL of culture medium. The medium in both compartments of the inserts was changed three times a week until cells formed a confluent layer. After cells had reached confluency, apical culture medium was removed and cultures were maintained at the ALI for 3, 7 or 14 d. Meanwhile, the medium at the basolateral side of the inserts was changed three times a week. For some experiments, Calu-3 cell cultures on inserts were maintained at the liquid-liquid interface (LLI) for 14 d after reaching confluency.

Induction of mucus production in the NCI-H292 2D-models

NCI-H292 cells were seeded in 96-well plates as described above and grown for 72 h until culture confluency. Then, they were starved of serum for 24 h by changing complete medium for FBS-free medium. Afterwards, mucus production was induced by exposing cells to EGF (25 ng/mL in FBS-free culture medium) or a HDM allergen extract (1 µg Der p 1/mL in FBS-free culture medium) for 24 h. Cells exposed to FBS-free culture medium were used as control (untreated cells). Mucus sample collection or transfection experiments in these 2D-models were carried out at the end of the 24-h incubation period as described below.

Induction of mucus production in the Calu-3 3D-models

Calu-3 cells were seeded on 24-well inserts as described above and grown for 72 h until culture confluency. Then, apical culture medium was removed and cells were grown at the ALI for 3, 7 or 14 d. Mucus sample collection or transfection experiments in these 3D-models were conducted afterwards as described below.

Transfection experiments

Transfection experiments were conducted on 2D-models, 3D-models, 3D-models after mucus removal, and on cell cultures on inserts at the LLI. In the 2D-models, cells grown in plates were exposed to freshly prepared lipoplexes at the desired N/P ratio by adding 10 µL of preparation to the cell culture supernatant. In the 3D-models, 100 µL of culture medium were deposited in the apical compartment of inserts just before adding 10 µL of lipoplexes. For mucus removal experiment in the 3D-models, 100 µL of culture medium were deposited in the apical compartment of the insert at day 14 of ALI culture. The day after, 10 µL of the lipoplex preparations were added in the apical compartment of the insert without mucus removal (controls) or the culture medium in the apical compartment was removed with the mucus and replaced by 100 µL of fresh culture medium, just before adding 10 µL of the lipoplex preparations (mucus removal). On insert cultures maintained at the LLI, 10 µL of the lipoplex preparations were added to the culture medium present in the apical compartment of the inserts. In all cases, the pDNA quantity added to the cultures was 0.2 µg/well or insert.

Cultures exposed to an equal volume of a 5% glucose solution were used as negative control.

Transfection efficiency was evaluated after a 24-h exposure by measuring luciferase activity in the culture supernatant. To do so, coelenterazine (1.5 µM) was added to an aliquot of culture supernatant and light production was measured for 1 s using a Centro LB960 XS luminometer (Berthold Technologies, Thoiry, France). A time course experiment was also performed on the 14-d Calu-3 3D-model without mucus removal, as described above, with transfection efficiency measurements after 24, 48, or 72-h of exposure to lipoplex preparations. In all experiments, each sample was assessed in triplicate and luminescence values (expressed as RLU/well or insert) were normalized to the basal luminescence measured in negative controls.

Mucus sample collection and characterization

Mucus sample collection

Mucus samples were collected from NCI-H292 2D-models, Calu-3 2D-and 3D-models and Calu-3 cultures on inserts at the LLI for MUC5AC and mucin assessments or rheological and MPT analyses as follow. For MUC5AC assay, in the 2D-models, culture supernatant was collected 24 h after cell seeding (Calu-3 cells) or after a 24-h mucus induction by EGF or allergen (NCI-H292 cells). In the 3D-models, culture medium (100 µL for 24-well inserts) was deposited in the apical compartment of the inserts and collected back 24 h later by gently pipetting. In the Calu-3 cultures on inserts at the LLI, culture medium supernatant was collected after 14 d of culture. All collected samples were centrifuged at 3,000 × g for 5 min to discard cell debris and stored at -80 °C for MUC5AC assay. For rheological or MPT analyses, mucus samples were extemporaneously collected from Calu-3 3D-cultures at the ALI in 6-well inserts (4 inserts per condition). Culture medium (1 mL) was added in the apical compartment of each insert and was collected back 24 h later by gently pipetting with a tip with a wide orifice (2-3 mm in diameter) in order to reduce shear stress. After a 1-h decantation at rt in conic plastic tubes, the upper clear liquid corresponding to culture medium (ca., 950 µL /insert) was discarded from each sample. The lower opaque phases (50 µL/insert) corresponding to mucus were then pooled before experiments.

MUC5AC assay

MUC5AC protein was assayed in mucus samples by ELISA. Frozen samples were thawed, distributed into the wells of a titration plate (Maxisorp Nunc immuno plate, Thermo Scientific) and incubated overnight at 40 °C. The coated plate was then washed 3 times with PBS-0.05% Tween ® 20 and blocked at rt for 1 h with a 2% BSA solution prepared in PBS.

Wells were washed again 3 times and exposed for 2 h to a polyclonal mouse IgG1 directed against the human MUC5AC. After another step of washings, samples were incubated for 1 h with a peroxidase-conjugated polyclonal goat anti-mouse IgG. Finally, after a last washing step, the peroxidase substrate, TMB, was added to each well for peroxidase measurement.

The reaction was stopped with 0.5 N H 2 SO 4 and absorbance of the samples was measured at 450 nm with a plate reader (Varioskan Lux, Thermo Scientific). Results were expressed as arbitrary units. They correspond to the mean ± SEM of at least 3 independent experiments.

Mucus observation by confocal laser scanning microscopy

Mucus on 3D-cultures was observed by confocal laser scanning microscopy (CLSM) after mucin labelling with the WGA. On the day of observation, the culture was gently washed with PBS and Alexa-647 ® -labeled WGA (100 µL of a 5 µg/mL solution in PBS) was added on the top of the cells for a 10-min incubation period at 37 °C. After WGA removal and cell washing with PBS, the insert membrane was cut and mounted onto a microscope slide using a permanent aqueous mounting medium (Bio-Rad). Samples were then observed (NA = 1.4) after excitation at 635 nm and detection from 650 to 735 nm, using a Leica SP2 confocal microscope (Leica, Wetzlar, Germany) equipped with a 63 × oil immersion objective. Images were acquired every 0.8 µm from the top of the mucus layer (defined as z = 0 µm) to 40 µm depth. To avoid excitation intensity decrease with deepness, laser power was configured to progressively increase while scanning deeper into the samples. The same increase scale and observation parameters were used for all samples. For each section, two images were acquired and averaged. 3D representations were reconstructed from image stacks using the Fiji image processing software and its 3D Viewer plug-in.

