Mesut Demirelli

Véronique Peyre

Juliette Sirieix-Plénet

Natalie Malikova

Jérôme Fresnais

Influence of polycation/cation competitionon the aggregation threshold of magnetic nanoparticles

Keywords: Magnetic Nanoparticles, Polyelectrolyte, electrostatic complexation, Hofmeister series, iron oxide, aggregation, ammonium cations, screening, light scattering

Precise control of the aggregation of magnetic nanoparticles in aqueous environment is a key to their successful application in the biological and medical fields. We investigate here the critical aggregation threshold of PAA-coated maghemite nanoparticles in aqueous solution, in the presence of ionenes, polycations with a regular, tunable and pH-insensitive chain charge density, and a variety of ammonium-based salts. Aggregation of the nanoparticles is followed by a combination of dynamic light scattering and light absorbance measurements, along a dilution pathway from initially dispersed systems at high salt concentration (3M). The position of the aggregation threshold, or critical salt concentration, is found to depend on two parameters: volume of the salt cation (V cation ) and volume of the charged monomer unit of the polycation (V mono + , reflecting the polycation charge density).

Combining these two parameters, a master curve emerges, showing that the critical salt concentration follows in a linear fashion the ratio of the above two volumes, V cation /V mono + .

Interpreted within the theory of Collins and coworkers, the master curve is a clear demonstration of the importance of local ion-pairing in governing the stability of the system, which remains unaccounted for in the continuum electrostatic theories attempting to describe polyelectrolyte complex formation. Moreover, such a master curve provides a very simple rule for the control of interactions between charged polyelectrolytes moderated by salts.

INTRODUCTION

2 Magnetic nanoparticles have many potential applications in medicine as nanoheaters [START_REF] Cazares-Cortes | Recent Insights in Magnetic Hyperthermia: From the "Hot-Spot" Effect for Local Delivery to Combined Magneto-Photo-Thermia Using Magneto-Plasmonic Hybrids[END_REF] , nanoplatforms for active molecule transport [START_REF] Chanana | Using Polymers to Make Up Magnetic Nanoparticles for Biomedicine[END_REF] , and contrast agents in magnetic resonance imaging [START_REF] Peng | Nanostructured Magnetic Nanocomposites as MRI Contrast Agents[END_REF] .

In some specific cases, the small size of the nanoparticles is a strength due to the magnetic monodomain they contain [START_REF] Hugounenq | Iron Oxide Monocrystalline Nanoflowers for Highly Efficient Magnetic Hyperthermia[END_REF] , but this small size (few nanometres in diameter) makes them difficult to manipulate. As such, controlled aggregates can be a way to combine the superparamagnetic properties of single nanoparticles and low magnetic fields needed to manipulate them [START_REF] Yan | Magnetic Nanowires Generated via the Waterborne Desalting Transition Pathway[END_REF] . Aggregates based on electrostatic complexation are promising in this sense, as this assembly process is highly tunable [START_REF] Harada | Chain Length Recognition: Core-Shell Supramolecular Assembly from Oppositely Charged Block Copolymers[END_REF][START_REF] Leclercq | A Physico-Chemical Approach of Polyanion-Polycation Interactions Aimed at Better Understanding the In Vivo Behaviour of Polyelectrolyte-Based Drug Delivery and Gene Transfection[END_REF][START_REF] Lucas | Surface Charge Density of Maghemite Nanoparticles: Role of Electrostatics in the Proton Exchange[END_REF][START_REF] Fresnais | Acrylic Acid)-Coated Iron Oxide Nanoparticles: Quantitative Evaluation of the Coating Properties and Applications for the Removal of a Pollutant Dye[END_REF][START_REF] Fresnais | Nanoparticle Aggregation Controlled by Desalting Kinetics[END_REF][START_REF] Van Der Burgh | Complex Coacervation Core Micelles. Colloidal Stability and Aggregation Mechanism[END_REF][START_REF] Voets | Irreversible Structural Transitions in Mixed Micelles of Oppositely Charged Diblock Copolymers in Aqueous Solution[END_REF][START_REF] Yadel | Hyperthermia Efficiency of Magnetic Nanoparticles in Dense Aggregates of Cerium Oxide/Iron Oxide Nanoparticles[END_REF][START_REF] Ou | Entropy and Enthalpy of Polyelectrolyte Complexation: Langevin Dynamics Simulations[END_REF] and allows modulating not only the structure/density of the final aggregate but also its region of stability.

Maghemite nanoparticles (NP) [START_REF] Massart | Preparation of Aqueous Magnetic Liquids in Alkaline and Acidic Media[END_REF][START_REF] Bee | Synthesis of Very Fine Maghemite Particles[END_REF][START_REF] Lefebure | Monodisperse Magnetic Nanoparticles: Preparation and Dispersion in Water and Oils[END_REF][START_REF] Lucas | Surface Charge Density of Maghemite Nanoparticles: Role of Electrostatics in the Proton Exchange[END_REF] , and their significantly increased colloidal stability through electrosteric repulsion [START_REF] Fresnais | Acrylic Acid)-Coated Iron Oxide Nanoparticles: Quantitative Evaluation of the Coating Properties and Applications for the Removal of a Pollutant Dye[END_REF][START_REF] Biggs | Electrosteric Stabilisation of Colloidal Zirconia with Low-Molecular-Weight Polyacrylic Acid. An Atomic Force Microscopy Study[END_REF] due to a polymer coating [START_REF] Fresnais | Acrylic Acid)-Coated Iron Oxide Nanoparticles: Quantitative Evaluation of the Coating Properties and Applications for the Removal of a Pollutant Dye[END_REF][START_REF] Sehgal | Precipitation-Redispersion of Cerium Oxide Nanoparticles with Poly(Acrylic Acid): Toward Stable Dispersions[END_REF][START_REF] Guibert | Hyperthermia of Magnetic Nanoparticles: Experimental Study of the Role of Aggregation[END_REF] such as poly (acrylic) acid (PAA), have been extensively studied. One route for tuning the stability of coated maghemite nanoparticles (CNPs) in aqueous solution is the addition of oppositely charged polymer chains. In the case of PAA-coated particles at high pH, these polymer chains are polycations (PCs). The underlying phenomenon is the electrostatic complexation between the polyanions forming the NP coating and the free polycations: at low ionic strength, PAA-coated maghemite nanoparticles in the presence of polycations form clusters and aggregates, as a result of attractive electrostatic forces, [START_REF] Fresnais | Nanoparticle Aggregation Controlled by Desalting Kinetics[END_REF] while the same mixtures show remarkable stability at high ionic strength (salt concentrations of 1-3 mol L -1 ), owing to electrostatic screening. [START_REF] Yan | Magnetic Nanowires Generated via the Waterborne Desalting Transition Pathway[END_REF][START_REF] Fresnais | Nanoparticle Aggregation Controlled by Desalting Kinetics[END_REF] One way of exploring the transition from a dispersed to an aggregated state of PAAcoated maghemite particles, in the presence of polycations, is to gradually decrease the ionic strength through a dilution pathway. [START_REF] Fresnais | Nanoparticle Aggregation Controlled by Desalting Kinetics[END_REF] Several groups have shown measurements of this transition for systems with different types of coated nanoparticles [START_REF] Lucas | Surface Charge Density of Maghemite Nanoparticles: Role of Electrostatics in the Proton Exchange[END_REF][START_REF] Fresnais | Acrylic Acid)-Coated Iron Oxide Nanoparticles: Quantitative Evaluation of the Coating Properties and Applications for the Removal of a Pollutant Dye[END_REF][START_REF] Biggs | Electrosteric Stabilisation of Colloidal Zirconia with Low-Molecular-Weight Polyacrylic Acid. An Atomic Force Microscopy Study[END_REF][START_REF] Sehgal | Precipitation-Redispersion of Cerium Oxide Nanoparticles with Poly(Acrylic Acid): Toward Stable Dispersions[END_REF][START_REF] Guibert | Hyperthermia of Magnetic Nanoparticles: Experimental Study of the Role of Aggregation[END_REF] , which occurs at what is referred to as the critical salt concentration. Ion valence is of outmost importance, however moving from monovalent to multi-valent ions usually changes the entire picture. In other words, monovalent and multivalent ions cannot be considered in a continuous scheme. Addition of multivalent salts into dispersions of PAA-coated nanoparticles leads to a rapid and irreversible aggregation of the particles, even in the absence of polycations. Thus, we consider here only monovalent salts and polycations with one charge per monomer.

Understanding electrostatic complexation of oppositely charged polyelectrolytes (PEs) remains a formidable task. These systems are strongly correlated [START_REF] Ou | Entropy and Enthalpy of Polyelectrolyte Complexation: Langevin Dynamics Simulations[END_REF] and described by a large number of parameters. [START_REF] Adhikari | Polyelectrolyte Complex Coacervation by Electrostatic Dipolar Interactions[END_REF][START_REF] Shi | Control over the Electrostatic Self-Assembly of Nanoparticle Semiflexible Biopolyelectrolyte Complexes[END_REF][START_REF] Delorme | Experimental Evidence of the Electrostatic Contribution to the Bending Rigidity of Charged Membranes[END_REF][START_REF] Tóth | Clustering of Carboxylated Magnetite Nanoparticles through Polyethylenimine: Covalent versus Electrostatic Approach[END_REF] Some of the parameters are easy to quantify (polymer concentration, charge density, mixing ratio, molecular weight), while for others an empirical classification has to suffice for now. Among the latter falls clearly the chemical specificity of the ionic groups involved (not only those on the polymer chains, but also the surrounding salt ions) as embodied by the Hofmeister series [START_REF] Kunz | Specific Ion Effects in Colloidal and Biological Systems[END_REF][START_REF] Collins | Why Continuum Electrostatics Theories Cannot Explain Biological Structure, Polyelectrolytes or Ionic Strength Effects in Ion-Protein Interactions[END_REF][START_REF] Salis | Models and Mechanisms of Hofmeister Effects in Electrolyte Solutions, and Colloid and Protein Systems Revisited[END_REF] . This remains to date an empirical ordering of cations/anions and reflects their relative effect on the stability of organic (biological) macromolecules and inorganic colloids in water.

