Maria Enrica Di

Greta Colombo Dugoni

Monica Ferro

Alberto Mannu

Franca Castiglione

Margarida Costa Gomes

Sophie Fourmentin

Andrea Mele
email: andrea.mele@polimi.it

Do Cyclodextrins Encapsulate Volatiles in Deep Eutectic Systems?

Keywords:

Efficient renewable and non-toxic absorbents can now be designed to eliminate air pollutants such as volatile organic compounds (VOCs) from confined atmospheres. New hybrid materials result from the combination of Deep Eutectic Systems (DES) with well-known VOCs capture agents like β-cyclodextrin (βCD). Yet, a question arises: does βCD retain its encapsulation ability in DES? Multiple NMR techniques are used here to demonstrate the formation of inclusion complexes of βCD with two VOCs, aniline and toluene, in the pure DES reline and in reline/water mixtures. Complexation-induced chemical shift changes and intermolecular hostguest NOEs in the rotating frame give evidence of a genuine encapsulation in the βCD cavity, and complementary information on the dynamics of the VOC is gathered via relaxation and diffusion experiments. This work shows how different NMR techniques can contribute to the design of task-specific sustainable materials for absorption/extraction processes.

INTRODUCTION

Volatile Organic Compounds (VOCs) are among the most common air pollutants emitted from chemical and petrochemical industries, transportation vehicles and commercial products such as solvents, paints, cleaners, and lubricants. [START_REF] Faisal | Removal of volatile organic compounds from polluted air[END_REF][START_REF] Alzate-Sánchez | Cotton fabric functionalized with a β-cyclodextrin polymer captures organic pollutants from contaminated air and water[END_REF][START_REF] Celebioglu | Molecular entrapment of volatile organic compounds (VOCs) by electrospun cyclodextrin nanofibers[END_REF] It is today compulsory in many countries to limit and control VOCs emissions both in terms of environment, since they affect the climate change and the growth and decay of plants, and in terms of human and animal health, since VOC exposure causes respiratory distress, eye and throat irritation, neurological toxicity, and cancer, among other effects. [START_REF] Faisal | Removal of volatile organic compounds from polluted air[END_REF][START_REF] Alzate-Sánchez | Cotton fabric functionalized with a β-cyclodextrin polymer captures organic pollutants from contaminated air and water[END_REF][START_REF] Celebioglu | Molecular entrapment of volatile organic compounds (VOCs) by electrospun cyclodextrin nanofibers[END_REF] In the presence of sunlight, VOCs may react with nitrogen oxides and other airborne chemicals to form ozone, leading to various environmental hazards. [START_REF] Bj | Tropospheric Air Pollution: Ozone, Airborne Toxics, Polycyclic Aromatic Hydrocarbons, and Particles[END_REF] VOCs removal is hence a major concern of the society's commitment towards the ecosystem. [START_REF] Faisal | Removal of volatile organic compounds from polluted air[END_REF] Among the materials used as VOC capture agents, β-cyclodextrin (βCD) is one of the most attractive. [START_REF] Alzate-Sánchez | Cotton fabric functionalized with a β-cyclodextrin polymer captures organic pollutants from contaminated air and water[END_REF][START_REF] Celebioglu | Selective and Efficient Removal of Volatile Organic Compounds by Channel-type Gamma-Cyclodextrin Assembly through Inclusion Complexation[END_REF][START_REF] Landy | Remediation technologies using cyclodextrins: An overview[END_REF][START_REF] Kfoury | Characterization of cyclodextrin/volatile inclusion complexes: A review[END_REF][START_REF] Blach | Cyclodextrins: A new efficient absorbent to treat waste gas streams[END_REF] βCD is a torus-shaped cyclic oligosaccharide made up of seven α-1,4-linked Dglucopyranose units. It can form inclusion complexes by entrapping small hydrophobic molecules (guests) in the hydrophobic cavity of the macrocyclic sugar (host) through noncovalent host-guest interactions. This is especially true if the target molecules contain hydrophobic groups, such as phenyl groups that are present in many VOCs. [START_REF] Srinivasan | Study of inclusion complex between 2,6-dinitrobenzoic acid and βcyclodextrin by 1H NMR, 2D 1H NMR (ROESY), FT-IR, XRD, SEM and photophysical methods[END_REF] βCD-based materials have been reported to be efficient in inclusion complex formation towards several organic pollutants. [START_REF] Alzate-Sánchez | Cotton fabric functionalized with a β-cyclodextrin polymer captures organic pollutants from contaminated air and water[END_REF][START_REF] Celebioglu | Molecular entrapment of volatile organic compounds (VOCs) by electrospun cyclodextrin nanofibers[END_REF][START_REF] Celebioglu | Selective and Efficient Removal of Volatile Organic Compounds by Channel-type Gamma-Cyclodextrin Assembly through Inclusion Complexation[END_REF][START_REF] Landy | Remediation technologies using cyclodextrins: An overview[END_REF][START_REF] Alsbaiee | Rapid removal of organic micropollutants from water by a porous β-cyclodextrin polymer[END_REF][START_REF] Wang | Β-Cyclodextrin based air filter for high-efficiency filtration of pollution sources[END_REF][START_REF] Szaniszló | Structure-stability study of cyclodextrin complexes with selected volatile hydrocarbon contaminants of soils[END_REF][START_REF] Morin-Crini | Environmental applications of water-insoluble β-cyclodextrinepichlorohydrin polymers[END_REF][START_REF] Mauri-Aucejo | Samplers for VOCs in air based on cyclodextrin-silica hybrid microporous solid phases[END_REF] Deep Eutectic Systems (DES) have been recently suggested as innovative materials with potential in gas absorption. [START_REF] Moura | Deep eutectic solvents as green absorbents of volatile organic pollutants[END_REF][START_REF] Sze | Ternary deep eutectic solvents tasked for carbon dioxide capture[END_REF][START_REF] Li | Absorption of Carbon Dioxide Using Ethanolamine-Based Deep Eutectic Solvents[END_REF][START_REF] Zhong | Phenol-Based Ternary Deep Eutectic Solvents for Highly Efficient and Reversible Absorption of NH3[END_REF] Practically speaking, a DES is the result of the combination in the proper ratio of an opportune hydrogen-bond donor (HBD) and a hydrogen-bond acceptor (HBA). [START_REF] Francisco | Low-transition-temperature mixtures (LTTMs): A new generation of designer solvents[END_REF][START_REF] Abbott | Novel solvent properties of choline chloride / urea mixtures[END_REF][START_REF] Ruß | Low melting mixtures in organic synthesis -An alternative to ionic liquids?[END_REF] Self-association of the HBD and HBA lowers the entropic difference of the phase transition, so that basically a eutectic is formed with a depressed freezing point that lies well below that of the individual components. DES show many beneficial characteristics: among other, they can be prepared from cheap, readily available, and toxicologically well characterized starting materials, which also implies low cost. A large number of different combinations of HBA and HBD has been reported and their use proposed in many fields of application. [START_REF] Zhang | Deep eutectic solvents: syntheses, properties and applications[END_REF][START_REF] Liu | Natural Deep Eutectic Solvents: Properties, Applications, and Perspectives[END_REF][START_REF] Pena-Pereira | Ionic liquids and deep eutectic mixtures: Sustainable solvents for extraction processes[END_REF][START_REF] Cruz | Deep eutectic solvents (DESs) as low-cost and green electrolytes for electrochromic devices[END_REF] Interestingly, studies using static headspace gas chromatography (SH-GC) on choline chlorideor tetrabutylphosphonium bromide-based DES showed they can be good candidates for VOC absorption processes. [START_REF] Moura | Deep eutectic solvents as green absorbents of volatile organic pollutants[END_REF] In this context, DES/βCD mixtures are likewise of interest as new hybrid materials, since in principle their individual absorbing/sequestrating properties may be mutually reinforced. Indeed, the complexation ability of several native and substituted cyclodextrins in the DES reline (choline chloride:urea at 1:2 molar ratio, ChU) towards four VOCs (dichloromethane, toluene, tert-butylcyclohexane and limonene) has been recently investigated via SH-GC. [START_REF] Moufawad | First Evidence of Cyclodextrin Inclusion Complexes in a Deep Eutectic Solvent[END_REF] The authors showed a decrease of the chromatographic peak area, which indicates a higher solubilization of the VOC in the mixture. No further information on the retention mechanism -whether genuine inclusion or aggregation or other -is unfortunately accessible by means of SH-GC. Yet, to concretely exploit reline/βCD mixtures for capturing toxic contaminants, a better characterization of the guest-host inclusion complex at the molecular level is crucial.When searching for an alternative and/or complementary technique to study interactions of small molecules with supramolecular hosts and in particular for investigating the formation of inclusion complexes, Nuclear Magnetic Resonance (NMR) spectroscopy is probably the best option. It is not only possible to directly probe the genuine inclusion of the guest in the CD cavity, but the large number of spectral parameters that can be measured and analysed give also access to unique qualitative and quantitative information. [START_REF] Kfoury | Characterization of cyclodextrin/volatile inclusion complexes: A review[END_REF][START_REF] Srinivasan | Study of inclusion complex between 2,6-dinitrobenzoic acid and βcyclodextrin by 1H NMR, 2D 1H NMR (ROESY), FT-IR, XRD, SEM and photophysical methods[END_REF][START_REF] Schneider | NMR Studies of Cyclodextrins and Cyclodextrin Complexes[END_REF][START_REF] Li | Interactions of Lycopodium alkaloids with acetylcholinesterase investigated by 1H NMR relaxation rate[END_REF][START_REF] Reddy | Molecular Level Insights on Collagen-Polyphenols Interaction Using Spin-Relaxation and Saturation Transfer Difference NMR[END_REF][START_REF] Marques | A review on cyclodextrin encapsulation of essential oils and volatiles[END_REF] In the present work, we demonstrate that the measurement of different NMR parameters is extremely beneficial to investigate the sorption behaviour of βCD towards selected VOC in the DES reline (see Fig. 1 for structures). First, complexation-induced chemical shift changes and intermolecular host-guest NOEs in the rotating frame allow us to assess whether or not a genuine encapsulation occurs in ChU/βCD mixtures. Moreover, Diffusion Ordered SpectroscopY (DOSY) experiments and measurements of non-selective and selective spin-lattice relaxation times (T 1 NS and T 1 SE ) are applied to glean additional insights into the dynamics of the guest molecule.