Mucus rheology

Rheological properties of mucus samples collected from Calu-3 3D-cultures were analyzed on a Haake Mars 60 Rheometer (Thermo Electron, Karlsruhe, Germany) equipped with a 25-mm plate-plate geometry. The temperature-controlled Peltier plate was allowed to equilibrate at 37 °C for 2 min before sample deposition (180 µL). After another equilibration period, oscillatory measurements were performed at 37 °C, using a gap width of 0.3 mm between the two plates. The linear viscoelastic region of the samples was determined by performing a stress sweep (0.0001-10) at 1 Hz (6.28 rad/s). The elastic or storage moduli (G'), the viscous or loss moduli (G"), and the phase angle (δ) dependence to frequency were then evaluated by performing frequency sweeps (0.1-17.8 Hz) in the viscoelastic region (constant stress of 0.01). The phase angle or loss tangent value d, calculated from the inverse tangent of the ratio G"/G' (with tan(d) = G"(w)/G'(w)), is usually determined at the frequency (w) of 1 rad/s to approximate the low velocity of mucociliary clearance in mucus rheological studies [START_REF] Innes | Ex vivo sputum analysis reveals impairment of protease-dependent mucus degradation by plasma proteins in acute asthma[END_REF]. The phase angle d ranges between 0° for a Hookean solid and 90° for an ideal liquid, thereby d < 45° characterizes viscoelastic solids. Measurements were performed on 3 independent mucus samples.

Multiple particle tracking experiments

MPT was used to study lipoplex diffusion in the mucus collected from Calu-3 3D-cultures. Fresh mucus samples (200 µL) were deposited into each well of an IbiTreat ® µ-slides (1.5 polymer coverslip, IBIDI, Gräfelfing, Germany). Freshly prepared lipoplexes (50 µL) labelled by adding 1:100 molar ratio of Rh-PE in the lipid ethanolic solution at the time of lipoplex preparation (see protocol above) were subsequently deposited on mucus samples and incubated for 2 h at 37 °C. Prior to particle tracking analysis, the remaining liquid on the mucus layer was slowly removed by pipetting. Complete cell culture medium was used as a negative control in each experiment to evaluate the diffusion of lipoplexes in the absence of mucus. Widefield imaging of the samples was performed on a home-built setup based on a Nikon Eclipse Ti microscope (Nikon Instruments, Melville, NY, USA) equipped with a 100 × oil-immersion objective (NA = 1.49) and coupled to a 532 nm cw laser. Emission from the samples was spectrally filtered with the help of a dichroic mirror (Di02-R532-25x36) and a long pass filter (Semrock BLP01-532R-25), and was then imaged on an ImagEM CCD camera (Hamamatsu Photonics, Massy, France). An additional lens was used to obtain a final magnification of 150 × corresponding to a pixel size of 106.67 nm.

Immobilized particles at the bottom of the well were discarded from observation by defining the baseline of the observation areas. Then, mucus observation areas were randomly chosen between 0 up to 40 µm after checking the absence of residual cells by transmitted light observation to avoid tracking potential intracellular lipoplexes. Five thousand frame videos were acquired in the chosen observation areas. At least 100 lipoplexes were tracked for at least 200 frames in each experimental condition. Thereafter, acquired videos were analyzed using the Fiji software after application of a gaussian blur to facilitate the subsequent tracking. Lipoplexes were tracked and their trajectories were extracted by the particle tracker plug-in [START_REF] Sbalzarini | Feature point tracking and trajectory analysis for video imaging in cell biology[END_REF] using the following set of parameters: radius 6-18, cut-off 0.001, Per/Abs 0.1 to 0.5, link range 2 and displacement 10. Positions of the lipoplexes at each time interval were then exported to the Igor Pro 8 software to calculate their mean squared displacements (MSDs) using the following equation:

𝑀𝑆𝐷 = 1/(𝑁 -𝑛) -|𝑥 !"# -𝑥 ! | $ , 𝑛 = 1, … , 𝑁 -1 %&# !'(
where N is the number of consecutive two-dimensional positions of a particle 𝑥 ! = (𝑥 ! , 𝑦 ! )

recorded with a constant time interval ∆t over a period of time 𝑇 = (𝑁 -1)∆𝑡. MSDs were fitted with a model of confined diffusion [START_REF] Saxton | Single-particle tracking: applications to membrane dynamics[END_REF][START_REF] Wagner | Classification and segmentation of nanoparticle diffusion trajectories in cellular micro environments[END_REF], defined by:

𝑀𝑆𝐷 = 𝑟 ) $ × (1 -𝐴 ( exp (-4𝐴 $ 𝐷𝑡/𝑟 ) $ ))
where r c corresponds to the radius of the confinement, D to the diffusion coefficient, and the constants A 1 and A 2 (here both fixed at 1) characterize the shape of the confinement. From the MSD fit of each individual particle, the D and the r c of tracked lipoplexes were then calculated. A cut-off was established for the r c values based on mucus structure analysis in the literature in order to exclude non-physiologic values. A 5 µm cut-off was applied to the r c from trajectories obtained in the mucus before data analyses and representations [START_REF] Meziu | Visualization of the structure of native human pulmonary mucus[END_REF].

Statistical analysis of the results

Data were analyzed with the GraphPad Prism 6.0 software. In the bar chart representations, statistical differences between groups were evaluated by one or two-way ANOVA followed by the Dunnett's or the Tukey test. The Pearson's correlation test was applied to evaluate the correlation and calculate the Pearson's r. Data were considered as significantly different when p value was less than 0.05.

Results

Lipid synthesis

In 1997, Gorman et al. were the first to demonstrate that a phospholipid-derived phosphotriester (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, EDMPC, Fig. 1) can mediate intracellular gene delivery [START_REF] Gorman | Efficient in vivo delivery of DNA to pulmonary cells using the novel lipid EDMPC[END_REF]. Thereafter, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC, Fig. 1) and other PC derivatives have been developed by MacDonald et al. and their physical and transfection properties extensively investigated [START_REF] Kennedy | Factors governing the assembly of cationic phospholipid-DNA complexes[END_REF][START_REF] Koynova | Cationic O-ethylphosphatidylcholines and their lipoplexes: phase behavior aspects, structural organization and morphology[END_REF][START_REF] Koynova | Hydrophobic moiety of cationic lipids strongly modulates their transfection activity[END_REF][START_REF] Koynova | An intracellular lamellar-nonlamellar phase transition rationalizes the superior performance of some cationic lipid transfection agents[END_REF][START_REF] Macdonald | Physical and biological properties of cationic triesters of phosphatidylcholine[END_REF][START_REF] Macdonald | Surface properties of dioleoyl-sn-glycerol-3-ethylphosphocholine, a cationic phosphatidylcholine transfection agent, alone and in combination with lipids or DNA[END_REF][START_REF] Rakhmanova | Effects of dioleoylphosphatidylethanolamine on the activity and structure of O-alkyl phosphatidylcholine-DNA transfection complexes[END_REF][START_REF] Rosenzweig | O-alkyl dioleoylphosphatidylcholinium compounds: The effect of varying alkyl chain length on their physical properties and in vitro DNA transfection activity[END_REF][START_REF] Tenchov | Modulation of a membrane lipid lamellar-nonlamellar phase transition by cationic lipids: A measure for transfection efficiency[END_REF][START_REF] Wang | Transfection activity of binary mixtures of cationic O-substituted phosphatidylcholine derivatives: The hydrophobic core strongly modulates physical properties and DNA delivery efficacy[END_REF]. Later on, DOPC-detergent conjugates have been proposed that showed extended fusogenic properties and improved gene delivery capabilities [START_REF] Pierrat | A cationic phospholipid-detergent conjugate as a new efficient carrier for siRNA delivery[END_REF]Pierrat et al., 2016b). And more recently, a sophisticated version of these DOPC-based compounds, with programmed biodegradability and lower cytotoxicity, has been described. These last DOPC-derived carriers incorporate a biolabile phospho(alkyl)enecarbonate moiety that pushes transfection efficiency to another level (Pierrat et al., 2013a;Pierrat et al., 2013b;Pierrat et al., 2016a;Pierrat et al., 2016b;Pierrat et al., 2016c). As was demonstrated, these carriers are sensitive to hydrolysis depending on pH conditions and are potential substrates for various enzymes. Upon hydrolysis or metabolization, they are transformed into DOPC. This zwitterionic phospholipid is a major constituent of the cell membranes and thus lacks any toxicity. Furthermore, the electrostatic interactions with the anionic nucleic acid payload cannot be maintained and the latter is slowly decondensed and released. On the timing of this decondensation/release, in the extracellular milieu, in the endosome or lysosome compartments, in the cytosol or the nucleus, depends the overall transfection rate.