Theoretical approaches to PE complex formation span a wide conceptual range. Firstly, we may distinguish those stemming from continuum electrostatics, which originate in the classical Voorn-Overbeek (VO) mean-field model [START_REF] Spruijt | Reversible Assembly of Oppositely Charged Hairy Colloids in Water[END_REF][START_REF] Spruijt | Binodal Compositions of Polyelectrolyte Complexes[END_REF][START_REF] Salehi | A Molecular Thermodynamic Model of Complexation in Mixtures of Oppositely Charged Polyelectrolytes with Explicit Account of Charge Association/Dissociation[END_REF] , secondly, those based on liquid state theory allowing a more explicit description of charge-charge correlations [START_REF] Limo | Thermodynamic Study of Interactions Between ZnO and ZnO Binding Peptides Using Isothermal Titration Calorimetry[END_REF] and, lastly, those departing from local phenomena of ion pairing, counterion release and solute-solvent interactions [START_REF] Fu | Ion-Pairing Strength in Polyelectrolyte Complexes[END_REF][START_REF] Schlenoff | Ion Content of Polyelectrolyte Complex Coacervates and the Donnan Equilibrium[END_REF][START_REF] Olvera-De-La-Cruz | Precipitation of Highly Charged Polyelectrolytes Solutions in Presence of Multivalents Salts[END_REF] . The latter approach is the more natural for consideration of the chemical specificity of the ionic groups involved.

A great deal of attention has been paid to evaluate the relative weight of the enthalpy and entropy contributions in the process of PE complex formation. Currently, the majority of studies points towards the process being entropically driven, stemming from the release of the polyelectrolyte counterions [START_REF] Ou | Entropy and Enthalpy of Polyelectrolyte Complexation: Langevin Dynamics Simulations[END_REF][START_REF] Adhikari | Polyelectrolyte Complex Coacervation by Electrostatic Dipolar Interactions[END_REF][START_REF] Fu | Ion-Pairing Strength in Polyelectrolyte Complexes[END_REF][START_REF] Schlenoff | Ion Content of Polyelectrolyte Complex Coacervates and the Donnan Equilibrium[END_REF][START_REF] Muthukumar | 50th Anniversary Perspective : A Perspective on Polyelectrolyte Solutions[END_REF][START_REF] Rathee | Role of Associative Charging in the Entropy-Energy Balance of Polyelectrolyte Complexes[END_REF] . Most recently, Schlenoff and collaborators have used isothermal titration calorimetry to systematically evaluate the interactions between oppositely charged polyelectrolytes [START_REF] Salis | Models and Mechanisms of Hofmeister Effects in Electrolyte Solutions, and Colloid and Protein Systems Revisited[END_REF][START_REF] Spruijt | Reversible Assembly of Oppositely Charged Hairy Colloids in Water[END_REF] . They have confirmed that the electrostatic complexation (either coacervation or precipitation) is entropically driven, not only due to the release of counterions but also due to the restructuring of the solvent water molecules. They also provided a hybrid model where both entropic effect and the Donnan pressure can be considered to highlight the role of counterion release in the process of complex formation [START_REF] Schlenoff | Ion Content of Polyelectrolyte Complex Coacervates and the Donnan Equilibrium[END_REF] , through the use of different anions along the Hofmeister series. They were able to establish that the equilibrium constant of the polyelectrolyte pairing was dependent on the nature of the added ions (chaotropic or kosmotropic).

The specificity of the salt ion had an impact on the enthalpic component of the complex formation, while the entropic component was still dominated by the release of counterions and water molecules.

Figure 1.

Schematic representation of the system: spherical maghemite NPs 43 with an anionic PAA brush and polycations, ionenes. [START_REF] Malikova | Aqueous Solutions of Ionenes: Interactions and Counterion Specific Effects as Seen by Neutron Scattering[END_REF] PAA is strongly complexed onto NPs and, at pH=9, leads to a brush layer at full ionisation of the carboxylate groups of PAA. [START_REF] Fresnais | Acrylic Acid)-Coated Iron Oxide Nanoparticles: Quantitative Evaluation of the Coating Properties and Applications for the Removal of a Pollutant Dye[END_REF] Ethylammonium cations and bromide anions are, respectively, the counterions of PAA and ionenes. Added salt is present at a concentration c salt .

In this study we investigate the role of salt ions in screening the interactions between PAA coated maghemite nanoparticles and ionene polycations. Our focus is on the PAA coated maghemite nanoparticles and we work under conditions of excess of ionene polycations. We seek to tune the location of the dispersed-aggregated transition, represented by the critical salt concentration,

[Salt] critical . We start with stable nanoparticle dispersions at a high ionic strength (c salt =3 mol.L -1 ) and use the aforementioned dilution pathway. As the nanoparticle polymer coating is negatively charged under the chosen experimental conditions (pH=9), the primary role in tuning the transition is that of the salt cations and the polycations, as opposed to any anions present. As shall be seen, we have clear evidence that for a common anion (Br-), two different ammonium-based salt cations lead to a very different nanoparticle aggregation threshold. The tuning of the aggregation threshold is thus achieved via a combination of two parameters: (i) the charge density of the ionene polycations and (ii) the nature of the salt cation. The salt cations, as well as the ionene polycations, are all based on charged ammonium centres. Ionene polycations are strong polyelectrolyte chains and thus possess a permanent charge, which is pH-insensitive. Additionally, the interest behind ionene polycations is the strict regularity and possibility of exact tuning of their linear charge density [START_REF] Malikova | Aqueous Solutions of Ionenes: Interactions and Counterion Specific Effects as Seen by Neutron Scattering[END_REF][START_REF] Malikova | On the Crossroads of Current Polyelectrolyte Theory and Counterion-Specific Effects[END_REF] . We arrive at a simple relationship for the tuning of the particle aggregation threshold, which is based on the relative volume of the salt cations and the charged monomer units of ionene polycations.

Abbreviation

Full Name Structure PAA - Poly (acrylic acid)

x-ionene [START_REF] Rembaum | Reactions of N,N,N',N'-Tetramethyl-α,ι-Diaminoalkanes with α,ι-Dihaloalkanes. II. x-y Reactions[END_REF][START_REF] Layman | Synthesis and Characterization of Aliphatic Ammonium Ionenes: Aqueous Size Exclusion Chromatography for Absolute Molecular Weight Characterization[END_REF] (x=3, 6, 9)

x, x-ionene 3-Bz 3,3-p-BzBr (90:10)

PTEA-PAM [START_REF] Fresnais | Nanoparticle Aggregation Controlled by Desalting Kinetics[END_REF] Poly (trimethylammonium ethyacrylate) 11 K -b-poly (acrylamide) 30K PDADMAC [START_REF] Fresnais | Nanoparticle Aggregation Controlled by Desalting Kinetics[END_REF] Poly (diallyldimethylammonium chloride)

EXPERIMENTAL 1.Materials

The following materials were purchased and used without further purification.

Iron Oxide Nanoparticles

The synthesis of bare magnetic nanoparticles (BNP, maghemite, γ-Fe 2 O 3 ) through coprecipitation of Fe II and Fe III in water was established by R. Massart. [START_REF] Massart | Preparation of Aqueous Magnetic Liquids in Alkaline and Acidic Media[END_REF] Their size polydispersity was reduced by size sorting through selective flocculation of the larger particles by increase of the ionic strength. [START_REF] Lefebure | Monodisperse Magnetic Nanoparticles: Preparation and Dispersion in Water and Oils[END_REF] The nanoparticles were characterised by Transmission Electron Microscopy (TEM). The picture analysis showed a log-normal size distribution of median diameter d 0 = 7 nm and a polydispersity index σ = 0.2 (SI 1). Small angle X-ray scattering results are reported in the Supporting Information (SI-2) and confirmed the particle size determined by the former methods.

The concentration of maghemite particles was consistently determined via UV-visible spectroscopy (Section 2.2.2) and atomic absorption spectroscopy (Section 2.2.3). For the first method, a master curve (500-700 nm, Supporting Information, SI 3) was used for dilutions in distilled water and for the second the NP were dissolved in concentrated HCl.