A practical limitation in ChU/βCD systems is the relatively high density and viscosity of the mixtures, which strongly reduces the resolution in NMR spectra. Luckily, DES readily lend themselves to be added with a considerable amount of water without losing their unique properties. The effect of water on the hydrogen-bonding network of a DES is still debated and it is generally accepted that reline keeps its nanostructure up to water concentrations of about 35 wt% and a transition from a "water-in-DES" to a "DES-in-water" regime only occurs at ca. 50 wt%. [START_REF] Posada | Reline aqueous solutions behaving as liquid mixtures of H-bonded co-solvents: Microphase segregation and formation of co-continuous structures as indicated by Brillouin and1H NMR spectroscopies[END_REF][START_REF] Hammond | The Effect of Water upon Deep Eutectic Solvent Nanostructure: An Unusual Transition from Ionic Mixture to Aqueous Solution[END_REF] The possibility to reduce DES viscosity while preserving its molecular interactions and structure is an extremely advantageous feature, if one considers that DESs are highly hygroscopic and latent absorbed water is unavoidable, especially in the context of industrial applications. This holds true also in the presence of βCD. Indeed, some of us have recently demonstrated that in reline/βCD/H 2 O systems, β-cyclodextrin interacts primarily with the added water, while reline preserves a residual molecular network. [START_REF] Dugoni | Effect of Water on Deep Eutectic Solvent/β-Cyclodextrin Systems[END_REF] In this mixture, βCD benefits of enough mobility to retain its encapsulation properties, as proved by selective variation of the chemical shifts and ROESY host-guest intermolecular correlations in the case of a model drug, namely the anti-inflammatory drug piroxicam.

Here we intend to apply multiple NMR experiments to investigate more deeply the potential of reline/βCD mixtures for VOC absorption in the absence and presence of water. As model compounds, we selected two major volatile organic compounds in the environment, aniline and toluene (Figure 1), which are listed as hazardous air pollutants in the Clean Air Act by the US Environmental Protection Agency (https://www3.epa.gov/ttn/atw/orig189.html). The formation of genuine inclusion complexes is evaluated and the dynamic properties of the guest molecules investigated.

EXPERIMENTAL SECTION

A summary of all samples used is reported in Hence, comparing the observed chemical shifts of guest protons as well as of protons located in the CD cavity (H 3' and H 5 ') in the 1 H 1D spectra of their mixture compared to pure components give a first clue on the formation of an inclusion complex. Figure 2 shows selected regions of 1 H spectra corresponding to VOC's signals for the case of aniline (AN) and toluene (Tol) in reline, with or without βCD. In the presence of βCD, it is observed the expected downfield shift of the peaks of the guest molecule, which suggests the formation of βCD/VOC inclusion complexes (see Table S1 for complexation-induced chemical shifts). [START_REF] Marques | A review on cyclodextrin encapsulation of essential oils and volatiles[END_REF] Moreover, a considerable line broadening of the aromatic peaks is observed after the addition of βCD, which might indicate an exchange regime between free and encapsulated species. Overall, the variation of the chemical shifts as well as the line broadening are more prominent in the case of toluene than aniline, which might point towards a stronger interaction of the former with βCD. Unfortunately, nonhydrated reline mixtures are highly viscous, which causes short relaxation times, broad lines and overall poorly resolved spectra. The effect is even more relevant of βCD's signals, which also suffer from severe overlap with peaks of choline (see Figs. S1-S2). Therefore, any further analysis of 1 H-NMR spectra is not possible. Aiming at confirming the formation of βCD/VOC inclusion complexes, we performed rotating-frame Overhauser (ROESY) experiments (Fig. S3).

Again, the signal loss due to the short relaxation time in the highly viscous DES solutions made it impossible to distinguish any intermolecular host-guest cross peaks. The use of relaxation NMR spectroscopy to study aggregation and/or complexation is well known and relies mostly on the use of inversion recovery experiments for the measurement of proton non-selective and selective spin-lattice relaxation rates, R 1 NS and R 1 SE . [START_REF] Schneider | NMR Studies of Cyclodextrins and Cyclodextrin Complexes[END_REF][START_REF] Reddy | Molecular Level Insights on Collagen-Polyphenols Interaction Using Spin-Relaxation and Saturation Transfer Difference NMR[END_REF][START_REF] Tošner | Rotational dynamics of adamantanecarboxylic acid in complex with β-cyclodextrin[END_REF] Indeed, assuming that the bound and free states interconvert quickly with respect to both chemical shift difference and proton relaxation rate, the formation of inclusion complexes affects R 1 NS and R 1 SE at different extents. In particular, it has been shown that R 1 SE is more sensitive to the slower rotational tumbling of the complex than R 1 NS . [START_REF] Li | Interactions of Lycopodium alkaloids with acetylcholinesterase investigated by 1H NMR relaxation rate[END_REF][START_REF] Freeman | Dipolar contribution to NMR spinlattice relaxation of protons[END_REF][START_REF] Hall | Spin-Lattice Relaxation of Protons. A General, Quantitative Evaluation of Contributions from the Intramolecular Dipole-Dipole Mechanism[END_REF][START_REF] Gaggelli | 1H Relaxation Investigation of the Interaction of Vinblastine with Tubulin[END_REF][START_REF] Tinoco | Determination of Correlation Times from Selective and Non-Selective Spin-Lattice Relaxation Rates and their Use in Drug-Drug and Drug-Albumin Interaction Studies[END_REF][START_REF] Fielding | NMR methods for the determination of protein-ligand dissociation constants[END_REF] Therefore, the combination of non-selective and selective inversion recovery measurements is a rich source of information in dynamic studies. It has been used extensively to measure chemical exchange rate constants separated from the spin-lattice relaxation rates in slow exchanging systems, [START_REF] Bain | Chemical exchange in NMR[END_REF] to describe molecular conformational motion in solution, [START_REF] Rossi | Proton magnetic relaxation mechanisms and solution dynamics of L-histidine[END_REF][START_REF] Sai | Application of 'H NMR Selective and Biselective Relaxation Times[END_REF] as well as to study interactions between small molecules with macromolecules, [START_REF] Li | Interactions of Lycopodium alkaloids with acetylcholinesterase investigated by 1H NMR relaxation rate[END_REF][START_REF] Reddy | Molecular Level Insights on Collagen-Polyphenols Interaction Using Spin-Relaxation and Saturation Transfer Difference NMR[END_REF][START_REF] Gaggelli | 1H Relaxation Investigation of the Interaction of Vinblastine with Tubulin[END_REF][START_REF] Tinoco | Determination of Correlation Times from Selective and Non-Selective Spin-Lattice Relaxation Rates and their Use in Drug-Drug and Drug-Albumin Interaction Studies[END_REF] and estimating the binding affinities of ligands with protein. [START_REF] Fielding | NMR methods for the determination of protein-ligand dissociation constants[END_REF][START_REF] Rossi | Ligand-macromolecule complexes: Affinity index determination by selective nuclear relaxation analysis[END_REF][START_REF] Bonechi | Nuclear magnetic resonance for studying recognition processes between anandamide and cannabinoid receptors[END_REF] In the case of inclusion complexes with cyclodextrins, the application of 1 H nonselective, selective, and bi-selective spin-lattice relaxation rates can be used to address intermolecular interactions and motional dynamics of drugs encapsulated within the βCD cavity. [START_REF] Kumar | Analysis of Molecular Interaction of Drugs within β-Cyclodextrin Cavity by Solution-State NMR Relaxation[END_REF] A theoretical description is provided for the interested reader in the SI. Basically, measuring R 1 NS and R 1