Focusing on the biodegradability of such gene carriers, we prepared a series of 6 related phosphotriesters in order to perform extensive evaluation in characterized mucus-producing airway epithelium models (Fig. 1). Varying the structure of substituents R 1 and R 2 in the compounds allows for the modulation of their hydrolytic stability, as was shown previously (Pierrat et al., 2013a;Pierrat et al., 2016a). Cationic lipids 1-3 were prepared by reacting DOPC with chloromethyl i-propyl carbonate, chloromethyl n-butyl carbonate, and chloromethyl n-octyl carbonate, respectively. The detailed synthetic procedures and full characterization of these compounds are provided as supplementary materials. The synthesis of compounds 4-6 has already been reported elsewhere (Pierrat et al., 2013a).

Physicochemical characterization of the lipoplexes

The ability of cationic lipids 1-6 to interact with nucleic acids and form lipoplexes was determined by conventional electrophoretic retardation assay. Charge ratios ranging from 0.8 to 5 were evaluated to identify for each lipid the minimal N/P value at which pDNA is integrally condensed into particles. All the lipids fully retarded pDNA at N/P 2, except compound 5 that required a N/P ratio of 4 (Fig. S1). This can be explained by the lower stability of this compound in aqueous media (Pierrat et al., 2013a). Lipoplexes formed at N/P ratio of 4 exhibited Z-averaged sizes ranging from 400 to 750 nm with a polydispersity index (PdI) higher than 0.2 revealing polydisperse distributions, regardless the lipid (Table 2). NTA size measurements gave similar values with sizes between 450 and 570 nm. Polydisperse distributions were also revealed by span indices superior to 1.2. The largest pDNA complexes were obtained with cationic lipid 4, either measured by DLS or NTA, with values of 751 and 567 nm, respectively. This is consistent with the lowest particle concentration achieved with that compound (0. 4 × 10 10 particles/mL). Also consistent with the electrophoresis data, all the lipoplexes were positively charged at the N/P ratio of 4 and exhibited a z-potential value around +35-45 mV.

Mucus production in the different culture models

Mucus production by the NCI-H292 and Calu-3 2D-and/or 3D-models (Table 1) was characterized by measuring MUC5AC protein levels by ELISA. The MUC5AC production varied significantly in the three NCI-H292 2D-models. It was low in untreated NCI-H292

cultures, but increased significantly by 8-(p < 0.01) and 16-fold (p < 0.001) when the cultures were exposed to EGF and allergen, respectively (Fig. 2A). MUC5AC production by Calu-3 cells grown in plates without further treatment (2D-model) was 137 times greater than the one of the untreated NCI-H292 2D-model. These data were confirmed by quantifying the total mucin content in the culture supernatants by enzyme-linked lectin assay (ELLA), as this content increased with the levels of MUC5AC protein (Fig. S2). In the Calu-3 3D-models, the amount of MUC5AC protein increased significantly with the number of culture days at the ALI, namely 3, 7 or 14 (r = 0.9996, p = 0.0173) (Fig. 2B).

The mucus layer covering the cells cultured for 3 and 14 d was observed after insert staining with Alexa-647 ® -labeled WGA (red fluorescence). For each condition, optical sections acquired by CLSM were processed in order to create an orthogonal projection (Fig. 2C). The lectin WGA is not mucin-specific and can also bind other O-glycosylated proteins associated to the cell membranes. Nevertheless, this staining has negligible impact on the determination of the thickness of the mucus layer in our models. The mucus layer increased in thickness with the number of culture days at the ALI. Although irregular (Fig. S3), the mucus layer was clearly thicker in the 14-d culture, reaching approximately 20 µm in some areas. This observation was consistent with the MUC5AC protein levels measured by ELISA (Fig. 2B) and was further supported by Western blot data (Fig. S4). Thus, using two cell lines and different culture conditions and treatments, we developed in vitro models with increasing quantity of secreted mucus, forming layers of different thicknesses.

Lipid-mediated transfection activity in the different culture models

We then evaluated how mucus secretion observed in the above described culture models impairs the pDNA delivery activity of our DOPC-based lipids, and of Lipofectamine ® 2000 used as a control. As a preliminary study, we assessed the transfection activity of the various lipids at different N/P ratios, to determine for each one the N/P ratio offering the optimal transgene expression. This study was carried out in the Calu-3 2D-model which is characterized by a mucus secretion of middle intensity, i.e., higher than in the NCI-H292 2Dmodels but lower than in the Calu-3 3D-models (Fig. 2). The tested N/P ratios ranged from 1 to 4, based on pDNA complexation experiments (Fig. S1). Viability of transfected cells was determined in parallel to assess safety of the lipoplexes. All the lipids exhibited a transfection activity that increased with the N/P ratio up to N/P of 4, with minimal viability loss (< 30%) whatever the N/P ratio (Fig. 3). A test with lipid 3 at higher N/P ratios confirmed the ratio of 4 to be optimal to obtain maximal gene expression and minimal toxicity (data not shown).