Coating of NP

A coating of poly (acrylic acid) around the maghemite nanoparticles was performed via electrostatic complexation following the precipitation-redispersion process. [START_REF] Sehgal | Precipitation-Redispersion of Cerium Oxide Nanoparticles with Poly(Acrylic Acid): Toward Stable Dispersions[END_REF] The precipitation step is conducted at pH 1.5. During the redispersion step, ethylamine (7 wt %) is used to increase the pH to 9.0, ethylammonium is the counterion for the anionic carboxylate groups of PAA. The PAA-coating is irreversible and stays in place between pH 1.5 and 10.0. The pKa of PAA is 4.5 [START_REF] Michaels | Polyelectrolyte Adsorption by Kaolinite[END_REF][START_REF] Wiśniewska | Influence of Temperature and Purity of Polyacrylic Acid on Its Adsorption and Surface Structures at the ZrO2/Polymer Solution Interface: Adsorption Science & Technology[END_REF] , at pH below 4.5 the functional groups in the polymer chain are predominantly undissociated, while at pH above 4.5 polyanion (PA) dissociation increases and reaches full dissociation close to pH 9.0. Therefore, the experiments involving CNP were carried out at a pH = 9.0. The hydrodynamic diameter of these particles was determined at 39 nm via DLS. The density of PAA on the nanoparticle surface is 2 chains per nm 2 . 18

Ionenes

The synthesis and the characterisation in terms of chain length and charge density of 3-, 6-and 9-ionene were previously reported by Malikova et al. [START_REF] Malikova | Aqueous Solutions of Ionenes: Interactions and Counterion Specific Effects as Seen by Neutron Scattering[END_REF][START_REF] Sakhawoth | Flocculation of Clay Colloids Induced by Model Polyelectrolytes: Effects of Relative Charge Density and Size[END_REF] For the synthesis of 3-Bz, N, N, N', N'-tetramethyl-1,3-propanediamine (20 mmol), 1,3dibromopropane (18 mmol) and 1,4-bis-bromomethyl-benzene (2 mmol) were added into DMF (20 mL) in a round bottom flask. The reaction mixture was stirred and heated at 80 °C. When white precipitate appeared, 10 mL of water was added to the mixture to ensure the solubility of the components. The heating was maintained for 50h. After removal of solvents under reduced pressure, the residual product was dissolved in water and washed with chloroform 3 times. The aqueous phase was collected and the water removed via lyophilisation to give a white solid (80%). The polycations for this study are summarised in Table 1. Based on the structures of these PC and their molar volumes listed by Fedors [START_REF] Fedors | A Method for Estimating Both the Solubility Parameters and Molar Volumes of Liquids[END_REF] , the following charge density classification is made: 3ionene > 3-Bz > PDADMAC > 6-ionene > PTEA-PAM > 9-ionene (SI 4).

Synthesis of EAN

Ethylammonium nitrate (EAN) was synthesised according to procedures described previously. [START_REF] Evans | Micelle Formation in Ethylammonium Nitrate, a Low-Melting Fused Salt[END_REF][START_REF] Mamusa | Microstructure of Colloidal Dispersions in the Ionic Liquid Ethylammonium Nitrate: Influence of the Nature of the Nanoparticles' Counterion[END_REF] Equimolar quantities of nitric acid (68 wt % aqueous solution) in a dropping funnel was added slowly to ethylamine (70 wt % aqueous solution) in a cooling bath at -15 °C. After the completion of addition, some excess ethylamine is added to reach basic pH and ensure completion of the reaction.

The excess ethylamine is then eliminated under reduced pressure. The elimination of the water was achieved via lyophilisation. The residual water content was determined by Karl Fischer coulometric titration to give less than 0.4 wt %. The obtained ionic liquid is a viscous, odourless and transparent fluid with a pH 7.5, measured by the same method as in ref [START_REF] Guibert | Controlling Nanoparticles Dispersion in Ionic Liquids by Tuning the PH[END_REF] .

Sample Preparation

In aqueous solution: The CNP-PC complexes were obtained by mixing stock solutions prepared at the same weight concentration (0.05 wt %) and the same pH (pH 9.0). The mixing of the two initial solutions was characterised by the charge ratio Z = Z +/ Z -, where Z + is the total number of positive charges of the PC in solution and Z -is the total number of negative charges of the CNPs in solution. [START_REF] Fresnais | Nanoparticle Aggregation Controlled by Desalting Kinetics[END_REF] In aqueous salt solution: The individual stock solutions of CNPs and ionenes were prepared at the same weight concentration (0.5 wt %) and pH (pH 9.0) in 3 mol L -1 aqueous salt solution, the molar volumes of salts were included in the calculations of sample preparation (SI 4). The two initial solutions were mixed at a charge ratio Z = Z +/ Z -.

Dilution: For the dilution process, distilled water was added to the CNP-PC mixtures stepwise. The concentration determination of well-dispersed CNPs was followed by UV-absorption spectroscopy (See Section 2.2.2). The size of the objects was determined by dynamic light scattering (See Section 2.2.1). Note that the measured [Salt] critical values were independent of the initial concentrations of CNPs in stock solutions between 0.05 and 0.5 wt %.

Techniques

Dynamic Light Scattering

Dynamic light scattering (DLS) measurements were performed with a MALVERN NanoZS (Malvern Instruments), at λ = 632 nm and backscattering detection at angle θ = 170°. An autocorrelation function is determined by measurements of intensity at different times. The autocorrelation functions of the scattered light are interpreted by using the peaks of the cumulants (Z-average) in order to determine the diffusion coefficient. For this procedure, we approximated that the refractive indices of solutions remained unchanged during the dilution procedure.

The diffusion coefficient D under non-interacting conditions is related to the hydrodynamic radius RH through the Stokes-Einstein equation:

𝐷 = 𝑘 ! 𝑇 6𝜋𝜂𝑅 ! (1)
where kB is the Boltzmann constant, η the solvent viscosity and T the absolute temperature, all experiments were conducted at 298 K.

For the determination of hydrodynamic diameter in aqueous salt solutions, the viscosities of stock salt solutions from 3 to 0 mol L -1 were measured (SI 5 and SI 6) and applied to the size determination via the Stokes-Einstein Equation (Equation 1).

UV-visible-Absorption Spectroscopy

The measurements were conducted on an Avantes Avaspec 2048/Avalight DHC UV-Visible spectrometer. This technique is a simple and quick method and allows to quantitatively determine the concentration for solely well-dispersed nanoparticles solutions by using a master curve from 500 to 700 nm (SI 3) and determine the transition from a dispersed to an aggregated state (SI 7).

Atomic Absorption Spectroscopy (AAS)

The iron concentration of samples was determined by measurements of iron absorbance by Atomic Absorption Spectroscopy (AAS) on a Perkin-Elmer PinAAcle 500 instrument, after the dissolution of NP in concentrated hydrochloric acid and adapted dilutions. The expressed iron concentration [Fe] in mol L -1 converts to the volume fraction using the molar weight and density of maghemite as follows: [START_REF] Lucas | Surface Charge Density of Maghemite Nanoparticles: Role of Electrostatics in the Proton Exchange[END_REF][START_REF]The Iron Oxides[END_REF] The concentration determination was made both via UV-Absorption and AAS and gave identical results. For reasons of simplicity, rapidity and convenience of the method, UV-absorption was used during dilutions. For the characterisation of electrostatic interactions of various mixtures, hydrodynamic size and UV-absorption measurements of samples were performed. As described previously, PAA-coated nanoparticles prepared and stored in water were used for all the mixtures. Their initial hydrodynamic diameter is 39 nm at 0.5 wt % in distilled water at pH 9.0. First, the hydrodynamic diameter of CNPs was measured at various concentrations of different salts to verify their stability without PC (Figure 2). Solutions of CNPs at 0.5 wt % were prepared in aqueous salt solutions of NH 4 Cl, EAN, TMABr and TEABr. The initial aqueous salt solutions (c salt = 3 mol L -1 ; c CNP = 0.5 wt %) were diluted with distilled water step by step and the hydrodynamic diameter was measured, while maintaining an overall pH 9.0. Firstly, for the initial solutions (c salt = 3 mol L -1 ) we do observe an increase of the hydrodynamic diameter of the CNPs depending on the nature of the salt in solution. Only in the case of EAN, do we recover the hydrodynamic radius of CNPs in pure water. Therefore, some partial CNP aggregation takes place in the presence of NH 4 Cl, TMABr and TEABr to begin with. Secondly, and more importantly, for any given salt, the hydrodynamic diameter of CNPs does not change over the whole dilution range indicating that the (partially aggregated) CNPs were stable in the range of 3 down to ~ 0.2 mol L -1 , whatever the nature of the salt. Indeed, despite the presence of high concentration of salts, which in general induces electrostatic screening between charges on the PAA chains and hence a collapse of the polymer brush [START_REF] Akkilic | 5 -Responsive Polymer Brushes for Biomedical Applications[END_REF][START_REF] Ballauff | Polyelectrolyte Brushes[END_REF] , here the CNPs were well dispersed due electrostatic and steric repulsion. Hydrodynamic diameter of aggregates in mixtures of ionenes (3-, 6-and 9-ionene) and CNPs as a function of the charge ratio Z (Z = Z +/ Z -) are shown in Figure 3. Z is the charge ratio between the positively charged PCs (Z + ) and the negatively charged CNPs (Z -). At Z = 1, we expect and indeed observe the largest aggregates (340-570 nm) in case of all ionenes, a stoichiometric compensation of the opposite charges is achieved at this charge ratio. For very small values of Z (Z <<1, excess of CNPs) we approach the hydrodynamic size of CNPs at 0.05 wt % in distilled water (measured as 22 nm). In this low-ionene limit, the majority of CNPs are well dispersed. For large values of Z (Z>>1, excess of ionenes) the hydrodynamic size oscillates between 70-160nm. Thus more extended aggregation is observed in this high-ionene limit, due to co-assembly of available CNPs with the PCs. [START_REF] Fresnais | Nanoparticle Aggregation Controlled by Desalting Kinetics[END_REF] For the study of CNP-ionene mixtures in salty aqueous solutions, we chose to work at a slight excess of ionenes (Z slightly greater than 1) to ensure that all CNPs are involved in aggregation but at the same time avoiding the Z=1 region, for which size determination by DLS becomes very inaccurate due to large aggregates formed.