SE

within the initial rate approximation it is possible to infer the molecular rotational correlation time 𝜏 𝐶 of the encapsulated drug, which may be thought of as the average time required for the molecule to rotate by approximately 1 radian. [START_REF] Hall | Spin-Lattice Relaxation of Protons. A General, Quantitative Evaluation of Contributions from the Intramolecular Dipole-Dipole Mechanism[END_REF][START_REF] Tinoco | Determination of Correlation Times from Selective and Non-Selective Spin-Lattice Relaxation Rates and their Use in Drug-Drug and Drug-Albumin Interaction Studies[END_REF][START_REF] Campbell | Influence of Cross-Relaxation on NMR Spin-Lattice Relaxation Times[END_REF] Both the R 1 NS /R 1 SE ratio and 𝜏 𝐶 are related to the motional regime of the guest, which in turns reflects its equilibrium between the free and bound states. [START_REF] Li | Interactions of Lycopodium alkaloids with acetylcholinesterase investigated by 1H NMR relaxation rate[END_REF][START_REF] Freeman | Dipolar contribution to NMR spinlattice relaxation of protons[END_REF][START_REF] Kumar | Analysis of Molecular Interaction of Drugs within β-Cyclodextrin Cavity by Solution-State NMR Relaxation[END_REF] Here we applied this method to get information about the dynamics of the selected VOCs in reline and on how it changes in the presence and absence of β-cyclodextrin. As overlapping peaks are not suitable for selective inversion, after investigation of 1 H NMR spectrum, the most isolated peaks were chosen for observation, namely protons H 3 ,H 5 for aniline and methyl protons for toluene (see Figs. In all four samples the R 1 NS /R 1 SE ratio is far smaller than 1. This is symptomatic of slow motion regime, which was expected for such viscous systems. For both aniline and toluene the R 1 NS /R 1 SE ratio decreases from the pure reline to the reline/βCD mixture. This indicates that in the presence of βCD the rotational motion of the guest gets even smaller, which is compatible with the formation of an inclusion complex. Even if it is tempting to compare the two VOCs, it should be remembered that different protons were selected for the two molecules (namely aromatic protons for aniline and methyl protons for toluene) and a direct comparison might not be fair.

From the ratio of the nonselective and selective relaxation rates, it was possible to get an estimate of the correlation times of VOCs in the ChU/βCD mixtures and to compare them with the systems without βCD. Results are reported in Table 2 and Fig 4 . For both AN and Tol, 𝜏 𝐶 in the ChU/βCD mixture is bigger than the corresponding value in the pure reline. Since the correlation time is inversely proportional to the degree of molecular mobility, that is the smaller values of 𝜏 𝐶 correspond to higher molecular mobility, [START_REF] Tinoco | Determination of Correlation Times from Selective and Non-Selective Spin-Lattice Relaxation Rates and their Use in Drug-Drug and Drug-Albumin Interaction Studies[END_REF] this is again compatible with a slowdown in dynamics of the guest molecule because of its encapsulation in the βCD cavity. Diffusion experiments have received attention since the mid-1990s thanks to the highly representative processing called Diffusion Ordered SpectroscopY (DOSY). [START_REF] Fielding | NMR methods for the determination of protein-ligand dissociation constants[END_REF][START_REF] Morris | Diffusion-Ordered Two-Dimensional Nuclear Magnetic Resonance Spectroscopy[END_REF][START_REF] Stilbs | Fourier transform pulsed-gradient spin-echo studies of molecular diffusion[END_REF] In a DOSY map, the diffusion experiment is processed in a 2D spectrum in which one dimension is related to the chemical shift information, while the other represents the diffusion coefficient. [START_REF] Pagès | Pulsed-field gradient nuclear magnetic resonance measurements (PFG NMR) for diffusion ordered spectroscopy (DOSY) mapping[END_REF][START_REF] Brand | Intermolecular interaction as investigated by NOE and diffusion studies[END_REF] Since the translational diffusion coefficients (D) of molecular species reflect their effective sizes and shapes, DOSY NMR allows both the identification and separation of the chemical entities in multicomponent systems, and provides information on their intermolecular interactions as well as on the dynamics of the system. [START_REF] Pagès | Pulsed-field gradient nuclear magnetic resonance measurements (PFG NMR) for diffusion ordered spectroscopy (DOSY) mapping[END_REF][START_REF] Senra | Interactions between quaternized chitosan and surfactant studied by diffusion NMR and conductivity[END_REF][START_REF] Barhoum | Diffusion NMR studies of macromolecular complex formation, crowding and confinement in soft materials[END_REF] In other words, a small molecule diffuses faster than a large one and the binding of a freely diffusing molecule to another species leads to a decrease of D. Indeed, in the fast exchange limit, the observed diffusion coefficient of the guest is the mole fraction weighted average of the diffusion coefficient of the free and bound states. This means that, in the presence of a complex, the diffusion of the encapsulated molecule will be slower than that measured for the free compound. DOSY maps are well suited to study aggregation and quantify molecular interactions in chemical systems, [START_REF] Pagès | Pulsed-field gradient nuclear magnetic resonance measurements (PFG NMR) for diffusion ordered spectroscopy (DOSY) mapping[END_REF][START_REF] Barhoum | Diffusion NMR studies of macromolecular complex formation, crowding and confinement in soft materials[END_REF][START_REF] Fernandes | Investigation of tetracaine complexation with beta-cyclodextrins and p-sulphonic acid calix[6]arenes by nOe and PGSE NMR[END_REF][START_REF] Simova | Diffusion measurements vs. chemical shift titration for determination of association constants on the example of camphor-cyclodextrin complexes[END_REF] and many examples can be found in the literature where DOSY NMR has been used to investigate structure, stoichiometry, host bound and unbound fractions to guest, association binding constant, and host/guest relative positioning in CD complexes. [START_REF] Tošner | Rotational dynamics of adamantanecarboxylic acid in complex with β-cyclodextrin[END_REF][START_REF] Pagès | Pulsed-field gradient nuclear magnetic resonance measurements (PFG NMR) for diffusion ordered spectroscopy (DOSY) mapping[END_REF][START_REF] Lis-Cieplak | Comparative proton nuclear magnetic resonance studies of amantadine complexes formed in aqueous solutions with three major cyclodextrins[END_REF][START_REF] Martins | 1H NMR studies of binary and ternary dapsone supramolecular complexes with different drug carriers: EPC liposome, SBE-β-CD and β-CD[END_REF][START_REF] Uccello-Barretta | Combined NMRcrystallographic and modelling investigation of the inclusion of molsidomine into α-, βand γ-cyclodextrins[END_REF][START_REF] Kfoury | Determination of formation constants and structural characterization of cyclodextrin inclusion complexes with two phenolic isomers: Carvacrol and thymol[END_REF] DOSY maps were acquired for samples 1 to 4 (Fig. S4).

Unfortunately, signals of βCD were not detectable in any samples and VOC's signals were observed only in pure reline. This indirectly suggest that in the presence of βCD, the guest (AN or Tol) are probably encapsulated, so that their lines get broader and not visible anymore in the 2D spectra. However, no additional considerations can be drawn.

Does β-cyclodextrin form inclusion complexes with VOCs in hydrated reline mixtures?

In the previous section, practical limitations to the NMR investigation of reline/βCD emerged because of the inherent viscosity of the samples. Even though both the changes in 1 H pattern of the guest as well as the modification of its dynamic behaviour suggest that the formation of βCD/VOC complexes really takes place, we do not have a direct proof of a genuine encapsulation of the VOC in the βCD cavity. To overcome this problem, we decided to add 210 equivalents of water with respect to the β-cyclodextrin in the mixture. The same NMR methodology was then applied to hydrated reline samples. Here slight changes are visible, more prominent on H 3' than on H 5' . To unambiguously confirm the encapsulation, rotating-frame Overhauser spectroscopy (ROESY) is the preferred NMR tool. ROESY studies have been extensively used to get information on inclusion complexes with βCD through analysis of intermolecular peaks between cavity protons and part of the guest involved in complexation. [START_REF] Srinivasan | Study of inclusion complex between 2,6-dinitrobenzoic acid and βcyclodextrin by 1H NMR, 2D 1H NMR (ROESY), FT-IR, XRD, SEM and photophysical methods[END_REF][START_REF] Schneider | NMR Studies of Cyclodextrins and Cyclodextrin Complexes[END_REF][START_REF] Fernandes | Investigation of tetracaine complexation with beta-cyclodextrins and p-sulphonic acid calix[6]arenes by nOe and PGSE NMR[END_REF][START_REF] Simova | Diffusion measurements vs. chemical shift titration for determination of association constants on the example of camphor-cyclodextrin complexes[END_REF][START_REF] Lis-Cieplak | Comparative proton nuclear magnetic resonance studies of amantadine complexes formed in aqueous solutions with three major cyclodextrins[END_REF][START_REF] Ali | Structure elucidation of β-cyclodextrin-xylazine complex by a combination of quantitative 1H-1H ROESY and molecular dynamics studies[END_REF][START_REF] Ali | Analysis of computational models of β-cyclodextrin complexes: Structural studies of morniflumate hydrochloride and β-cyclodextrin complex in aqueous[END_REF] Nonselective and selective relaxation experiments were performed on samples 5 to 8 to get dynamic information on the encapsulated VOC. Results are summarized in Table 2 and Fig. 4.