Consequently, this N/P ratio was selected for further evaluation of the pDNA delivery activity of lipids 1-6 in the NCI-H292 and Calu-3 2D-and/or 3D-models, as well as in all subsequent transfection experiments. In the different NCI-H292 2D-models, all the investigated lipids as well as L2k mediated significant transgene expression (Fig. 4A). Transfection was of similar rate for all the lipids in untreated cells and in the EGF-exposed model. Activity of lipids 1-6 and L2k tend however to decrease in allergen-exposed cultures that exhibited a greater increase in MUC5AC secretion (16-fold) than EGF-treated ones (8-fold). In this model, lipid 3 performed slightly better than the other carriers. In the Calu-3 2D-model, the general transfection activity profile was roughly preserved when comparing the carriers with each other, but transgene expression was decreased by one to two orders of magnitude with respect to the allergen-treated NCI-H292 2D-model (Fig. 4B), which is consistent with the greater MUC5AC production by Calu-3 cells when compared to allergen-treated NCI-H292 cells (cf. Fig. 2A). Lipids 3 and 6 were slightly less impacted, whereas lipids 1, 2, 4 and 5 exhibited similar activity. At last, in the Calu-3 3D-models, transfection activity of the lipids and L2k was much severely affected as a function of mucus production (Fig. 4B). In the 3-d ALI model, activity of 3, 4, 5 and 6 revealed similar to that in the Calu-3 2D-model, but that of 1 and 2 was significantly lowered.

B A 1,E+00 1,E+01 1,E+02 1,E+03 1,E+04 1,E+05 1,E+06 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4 L iC3 C4 C8 C12
In the 14-d ALI model, none of the lipids, nor L2k, were able to transfect cells. It is worth to note that in all models, cell viability was not affected by any of the lipids, nor L2k (Fig. S5).

Thus, in the 14-d ALI Calu-3 model, characterized by the more abundant MUC5AC secretion forming a mucus layer of 20 µm thickness, none of the investigated carriers could mediate any measurable transfection signal. To determine whether mucus produced by Calu-3 cells grown on inserts at the ALI for 14 d was solely responsible for the drastic decrease of gene delivery efficiency of our carriers, we first checked that Calu-3 cells had kept their ability to be transfected after 14 d of culture on inserts. To do so, transfection rate of the 3/pDNA complexes, which proved the most potent in the different models, was measured on Calu-3 cells grown on inserts at the LLI for 14 d and compared to the rate obtained in the Calu-3 2D-model (Fig. S6A). Results showed

1,E+00 1,E+01 1,E+02 1,E+03 1,E+04 1,E+05 1,E+06 L iC3 C4 C8 C12 C12' Suc12 L iC3 C4 C8 C12 C12' Suc12 L iC3 C4 C8 C12 C12' Suc12 Untreated EGF Allergen
Luciferase activity (RLU/well) that 3/pDNA complexes generated a significant transfection signal in Calu-3 cells grown on inserts at the LLI for 14 d, as did L2k/pDNA, suggesting that 14-d culture duration on insert had no impact on the sensitivity of Calu-3 cells to the transfection reagents. Interestingly, levels of luciferase activity measured in these cultures were similar to that in the Calu-3 2Dmodel (Fig. S6A). As the two models were characterized by similar levels of MUC5AC secretion (Fig. S6B), the correlation previously found between transfection activity and mucus production is further supported. Besides, transfection activity of 3/pDNA and L2k/pDNA complexes was evaluated in the Calu-3 14-d ALI model over an extended period of time. Some activity was observed at 48 and/or 72 h (Fig. S7), comforting the idea that ALI culture was not responsible for the loss of activity of the vectors. At last, partial removal of mucus on the Calu-3 cultures after a 14d-period at the ALI was directly associated with a restoration of the transfection activity of both 3/pDNA and L2k/pDNA complexes (Fig. S8), fully demonstrating that total loss of transfection activity in the Calu-3 14d-ALI model after a 24-h exposure period resulted from increased mucus secretion acting as a barrier for carrier/pDNA complexes.

1 2 3 4 5 6 L 1 2 3 4 5 6 L 1 2 3 4 5 6 L Untreated EGF Allergen 1,E+00 1,E+01 1,E+02 1,E+03 1,E+04 1,E+05 1,E+06 L iC3 C4 C8 C12 C12' Suc12 L iC3 C4 C8 C12 C12' Suc12 L iC3 C4 C8 C12 C12' Suc12

Effect of mucus thickness on gene delivery in various 3D-models

In the above described data, 3D-models established with Calu-3 cells exhibited a mucus layer which thickness increased with culture duration at the ALI, and the 14-d ALI model that was characterized by the presence of a thick layer resulted in the most potent impairment of gene transfer. To further investigate the effect of mucus thickness on transfection, we established similar 3D-models with three other pulmonary cell lines, namely NCI-H292, 16HBE and A549 (Fig. 5). These models were characterized by measuring MUC5AC secretion, visualizing mucus layer by CLSM, and assessing transfection activity of the L2k/pDNA formulation. Calu-3 and A549 models secreted higher amounts of MUC5AC than the NCI-H292 model, whereas the 16HBE model was characterized by almost no MUC5AC secretion (Fig. 5A). Along with these data, CLSM observations allowed separation of the 4 models into 2 groups: thin mucus layer models including the NCI-H292 and 16HBE models, and thick mucus layer models including the Calu-3 and A549 models (Fig. 5C). Transfection assays performed with L2k/pDNA complexes showed some significant luciferase activity in the NCI-H292 and 16HBE models and no activity in the Calu-3 and A549 models (Fig. 5B).

These data provide another confirmation that mucus layer thickness is the major obstacle limiting gene delivery to the airway epithelium.

Macro-and micro-rheological properties of mucus produced by Calu-3 3D-models

The rheological behavior of the mucus can be determined at the macroscale, useful to characterize its physical properties, and at the microscale, necessary to identify the effective characteristics of its 3D network which both are decisive for the diffusion of transfection particles. To further characterize the potential of the Calu-3 3D-models as highly relevant models for investigating intracellular delivery of transfection particles through the airways, the mucus covering Calu-3 cells grown on inserts at the ALI for 7 or 14 d was collected and its macro-rheological properties were investigated using a rheometer equipped with a plateplate geometry. The frequency-dependent elastic (G') and viscous (G") moduli were used to characterize the viscoelastic nature of the collected mucus. They were first tested over a large scale of strain (g) to determine the viscoelastic linear range that revealed to extend up to ca.

10% (Fig. S9). A strain of 1% was therefore chosen to investigate the frequency dependence of G' and G" (Fig. 6). The general viscoelastic properties of the Calu-3 mucus increased with the ALI culture duration. Indeed, the elastic and the viscous moduli of the 14-d ALI mucus were 12.6-and 12.0-times greater, respectively, than those of the 7-d ALI mucus. However, it is to note that the mucus collected after 7-d cultures already presented a solid viscoelastic behavior (defined by a phase angle δ below 45°), as its δ value was 19.5 ± 4.5°, and similar to the δ value of the mucus collected after 14 d of culture (18.5 ± 0.6°). Besides, the elastic modulus was always higher than the viscous one (G' > G") revealing that mucus already showed an elastic behavior after 7 d of culture. Overall, rheological properties of both mucus presented a G' predominance and a weak dependence of G' and G" on frequency indicative of cross-linked gels, as previously reported for human airway mucus [START_REF] Innes | Ex vivo sputum analysis reveals impairment of protease-dependent mucus degradation by plasma proteins in acute asthma[END_REF][START_REF] Murgia | Modelling the bronchial barrier in pulmonary drug delivery: A human bronchial epithelial cell line supplemented with human tracheal mucus[END_REF][START_REF] Schuster | Nanoparticle diffusion in respiratory mucus from humans without lung disease[END_REF]. Nevertheless, the increase in G' and G" between the 7-d and 14-d ALI mucus indicated both increased cross-linking and concentration of mucins.