Nuclear Magnetic Resonance

RESULTS AND DISCUSSION

Coated maghemite nanoparticles in the presence of polycations in salty aqueous solutions

To study the electrostatic interactions between coated maghemite nanoparticles and ionene polycations in the presence of different salts, mixtures of CNPs and 3-, 6-and 9-ionenes were prepared at charge ratios Z (Z = Z + /Z -) of 5.1, 4.1 and 3.4 respectively, in 3 mol L -1 aqueous salt solutions of NH 4 Cl, EAN, TMABr and TEABr in turn. These concentrated mixtures were then diluted stepwise with distilled water and characterized with DLS (Figure 4). To verify the dispersion state of the CNPs during the dilution process, the hydrodynamic diameter was compared to the values in The results allow to state that [Salt] critical values depend strongly both on the charge density of the ionene polycation and the nature of the salt present. For all four salts, [Salt] critical increases with increasing polycation charge density (even for modified I3-Bz ionene, SI 8). Regarding the nature of salt, we suggest it is the nature of the salt cation, as opposed to the salt anion, that is crucial in tuning the [Salt] critical . In line with this, note that for TMABr and TEABr salts (different cations with the same bromide anion), the [Salt] critical values increase significantly for all ionenes. In the mixtures, there is an excess of the ionenes (Z>1) and thus we depart from the picture of salt cations and positive charges on the polycation competing for the complexation with the negatively charged PAAcoated maghemite nanoparticles. The four salt cations considered are all based on an ammonium ion with a varying degree of hydrophobicity, according to the number and length of alkyl chains attached to the ammonium ions. In a simple first approach, these ions can be ordered in terms of their size, which reflects in broad terms also their hydration enthalpy and thus is a pertinent parameter to consider, as shall be seen from the following discussion. In Figure 5, the [Salt] critical values are plotted as a function of the molar volumes of the four salt cations (V cation , SI 4), determined on the basis of Millero et al. [START_REF] Millero | Molal Volumes of Electrolytes[END_REF] This representation highlights two facts: (1) for a given PC charge density, mixtures with larger salt cations feature higher values of [Salt] critical , in other words, the transition from a dispersed to an aggregated state along the dilution pathways occurs earlier; (2) mixtures with larger salt cations are increasingly more sensitive to the PC charge density. Overall, the worst combination for maintaining a well-dispersed CNP-PC mixture is the use of a salt with large cations in the presence of a densely charged PC. We go now a step further and combine the two parameters (volume of salt cation and PC charge density), in search of a simple relationship to [Salt] critical . If salt cation size (or volume) seems to be a critical parameter, the charge density of the polycation can be reconsidered in terms of a volume. For ionene polycations with tunable and perfectly regular charge distribution along the chain backbone [START_REF] Malikova | Aqueous Solutions of Ionenes: Interactions and Counterion Specific Effects as Seen by Neutron Scattering[END_REF][START_REF] Malikova | On the Crossroads of Current Polyelectrolyte Theory and Counterion-Specific Effects[END_REF] , it is trivial to express the charge density in terms of a volume per unit of charge (denoted here V mono + ). The higher the chain charge density, the lower the volume per unit of charge.

In Figure 6, [Salt] critical is expressed as a function of the ratio V cation /V mono + and a simple linear dependence emerges. For completeness, the best linear fit of the entire data set leads to [Salt] critical plotted as a function of the ratio V cation /V mono + for all data presented in Figure 5 ("x-ionene" data series), together with additional data from supplementary measurements and the literature.

[𝑆𝑎𝑙𝑡] !"#$#!%& = 1.2 ! !"#$%& ! !!"! !
[Salt] critical is the critical salt concentration at which aggregation of PAA-coated nanoparticles in the presence of polycations appears, V cation is the molar volume of salt cations and V mono + is the average molar volume per charged monomer unit of the polycation (SI 4 and SI 9). The dashed line is the linear fit of all the [Salt] critical values for ionenes.

The master curve in Figure 6 seems to be a very simple tool for designing a system with a desired

[Salt] critical . The critical salt concentration is proportional to the ratio of volumes of the salt cation and the polycation monomer. The passage of the master curve through the origin corresponds to the zero salt case, for which an aggregated state is observed. In order to understand the appearance of the master curve we start with a picture based on the competition of these two species in the complexation with the PAA brush of the CNPs. The reason why the size of the monomer is considered here is related to the high salt concentration, for which no Debye Length can be defined (smaller than the size of a single atom). Although the structure of the polymer chain has a role on the particle aggregation, the local interactions are considered at the level of the monomer unit.

Let us consider at first the effect of size and hydration of salts, as Collins and coworkers did [START_REF] Collins | Why Continuum Electrostatics Theories Cannot Explain Biological Structure, Polyelectrolytes or Ionic Strength Effects in Ion-Protein Interactions[END_REF][START_REF] Collins | Charge Density-Dependent Strength of Hydration and Biological Structure[END_REF] . The point developed in Collins's models derives from the impossibility to describe the local effect of salt from classical mean field theory, and especially at high salt concentration. Collins uses the classical ordering of salts dealing with their ability to balance preferentially their interactions with water molecules (kosmotropes) or with their counterion (chaotropes). Further, Collins reports on the affinity for two oppositely charged kosmotropes to make ions pairs, as well as two chaotropes, but low probability of ion pairing for chaotrope-kosmotrope pairs. These considerations are at the origin of the phase separation in mixture of two anions (Cl -and TFSi -) sharing the same cation (Li + ) [START_REF] Dubouis | Chasing Aqueous Biphasic Systems from Simple Salts by Exploring the LiTFSI/LiCl/H 2 O Phase Diagram[END_REF] .

Kosmotropes are described as small highly charged ions (carboxylate anions, characterized by a molar volume of 43.6 cm 3 .mol -1 , in the present system) and chaotropes as large and weakly charged ions (ammonium cations in the present system). [START_REF] Collins | Why Continuum Electrostatics Theories Cannot Explain Biological Structure, Polyelectrolytes or Ionic Strength Effects in Ion-Protein Interactions[END_REF] Considering our system, the cations are ranging from NH 4 + to TEA + , with increasing size ranging from NH4 + (17.86 cm 3 .mol -1 ), EA + (52.94 cm 3 .mol -1 ), TMA + (89.57 cm 3 .mol -1 ), and to TEA + (149.12 cm 3 .mol -1 ), thus a more pronounced chaotropic behavior. The determination of the Jones-Dole viscosity B coefficient was achieved from the variation of viscosity of salt solution, for salt concentration ranging from 0.5 mol.L -1 to 3 mol.L -1 (figure SI-10).

The global trend shows that the larger the size of the counterion, the larger the Jones-Dole viscosity B coefficient, which confirms the increasingly chaotropic behavior along the above cation series.

Our conceptual picture behind the representation in Figure 6, is based on salt cations competing with monomer units of the polycation for complexation to the PAA-coating and the relative volumes of the two competitors are being decisive. The V cation /V mono + ratio is an important parameter if we consider it under the ion-pairing scheme proposed by Collins and coworkers 32 :

oppositely charged species with similar sizes have a stronger ion binding tendency than those with dissimilar size. Under the Collins scheme, carboxylate ions are considered as small kosmotropic anions and thus of favourable ion pairing with the small cations of the series considered, NH 4 + and EA + , and less so as the V cation increases, case of TMA + and TBA + . The ratio V cation /V mono + thus reflects the relative ion-pairing tendency of the PAA carboxylate group with the salt cation on one side and the monomer of the polycation on the other: the higher the V cation /V mono + ratio, the more favourable is the "polycation-PAA" pairing relative to "salt cation-PAA" pairing. The more favourable the polycation-PAA pairing, the wider is the salt concentration range over which aggregation is seen, i.e.

the higher is the critical salt concentration or the aggregation threshold. For a given salt type, the increase of the polycation (ionene) monomer size (from 3-ionene to 9-ionene) leads to a decrease in

[Salt] critical , as the ionene-PAA pairing is increasingly unfavorable.

At this point, it is interesting to link the above picture with that developed by Schlenoff et al [START_REF] Schlenoff | Ion Content of Polyelectrolyte Complex Coacervates and the Donnan Equilibrium[END_REF] , based on salt ions "doping" a polycation-polyanion complex and ultimately dissolving the complex for high degrees of such doping. By using the Collins concept based on "matching" ion sizes, we have constructed a way to express the degree of "doping" by salt ions in the form of the V cation /V mono + ratio. As V cation /V mono + increases (moving along a horizontal line from left to right in Figure 6) the degree of salt doping decreases, polycation-polyanion complexation is more favourable and a transition from a dispersed to an aggregated state is observed. From the Schlenoff point of view, it is more natural to consider a decreasing V cation /V mono + (right to left in Figure 6), which is accompanied by an increase in salt "doping" and a transition from a polyelectrolyte complex (called here "aggregated" state) to a dissolved polyelectrolyte complex (here "dispersed" state) is observed.

3

CONCLUSIONS

In conclusion, we provide here a simple tool for tuning the dispersed-aggregated transition in salty aqueous solutions of PAA-coated NPs in the presence of polycations. This tuning is achieved via a combination of two parameters: volume of the salt cation (V cation ) and volume of the charged monomer unit of the polycation (V mono + , reflecting the polycation charge density). The critical salt concentration, corresponding to the dispersed-aggregated transition of the NPs, follows in a linear fashion the ratio of the above two volumes, V cation /V mono + . The higher the V cation /V mono + ratio, the narrower the region of dispersed NP state at high salt concentration, i.e. the earlier take place the onset of aggregation as the system is diluted from the high salt concentration limit. In other words, the worst combination for maintaining a well-dispersed NP-PC mixture over a wide salt concentration range is the use of a salt with large cations in the presence of a densely charged polycation (small V mono + ). The master curve was built from a data set of PAA-coated maghemite nanoparticles in the presence of linear polycations (ionenes) and salt cations both based on ammonium charged centres, supplemented by a few neighbouring systems. A generalisation of the master curves to other types of cations and polycations necessarily involves consideration going beyond that of ion size, and their position in the Hofmeister series is the best existing, albeit empirical, starting point.

Phenomena at high salt concentrations are at play and the conceptual picture rationalizing the observed master curve focuses on local ion-pairing tendencies of the species involved. The central idea is that salt cations and polycation monomers compete in the complexation with the polyanion brush on the nanoparticles. The originality comes from using the Collins concept based on "matching" ion sizes, to express, via the V cation /V mono + ratio, the relative ion-pairing tendency of the polyanion group with the salt cation on one side and the monomer of the polycation on the other.