Clearly, all R 1 NS /R 1 SE ratios and 𝜏 𝐶 values for hydrated reline mixtures are bigger than the corresponding values in non-hydrated samples. More in detail, for aniline in ChU/βCD/H 2 O, the

R 1 NS /R 1 SE ratio is around 1.4.
This value is expected when the molecule is in the extreme narrowing region (𝜔 0 𝜏 𝐶 ≪ 1). [START_REF] Tinoco | Determination of Correlation Times from Selective and Non-Selective Spin-Lattice Relaxation Rates and their Use in Drug-Drug and Drug-Albumin Interaction Studies[END_REF][START_REF] Kumar | Analysis of Molecular Interaction of Drugs within β-Cyclodextrin Cavity by Solution-State NMR Relaxation[END_REF] For toluene in ChU/βCD/H 2 O, the value of the R 1 NS /R 1 SE ratio is just close to 1, meaning that it is in the intermediate motion limit. This would indicate that in the mixture composed of reline and water, aniline rotates more freely than toluene. Nevertheless, as different protons were selected for the two VOCs (aromatic protons for AN vs methyl protons for toluene) any further comparison would sound forced. When β-cyclodextrin is added to the mixture, the R 1 NS /R 1 SE ratio and the 𝜏 𝐶 values for both AN and Tol get smaller. Since the correlation time is inversely proportional to the degree of molecular mobility (the smaller values of 𝜏 𝐶 correspond to higher molecular mobility), this is compatible with a slowdown in dynamics of the guest molecule (Tol or AN) because of its encapsulation in the βCD cavity.

Finally, diffusion experiments were performed on samples 5 to 8. DOSY maps obtained for AN and Tol in reline/water mixtures, with or without βCD, are reported in Figure 7. It can be seen that the diffusion of the VOC in the samples with βCD (left column) is in both cases slower that in the corresponding samples without βCD (right column). This is a consequence of formation of inclusion complexes with the βCD. A graphical summary of diffusion coefficients for the different species is reported in Fig. S5.

To confirm that the reduced diffusion of the guest in the presence of βCD is not due to viscosity changes caused by the addition of the macrocyclic oligosaccharide, the self-diffusion coefficient of H 2 O was used as an internal reference. 61 A look at Fig. S5 and Table S2 reveals that the diffusion coefficient of water remains constant upon addition of βCD in the reline system. This means that the variations in solution viscosity are minimal and do not account for the change in diffusion of the guest molecules, toluene or aniline. observed using static headspace gas chromatography (SH-GC), that association constant for the VOC/βCD of in reline are lower than the corresponding values in water. [START_REF] Moufawad | First Evidence of Cyclodextrin Inclusion Complexes in a Deep Eutectic Solvent[END_REF] However, it should be noted that also in reline/H 2 O mixtures studied here, toluene binds more strongly to βCD than aniline, which is in agreement with data from the literature. Moreover, the 𝐾 𝑎 value found here for βCD/toluene complex is higher than the one calculated by SH-GC (79 M -1 vs 11 M -1 ). This could be ascribed to the presence of water in the systems used in this work.

Conclusions

DES/βCD mixtures are interesting as new hybrid materials for VOC removal. To design the most performant DES-based absorbents, it is imperative to probe the formation of inclusion complexes and to have access to the dynamics of the system at the molecular level. Here we have illustrated the complementarity of the structural and dynamic information obtained from the 1D

1 H and ROESY spectra with diffusion and relaxation NMR experiments, and shown that their combination provides unique information to understand the formation of inclusion complexes with β-cyclodextrin in complex media such as deep eutectic systems.

Interestingly, we have proved that a certain amount of water is beneficial in reline systems, since the viscosity of the medium is reduced, while preserving both the DES network and the complexing ability of β-cyclodextrin. In non-hydrated systems, NMR results suggest the formation of inclusion complexes but given the impossibility to obtain correlation peaks in the ROESY spectra, a direct evidence is not available. When water is added to the mixture, intermolecular peaks are clearly visible in the ROESY spectra, confirming unequivocally the inclusion of the VOC.

In both hydrated and non-hydrated samples, selective T 1 measurements turned out to be very sensitive and convenient for the investigation of the complexation of VOCs into the βcyclodextrin, allowing the calculation of the correlation times. The R 1 NS /R 1 SE ratio is a marker for the transition between dynamic states of solutes in DES systems: the border value 1.5 for molecules in the extreme narrowing limit can be exploited to monitor the transition between fast and slow tumbling solutes. The data of Table 2 related to samples 5 and 6 clearly point out that the formation of an inclusion complex between βCD and AN causes the transition between fast and slow regime, not observed in the other examples of Table 2. Additionally, DOSY experiments allowed a rough but simple estimate of the molar fraction of the bound guest and the association constant of the complex in reline/water mixtures.

ASSOCIATED CONTENT Supporting Information.

The following files are available free of charge.

Experimental details, additional NMR spectra, graphical summary of diffusion coefficients and

Figure 1 .

 1 Figure 1. Structure and numbering of compounds used in this work.

Figure 2 .

 2 Figure 2. Selected regions of 1 H NMR spectra corresponding to aromatic protons of (a) AN

  2 and S1-S2). Figure3shows as an example the stack plots obtained with conventional nonselective pulses in the 180°-t-90° IR sequence (a) and with a selective IR experiment with the selective 180° centered on protons H 3 ,H 5 of aniline (b). In Fig.3(b), the signals of unselected protons remain essentially unperturbed, but the resonance of the selected protons relaxes faster. The values of the R 1 NS /R 1 SE ratio obtained for samples 1 to 4 are summarized in Table 2.

Figure 3 .

 3 Figure 3. Stack plots of (a) nonselective and (b) selective for H 3 , H 5 protons of AN inversion

Figure 4 .

 4 Figure 4. Summary of 𝜏 𝐶 values obtained for aniline (AN) and toluene (Tol) dissolved in reline mixtures. Maximum errors are estimated to be 10% and 20% for the hydrated and non-hydrated

Fig. 5

 5 Fig.5shows selected regions of 1 H spectra of samples 5 to 8. Both VOC's and βCD's signals

Figure 5 .

 5 Figure 5. Comparison of 1 H NMR signals of the guests aniline (AN) and toluene (Tol) and the

Fig. 6

 6 shows representative ROESY spectra obtained for the sample ChU/βCD/H 2 O/AN and ChU/βCD/H 2 O/Tol. The host-guest interactions are displayed as intermolecular correlation peaks between aromatic protons of aniline and toluene and protons H 3' and H 5' of βCD. This unequivocally confirms the formation of the inclusion complex of the VOC in βCD. Both AN and Tol produce larger NOEs on the H 3' proton, located in the larger diameter internal part of βCD, than they did on the other internal H 5' proton nearer the smaller diameter rim. This would indicate they are included through the larger diameter cavity βCD.

Figure 6 .

 6 Figure 6. Selected regions of 1 H-1 H ROESY spectra showing intermolecular correlation peaks

Figure 7 .

 7 Figure 7. DOSY maps acquired for hydrated reline mixtures with (left column) and without

  theoretical background for the treatment of relaxation and diffusion data (PDF) solution by quantitative ROESY analysis. J Incl Phenom Macrocycl Chem. 2015;83(1-2):19-26. doi:10.1007/s10847-015-0534-7 61. Silva M, Figueiredo AM, Cabrita EJ. Epitope mapping of imidazolium cations in ionic liquid-protein interactions unveils the balance between hydrophobicity and electrostatics towards protein destabilisation. Phys Chem Chem Phys. 2014;16(42):23394-23403. doi:10.1039/c4cp03534h SYNOPSIS The combination of the 1D 1 H and ROESY spectra with diffusion and relaxation NMR experiments provides unique structural and dynamic information on inclusion complexes in DES/βCD mixtures, crucial for the design of task-specific sustainable absorbents.

Table 1

 1

	. The deep eutectic solvent reline (ChU)

Toledo and was found equal to 2.1% for ChU/βCD and 0.5% for ChU. Overall this translates into a total water content in hydrated samples between 25 and 27 wt%, which is below the expected transition to an aqueous solution. Samples 1 to 4 were prepared by adding either aniline or toluene to the ChU/βCD and ChU solvents. Samples 5 to 8 were prepared by adding the same VOCs to the corresponding hydrated systems, ChU/βCD/H 2 O and ChU/H 2 O. All samples containing βCD (1, 3, 5 and 7) were prepared to have a final host/guest molar ratio of 1:1 and the same weight percentage of VOC was also used in samples without βCD (2, 4, 6 and 8). Samples 9 and 10 without VOCs were used as references. The samples for NMR analysis were placed in standard 5 mm tubes and multiple NMR experiments were performed (details are given in the SI).

Table 1 .

 1 Composition of samples used in this work.It is well known that NMR chemical shift is sensitive to changes in electronic environment.

	Sample	Short name	DES	βCD	Added H 2 O	Guest molecule
	1	ChU/βCD/AN	ChU	10%	-	AN (1 eq.)
	2	ChU/AN	ChU	-	-	AN (1 eq.)
	3	ChU/βCD/Tol	ChU	10%	-	Tol (1 eq.)
	4	ChU/Tol	ChU	-	-	Tol (1 eq.)
	5	ChU/βCD/H 2 O/AN	ChU	10%	210 eq.	AN (1 eq.)
	6	ChU/H 2 O/AN	ChU	-	210 eq.	AN (1 eq.)
	7	ChU/βCD/H 2 O/Tol	ChU	10%	210 eq.	Tol (1 eq.)
	8	ChU/H 2 O/Tol	ChU	-	210 eq.	Tol (1 eq.)
	9	ChU/βCD	ChU	10%	-	-
	10	ChU/βCD/H 2 O	ChU	10%	210 eq.	-

RESULTS AND DISCUSSION

Does β-cyclodextrin form inclusion complexes with VOCs in pure (non-hydrated) reline?

Table 2 .