Consequently, varying the duration of the Calu-3 cell culture at the ALI offers models of bronchial epithelium mucus with different physical characteristics. The 3/pDNA lipoplex trajectories were more diffusive in the culture medium and the mucus collected in the 2D-model than in the mucus collected from Calu-3 3D-models. In the mucus from the 14-d Calu-3 3D-model, the lipoplexes exhibited constrained trajectories. Fig. 7B presents calculated individual MSDs of 100 lipoplexes which were randomly and equally selected among independent experiments performed in culture medium, and in mucus collected from 2D-or 3D-models. These qualitative data reveal a trend toward an increasing hindrance of lipoplex diffusion in mucus produced by the 3D-models, with the 14-d model showing the highest hindrance. Distribution of the individual lipoplex diffusion coefficients calculated from the MSDs are presented in Fig. 7C. Diffusion properties of lipoplexes in the mucus collected from the 2D-model were quite similar to those observed in culture medium, supporting the idea of a lack of 3D structure of this mucus. In this mucus, 43% of the lipoplexes displayed diffusion coefficient values below the modal interval of their distribution. In the mucus collected from the 7-d and 14-d 3D-models, distribution analysis underlined that 67% and 84% of lipoplexes, respectively, displayed diffusion coefficients below this modal interval considered as a threshold. Therefore, 41% more lipoplexes were hindered in the 14-d ALI mucus compared to mucus collected from the 2D-model.

Reinforcing these qualitative and descriptive data, the mean diffusion coefficients of the lipoplexes in the 7-d and 14-d ALI mucus were statistically decreased when compared to the mucus produced in the 2D-model (Fig. 7D). This decrease did perfectly correlate with MUC5AC levels in the different mucus (squared linear correlation coefficient of 0.97) (Fig. 7E). Confinement radiuses were calculated from the MSDs of the 3/pDNA lipoplexes after being fitted with the model of confined diffusion described above (Fig. 8). Distribution analysis indicated high polydispersity of confinement areas of lipoplexes in the mucus from the 2D-model (Fig. 8A). These results reinforced the hypothesis that this mucus lacks 3D structure. On the contrary, confinement radiuses of transfection particles in the mucus from Calu-3 cells cultured on inserts at the ALI for 14 d (14-d 3D-model) displayed a narrower distribution with a predominant size of confinement areas between 0.5 and 0.6 µm.

Furthermore, the mean confinement radius calculated for this mucus was statistically decreased when compared to the mean confinement radius calculated for the two other mucus tested (Fig. 8B). In order to represent graphically these differences, we calculated an MSD fit for the trajectory of each lipoplex and an average MSD fit for each experimental condition, using 334 (n = 2 independent experiments), 504 (n = 3) and 309 trajectories (n = 4) for the mucus collected from Calu-3 cells grown in plate, and on insert at the ALI for 7 and 14 d, respectively (Fig. 8C). Reduced confinement areas of the lipoplexes in Calu-3 mucus were observed with increasing culture duration at the ALI. Thus, with lower diffusion of lipoplexes and smaller confinement areas, the mucus produced by Calu-3 cells grown on inserts at the ALI for 14 d was characterized by higher mucus density and a 3D structure with a tighter mesh. These overall results confirmed the difference of mucus structure when modulating mucus production by adapting cell culture conditions. The Calu-3 cells grown on inserts at the ALI for 14 d and its mucus may thus constitute a useful complete tool to study impairment of diffusion and transfection activity of gene delivery systems intended for airway administration.

Discussion

The therapeutic efficiency of nucleic acid delivery to the lungs following inhalation depends on the capacity of the transfection particles to overcome barriers, particularly the mucus layer covering the airways and forming an efficient extracellular fence. We have set up and characterized different epithelial cellular models with adjustable levels of mucus of various quality in order to more reliably evaluate the efficiency of nucleic acid carriers for delivering their therapeutic payload in the airways. Using these models, we evaluated a series of 6 biodegradable gene carriers derived from the major constituent of cell membranes, DOPC. The transfection particles made thereof did transfect pulmonary cells, albeit with variable efficiency depending on the cellular type, NCI-H292 or Calu-3 cells, and the amount and properties of mucus they had produced. We observed that mucus produced by Calu-3 cells grown on inserts at the ALI for 14 d best mimicked the situation found in the lung tissue, in terms of thickness and rheological properties, and resulted in the strongest impairment of the gene delivery activity of the transfection particles.

Mucus characteristics result from a cross-linked structure due to numerous disulfide bridges between mucins, a stiffness of this structure due to hydrophobic domains and charge repulsion, and a strong net negative charge of mucins due to the high sialic acid and sulfate content. Taken all together, these properties induce filtering processes based on the size and charge of transfection particles, drastically altering penetration of these particles into the mucus for reaching the airway epithelium [START_REF] Lieleg | Characterization of particle translocation through mucin hydrogels[END_REF]. The structural analysis of airway mucus by cryo-scanning electron microscopy reported a highly heterogenous meshlike structure with pore sizes in the range of 1-10 µm [START_REF] Kirch | Optical tweezers reveal relationship between microstructure and nanoparticle penetration of pulmonary mucus[END_REF][START_REF] Vukosavljevic | Tracing molecular and structural changes upon mucolysis with N-acetyl cysteine in human airway mucus[END_REF]. However, a questioning on the porous network has progressively emerged and some authors have recently shown that it could be artificially generated from a non-porous structure during the sublimation step of a mucus sample while prepared for cryo-scanning electron microscopy analysis [START_REF] Meziu | Visualization of the structure of native human pulmonary mucus[END_REF]. Furthermore, in this study, Meziu et al. analyzed the human airway mucus in its hydrated state using multiple labeling and CLSM, and described this mucus as a rather compact material, which does not exhibit a regular pattern of pores.

Some studies have previously shown that negatively charged particles less than 200 nm in diameter can diffuse easily in the mucus, contrary to larger particles (500 nm) which were trapped [START_REF] Murgia | Size-limited penetration of nanoparticles into porcine respiratory mucus after aerosol deposition[END_REF][START_REF] Schuster | Nanoparticle diffusion in respiratory mucus from humans without lung disease[END_REF]. Nevertheless, a rapid transport of nanoparticles of 500 nm in diameter with a dense poly(ethylene glycol) coating has been described in the cervicovaginal mucus, while the mesh spacing for this material was reported to range from 20 to 200 nm [START_REF] Lai | Rapid transport of large polymeric nanoparticles in fresh undiluted human mucus[END_REF][START_REF] Meziu | Visualization of the structure of native human pulmonary mucus[END_REF]. This supports the possibility of their diffusion through low viscosity channels in mucus, which is consistent with the results of Meziu et al. (Lai et al., 2007;[START_REF] Meziu | Visualization of the structure of native human pulmonary mucus[END_REF]. In the present study, lipoplexes prepared with the cationic DOPC-based carriers at the N/P ratio of 4 exhibited a diameter ranging from 400 to 750 nm with polydisperse distribution and a z-potential of +35 to +45 mV. Therefore, the size and positive charge of the transfection particles produced with our carriers, and their polydispersity, confer on them properties inducing size and charge filtration and, therefore, make them good candidates to test the relevance of the mucus properties in our cellular models.