The higher the V cation /V mono + ratio, the more favourable is the "polycation-PAA" pairing relative to "salt cation-PAA" pairing. This simple concept rationalizes the ensemble of the observed aggregation thresholds and highlights the importance of local ion-pairing phenomena in polyion complexation, governing here the aggregation of polymer-coated nanoparticles. Our approach leads successfully to a master curve predicting the phase diagram of polymer-coated magnetic nanoparticles.

1 H

 1 NMR spectra were recorded on a Bruker Advance 300 MHz spectrometer in D 2 O. The following abbreviations were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, br = broad.

2. 1 Figure 2 .

 12 Figure 2. Hydrodynamic diameters of CNP in different aqueous solutions of NH 4 Cl, EAN, TMABr and TEABr following a dilution pathway from 3 to ~0.2 mol L -1 at a constant pH of 9.0. The size of CNP in pure water is marked as a Blue Cross. The initial concentration of CNPs in solution was 0.5 wt %. The

58

 58

2. 2 Figure 3 .

 23 Figure 3. Hydrodynamic diameter of aggregates in mixtures of x-ionene (x=3, 6, 9) and CNPs as a function of the charge ratio Z = Z +/ Z -at pH 9.0.

Figure 2 ,

 2 Figure 2, which features the CNP behaviour in the respective aqueous salt solutions in the absence of polycations.

Figure 4 .

 4 Figure 4. Dependence of the hydrodynamic diameter of CNPs in their mixtures with x-ionene (x=3, 6, or 9) PCs as a function of the salt concentration, for four different salts: NH 4 Cl, EAN, TMABr andTEABr. CNPs and PCs were prepared at initial concentrations of 0.5 wt % at charge ratios Z (Z = Z + /Z -) 5.1, 4.1 and 3.4, respectively. The initial salt concentration was 3 mol L -1 in each case, the concentration was gradually decreased through a dilution process with distilled water, while pH was kept constant at 9.0.

Figure 5 .

 5 Figure 5. Critical salt concentration ([Salt] critical ) values for mixtures derived from Figure 4 as a function of molar volume of salt cations (V cation ). Transition values for 3-(full line), 6-(dotted line) and 9-ionene (dashed line) in different aqueous salt solutions: NH 4 Cl (blue triangle), EAN (black round), TMABr (green diamond) and TEABr (red square).

. 5 Figure 6 .

 56 Figure 6.[Salt] critical plotted as a function of the ratio V cation /V mono + for all data presented in Figure5

Table 1 .

 1 Abbreviations, full names and molecular structures of the polyanion PAA and polycations studied.

  Hz,4H, n.2H, -N + -CH 2 -CH 2 -CH 2 -N + ),4.61 (s, 4H, N + -CH 2 -C Harom ), 7.66 (s, 4H arom ,),

	1 H
	NMR (D 2 O, 300 MHz,) δ (ppm) 2.37 (br, n.2H, -N + -CH 2 -CH 2 -CH 2 -N + ), 3.18 (s, n.12H, CH 3 N + ), 3.49 (t,
	J=8,1

Table 2 .

 2 Critical Salt concentrations ([Salt] critical ) for mixtures of CNPs with ionene PCs in different aqueous salt solutions, extracted from data in Figure 4.

	[Salt] critical (mol L -1 )	NH 4 Cl	EAN	TMABr	TEABr
	3-ionene	0.54 ± 0.01	0.55 ± 0.01	0.90 ± 0.01	1.85 ± 0.05
	6-ionene	0.48 ± 0.01	0.48 ± 0.02	0.83 ± 0.02	1.18 ± 0.03

Acknowledgements

Authors wish to acknowledge Yasmina Bouharicha for her assistance during benzylic ionene synthesis and Aude Michel-Tourgis for the synthesis of iron oxide nanoparticles.


Mattia ; Pasqua
email: mattia.pasqua@utc.fr

Ulysse Pereira
email: ulysse.pereira@utc.fr

Claire De Lartigue
email: claire.de-lartigue@utc.fr

Jonathan Nicolas
email: jonathan.nicolas@etu.utc.fr

Pascale Vigneron
email: pascale.vigneron@utc.fr

Quentin Dermigny
email: quentin.dermigny@utc.fr

Cécile Legallais

Cecile Legallais
email: cecile.legallais@utc.fr

Preclinical characterization of alginate-Poly-L-Lysine encapsulated HepaRG for extracorporeal liver supply

Keywords: Acute liver failure, Bioartificial liver, Liver tissue engineering, Bioreactor, Organoid Acute liver failure, bioartificial liver, liver tissue engineering, bioreactor, organoid

Introduction

Acute live failure (ALF) is a devastating syndrome involving severe injury to liver cells. It affects previously healthy individuals, and has a frequently fatal outcome [START_REF] Keeffe | Acute liver failure[END_REF]. Following diagnosis of ALF, the optimal treatment is orthotopic liver transplantation (OLT) needed within a few days, making the availability of donor organs far lower than the demand [START_REF] Van De Kerkhove | Liver support therapy: An overview of the AMC-bioartificial liver research[END_REF]. It is therefore urgent to develop new approaches capable of improving the management of ALF. In this sense, artificial livers (ALs) have been proposed as temporary therapy with purely detoxification functions. However, while analysis of clinical studies showed improvements in patients' clinical pictures, there was no such improvement in their survival [START_REF] Kandiah | Extracorporeal devices[END_REF]. On the other hand, bioartificial livers (BALs) also provide metabolic and synthetic activities, which could better replace the liver functions as extracorporeal therapy. The main goal of this therapy is either to allow the self-regeneration of the diseased liver, thus avoiding OLT and its long-term complications, or to serve as a bridge until a donor organ becomes available.

In the context of BALs, the bioreactor represents one of the key components as it hosts the active biomass. In general, a bioreactor is a volume where biochemical and biological processes take place [START_REF] Catapano | Bioreactors for liver tissue engineering[END_REF]. A bioreactor should provide cells with adequate amounts of oxygen and nutrients, favour mass transfer between cells and fluids (the blood or plasma of the patient in ALF), and support the maintenance of cell viability and functions. To meet these requirements, the best option consists in designing a system in which convection (i.e. flux) from and to the plasma or surrounding medium takes place. Nevertheless, direct contact and shear stress may be harmful to the cells. Encapsulation of the biomass in semi-permeable hollow fibres or porous beads is thus often adopted so as to protect it from the hydrodynamic shear stress imposed by the flux [START_REF] Rebelo | HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism[END_REF] while simultaneously preserving cell activity [START_REF] Pasqua | HepaRG Self-Assembled Spheroids in Alginate Beads Meet the Clinical Needs for Bioartificial Liver[END_REF], and promoting cell-cell interaction, thus enhancing hepatic-specific functions [START_REF] Elkayam | Enhancing the drug metabolism activities of C3A -a human hepatocyte cell line-by tissue engineering within alginate scaffolds[END_REF].

The fluidized bed bioreactor hosting alginate beads previously developed in our group ( [START_REF] Selden | A clinical-scale BioArtificial Liver, developed for GMP, improved clinical parameters of liver function in porcine liver failure[END_REF]Zhou et al., 2016) with HepG2 cells or primary porcine hepatocytes. However, with a very active biomass, such as HepaRG spheroids (6), we recently observed a phenomenon of severe deterioration of the alginate beads in the bioreactor, when they were exposed to a human equivalent pathological plasma medium (containing some ALF toxins), leading to an unacceptable loss of biomass and the associated risk of cells' release to the patient. This phenomenon has already been noted by other authors [START_REF] Simó | Research progress in coating techniques of alginate gel polymer for cell encapsulation[END_REF] and was attributed to the presence of Ca 2+ chelators (such as lactate) or anti-gelling cations (Na + and Mg 2+ ) in the surrounding media. For safety reasons, reinforcement of the alginate beads by means of an external coating becomes essential when using encapsulated HepaRG or other very active hepatocyte constructs in view of clinical application.

Several types of coatings have been proposed to date in the literature [START_REF] De Vos | Effect of the alginate composition on the biocompatibility of alginate-polylysine microcapsules[END_REF][START_REF] Edwards-Lévy | Serum albumin-alginate coated beads: Mechanical properties and stability[END_REF][START_REF] Joly | Survival, proliferation, and functions of porcine hepatocytes encapsulated in coated alginate beads: a step toward a reliable bioartificial liver[END_REF][START_REF] Simó | Research progress in coating techniques of alginate gel polymer for cell encapsulation[END_REF]. They are often developed to formulate capsules, whose core is liquid in contrast to beads. These techniques involve either ionic or covalent [START_REF] Dusseault | Microencapsulation of living cells in semi-permeable membranes with covalently cross-linked layers[END_REF][START_REF] Lou | Fabrication of stable galactosylated alginate microcapsules via covalent coupling onto hydroxyl groups for hepatocytes applications[END_REF][START_REF] Simó | Research progress in coating techniques of alginate gel polymer for cell encapsulation[END_REF] interactions between the surface of the alginate gel and the coating materials. In particular, ionic cross-linking coating of the beads can be performed thanks to the alginate's ability (with a negatively charged surface) to form strong electrostatic complexes with polycations [START_REF] Simó | Research progress in coating techniques of alginate gel polymer for cell encapsulation[END_REF]. Of the latter, the natural polymers Poly-L-lysine (PLL) and Poly-L-ornithine (PLO) are most frequently used to reinforce the mechanical stability of the alginate beads [START_REF] Bhujbal | Factors influencing the mechanical stability of alginate beads applicable for immunoisolation of mammalian cells[END_REF][START_REF] Du | Cell Microencapsulation for Tissue Engineering and Regenerative Medicine[END_REF][START_REF] Goosen | Optimization of microencapsulation parameters: Semipermeable microcapsules as a bioartificial pancreas[END_REF][START_REF] Simó | Research progress in coating techniques of alginate gel polymer for cell encapsulation[END_REF].