 2 R 1 NS /R 1 SE ratio and 𝜏 𝐶 values obtained for aniline (AN) and toluene (Tol) dissolved in reline mixtures. Maximum errors are estimated to be 10% and 20% for the hydrated and nonhydrated samples, respectively.

	Sample	Short name	Guest	Selected proton(s)	𝐑 𝟏 𝐍𝐍 /𝐑 𝟏 𝐍𝐒	𝝉 𝑪 (s)
	1	ChU/βCD/AN	AN	H 3 ,H 5	0.13	2.0x10 -9
	2	ChU/AN	AN	H 3 ,H 5	0.50	8.4x10 -10
	3	ChU/βCD/Tol	Tol	CH 3	0.04	3.9x10 -9
	4	ChU/Tol	Tol	CH 3	0.39	1.0x10 -9
	5	ChU/βCD/H 2 O/AN	AN	H 3 ,H 5	0.83	4.8x10 -10
	6	ChU/H 2 O/AN	AN	H 3 ,H 5	1.37	1.2x10 -10
	7	ChU/βCD/H 2 O/Tol	Tol	CH 3	0.74	5.7x10 -10
	8	ChU/H 2 O/Tol	Tol	CH 3	0.93	4.1x10 -10

Table 3 .

 3 Diffusion coefficients measured in hydrated reline mixtures and corresponding molar fraction of the bound guest x G 𝑜𝑜𝑏𝑏𝑏 and apparent association constant 𝐾 𝑎 estimated for AN and

	Tol
	Guest	𝐃 𝐆 𝒐𝒐𝒐 (m 2 s -1 )	𝐃 𝐆 𝒇𝒇𝒇𝒇 (m 2 s -1 ) 𝐃 𝐇 (m 2 s -1 )	𝐱 𝐆 𝒐𝒐𝒃𝒃𝒃	𝑲 𝒂 (M -1 )
	AN	1.7x10 -10	2.6x10 -10	5.6x10 -11	0.46±0.05	19±4
		±0.1x10 -10	±0.1x10 -10	±0.6x10 -11
	Tol	1.4x10 -10	3.2x10 -10	5.6x10 -11	0.70±0.07	79±16
		±0.1x10 -10	±0.2x10 -10	±0.6x10 -11

ACKNOWLEDGMENT

The work has been carried out within the frame of the bilateral France-Italy Galileo Programme (Grant G18-392 and 39467XJ). G.C.D. thanks Politecnico di Milano for her Inter-Departments PhD scholarship. The authors thank Tarek Moufawad for helpful discussion.

AUTHOR INFORMATION

Corresponding Author * Dr. Maria Enrica Di Pietro. Phone +39 02 2399 3045. Email mariaenrica.dipietro@polimi.it * Prof. Andrea Mele. Phone +39 02 2399 3006. Email andrea.mele@polimi.it

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.


Sabrina Madiedo-Podvrsan

Louise Sebillet

Thomas Martinez

Salimata Bacari

Fengping Zhu

Marie Cattelin

Eric Leclerc

Franck Merlier

Rachid Jellali

Ghislaine Lacroix

Muriel Vayssade
email: muriel.vayssade@utc.fr

Development of a lung-liver in vitro coculture model for inhalation-like toxicity assessment

Keywords: bioengineering, multiorgan-on-a-chip, toxicology, coculture, lung-liver crosstalk

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

Modern urbanization and globalization phenomena multiply and complexify pollution sources.

Among them, air pollution has an increasingly significant impact on global health due to its transboundary nature [START_REF] Cohen | Estimates and 25-year trends of the global burden of disease attributable to ambient air pollution: An analysis of data from the Global Burden of Diseases Study 2015[END_REF][START_REF] O'neill | Air pollution and health: Emerging information on susceptible populations[END_REF][START_REF] Solomon | Air pollution and health: Bridging the gap from sources to health outcomes: Conference summary[END_REF]. A recently published review reports that air pollution causes over 6.5 million deaths each year globally, and that this number is increasing [START_REF] Fuller | ollution and health: A progress update[END_REF]. Air quality is closely linked to anthropogenic pollution sources mainly defined by industrial processes, domestic pollution, transportation and agricultural systems.

The lungs are the main route of entry of airborne xenobiotics. Upon internalization, inhaled compounds undergo absorption, distribution, metabolism, and excretion (ADME) processes which lead them to interact with pulmonary and extra-pulmonary cells [START_REF] Castell | Metabolism and bioactivation of toxicants in the lung. The in vitro cellular approach[END_REF].

Associated toxicity mechanisms are complex and involve local and systemic responses that can aggravate, stabilize, or protect the body from adverse effects. Because of this complexity, animals are considered prime study models. However, in the European context of animal experimentation reduction (3Rs) the development of reliable alternative methods has become a necessity. In vitro models are particularly interesting because they are cost-effective and easier to implement than vivo models. However, since tissues and organs reside in vivo in highly integrated and dynamically interactive environments, extrapolation from vitro to vivo is limited if inter-organ crosstalk is not considered. Multi-organ-on-a-chip technologies seek to overcome these limitations by connecting multiple metabolically active organoids within a single culture circuit to mimic systemic-like interactions. The literature reports numerous cases of multi-organ platforms comprising a pulmonary compartment [START_REF] Hübner | Simultaneous evaluation of anti-EGFR-induced tumour and adverse skin effects in a microfluidic human 3D co-culture model[END_REF][START_REF] Kimura | An On-Chip Small Intestine-Liver Model for Pharmacokinetic Studies[END_REF][START_REF] Skardal | Multi-tissue interactions in an integrated three-tissue organ-on-a-chip platform[END_REF], however only a minority considers and studies the lungs as a key route of entry in the ADME process.

Since hepatic xenobiotic metabolism is strongly tied to systemic toxicity in vivo, and the literature reports instances of lung-liver crosstalk in stress-induced environments, investigation of hepatic responses to respiratory stress was given consideration [START_REF] Guo | Prenatal exposure to pyrrolizidine alkaloids induced hepatotoxicity and pulmonary injury in fetal rats[END_REF][START_REF] Kim | The Role of Air Pollutants in Initiating Liver Disease[END_REF][START_REF] Ya | Liver injury induced in Balb/c mice by PM 2.5 exposure and its alleviation by compound essential oils[END_REF]. Here, we describe a coculture model designed to investigate inhalation-like toxicity in a further systemic manner by connecting in vitro, through microfluidics, a pulmonary barrier to a detoxifying organ such as the liver. Our approach considers the lungs as a route of entry and intends to study the metabolic fate of inhaled compounds by considering inter-organ crosstalk as possible modulators of toxicity.

Even though a few other lung-liver coculture systems have previously been described [START_REF] Bovard | A lung/liver-on-a-chip platform for acute and chronic toxicity studies[END_REF][START_REF] Coppeta | A portable and reconfigurable multi-organ platform for drug development with onboard microfluidic flow control[END_REF][START_REF] Schimek | Human multi-organ chip co-culture of bronchial lung culture and liver spheroids for substance exposure studies[END_REF], the metabolic fate of xenobiotics has not been studied.

The present lung-liver model was characterized using a well-documented hepatotoxicant: acetaminophen also known as paracetamol (APAP). The coculture was exposed to APAP through the pulmonary barrier, to imitate an inhalation type exposure. APAP exposure allows us to visibly assess the passage and circulation of a model substance through the device, as it interferes with hepatic viability and metabolic performances.

Material & Methods

Calu-3 tissue culture

Cryopreserved Calu-3 (ATCC, reference HTB-55) cells were grown and expanded using Roswell Park Memorial Institute 1640 supplemented with stable L-Glutamine (RPMI 1640 GlutaMAX Supplement, ThermoFischer Scientific), and completed with 10% of Fetal Bovine Serum (FBS, Gibco) and 1% of 100 units/mL Penicillin-Streptomycin (PAN Biotech). Cells were amplified in 75 cm 2 flasks (Corning, Falcon) seeded at an initial density of 20 000 cells per cm 2 , in a 37°C, 5% CO 2 incubator. Once cells reached 80% of confluence, they were detached using trypsin-EDTA (1X) (Gibco) and seeded, at 200.10 3 cells per 6-plate sized Transwell culture insert. Tissues were grown under static submerged conditions, i.e. tissues were apically covered by culture media, for 11 days when trans-epithelial resistance (TEER) measurements exceeded 1000 Ω.cm 2 (Figure 1A). As soon as the bronchial barrier completes reconstruction, Calu-3 inserts were transferred in the coculture platform and submerged tissues underwent 72-hour of perfused culture at a continuous microfluidic flow rate of 25 μL per min (Figure 1B andC). Medium perfusion was perceived on the basolateral side of the culture insert. Calu-3 tissue culture was carried out for a total of 14 days.

HepG2/C3A biochip cultures

Biochip design and microfabrication.

The design and fabrication of the biochip has been previously documented [START_REF] G467 -Baudoin | Behavior of HepG2/C3A cell cultures in a microfluidic bioreactor[END_REF]. Briefly, the inner microarchitecture of biochips is printed by photolithography onto wafer containing SU-8 photoresist. The design comprises two sides, a hollow media reservoir for the upper compartment and a series of microstructured chambers and channels for the bottom cell cultivating side. 10:1 (w/w) polydimethylsiloxane (PDMS) and cross-linking agent (Sylgard 184, Dow Corning) mixture was used to manufacture biochips. The mix was poured onto the microdesigned molds and cured for 2 hours at 70°C. Reactive air plasma treatment of surfaces allowed to seal both sides together to achieve the completed biochip. The final assembled biochip holds a total volume of 40 μL and covers a cell growth area of 2cm 2 . Prior to cell culture, biochips are sterilized by autoclave, and coated with a 0.36 mg/ml Collagen I (Corning Life Science) solution to allow cell adhesion.