The role of mucus as a barrier in the airways has been extensively studied in the field of lung gene therapy, and various models have been set up to evaluate the activity of gene carriers. One strategy was to investigate separately the influence of mucus main components (linear DNA, bovine or porcine mucin, phosphatidylcholine, phosphatidylglycerol, albumin…) on the stability of the transfection particles, or to add, one by one, mucus components to the culture medium in transfection experiments [START_REF] Ibrahim | A strategy to deliver genes to cystic fibrosis lungs: a battle with environment[END_REF][START_REF] Sanders | Structural alterations of gene complexes by cystic fibrosis sputum[END_REF]. Another strategy was to cover a cell culture with a mucus model, such as CF sputum, bronchial secretions or tracheal mucus from patients, to study its influence on reporter gene expression or internalization of transfection particles [START_REF] Murgia | The role of mucus on drug transport and its potential to affect therapeutic outcomes[END_REF][START_REF] Rosenecker | Interaction of bronchoalveolar lavage fluid with polyplexes and lipoplexes: analysing the role of proteins and glycoproteins[END_REF]. Limitations of gene transfer efficiency has also been studied ex vivo in a sheep tracheal epithelium explant maintained at the ALI [START_REF] Ferrari | Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium[END_REF]. At last, fully differentiated and polarized bronchial epithelium model obtained by culturing primary cells on semi-permeable membranes at the ALI for a prolonged period of time were proposed to constitute particularly suitable in vitro models of human airways [START_REF] Chu | Binding and uptake of cationic lipid:pDNA complexes by polarized airway epithelial cells[END_REF][START_REF] Griesenbach | Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer[END_REF][START_REF] Jiang | Efficiency of cationic lipid-mediated transfection of polarized and differentiated airway epithelial cells in vitro and in vivo[END_REF]. If several reports did describe interesting transfection efficiency with viral carriers in these models, non-viral vectors showed low transfection efficiency [START_REF] Chu | Binding and uptake of cationic lipid:pDNA complexes by polarized airway epithelial cells[END_REF][START_REF] Jiang | Efficiency of cationic lipid-mediated transfection of polarized and differentiated airway epithelial cells in vitro and in vivo[END_REF][START_REF] Liu | Species-specific differences in mouse and human airway epithelial biology of recombinant adeno-associated virus transduction[END_REF][START_REF] Zhang | CFTR delivery to 25% of surface epithelial cells restores normal rates of mucus transport to human cystic fibrosis airway epithelium[END_REF]. Nevertheless, replacement of the standard Firefly luciferase reporter gene by the Gaussia luciferase reporter gene enabled transfection experiments with non-viral vectors in these models [START_REF] Griesenbach | Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer[END_REF]. Even so, access to primary cells can be limited and the variability between donors has to be considered, while immortalized cell lines offer the advantages to be an unlimited and reproductible cell source when watching out carefully to the number of culture passaging. In the present study, we have chosen the two mucinsecreting cell lines, NCI-H292 and Calu-3 [START_REF] Kreft | The characterization of the human cell line Calu-3 under different culture conditions and its use as an optimized in vitro model to investigate bronchial epithelial function[END_REF][START_REF] Takeyama | Epidermal growth factor system regulates mucin production in airways[END_REF], to set up a bench test for a reliable evaluation of the gene transfer capabilities of our DOPC-based gene carriers.

Previous transfection experiments had shown that NCI-H292 and Calu-3 cells could be transfected by biodegradable nucleic acid carriers derived from DOPC (Pierrat et al., 2016a).