However, to our knowledge, it has never been applied in the context of an external bioartificial liver application. On the other hand, an external coating modifies the permeability of the beads without heavily affecting the cells' metabolic performances and viability, as observed by Capone et al. using encapsulated HepG2/C3A in alginate-PLL beads [START_REF] Capone | Impact of Alginate Composition: From Bead Mechanical Properties to Encapsulated HepG2/C3A Cell Activities for In Vivo Implantation[END_REF].

Therefore, in this study we proposed analysing the impact of a PLL coating on alginate beads' physical and mechanical properties, and its role on encapsulated HepaRG cells' metabolic performances. The encapsulated biomass was then transferred to a perfused bioreactor to mimic

Materials and Methods

2D HepaRG cell culture

HepaRG cells from Biopredic (Rennes, France) were expanded into two-dimensional (2D) monolayers following the indications given by the supplier. The cells were passaged every 2 weeks until passage 18, with HepaRG proliferation culture medium (William's E-WE-, with sodium bicarbonate, without L-glutamine and phenol red, Sigma-Aldrich, with added Biopredic 710 proliferation media) replenished three times a week. The cultures were maintained in a humidified environment at 37 °C, 5 % CO 2 . Cells were then detached with trypsin-EDTA 0.25 % (ThermoFisher scientific) from the culture flasks and used for the encapsulation process.

Alginate microencapsulation and PLL coating

The cells were mixed in sodium alginate (Manucol-LKX from FMC BioPolymer, Guluronic / Mannuronic ratio 30-70, viscosity Cps(1 %) 60-170) 1.5 % (w/v), sterilized with successive filtrations (0.8, 0.45, and 0.22 µm pore size membrane filters). The encapsulation was performed using a homemade system based on a coaxial airflow extrusion method [START_REF] Gautier | Impact of alginate type and bead diameter on mass transfers and the metabolic activities of encapsulated c3a cells in bioartificial liver applications[END_REF]. Briefly, the alginate solution (1mL) containing cells (5 million) was rapidly extruded through a 24 G nozzle and the droplets fell into a gelation bath (NaCl 154 mM, HEPES 10 mM, CaCl 2 115 mM, pH 7.4). This small volume ensured both even distribution of the cells throughout the beads and the absence of empty beads. The droplets produced were left to settle for 15 min in the gelation bath to ensure gel formation.

Then, the microbeads were washed three times in WE medium and resuspended in HepaRG proliferation culture medium. At day 1 post-encapsulation, a PLL coating (Poly-L-lysine hydrobromide, molecular weight 30,000-70,000, Sigma-Aldrich) was added to the beads, relying on the protocol developed by [START_REF] Khanna | Generation of alginate microspheres for biomedical applications[END_REF] with few modifications. Briefly, alginate beads containing cells were washed three times in WE medium, resuspended in a 0.1 % PLL solution and then transferred to culture dishes in continuous orbital shaking (60 rpm), for 30 minutes. After that, the beads were washed again 3 times with WE medium. Finally, they were incubated with proliferation culture medium and transferred to culture dishes in F o r P e e r R e v i e w 6 continuous orbital shaking in a humidified environment at 37 °C, 5 % CO 2 until day 14. The HepaRG proliferation culture medium was replaced every 2 days.

Culture setups and experimental design

The hepatic biomass was tested in two different culture conditions (shaken and perfused), shown in Figure 1A. Experiments designed to assess the cells' metabolic performance are detailed in Figure 1B.

At the end of each experiment, microbeads were recovered and assessed for cell viability. The quantity of marker analysed was calculated and normalized by the number of hours of incubation and the number of cells seeded. Moreover, in order to avoid artefacts, all the metabolic activities tested were normalized by the quantity present at time zero and by carrying out controls with empty microbeads.

Any influence of the empty beads on the cellular activities assessed was therefore corrected during analysis of the results.

2.4.2.Albumin synthesis

Albumin synthesis was quantified using the ELISA test (Human Albumin ELISA kit, Bethyl Laboratories, Inc.). Analyses were performed in conformity with the manufacturer's instructions.

2.4.3.ICG assay

The indocyanine green (ICG, Sigma-Aldrich) assay was carried out as previously described [START_REF] Pasqua | HepaRG Self-Assembled Spheroids in Alginate Beads Meet the Clinical Needs for Bioartificial Liver[END_REF]. Briefly, beads were incubated with ICG 1 mg/mL in proliferation culture medium for 30 minutes at 37 °C and 5 % CO 2 to favour the uptake of ICG by the transporters OATP1B3 (solute carrier organic anion transporter family member 1B3) and NTCP (sodium/taurocholate cotransporting-polypeptide) [START_REF] Graaf | Transporters involved in the hepatic uptake of and indocyanine green Tcmebrofenin[END_REF]. ICG was then removed by washing with WE five times and the microbeads were incubated with proliferation culture medium for 1 hour to study the release of ICG by the transporters MDR3 (multidrug-resistance protein 3) and MRP2 (multidrug-resistanceassociated protein 2) [START_REF] Cusin | Hepatocyte Concentrations of Indocyanine Green Reflect Transfer Rates Across Membrane Transporters[END_REF]. The quantity of ICG released was obtained by measuring its absorbance (at 820 nm) based on a standard curve generated from a solution containing ICG 1 mg/mL.

2.4.4.EROD assay

The activity of the cytochrome P450-1A1/2 (CYP1A1/2) was studied with the ethoxyresorufin-Odeethylase (EROD) assay as previously described [START_REF] Diaz | Basolateral and canalicular transport of xenobiotics in the hepatocyte: A review[END_REF][START_REF] Pasqua | HepaRG Self-Assembled Spheroids in Alginate Beads Meet the Clinical Needs for Bioartificial Liver[END_REF]. The microbeads were incubated in ethoxyresorufin (10 μM), prepared in WE, for 1 hour at 37°C. The substrate included salicylamide (3 mM) + dicumarol (40 µM) in order to inhibit phase II enzymes. The substrate was converted into resorufin then measured in the supernatant by spectrometry (excitation wavelength of 535 nm and emission wavelength of 595 nm, Spectafluor Plus, TECAN). The standard curve was generated with a solution containing exogenous resorufin (10μM).

2.4.5.Ammonia and lactate quantification

Supernatants of test medium and pathological plasma medium were collected and analysed with Indiko™ (Thermofisher) to determine the concentration of ammonia and Lactate Assay Kit (Sigma-Aldrich) to measure the concentration of lactate, following the indications reported by the supplier.

Physical and mechanical characterization of empty alginate microbeads

2.5.1.PLL distribution in the alginate beads

In order to visualize the distribution of the PLL on the empty alginate beads (i.e. without cells, produced using the same protocol previously presented), PLL staining with rhodamine was performed.

Firstly, we prepared an NHS-Rhodamine (Thermofisher) solution in DMSO at concentration 50 mg/mL and a solution of sodium bicarbonate at 8.3 mg/mL in double distilled H 2 O, pH 8.2. PLL was solubilized in the sodium bicarbonate solution at a final concentration of 0.1 %w/v, containing NHS-Rhodamine solution at a final concentration of 500 µg/mL. In order to favour the conjugation between the NHS-Rhodamine and epsilon-NH 2 of lysine residues, the solution was incubated in the dark for 3 hours at room temperature. The control solution did not contain PLL. After that, the PLL coating was achieved by putting the beads in contact with this solution, according to the protocol already described. The beads were finally washed 3 times in DPBS and visualized by confocal microscopy.

.Mechanical characterization

The elastic modulus of the outer surface of the empty beads was measured by microindentation (ChiaroOptics 11, Amsterdam, Netherlands). The probes used for surface indentation had a radius of around 25 µm and a spring constant of 0.5 N/m and 5 N/m, for pure alginate beads and PLL coated ones, respectively. Before testing, the optical sensitivity and the geometrical factor were calibrated by indenting a hard surface (e.g. glass slide). Alginate beads were deposited on a glass slide and then immersed in proliferation culture medium. The probe was placed in contact with a single bead and a maximal indentation of 3 µm was applied. All experiments were performed at room temperature. For each condition, about 15-20 curves (load vs indentation depth) were acquired. Data were analysed using DataViewer 2.2 software (Optics 11, Amsterdam, Netherlands) and the Hertzian theory, which provided the elastic modulus of the indented area.

2.5.3.Bead permeability

The permeability of empty alginate beads, with and without a PLL coating, was studied by putting them in contact with fluorescent molecules of different molecular weights and sizes on day 13 postencapsulation process, in orbital agitation. On day 14, the beads were washed once in WE medium and visualized using confocal microscopy.

Table 1

Statistical analysis

All the results presented were obtained from at least three independent cultures (N ≥ 3). The statistical analysis was performed using GraphPad InStat v.3.10. Unpaired data were subjected to a Mann Whitney test or Dunn's Multiple Comparisons Test, with a 95 % confidence level considered significant. P values are presented as follows: no stars: p > 0.05; **p < 0.01; ***p < 0.001.

Results

Physical and mechanical characterization of the PLL coated beads

This step was performed with empty beads that were produced following the same protocol as that exposed in the Materials & Methods section for cell encapsulation. On day 1, after PLL coating, a variation in the sphericity of the beads was visible (Figure 2A), due to a shrinking effect. The diameter of the beads decreased by about 20 %, from 949 ± 49 µm postencapsulation to 785 ± 25 µm after PLL coating. The diameter of the beads, with or without a PLL coating, then remained stable until the end of the experiment.

Phase contrast and confocal microscopy clearly showed that PLL was distributed only on the surface of the alginate beads, causing a slight morphological variation on their periphery (Figure 3). The thickness of the external coating was around 14.4 ± 6.4 µm.

3.1.2.Mechanical properties of the PLL layer

The mechanical properties of the surface of single empty beads were assessed by microindentation.