Biochip hepatic cell culture.

Hepatocellular carcinoma-derived HepG2/C3A cells (ATCC, reference CRL-10741) were grown and expanded using Minimum Essential Medium (MEM, Corning) supplemented with 10% FBS, 1% of Penicillin-Streptomycin, 1% of L-Glutamine (100X) (PAN Biotech), 1% of N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid (HEPES) buffer (1M) (Gibco), 1% of Sodium Pyruvate (100X) (Gibco) and 1% of MEM non-essential amino acids (BioWest). Cells were amplified in 75 cm 2 flasks (Corning, Falcon), seeded at an initial density of 20 000 cells per cm 2 , in a 37°C, 5% CO 2 incubator. Once cells reached 90% of confluence, they were detached using trypsin-EDTA (Gibco) and seeded, into culture ready-biochips at an initial density of 500.10 3 cells per biochip. Seeded biochips were kept at rest 24 hours, in a 37°C, 5% CO 2 incubator, to complete cell adhesion. Biochips were then boxed into an Integrated Dynamic Cell Culture in Microsystems (IDCCM) device (Patent -{US20130084632A1}), perfused culture was maintained for 72 hours at a continuous microfluidic flow rate of 25 μL per min thanks to a peristaltic pump.

Coculture

Coculture box: Integrated Insert in a Dynamic Microfluidic Platform (IIDMP).

Both compartments were serially connected using the Integrated Insert in Dynamic Microfluidic Platform (IIDMP) device [START_REF] Bricks | Development of a new microfluidic platform integrating co-cultures of intestinal and liver cell lines[END_REF]. This coculture platform is a polycarbonatebased box able to accommodate 3 parallelized cocultures (Figure 1D andE). The bottom side comprises wells for 6-plate sized Transwell culture inserts, attachments for biochips and associated media reservoirs, and a top lid for tubing. Both parts are joined and sealed together by a silicon seal and screws. Once mounted, the device is set at a continuous microfluidic flow rate of 25 μL per min, thanks to a peristaltic pump. Flow was directed from the insert to the biochip in a closed loop.

2.3.2. Bronchial and hepatic coculture conditions. Calu-3 inserts and HepG2/C3A biochips were matured separately, according to previously described protocols, before being joined in culture for 72 hours in RPMI-based medium, in a 37°C, 5% CO 2 incubator.

Acetaminophen (APAP, Sigma Aldrich) was dissolved in RPMI-based medium and APAP solutions (1 mL) were deposited apically on the pulmonary barrier at local exposure concentrations of 7.5 and 15 mM, which dilute into the circulating media respectively to systemic concentrations of 1.5 and 3 mM when exposure solution joins the dynamic circulation within the device.

Cell proliferation assays

Mitochondrial activity assessment. Calu-3 mitochondrial activity was assessed by

Prestoblue assay (Prestoblue TM Cell viability Reagent, Fischer Scientific). Non-cytotoxic fluorogenic probe passively entered living cells in its non-fluorescent form (reazurin) and reduced into resorufin by mitochondria, a soluble red fluorescent product, that diffused out of the cells and was directly measured by spectrophotometry (excitation wavelength of 535 nm and emission wavelength of 595 nm, Spectafluor Plus, TECAN).

Cell count.

HepG2/C3A cells within post-culture biochips were detached using trypsin-EDTA and counted using a Malassez' hemocytometer. Cell viability was assessed by Trypan blue dye exclusion.

Calu-3 tissue barrier properties

Trans-epithelial electrical resistance measurement.

The quantitative measurement of barrier integrity was achieved using double electrodes (STX2 set and EndOhm-24SNAP 147581 from World Precision Instruments) connected to Millicell-ERS (Electrical Resistance System) (Millipore) to measure passing current. Every series of measurements included measuring the blank resistance (R BLANK ) of an acellularized membrane and the resistance across a cellularized membrane (R TOTAL ). TEER values are reported (TEER REPORTED ) in units of Ω.cm 2 and calculated as: Where dQ/dt was the amount of compound transported per second (mg.s -1 ), A was the surface area of the culture membrane (cm 2 ) and C 0 the initial donor concentration (mg/mL). The mass balance (R, unit: %) was calculated as:

TEER REPORTED = (R TOTAL -R BLANK )(Ω) × M AREA (cm 2 )

Adherens and tight junction

Where A and D were the amounts of compounds in the apical and basal compartments respectively, and D 0 the initial amount of Lucifer Yellow introduced at t0 in the donor compartment. Mass balances of all compounds were between 46 and 86%.

Hepatic functionality assays

2.6.1. Albumin synthesis. Hepatic albumin secretion was quantified in post-culture media samples by ELISA assay according to manufacturer instructions (Human albumin ELISA Quantitation Set, Bethyl Laboratories).

EROD assay (CYPA1/2 and CYP2B detoxifying activity). CYP1A1/2 and CYP2B

activity levels of post-culture hepatic biochips were measured using 5-ethoxyresorufin (10 mM) as substrate. Resorufin formation by 7-ethoxyresorufin O-deethylation (EROD) was quantified by fluorescence intensity measurement (λ excitation = 5 5 nm, λ emission = 595 nm) (TECAN, Spectrafluor plus) after 1 h incubation in presence of salicylamide (3 mM), which inhibits phase II enzymes.

Mass spectrometry detection

An Agilent QQQ 6460 mass spectrometer with a jet stream electrospray ion source and an Agilent 1200 series fast resolution LC system (Wilmington, DE) was employed to detect acetaminophen and associated metabolites in culture medium samples. MassHunter software was used for system control, data acquisition, and data processing. LC separation was performed on an Agilent poroshel C18 reverse phase column (100 mm x 4.6 mm i.d., 2.6 μm particle size) with a gradient program at a flow rate of 1 mL/min. The mobile phase A consisted of 100% HPLC grade water with 0.1% formic acid and mobile phase B consisted of 100% HPLC grade acetonitrile. The gradient started with 2% solvent B, held at 2% B for 1 minute before being increased to 20% B then increased to 95% in 1 minute and was then held at 95% B for 2 additional minutes. The column was re-equilibrated with 2% B for 3 minutes.

Total run time was 12 minutes with a 10 μL injection volume. The mass spectrometer was operated in positive and negative jet stream ESI modes. Nitrogen was used as a nebulizer, turbo (heater) gas, curtain, and collision-activated dissociation gas. The capillary voltage was +3800 V and -3500 V. The ion source gas temperatures were 350°C with flows of 12 L/min.

Jetstream gas temperatures were 350°C with flows of 12 L/min. APAP and metabolites were measured by selective reaction monitoring (SRM). The calibration curve was performed with internal calibration using 0.5 μM APAP-D4 in acetonitrile. The samples are prepared by taking 20 μL of medium and adding 80 μL of the internal standard solution to the acetonitrile in a 1 mL glass vial, then centrifuged for 5 minutes at 13500 rpm before being transferred from a vial with a 200 μL glass insert.

Statistical analysis

Data are presented as the mean ± standard deviations.

Results

Characterization of Calu-3 behavior to coculture

For bronchial tissues to acquire proper barrier function, through the development of solid junctional networks, Calu-3 cells were cultured for 11 days until trans-epithelial resistance (TEER) measurements exceed 1000 .cm 2 . Mature constructs on inserts were then cultured in a dynamically-stimulated environment for 72h, in monoculture or coculture (with liver biochip) settings. To ensure accurate readout, viability and functionality parameters of cocultured tissues were monitored post-culture and results were compared to monoculture data.

A global overview of tissue integrity was assessed through phase-contrast microscopy observations (Figure 2A). Overall, coculture did not seem to disturb the morphological development of Calu-3 bronchial tissues: no difference in cell refringence was noted between monoculture and coculture. The culture mode did not visually perturb adhesion and proliferation trends of Calu-3 cells as both tissues remained homogenous and displayed similar confluence and cohesiveness.

Calu-3 tissues also seemed to seamlessly adapt to the coculture environment as mitochondrial activity data showed that the presence of the hepatic compartment had no significant impact on cell metabolic activity compared to that of monocultured cells (Figure 2B). From a functional standpoint, Calu-3 tissues formed and maintained strong barrier integrity (Figure 2C), as non-invasive TEER measurements revealed that values stabilized at 2500 Ω.cm ² , and continued to apically release gel-forming mucins MUC5AC (Figure 2D) regardless of culture conditions.

Intercellular junction immunostainings allowed to further depict the barrier integrity status of cocultured tissues (Figure 3). The staining of E-Cadherin remained localized at the cell-cell interface without any diffusion, whereas Claudin-1 expression appeared disrupted from its native peripheral localization compared to monocultured samples.

Despite the alteration of the tight junction network of cocultured Calu-3 tissues, Lucifer

Yellow assays revealed no significant permeability rates between tissues of different culture settings (Table 1). Overall, permeability remained minimal, under 1%, coculture did not disrupt the permeability of the barrier.