However, these cells only produce a low basal amount of mucus if not induced by specific compounds or culture conditions, and do not satisfactorily mimic the mucus layer met by the transfection particles in the lung. Numerous factors have been shown to induce mucus production, and in particular secretion of the main airway mucin MUC5AC in vitro. These factors include cytokines (tumor necrosis factor a), interleukins (IL-1b, IL-13, IL-17), cigarette smoke, growth factors (EGF), and allergens [START_REF] Zeng | Epidermal growth factor, from gene organization to bedside[END_REF], as well as cell culture on inserts at the ALI [START_REF] Kreft | The characterization of the human cell line Calu-3 under different culture conditions and its use as an optimized in vitro model to investigate bronchial epithelial function[END_REF]. In the present study, NCI-H292 cells cultured in 2D-conditions displayed low MUC5AC production as assessed by ELISA, which is consistent with results reported by other groups using immunocytochemical analysis or periodic acid Schiff staining [START_REF] Oguma | Induction of mucin and MUC5AC expression by the protease activity of Aspergillus fumigatus in airway epithelial cells[END_REF][START_REF] Takeyama | Epidermal growth factor system regulates mucin production in airways[END_REF]. As well, EGF and HDM allergen extract were successfully used to increase MUC5AC production in NCI-H292 2D-cultures. The magnitude of the MUC5AC induction with EGF (8-fold) is in agreement with results reported in previous studies [START_REF] Nishimoto | Glycyrrhizin attenuates mucus production by inhibition of MUC5AC mRNA expression in vivo and in vitro[END_REF][START_REF] Perrais | Induction of MUC2 and MUC5AC mucins by factors of the epidermal growth factor (EGF) family is mediated by EGF receptor/Ras/Raf/extracellular signal-regulated kinase cascade and Sp1[END_REF]. As well, HDM allergen extract has previously been reported to activate the EGF-R in epithelial cells, supporting the 16-fold increase in MUC5AC production observed in our model [START_REF] Heijink | Der p, IL-4, and TGF-beta cooperatively induce EGFR-dependent TARC expression in airway epithelium[END_REF][START_REF] Heijink | House dust mite-promoted epithelial-to-mesenchymal transition in human bronchial epithelium[END_REF]. In NCI-H292 2D-models, no decrease in transfection efficiency of our DOPC-derived carriers was observed when mucus was induced by EGF. Through activation of EGF-R, EGF triggers many biological responses including cell proliferation, which might have counterbalanced a possible limiting effect of NCI-H292 mucus on transfection [START_REF] Zeng | Epidermal growth factor, from gene organization to bedside[END_REF]. Upon mucus induction with HDM allergen extract, a decrease in luciferase activity was observed for all evaluated lipoplexes, revealing an impairment in the pDNA delivery activity of the DOPC-based carriers by the mucus of NCI-H292 cells. The Calu-3 2D-model was characterized by a mucus production that was 137-fold greater than the one of untreated NCI-H292 cells. This high basal mucus production in Calu-3 cells, in agreement with previous reports [START_REF] Kreft | The characterization of the human cell line Calu-3 under different culture conditions and its use as an optimized in vitro model to investigate bronchial epithelial function[END_REF], impacted the pDNA delivery activity of our DOPC-based carriers to different extent. The less impacted lipoplexes are those prepared from lipids 3 and 6. To modulate mucus production by Calu-3 cells, we cultured the cell line on inserts at the ALI, as previously described by Kreft and collaborators who achieved the formation of a pseudostratified epithelium of non-dividing cells with abundant secretory vesicles capable of mucus secretion [START_REF] Kreft | The characterization of the human cell line Calu-3 under different culture conditions and its use as an optimized in vitro model to investigate bronchial epithelial function[END_REF]. In keeping with this previous report, we obtained 3D-models exhibiting a significant increase in MUC5AC that was linked to the number of culture days at the ALI. In the model cultured at the ALI for 3 d, the lipoplexes prepared from lipids 1 and 2, i.e., those with the shorter additional carbon chains installed on the DOPC phosphate group, were the most impacted by the mucus. The lipoplexes built from the 4 other cationic lipids did in contrast well resist the mucus effects, offering almost not affected transfection rates. In the model cultured at the ALI for 14 d, the transgene expression rate measured 24 h after exposure was however fully extinguished whatever the lipid carrier. Therefore, in the Calu-3 3D-models, the increase in MUC5AC production as a function of ALI culture duration was closely related to a decreased transfection efficiency of the DOPC-based carriers. The transfection activity of lipofectamine measured in Calu-3 cells cultured on inserts at the LLI allowed us to ascertain that 14 d of culture on inserts was not in cause of the absence of detectable transfection activity of our gene carriers. We also verified that after 14 d of culture at the ALI, significant transgene expression could be obtained in Calu-3 cells cultured on inserts by 1-removing the mucus before culture incubation with the lipoplexes as used previously by some authors in an ex vivo model [START_REF] Ferrari | Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium[END_REF], and 2-measuring transfection activity at later time points (48 and 72 h), in agreement with results obtained with the same gene reporter in a Mucilair ® system [START_REF] Griesenbach | Secreted Gaussia luciferase as a sensitive reporter gene for in vivo and ex vivo studies of airway gene transfer[END_REF]. CLSM images of Calu-3 cells cultured at the ALI for 14 d revealed a mucus layer of 20 µm, which was thus thicker than the one observed on the 3-d ALI cultures. Therefore, the layer of mucus produced by Calu-3 cells after 14 d of culture at the ALI drastically impaired the pDNA delivery activity of the lipoplexes resulting in absence of transfection signal at the 24-h time point. This impairment of pDNA delivery could result from the incapacity of lipoplexes to diffuse fast enough in the 20 µm mucus layer. We also observed thickness irregularities in the mucus layer of the Calu-3 3D-model at the ALI for 14 d as previously reported [START_REF] Kreft | The characterization of the human cell line Calu-3 under different culture conditions and its use as an optimized in vitro model to investigate bronchial epithelial function[END_REF]. Those irregularities may lead to a lack of reproducibility in studies, which has to be considered [START_REF] Nafee | Mucus-penetrating solid lipid nanoparticles for the treatment of cystic fibrosis: Proof of concept, challenges and pitfalls[END_REF].

Mucus is a complex material made of many components, and is characterized by a viscoelastic behavior. This behavior obtained with a model of mucus is of crucial importance when attempting to accurately mimic physical properties implicated in clearance and lubrication in the airways. In order to characterize the viscoelastic properties of mucus produced in the Calu-3 3D-models, rheological measurements were performed on the bulk undiluted material collected from the cultures. Viscoelastic data on human mucus secretions obtained from in vitro human tissue cultures grown at the ALI are very scarce. Protocols for sample preparation previously mentioned by Hill et al. require extensive washing of the biopolymer for collection, dialysis for removal of cellular debris, and concentration [START_REF] Hill | Establishment of respiratory air-liquid interface cultures and their use in studying mucin production, secretion, and function[END_REF][START_REF] Hill | A biophysical basis for mucus solids concentration as a candidate biomarker for airways disease[END_REF]. The mucus-based gel obtained at the end of this process may not accurately mimic the bulk material [START_REF] Duncan | The Mucus Barrier to Inhaled Gene Therapy[END_REF]. Since cells and cellular debris are involved in the structure and the physical properties of the mucus, it seems impossible to remove these cells from the bulk mucus without altering its physical properties [START_REF] Lai | Micro-and macrorheology of mucus[END_REF][START_REF] Meziu | Visualization of the structure of native human pulmonary mucus[END_REF]. Therefore, we did perform all our rheological analyses on untreated mucus samples. The mucus produced by 7-d ALI cultures displayed a viscoelasticity with dominant elastic properties supported by the δ value at 19.5 ± 4.5°, which were maintained in the mucus collected from 14-d cultures (δ = 18.5 ± 0.6°). Interestingly, these properties are in line with previously reported rheological behavior of ex-vivo samples of human airway mucus [START_REF] Murgia | Modelling the bronchial barrier in pulmonary drug delivery: A human bronchial epithelial cell line supplemented with human tracheal mucus[END_REF][START_REF] Schuster | Nanoparticle diffusion in respiratory mucus from humans without lung disease[END_REF]. Consistently, no transfection activity was detected in Calu-3 cells cultured for 7 d (data not shown) or 14 d at the ALI, whatever the gene carrier used.

As macro-rheological properties of the mucus produced by the 14-d Calu-3 3D-model translated into a viscoelastic behavior of the bulk material, this latter was used to investigate transfection particle diffusion. Experiments was performed with lipid 3-based lipoplexes by MPT. MSDs determined from acquisition of particle trajectories were fitted with a model of confined diffusion. Comparison of the diffusion coefficient distributions confirmed that the mucus produced by the Calu-3 2D-model lacks a mesh-like structure, and revealed an increased hindrance to the diffusion of the lipoplexes in the mucus produced by the Calu-3 3D-models. The mean diffusion coefficient of lipoplexes in the mucus from Calu-3 7-d and 14-d ALI cultures was statistically decreased compared to that in the mucus from Calu-3 2Dcultures, and this decrease was directly related to the amounts of MUC5AC produced by the different models. A similar relation has been previously reported with a decreasing diffusion of 1 µm carboxylate-modified polystyrene particles in mucus from human bronchial epithelial cells grown at the ALI as a function of increasing concentration of mucus solids [START_REF] Hill | A biophysical basis for mucus solids concentration as a candidate biomarker for airways disease[END_REF]. Nevertheless, our lipoplexes were able to diffuse inside the mucus, supporting the idea that the thickness of the mucus played a key role in transgene expression by delaying the entry of transfection particle into the cells. Interestingly, the MSD data of the lipoplexes analyzed on 14-d ALI mucus are somehow similar to those reported by [START_REF] Murgia | Modelling the bronchial barrier in pulmonary drug delivery: A human bronchial epithelial cell line supplemented with human tracheal mucus[END_REF] for 500 nm negatively charged polystyrene nanoparticles. This supports the idea that, in this size range, diffusion of the particles is limited by their size rather than their charge.

With the fit modelling of confined diffusion, we were able to approximate dimensions of the zones within the limit of which lipoplexes were preferably diffusing. The confinement radiuses (r c ) calculated in the mucus from the Calu-3 2D-model displayed a large range of values, but got narrower in the Calu-3 3D-models, with a predominant size of 0.5-0.6 µm in the mucus produced by the 14-d 3D-model. Considering the heterogeneity and the complex structure of mucus, the r c distribution seems more accurate than the average confinement zone radius for evaluating the mucus structure [START_REF] Meziu | Visualization of the structure of native human pulmonary mucus[END_REF]. Nevertheless, the r c mean value calculated from 14-d 3D-model was statistically lower compared than those obtained for the Calu-3 2D-models and 7-d 3D-model revealing a real difference in the structure of the materials. Overall, these results demonstrated that the Calu-3 2D-and 3D-models are characterized by a gradual hindrance of the lipoplex diffusion, in parallel with narrower confinement zones for their diffusion. This combination of factors is resumed with the MSD fit of the lipoplexes in the various mucus which reflects a denser and more complex architecture of the mucus produced by the 14-d 3D-model. Therefore, the mucus produced by the Calu-3 cell cultures on inserts at the ALI for 14 d presents useful properties for an accurate characterization of the interactions between the airway mucus and transfection particles. The impact of a thick mucus layer on the pDNA delivery activity of our gene carriers definitely suggests that this model will be highly useful for finely investigating the properties of gene carriers intended for administration through the airways.

In summary, in the present study, we developed DOPC-based cationic lipids as gene carriers and elaborated cellular models with a mucus layer displaying adjustable properties in terms of thickness and viscoelasticity to better mimic in vivo physiological conditions and predict in vivo gene delivery efficiency of our carriers. Although high transfection efficiency was obtained in the 2D-models with low mucus level, the DOPC-derived carriers have shown reduced or even no detectable transfection efficiency in the 3D-models at the ALI. We thus demonstrated that Calu-3 2D-and 3D-models constitute useful tools for investigating the impact of mucus on gene delivery through the airways. Moreover, we showed that the mucus produced by Calu-3 3D-models can provide complementary insights by offering the possibility to assess multiple parameters necessary to fully characterize carrier interactions with the mucus, such as mucus viscoelasticity and transfection particle diffusion. Gene delivery activity of our carriers in the developed cellular models was increasingly altered as a function of mucus quantity and thickness. Thus, these models will be useful in the future to search for and evaluate new strategies to help our carriers to overcome the mucus barrier in the airways.

  3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), bovine serum albumin (BSA), 3,3',5,5'-tetramethylbenzidine (TMB) and epidermal growth factor (EGF) were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France). Allergen extract from the HDM Dermatophagoides pteronyssinus was from Greer Laboratories (Worcester, MA, USA). Culture media (DMEM/F12, Opti-MEM, and RPMI-1640), fetal bovine serum (FBS) and other medium supplements were from GIBCO-BRL (Cergy-Pontoise, France).Lipofectamine ® 2000 (L2k) was obtained from Invitrogen (Cergy-Pontoise, France). Plasmid encoding for Gaussia luciferase (pCMV-Gluc, 5.8 kbp) and Gaussia luciferase substrate, coelenterazine, were purchased from Nanolight Technology (Pinetop, Az, USA). Alexa-647 ®conjugated wheat germ agglutinin (WGA) was obtained from Molecular Probes (Eugene, OR, USA). Goat anti-mouse IgG1 and mouse anti-human MUC5AC IgG1 (clone 45M1) were from Jackson ImmunoResearch (Cambridgeshire, UK) and Abcam (Cambridge, UK), respectively. Horseradish peroxidase (HRP) conjugated sheep anti-mouse IgG were from GE Healthcare (Buc, France). 1,2-Dioleoyl-sn-glycero-3-phospho-ethanolamine-N-(lissamine rhodamine B sulfonyl) (Rh-PE) was purchased from Avanti Polar Lipids (Alabaster, AL, USA).

Fig. 1 .

 1 Fig.1. Chemical structure of EDMPC[START_REF] Gorman | Efficient in vivo delivery of DNA to pulmonary cells using the novel lipid EDMPC[END_REF]), of EDOPC (MacDonald et al., 1999),

Fig. 2 .

 2 Fig. 2. Mucus production in the NCI-H292 and Calu-3 2D-and/or 3D-models. (A) NCI-H292 or Calu-

Fig. 3 .

 3 Fig. 3. Transfection efficiency of the lipids 1-6, as a function of the N/P ratio in the Calu-3 2D-model.

Fig. 4 .

 4 Fig. 4. pDNA transfection mediated by the lipids 1-6 and L2k in the different culture models. (A)

Fig. 5 .

 5 Fig. 5. Mucus production and transfection efficiency in various 3D-models. Calu-3, A549, NCI-H292

Fig. 6 .

 6 Fig. 6. Bulk rheology of the mucus produced by Calu-3 cells grown at the ALI for 7 (open symbols)

EFig. 7 .

 7 Fig. 7. Diffusion of 3/pDNA lipoplexes (N/P of 4) in mucus collected from Calu-3 2D-and 3D-

Fig. 8 .

 8 Fig. 8. Confinement areas of 3/pDNA lipoplexes in mucus collected from Calu-3 2D-and 3D-models,

Table 2 .

 2 Physical characteristics of the lipid/pDNA complexes prepared from compounds 1 to 6 at N/P of 4. Data are mean values ± SD of at least 3 independent experiments.

			DLS			NTA
	Compound	Z-average	PdI	ζ-potential	Size	Span	Particles/mL
		(nm)		(mV)	(nm)		(× 10 10 )
	1	394 ± 12	0.248 ± 0.021	+44 ± 3	491 ± 17	1.2 ± 0.1	1.5 ± 0.1
	2	409 ± 45	0.320 ± 0.063	+40 ± 7	451 ± 40	1.2 ± 0.1	1.2 ± 0.2
	3	546 ± 88	0.385 ± 0.033	+44 ± 5	505 ± 20	1.3 ± 0.1	1.0 ± 0.1
	4	751 ± 151	0.309 ± 0.064	+36 ± 11	567 ± 32	1.2 ± 0.1	0.4 ± 0.2
	5	634 ± 107	0.438 ± 0.051	+43 ± 5	497 ± 46	1.3 ± 0.1	1.1 ± 0.1
	6	541 ± 62	0.288 ± 0.100	+41 ± 5	504 ± 36	1.4 ± 0.1	1.1 ± 0.3

Funding Sources

This work was supported by the Agence Nationale de la Recherche (ANR, ANR-17-CE18-0034), the Foundation for Frontier Research in Chemistry (LabEx CSC, ANR-10-LBX-026-CSC), the ITI InnoVec (IdEx, ANR-10-IDEX-0002; SFRI, ANR-20-SFRI-0012), and Vaincre la Mucoviscidose (VLM, RF20200502702/1/1/161).

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

Supplementary data to this article can be found online.