The results showed a significant increase in the elastic modulus of the external area of the beads coated with PLL, 6.6 times higher in comparison with the pure alginate condition (Figure 2B), ensuring surface mechanical reinforcement and thus better stability.

Figure 2

3.1.3.Bead permeability

The porosity of empty alginate beads with or without a PLL coating was evaluated by means of exposure to different fluorescent markers of increasing size (Table 1). The microscope observations (Figure 3) did not show any measurable differences between either condition. Albumin, IgG, and 41nm diameter NPs were able to penetrate the core of the beads. Larger NPs were fully retained entirely outside the beads, and did not colour their inner part green. The cut-off threshold for both conditions was thus between 41nm and 103nm.

Figure 3

Viability of encapsulated cells over 14 days

The production of alginate beads with homogeneous cell distribution was achieved with success and no empty beads were visible after the cell encapsulation process. We estimated approximately 1100 beads per encapsulation batch (1 mL of alginate solution) by counting them with the optical microscope. As the initial biomass was 5 million cells/mL of alginate, we calculated approximately The number of viable hepatocytes (Figure 4, in green) within the microbeads remained high over time, as very few dead cells (Figure 4, red stained nuclei) were observed, in comparison with the negative control where cells were killed by ethanol 70 % exposure. The PLL coating did not seem to affect cell viability. Therefore, neither the long-time culture nor the coating process had any significant impact on cellular viability. Qualitatively from day 1 to day 14, a phenomenon of cell-cell aggregation likely occurred, visible through the incorporation of Calcein-AM by living cells. We observed very similar results in beads without a PLL coating (data not shown). It should be noted that, once the PLL coating was present, the beads became opaque making the encapsulated cells difficult to visualize under the light microscope.

Figure 4

Albumin production is considered to be a quality marker for the hepatic biomass in a bioreactor (Van Wenum et al., 2014). We therefore evaluated the secretion rate of albumin in order to quantitatively determine biomass functionality over the 14 days of culture (Figure 1, Supplementary data). Albumin secretion rate increased from 0.20 ± 0.09 and 0.17 ± 0.05 µg/h/10 6 at day 1 for the conditions without and with PLL respectively, to 0.49 ± 0.15 and 1.01 ± 0.21 µg/h/10 6 at day 14. This increase was significant for the two weeks of culture (p<0.01) when the PLL coating was present.

Metabolic activity of cells on day 14

To investigate the impact of the PLL coating on the cells' metabolic performance, we designed culture media with increasing complexity in their composition (Table 2), in order to progressively mimic a clinical condition of ALF human plasma.

Table 2

The first batch of encapsulated cells was first evaluated in the proliferation culture medium (Table 3).

In the presence of the PLL coating, there was a slight tendency for metabolic activities to decrease, but this was only significant for CYP1A1/2, 3.5 times lower (p<0.01). However, even in the presence of a PLL coating, the cells remained metabolically active. The same biomass was then used in test medium (Table 3) to measure the rate of albumin production, and the ability of cells to detoxify typical ALF toxins (ammonia and lactate elimination rates). The metabolic activities measured remained equivalent between both conditions (PLL coated vs uncoated).

The second batch of biomass was tested on the equivalent pathological plasma medium, cultivating encapsulated cells in shaken culture and in a perfused dynamic bioreactor (PDB), the biological component of the BAL developed in our laboratory. The aim was to compare the two culture setups and above all to reproduce BAL therapy, using an ALF-human plasma model and a perfusion system.

Only the condition "alginate beads with PLL coating" could be exploited with this medium, as without PLL we observed severe bead deteroration.

Importantly, before testing in an equivalent pathological medium, the biomass was exposed to an equivalent plasma medium (HepaRG proliferation culture medium supplemented with 70 g/L bovine serum albumin-BSA-, to mimic healthy human plasma in a total protein concentration), in order to reproduce the BAL priming with a view to clinical therapy. After that, cells were exposed to the equivalent pathological plasma medium for 6 hours.

The ability of the cells to produce albumin and detoxify these toxins is shown in Figure 5. While the albumin secretion rate (Figure 5A) was favoured in the shaken culture, ammonia detoxification (Figure 5B) and lactate clearance rates (Figure 5C) were stronger in the PDB setup.

Figure 5

At the end of the experiments, we evaluated the cell viability for both batches of cells. Viability remained high and stable overall for all conditions (Figure 2, Supplementary data). As previously mentioned, a phenomenon of cell-cell aggregation inside the beads was observed.

Discussion

We recently demonstrated the benefits of using HepaRG cells, capable of differentiating into hepatocyte-like cells [START_REF] Cerec | Transdifferentiation of hepatocyte-like cells from the human hepatoma hepaRG cell line through bipotent progenitor[END_REF], encapsulated in alginate beads as the biomass for BAL [START_REF] Pasqua | HepaRG Self-Assembled Spheroids in Alginate Beads Meet the Clinical Needs for Bioartificial Liver[END_REF]. The present study therefore focused on the transfer of this biomass within a We hypothesized that the deterioration was due to the presence of an excess of BSA and lactate in the surrounding medium, both capable of sequestrating calcium ions involved in the formation of the alginate gel. A similar deterioration effect was observed by Benson et al. [START_REF] Benson | Towards the development of a bioartificial pancreas: Effects of poly-L-lysine on alginate beads with BTC3 cells[END_REF]). In the literature, a PLL coating has been proposed to increase the structural stability of alginate beads and to limit access of the immune system to the beads/capsules [START_REF] Benson | Towards the development of a bioartificial pancreas: Effects of poly-L-lysine on alginate beads with BTC3 cells[END_REF][START_REF] Capone | Impact of Alginate Composition: From Bead Mechanical Properties to Encapsulated HepG2/C3A Cell Activities for In Vivo Implantation[END_REF][START_REF] Strand | Current and future perspectives on alginate encapsulated pancreatic islet[END_REF]. For this specific application, if PLL was not additionally coated with a layer of alginate (i.e. alginate-PLLalginate beads), it demonstrated immunogenicity (Berit L. [START_REF] Strand | Current and future perspectives on alginate encapsulated pancreatic islet[END_REF] and caused fibrotic overgrowth when implanted in mice (B. L. [START_REF] Strand | Poly-L-lysine induces fibrosis on alginate microcapsules via the induction of cytokines[END_REF]. While this additional layer of alginate on the PLL coating appeared essential for implantation of the encapsulated cells, this is not the case for extracorporeal circulation devices such as BAL therapy. In fact, most BAL devices, including the one developed in our laboratory [START_REF] Figaro | SUPPLIVER: Bioartificial supply for liver failure[END_REF], include a plasma filter to separate the plasma from the elements formed of the blood. As a result, the cells of the immune system are discarded upstream, before making contact with the therapeutic biomass, making activation of a specific immune response unlikely.

Preliminary experiments conducted on our medium model of pathological plasma showed that a PLL coating successfully prevented beads from deteriorating. We analysed the physical/mechanical characteristics of the biomaterial with and without a PLL coating. Looking at the morphology, we observed an immediate and stable reduction of about 20 % in the beads' diameter after a PLL coating was applied, without any significant impact on cell viability. Analysis of the distribution of PLL showed that it was not able to enter the core of the alginate bead, remaining only on its shell. This phenomenon was also reported by Yahia et al. [START_REF] Tam | Biocompatibility and physicochemical characteristics of alginate-polycation microcapsules[END_REF]. In addition, the presence of the coating significantly increased the elastic modulus of the external area of the beads, making them 13 resistant to deterioration. Regarding the porosity, we did not notice any measurable difference between beads with or without a PLL coating. Both conditions had a cut-off between 41 and 103nm.

Fluorescent albumin, IgG, and nanoparticles of 41nm in diameter could effectively enter the beads, after 24 hours' incubation, but they were impermeable to polystyrene nanoparticles of 103nm, as also observed by [START_REF] Tran | Alginate hydrogel protects encapsulated hepatic HuH-7 cells against hepatitis C virus and other viral infections[END_REF]. Of note, some large molecules physiologically produced by hepatocytes fell within this cut-off value (such as fibrinogen, about 48nm in hydrodynamic diameter (Bratek-Skicki, Zeliszewska, & Ruso, 2016)) and their transfer between the polymer network and the surrounding medium could be hindered. Importantly, considering that liver sinusoidal endothelial cells (the cells that delimit the space of Disse together with hepatocytes, controlling the trafficking of macromolecules), have fenestrae with diameter between 50-300nm (Wisse, 2002), the cut-off of our beads fitted well with this physiological range. On the basis of our results, we concluded that a large set of molecules produced by the encapsulated hepatocytes (and those present in the culture medium) could leave (and enter) the alginate matrix, an essential factor for BAL therapy.

We then evaluated whether or not the presence of this additional polymer could affect the cells' metabolic performance. According to our observations, encapsulated cell viability was affected neither by the long-term culture of cells nor by the presence of PLL. Noticeably, one of the markers of liver cell functionality is albumin production. Monitoring this secretion between the two conditions (with or without PLL) in shaken conditions showed a progressive increase throughout the culture up to day 14 with an advantage for PLL coated beads, corresponding to efficient HepaRG differentiation. On day 14, we tested different metabolic activities required for BAL applications, such as ammonia and lactate elimination. We were not able to measure any significant difference between either condition, with the exception of a downward trend in xenobiotic activities, and more precisely for CYP1A1/2, when the coating was present. This might be due to an increase in the beads' core stiffness after shrinking. Chen et al. [START_REF] Chen | Modulation of hepatocyte phenotype in vitro via chemomechanical tuning of polyelectrolyte multilayers[END_REF] effectively cultured rat hepatocytes on polyelectrolyte multilayers and found that albumin production and CYP1A activity decreased with increasing substrate stiffness. In the next step, we tested the functionality of cells on an equivalent pathological plasma medium, reproducing clinical therapy. We estimated that this biomass (alginate-PLL encapsulated HepaRG) had the characteristics necessary for extracorporeal BAL application. To support this hypothesis, we decided to use it in the PDB, compatible with the system developed in our laboratory (Figaro, Pereira, Rada, et al., 2015). Similar bioreactor designs, such as stirred-tank bioreactors, have long been used in large-scale culture of mammalian cells to provide an environment allowing control of oxygen, pH, and the continuous addition and removal of medium. They are commonly used for the large-scale production of spheroids that can be maintained for extended periods [START_REF] Tostões | Human liver cell spheroids in extended perfusion bioreactor culture for repeated-dose drug testing[END_REF], classically used in toxicological applications [START_REF] Rebelo | HepaRG microencapsulated spheroids in DMSO-free culture: novel culturing approaches for enhanced xenobiotic and biosynthetic metabolism[END_REF]. Moreover, Dr. Nyberg's group, who recently proved the beneficial role of the spheroid-reservoir BAL in treating pigs experiencing posthepatectomy ALF (Chen HS., Joo DJ., Shaheen M., Li Y., WangY., Yang J., Nicolas CT., Predmore K., Amiot B., Michalak G., Mounajjed T., Fidler J., Kremers WK., 2019), highlighted how an impeller-agitated bioreactor could support high volumes of hepatocyte spheroid solutions, with a simple and compact design that is easy to scale up for BAL clinical applications [START_REF] Mcintosh | Engineering analysis and development of the spheroid reservoir bioartificial liver[END_REF]. Overall, we consider that the PDB hosting alginate-PLL encapsulated HepaRG fitted these important criteria and, to our knowledge, it is the first time that this design has been used for BAL applications.

HepaRG matured in PLL alginate beads were able to produce proteins, detoxify ammonia and metabolize lactate to levels comparable or superior to other published works with similar cells [START_REF] Nibourg | Increased hepatic functionality of the human hepatoma cell lineHepaRG cultured in the AMC bioreactor[END_REF] or primary porcine hepatocytes in the AMC-BAL [START_REF] Poyck | Functional and morphological comparison of three primary liver cell types cultured in the AMC bioartificial live[END_REF]. The latter two activities were superior in the PDB compared to the shaken conditions setup linked with net cellular activity, as the results were normalized by the controls with empty alginate-PLL beads. It is difficult to assume that the perfusion of medium was the key element for the enhanced activity observed in PDB, given the very low perfusion flow in our experiments. We thus hypothesized that such improvement was promoted by increased oxygenation of the culture medium. The latter is associated with the synergistic role of orbital shaking culture and the increased exchange surface between the medium and the external environment in PDB rather than the shaken setup. Adam et al. assumed that improved oxygenation when cultivating HepaRG cells under shaking conditions would lead to mitochondrial biogenesis, improving hepatic differentiation and cell metabolic activity (such as ammonia detoxification and CYP-3A4 activity) [START_REF] Adam | A practice-changing culture method relying on shaking substantially increases mitochondrial energy metabolism and functionality of human liver cell lines[END_REF]. In addition, the role of oxygen on HepaRG differentiation and functions is crucial, as observed by van Wenum et al., where the authors demonstrated that culturing HepaRG in hyperoxia (i.e. 40 % O 2 ) enhanced cell differentiation and improved metabolic functions (such as ammonia detoxification and CYP3A4 activity), when compared with normoxia conditions (i.e. 21 % O 2 ) [START_REF] Van Wenum | Oxygen drives hepatocyte differentiation and phenotype stability in liver cell lines[END_REF].

In PDB, we noticed a significant decrease of around 40 % in initial ammonia concentrations and around 30 % in lactate after 6 hours of cell perfusion in the equivalent pathological plasma medium.

At the end of the experiment, we did not notice any deterioration of the alginate-PLL beads or loss of cells, observing the supernatant on bright field microscopy. This feature, together with the presence of filters that avoid cells escaping from the bioreactor, is of key importance for conducting safe treatment for the patient.

Conclusion

We adapted the HepaRG biomass encapsulated in alginate beads to the conditions of ALF therapy by adding a PLL coating. This coating covered the shell of the beads without altering the porosity or viability of the cells. In terms of metabolic activity, we measured very interesting performances in line with those required of BAL in the treatment of ALF. The next step will be to implement the PDB in the BAL, currently being optimized in our laboratory, and start the preclinical experiments on rodent ALF models.

Acknowledgement

We thank Dr Antonietta Messina and Dr Anne Dubart-Kupperschmitt (INSERM U1193) for the fruitful discussion throughout this work, and Augustin Lerebours, for his technical assistance. The HepaRG cell line, media and supplements used were supported by Biopredic International (funded by ANR 16-RHUS-0005 in the framework of the iLite programme), for which the authors express their appreciation. This cell line was patented by Inserm under number WO 03/004627.

  demonstrating the features necessary for clinical use of BAL, in terms of cell metabolic activity and therapy safety.

  Figure1

  3.1.1.PLL coating and distribution around the alginate beads

  bead. As previously observed on empty beads, the PLL coating provoked a shrinking effect of about 20 % in the diameter of the beads.

  BAL therapy. However, during the first experiments carried out in an ALF-human equivalent pathological plasma medium, we observed severe and unacceptable deterioration of the beads. We thus highlighted the need to reinforce the mechanical properties of the biomaterial.

F

Figure 1 .

 1 Figure 1. A: Experimental setups allowing culture and metabolic characterization of the encapsulated biomass. Left: Shaken condition: Petri dish hosting 5x10 6 cells in 7.5 mL of surrounding medium kept

Figure 2 .

 2 Figure 2. A: Distribution of PLL-rhodamine (red staining) in empty beads in comparison with the

Figure 3 .

 3 Figure 3. Permeability of empty beads (coated or not with PLL) to fluorescent markers (albumin, IgG,

Figure 4 .

 4 Figure 4. Observation of beads without and with PLL coating morphology by bright field microscopy

Figure 5 .

 5 Figure 5. Metabolic activities of cells in alginate-PLL beads, measured on day 14, over 6 hours'

Figure 3 .Figure 4 .Figure 5 .Figure 1 ,

 3451 Figure1. A: Experimental setups allowing culture and metabolic characterization of the encapsulated biomass. Left: Shaken condition: Petri dish hosting 5x106 cells in 7.5 mL of surrounding medium kept in continuous orbital shaking (60 rpm). Right: Perfused dynamic bioreactor (PDB): double arm glass flask hosting 50x106 cells in 37.5 mL of total medium volume. The peristaltic pump (Ismatec) makes constant perfusion possible (flow rate = 0.4 mL/min) while the beads are kept in continuous orbital shaking (80 rpm). The needles for media recirculation are protected with a filter membrane to avoid biomass escaping from the PDB. Two outlets are dedicated to filtered air intake to preserve sterility and guarantee gas exchanges. B: Experimental setup. After 2D amplification, cells were encapsulated in alginate beads and then cultivated for 14 days. Cell culture evolution was monitored analysing cell viability (by Live/Dead staining on days 0, 1, 7, 14) and albumin synthesis rate (overnight between days 0-1, 6-7, 9-10, 13-14). On day 14 postencapsulation, cell performance was tested by means of metabolic and xenobiotic tests in different media (HepaRG proliferation culture medium, test medium, and pathological plasma medium).149x146mm (300 x 300 DPI)

Figure 2 ,

 2 Figure 2, Supplementary Data. Cell viability (live/dead assay) of cells in alginate beads without and

Table 1 :

 1 Fluorescent molecules with their characteristics used to investigate bead permeability. DH indicates the hydrodynamic diameter of the protein without the fluorophore; MW indicates the molecular weight.

	o r
	P
	e e r
	R e v
	i e w

Table 2 :

 2 Media for metabolic characterization on day 14 post-encapsulation and tests performed.

		HepaRG proliferation culture medium	Test medium	Human equivalent pathological plasma medium
	Medium composition	William's E medium + Biopredic 710 proliferation media	HepaRG proliferation culture medium + 1.5 mM ammonium chloride + 2 mM L-Lactate	HepaRG proliferation culture medium + 70 g/L bovine serum albumin + 1.5 mM ammonium chloride + 2 mM L-Lactate
		Albumin synthesis rate	Albumin synthesis rate	Albumin synthesis rate
	Metabolic	(2 h)	(2 h)	(6 h)
	performances	ICG clearance rate	Ammonia detoxification rate	Ammonia detoxification rate
	tested	(1 h)	(2 h)	(6 h)
	(Test duration)	CYP 1A1/2 activity	Lactate clearance rate	Lactate clearance rate
		(1 h)	(2 h)	(6 h)

Table 3 :

 3 Summary of metabolic activities between biomass in alginate beads with and without a PLL coating.

	Activities tested on day 14 post-encapsulation in shaken conditions, in HepaRG proliferation culture medium, and
	in test medium. N > 3
		HepaRG proliferation culture medium		Test medium
	Biomass (5 x 10 6 cells)	Albumin synthesis rate (µg/h/10 6 )	ICG releasing rate (µg/h/10 6 )	CYP 1A1/2 activity (pmol/h/10 6 )	Albumin synthesis rate (µg/h/10 6 )	Ammonia detoxification rate (nmol/h/10 6 )	Lactate clearance rate (nmol/h/10 6 )
	Alginate beads
	without PLL	0.85 ± 0.42	45.3 ± 6.9	6.5 ± 1.9	0.43 ± 0.19	90.4 ± 61.0	111.8 ± 68.3
	coating
	Alginate beads
	with PLL	0.62 ± 0.22	20.9 ± 7.8	1.8 ± 1.0	0.56 ± 0.24	73.7 ± 28.4	95.6 ± 50.4
	coating

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encapsulated hepatocyte spheroids. Proceedings of the Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBS, 1335-1338.