Characterization of HepG2/C3A behavior to coculture

Prior to coculture, common medium tests were conducted to ensure optimal maintenance of tissue viability and functionality of both compartments (data not shown). As HepG2/C3A cellular behavior in RPMI-based medium remained comparable to that of initial MEM-based medium (no cell death induction, stable CYP1A activity), RPMI-based medium was used for coculture purposes. Cocultured hepatic biochips were collected post-culture and underwent a variety of assays to assess the impact of coculture on hepatic functions.

Phase contrast microscopy showed that adhesion and proliferation of hepatocytes within the biochip were successful despite the culture mode, as cells were homogeneously spread throughout the microchambers and channels of the system (Figure 4A). Overall, morphological features seemed to remain unchanged, cocultured HepG2/C3A biochips continued displaying confluent and cohesive tissues.

Quantitative assessments of cell viability and functionality confirmed these qualitative observations: cocultured biochips maintained similar cell density (approximating 1.5.10 6 cells per biochip, Figure 4B) and hepatic differentiation (HepG2/C3A albumin secretion stabilized at 200 ng/10 6 cells/h, Figure 4C). In terms of hepatic xenobiotic response, the results showed that coculture induced an induction of CYP1A1/2 activity as rates nearly doubled compared to their monocultured counterparts (Figure 4D).

Stability and response of the lung/liver coculture to a stress-induced environment

Following the characterization of the suitability of both pulmonary and hepatic in vitro building blocks in a joint culture setting, the lung/liver model was characterized in a stressinduced environment using APAP. The chosen exposure concentrations relied on documented hepatotoxicity thresholds, 1.5 mM and 3 mM, to allow better quantification of biological responses. First we confirmed that the culture equipment (IIDMP coculture box, culture insert, biochip, tubing) did not induce possible biases by passively absorbing a part of the exposed APAP during the culture period. APAP was introduced in the coculture system but in an acellular environment and APAP concentrations were measured after 72 hours of dynamic culture (Supplementary data, Table A). Data indicated that initial APAP concentrations were found at the same levels after 72h.

Tissue-specific APAP-induced responses were compared to coculture data to highlight crosstalk-specific responses to xenobiotic stress. Following the 72-hour coculture period, media samples were collected from the apical and baso-lateral sides of the pulmonary barrier respectively corresponding to the inhalation-like APAP exposure site, and to the circulating common culture medium. To verify that APAP was able to cross the bronchial barrier and reach the systemic circulation, APAP levels were quantified through mass spectrometry (Table 2). Results revealed that the passage into the circulating medium was successful as APAP was detected on the apical and basal sides of the pulmonary barrier. In addition to APAP levels, secondary non-toxic metabolites (APAP-glucuronide and APAP-sulfate) were detected mainly in the basal compartments but in a non-dose dependent manner. This suggests that the lung/liver coculture was capable of metabolizing APAP at hepatotoxic exposure concentrations.

Calu-3 tissues were subject to Prestoblue assays, to determine the effect of APAP on metabolic activity (Figure 5A). APAP exposure induced a significant decrease of mitochondrial activity in Calu-3 tissues, with no dose-dependent effect. The intensity of APAP adverse effects was reduced in cocultured Calu-3 tissues: the mitochondrial activity became significant only at 3 mM and measured values were significantly higher to those obtained in monocultures.

Hepatic cell count data (Figure 5B) revealed similar response patterns, where the adverse effects induced by APAP in monoculture settings diminished in coculture. Statistical analysis revealed alleviated differences between APAP-exposed cell counts as cocultured exposed biochips featured smaller cell count variations.

Upon contact with APAP, the hepatic responses to stress differed according to culture conditions: cocultured HepG2/C3A biochips were subject to an earlier induction of CYP1A1/2 activity at 1.5 mM and decreased at 3 mM APAP exposure concentration whereas the stimulation of the activity in monocultured cells only occurs at 3 mM (Figure 5C).

Moreover, at the same exposure dose (1.5 mM), the cocultured cells respond significantly stronger. The maximum intensity of CYP activity perceived under coculture conditions was about 5 times greater than that under monoculture. Maximum CYP activity peaked at 256 ± 101 pmol of resorufin per hour per 10 6 cells, whilst in monoculture the maximum only reached 75 ± 21 pmol of resorufin per hour per 10 6 cells. Thus, the metabolic profile of HepG2/C3A biochips was impacted by the presence of a bronchial barrier in a stress-induced environment.

Discussion

Overall, present results show that the submerged Calu-3 bronchial barrier and the HepG2/C3A hepatic biochip can be successfully cocultured in vitro during 72 hours as they display stable and functional cellular behaviors. According to the collected data, the presence of a foreign tissue did not induce a stressful culture environment for either of the tissues, however, it did cause a reorganization of the Calu-3 tissue architecture. The alteration of Claudin-1 labeling at cell-cell contact sites should have resulted in increased permeability of the barrier, as they are responsible for regulating access to paracellular spaces [START_REF] Bibliography -Anderson | Tight junctions and the molecular basis for regulation of paracellular permeability[END_REF], yet this was not the case. As the involvement of the HepG2/C3A cells in the maintenance of Calu-3 tissue permeability has not yet been observed or studied in the literature, the study of other biomarkers (such as reactive oxygen species, inflammatory cytokines …) would improve the understanding of the crosstalk between both cell types.

The quantification of APAP concentration within the acellular coculture platform confirms the previously documented data [START_REF] Bricks | Investigation of omeprazole and phenacetin first-pass metabolism in humans using a microscale bioreactor and pharmacokinetic models[END_REF] which reveals that the materials of the present culture equipment don't absorb A A passively and that the bioavailability of paracetamol in coculture conditions remains the same as the initial exposure. The observed experimental biological effects can therefore be related to the theoretical exposure concentrations. While APAP exposures have usually only been considered on the apical side of the bronchial barrier, it is presumed that within the coculture platform APAP and its metabolites recirculate continuously throughout the culture period, which would mean that the bronchial compartment could suffer a continuous re-exposure through is basal cell pole. The quantification of the basal permeability of the Calu-3 barrier indicates that such an APAP reexposure is plausible as basal tissue permeability (0.071 ± 0.03 x 10 -6 cm.s -1 ) is similar to apical permeability (0.049 ± 0.01 x 10 -6 cm.s -1 ).

The coculture setting allowed the passage of APAP through the Calu-3 barrier and into the circulatory system, as APAP was detected in the circulating medium and likely reached the liver compartment as the hepatic homeostasis of the biochip was impaired. Overall APAP exposures elicited similar cellular behavior patterns, associated with apparent cytotoxicity.

However, the intensity of associated adverse effects was reduced, and tissue responses followed a different kinetic in the coculture setting. While the presence of APAP-glucuronide and APAP-sulfate in the medium is indicative of active phase II metabolism, CYP1A activity measurements allow following phase I oxidation metabolism. Results show that in the coculture setting, APAP induces earlier and 5-fold stronger CYP1A activities than in monoculture. CYP1A is involved in the biotransformation of APAP into reactive N-acetyl-pbenzoquinone imine (NAPQI) (Prot et al., 2011a) which is known to be the toxic metabolite of APAP [START_REF] Larson | Acetaminophen hepatotoxicity[END_REF][START_REF] Mazaleuskaya | PharmGKB summary: Pathways of acetaminophen metabolism at the therapeutic versus toxic doses[END_REF]. Increased CYP1A activity should be related to greater cytotoxicity, but because this was not the case, this could imply that NAPQI is actively detoxified. Possible detoxification would explain why the mitochondrial metabolism of cocultured Calu-3 is higher than in the monoculture setting, as literature reports how NAPQI targets mitochondrial proteins [START_REF] Qiu | Identification of the hepatic protein targets of reactive metabolites of acetaminophen in vivo in mice using two-dimensional gel electrophoresis and mass spectrometry[END_REF]. As the exposure concentration rises (3 mM), the lung/liver coculture seems to display disrupted xenobiotic metabolism respectively as CYP1A activity reduces and APAP-glucuronide and APAPsulfate metabolites are absent on the apical side of the bronchial barrier. As a result, produced NAPQI could freely circulate through the coculture, no longer undergoing enhanced detoxification processes previously described, which would explain at least partly why the adverse effects are more pronounced than for 1.5 mM-APAP exposed cocultures. Delayed cytotoxicity suggests that at least a part of the NAPQI was metabolized into a non-toxic metabolite, implying that the detoxification metabolism would have been at least momentarily active during the 72-hour culture period, before the exhaustion of cellular defense mechanisms. To verify this hypothesis, together with direct measurement of NPAQI and APAP-glutathione metabolites in the system, the state of Calu-3 and HepG2/C3A metabolisms could be punctually assessed throughout the coculture period to verify if a gradual decrease occurs. Our results coincide with [START_REF] Bovard | A lung/liver-on-a-chip platform for acute and chronic toxicity studies[END_REF] and [START_REF] Schimek | Human multi-organ chip co-culture of bronchial lung culture and liver spheroids for substance exposure studies[END_REF] who also highlighted that bronchial tissue/HepaRG spheroids cocultures induced a modulation of toxicity in a stress environment upon exposure to aflatoxin B1. Because the authors did not assess the presence of metabolites in their platform, they could not conclude with certainty that the xenobiotic was metabolized: this is not the case in the lung-liver coculture model we describe, which is metabolically competent (assessed with CYP1A activity induction and APAP-glucuronide and APAP-sulfate metabolites detection).

The biochip employed in the coculture has been previously developed, characterized and optimized for hepatic tissue culture. This microsystem was utilized for short-and long-term cultures of cell lines but also primary (rat and human hepatocytes) and stem cells (Essaouiba et al., 2022;Prot et al., 2011aProt et al., , 2011b;;[START_REF] Legendre | Metabolic Characterization of Primary Rat Hepatocytes Cultivated in Parallel Microfluidic Biochips[END_REF][START_REF] Jellali | Long-term human primary hepatocyte cultures in a microfluidic liver biochip show maintenance of mRNA levels and higher drug metabolism compared with Petri cultures[END_REF][START_REF] Danoy | Investigation of the hepatic development in the coculture of hiPSCsderived LSECs and HLCs in a fluidic microenvironment[END_REF][START_REF] Etxeberria | Validation of HepG2/C3A Cell Cultures in Cyclic Olefin Copolymer Based Microfluidic Bioreactors[END_REF]. It has already been used successfully for many toxicity and metabolic studies [START_REF] Baudoin | Evaluation of seven drug metabolisms and clearances by cryopreserved human primary hepatocytes cultivated in microfluidic biochips[END_REF][START_REF] Jellali | Effects of DDT and permethrin on rat hepatocytes cultivated in microfluidic biochips: Metabolomics and gene expression study[END_REF][START_REF] Jellali | Investigation of steatosis profiles induced by pesticides using liver organon-chip model and omics analysis[END_REF]Prot et al., 2011b), and numerous coculture models [START_REF] Bricks | Investigation of omeprazole and phenacetin first-pass metabolism in humans using a microscale bioreactor and pharmacokinetic models[END_REF][START_REF] Danoy | Investigation of the hepatic development in the coculture of hiPSCsderived LSECs and HLCs in a fluidic microenvironment[END_REF][START_REF] Essaouiba | Development of a pancreas-liver organ-on-chip coculture model for organ-to-organ interaction studies[END_REF][START_REF] Prot | First pass intestinal and liver metabolism of paracetamol in a microfluidic platform coupled with a mathematical modeling as a means of evaluating ADME processes in humans[END_REF][START_REF] Zeller | Hepatocytes cocultured with Sertoli cells in bioreactor favors Sertoli barrier tightness in rat[END_REF]. Recently, hydrogel and hydroscaffolds were integrated in the biochip to promote liver cell organization in 3D spheroids/organoids (Boulais et al., 2020;[START_REF] Messelmani | Development of Liver-on-Chip Integrating a Hydroscaffold Mimicking the Liver's Extracellular Matrix[END_REF]. Regarding the pulmonary barrier, in addition to simplistic model presented here, our team developed multicellular alveolo-capillary barrier including macrophages and pulmonary endothelial cells [START_REF] Dekali | Assessment of an in vitro model of pulmonary barrier to study the translocation of nanoparticles[END_REF]. Along with the potential that the liver biochip and lung barrier models offers, air-liquid aerosolized exposure could offer promising complexification opportunities to the present coculture model and therefore empower its relevance for the risk assessment of inhaled xenobiotics. Indeed the IIDMP platform is equipped with inlet port (top of insert compartment) allowing the connection to aerosol generator such as Vitrocell® system [START_REF] Loret | Predicting the in vivo pulmonary toxicity induced by acute exposure to poorly soluble nanomaterials by using advanced in vitro methods[END_REF]. Furthermore, the use of human primary cells and chronic exposures to well-known inhaled compounds would be needed to expand the significance of the current model.

Conclusions

Our data indicate that a Calu-3 bronchial barrier and a HepG2/C3A biochip can be successfully maintained viable and functional in a dynamic coculture setting for 3 days. The developed model is seemingly responsive to xenobiotic exposures, and emulates active and functional in vitro crosstalk between both compartments, allowing to obtain preliminary investigative predictions in 72 hours. The crosstalk induced a modulation of stress response kinetics, delaying cytotoxicity, proving that xenobiotic fate and biological behaviors can be modulated in a broader systemic-like environment. The present lung/liver coculture displays promising potential to empower the prediction of inhalation-like exposures.

Acknowledgments

The work was funded by the European Regional Development Fund (ERDF) 2014/2020 and the national research program Environnement-Santé-Travail (PNR EST) of the French National Food Safety Agency (Anses).

The graphical abstract was designed and realized by Dr Augustin Lerebours.

  immunostaining. Post-culture exposed and non-exposed samples were collected, rinsed 3 times with PBS (1X, pH 7.4, Gibco) and fixed with 4% paraformaldehyde (PFA) solution. Cells were permeabilized with 100X Triton (BDH) à 0.1% at room temperature. Non-specific sites were blocked by immersing the sections into 2% bovine serum albumin (BSA, Gibco). Primary antibodies (rat anti-CD324 antibody, Invitrogen 5µg/mL or rabbit anti-Claudin 1 antibody MH25, Invitrogen 20 µg/mL) were added to the sections for 1 hour at room temperature. Then, the sections were rinsed 3 times with PBS at room temperature. Secondary antibodies (Cyanine 2 AffiniPure goat anti-rat IgG secondary antibody, 1:100, Jackson ImmunoResearch or AlexaFluor 680 goat anti-rabbit IgG secondary antibody, 4 g mL, Thermo Scientific), were added to the sections for 1 hour at room temperature in a dark chamber. The samples were rinsed last times with S at room temperature and received a 1 mg mL 4 ,6-diamidino-2-phenylindole (DAPI) immunostaining (MBD0015, Merck) for nuclei staining. Immunofluorescence microscopy scans where achieved with confocal microscopy (Zeiss LSM 710).2.5.3. MUC5AC dosage.Quantitative detection of MUC5AC in post-culture supernatant samples was achieved using the Human MUC5AC ELISA kit (CliniSciences) according to manufacturer instructions.2.5.4. Lucifer Yellow permeability assay. Barrier function was quantified by Lucifer Yellowpermeability assay (LY) (Lucifer Yellow CH dipotassium salt, Sigma-Aldrich) on postculture Calu-3 tissues. Briefly, tissues were washed with pre-warmed (37°C) HBSS (with CaCl 2 and MgCl 2 , pH 7.4, Invitrogen) on both apical and baso-lateral compartments. Lucifer Yellow CH dipotassium salt was then diluted in HBSS and added to the donor compartment at a final concentration of 100 μg mL. After 1 hour of incubation at 7°C and 5% CO 2 , aliquots from the donor and receiver compartments were collected in a black 96-well micro-plate for determination of fluorescence leakage of the LY with a fluorescence microplate reader (TECAN, 71 Spectrafluor plus) (λ excitation = 485 nm, λ emission = 530 nm). The apparent permeability coefficient (Papp, unit: cm.s -1 ) was calculated as follows:

Figure captions Figure 1 :

 captions1 Figure captions

Figure 2 :

 2 Figure 2: Characterization of Calu-3 tissues in mono-and cocultures.

Figure 3 :

 3 Figure 3: Comparison of monocultured and cocultured Calu-3 bronchial-like barriers.

Figure 4 :Figure 5 :

 45 Figure 4: Characterization of liver biochip in mono-and cocultures.

  Histogram charts are complemented by scatter plots, which gather the samples that constitute the groups. n represents the number of independent experiments performed. Group comparison statistical tests were chosen based on analysis of dataset variance and normality. All data was unpaired. Two-group comparisons were carried out by unpaired t-test, and Mann-Whitney test respectively for normal and nonnormal data. Multiple group comparisons were carried out by ANOVA, and Kruskal-Wallis test respectively for normal and non-normal data. Statistical analysis was performed using GraphPad Instat v.3.10. P values less than 0.05 were considered statistically significant and are presented as follow: *p<0.05, **p<0.01, ***p<0.001.

Table 1 :

 1 Papp measurements of Calu-bronchial tissues on day 14 (n ≥ , 6 samples).

		Papp (x10 -6 cm.s -1 )	Permeability rate (%)
	Cell free culture insert	60.581	100
	Apical permeability
	Monoculture	0.028 ± 0.02	0.045
	Coculture	0.041 ± 0.04	0.067

Table 2 :

 2 Mass spectrometry measurements of APAP and non-toxic metabolites (APAPglucuronide and APAP-sulfate) levels in post-culture apical and basal supernatants of the lung liver cocultures according to A A exposure (n ≥ , 6 samples).

	APAP exposure (mM)	APAP (mM)	APAP-glucuronide	APAP-sulfate
				(mM)	(mM)
	Apical	0	0.013 ±0.008	0 ± 0	0 ± 0
	Basal		0.005 ±0.002	0 ± 0	0 ± 0
	Apical	1.5	1.844 ±0.587	0.039 ±0.092	0 ± 0
	Basal		1.872 ±0.378	0.135 ±0.0140	0.012 ±0.013
	Apical	3	2.267 ±1.259	0 ± 0	0 ± 0
	Basal		3.178 ±0.759	0.089 ±0.122	0.007 ±0.010

Highlights:

1. A bronchial barrier and a liver biochip were successfully maintained viable and functional in a dynamic coculture setting.

2.

The coculture model is responsive to xenobiotic exposures and emulates active and functional in vitro crosstalk between both tissue constructs.

3.

The lung-liver crosstalk induces a modulation of stress response kinetics, delaying cytotoxicity.

Declaration of interests

☒ 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.

☐ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: