Nadia Chaher
email: nadia.chaher@kcl.ac.uk

Giuseppe Digilio
email: giuseppe.digilio@uniupo.it

Sara Lacerda
email: sara.lacerda@cnrs.fr

René M Botnar
email: rene.botnar@kcl.ac.uk

Alkystis Phinikaridou
email: alkystis.1.phinikaridou@kcl.ac.uk

Optimized Methods for the Surface Immobilization of Collagens and Collagen Binding Assays

analyze, and interpret in vitro time-resolved fluorescence binding studies to investigate the interactions between collagens and candidate collagen-binding compounds (e.g., a peptide conjugated to a metal chelate carrying, for example, europium [Eu(III)]). Such an approach can be universally applied to various biomedical applications, such as in the field of molecular imaging to develop targeted imaging probes, drug development, cell toxicity studies, cell proliferation studies, and immunoassays.

INTRODUCTION:

The accumulation of fibrous connective tissue as part of the natural wound-healing process following tissue injury is known as fibrosis. However, if the deposition of fibrous tissue fails to terminate and continues beyond what is needed for tissue repair, then fibrosis becomes excessive [START_REF] Distler | Review: Frontiers of antifibrotic therapy in systemic sclerosis[END_REF][START_REF] Wynn | Fibrotic disease and the TH1/TH2 paradigm[END_REF] . Excessive fibrosis impairs organ physiology and function and could lead to organ damage and potentially organ failure [START_REF] Saha | Magnetic resonance T 1 relaxation time of venous thrombus is determined by iron processing and predicts susceptibility to lysis[END_REF][START_REF] Mirshahi | Defective thrombolysis due to collagen incorporation in fibrin clots[END_REF][START_REF] Comerota | The ATTRACT trial: Rationale for early intervention for iliofemoral DVT[END_REF] . Two main drivers of fibrosis are the extracellular matrix (ECM) proteins collagen type I and type III [START_REF] Bateman | Immunohistochemical study of collagen types in human foetal lung and fibrotic lung disease[END_REF] . Collagen is a structural protein found in various organs that makes up approximately one-third of the total protein content of the human body [START_REF] Distler | Review: Frontiers of antifibrotic therapy in systemic sclerosis[END_REF] . There are 28 different types of collagens identified by human genome sequencing, and the most abundant are the fibrillar collagens [START_REF] Pawelec | Collagen: A network for regenerative medicine[END_REF] . The primary fibrillary collagen is type I collagen, which provides the ECM with tensile strength and resistance to deformation [START_REF] Frantz | The extracellular matrix at a glance[END_REF] . Type III collagen is a structural component that provides elasticity and colocalizes with type I collagen. It is expressed during embryogenesis and is naturally found in small amounts in adult skin, muscle, and blood vessels [START_REF] Copes | Collagenbased tissue engineering strategies for vascular medicine[END_REF] .

In vivo collagen synthesis begins with an intracellular process in which mRNA is transcribed in the nucleus and then moves to the cytoplasm, where is it translated. After translation, the chain formed undergoes post-translational modification in the endoplasmic reticulum, where pro-collagen (the precursor of collagen) is formed. The pro-collagen then travels to the Golgi apparatus for final modification before being excreted to the extracellular space [START_REF] Veis | The biochemistry of collagen[END_REF] .

Through proteolytic cleavage, the procollagen is transformed into tropocollagen. This is then cross-linked either via an enzymatic-mediated cross-linking pathway catalyzed by the enzyme lysyl oxidase (LOX) or via a non-enzymatic-mediated cross-linking pathway involving the Maillard reaction [START_REF] Bielajew | Collagen: Quantification, biomechanics, and role of minor subtypes in cartilage[END_REF] . However, in vitro protocols to immobilize collagen mainly rely on the ability of collagen to self-assemble. Collagen is extracted from tissues based on its solubility, which largely depends on the extent of cross-linking of individual collagen fibrils [START_REF] Pawelec | Collagen: A network for regenerative medicine[END_REF] .

Fibrillar collagen is dissolved in acetic acid, and fibrils can reform when the pH and temperature are adjusted [START_REF] Zhao | Structural and functional plasticity of collagen fibrils[END_REF] . In vitro, the fibrillogenesis of collagen can be viewed as a twostage process [START_REF] Pawelec | Collagen: A network for regenerative medicine[END_REF] . The first stage is the nucleation phase, where collagen fibers form dimers and trimer fibrils before they are rearranged to form a triple helical structure. The second phase is the growth phase, whereby the fibrils start to grow laterally and result in the characteristic D-band formation, which is generally observed by changes in turbidity [START_REF] Pawelec | Collagen: A network for regenerative medicine[END_REF] .

Atomic force microscopy (AFM) studies have also revealed that type I and type III collagen have different characteristics (Erreur ! Source du renvoi introuvable.) [START_REF] Eryilmaz | In vitro analysis of the co-assembly of type-I and type-III collagen[END_REF] .

To study the binding interactions between collagen and other compounds, collagen needs to be reproducibly immobilized into the wells of microplates. There are various protocols for immobilizing soluble collagen [START_REF] Jagnow | Klebsiella pneumoniae MrkD-mediated biofilm formation on extracellular matrix-and collagen-coated surfaces[END_REF][START_REF] O'sullivan | Direct plasma deposition of collagen on 96-well polystyrene plates for cell culture[END_REF][START_REF] Caravan | Collagen-targeted MRI contrast agent for molecular imaging of fibrosis[END_REF] . Commercially available microplates that are pre-coated with collagen are typically used for cell culture. However, pre-coated microplates have a very thin layer of an unknown amount of collagen coated onto the wells, which makes them unsuitable for in vitro binding assays. There are several challenges when immobilizing collagen onto the plate wells. One of the key challenges is choosing a suitable type of microplate, because different types of collagens (e.g., type I and III) have different chemical properties and, therefore, immobilize more stably and effectively depending on the material of the microplate. Another challenge is the experimental conditions of the immobilization protocol, as the process of fibrillogenesis depends on multiple factors, including temperature, pH, the stock concentration of collagen, and the ionic concentration of the buffer [START_REF] Pawelec | Collagen: A network for regenerative medicine[END_REF] .

For studying the interactions between the collagen (the target) and other compounds (i.e., a targeting peptide), it is also necessary to develop a robust screening assay to investigate the specificity and selectivity of the compound toward the target by measuring the dissociation constant, K d . The position of the equilibrium of formation of a bimolecular complex between a protein (collagen) and a ligand is expressed in terms of the association constant K a , whose magnitude is proportional to the binding affinity. However, most commonly, biochemists express affinity relationships in terms of the equilibrium dissociation constant, K d , of the binary complex, which is defined as K d = 1/K a •K d and is the inverse of K a ; the lower the K d value, the stronger the binding strength between the protein and the ligand. The advantage of using K d to compare the binding affinity of different ligands for the same protein (and the other way around) is linked to the fact that the units of K d for a bimolecular complex are mol/L (i.e., concentration units). Under most experimental conditions, the K d value corresponds to the ligand concentration that leads to 50% saturation of the available binding sites on the target at the equilibrium [START_REF] Copeland | Enzymes: A Practical Introduction to Structure, Mechanism, and Data Analysis[END_REF][START_REF] Salahudeen | An overview of pharmacodynamic modelling, ligand-binding approach and its application in clinical practice[END_REF] . The dissociation constant is typically extracted by analyzing the receptor fractional occupancy (FO), which is defined as the ratio between the occupied binding sites and total available binding sites, as a function of ligand concentration. This can be done provided that an analytical assay able to distinguish and measure the amount of bound ligand is available.

In vitro ligand binding assays can be performed using various bioanalytical methods, including optical photometry, radioligand methods, inductively coupled plasma mass spectrometry (ICP-MS), and surface plasmon resonance (SPR). Amongst the photometric methods, those based on fluorescence emission typically require the labeling of ligands or proteins with fluorophores to increase the sensitivity and to improve the detection limit of the assay. Chelates of certain lanthanides(III) ions, such as Eu(III), are very attractive as fluorophores as they have large Stokes' shifts, narrow emission bands (providing a good signal-to-noise ratio), limited photobleaching, and long emission lifetimes. Importantly, the latter property enables the use of time-resolved fluorescence (TRF) from Eu(III) fluorophores to abolish background autofluorescence [START_REF] Bünzli | Taking advantage of luminescent lanthanide ions[END_REF] . In the dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) version of the Eu(III)-based TRF assay, ligands labeled with a non-luminescent Eu(III)-chelate are incubated with the receptor immobilized onto microplates. The labeled ligand/receptor complex is separated from the unbound ligand, and Eu(III) fluorescence is activated by dissociation of the Eu(III) complex at an acidic pH, followed by re-complexation with a fluorescence-enhancing chelator to form a micelleembedded, highly fluorescent Eu(III) complex [START_REF] Hemmilii | Luminescent lanthanide chelates -A way to more sensitive diagnostic methods[END_REF] .

The decomplexation step can be reasonably achieved with chelators, such as diethylenetriamine pentaacetate (DTPA), that show fast decomplexation kinetics. However, Eu(III) complexes with certain macrocyclic chelators, such as DOTA (1,4,7,10-tetraazacyclododecane1,4,7,10-tetraacetic acid) and its monoamide derivatives (DO3AAm), show high thermodynamic stability and very high kinetic inertness. In this case, the decomplexation steps must be accurately optimized to achieve sufficient and reproducible activation of Eu(III)-based TRF [START_REF] De Silva | Optimization of timeresolved fluorescence assay for detection of europium-tetraazacyclododecyltetraacetic acidlabeled ligand-receptor interactions[END_REF] . It is worth noting that lanthanide (Ln(III))-DOTA and Ln(III)-DO3AAm complexes are those most commonly employed as contrast agents for in vivo molecular imaging by magnetic resonance imaging (MRI) techniques [START_REF] Digilio | Extracellular matrix targeted MRI probes[END_REF] . Thus, the Ln(III)based TRF assay is the tool of choice to study in vitro the binding affinity of MRI molecular probes with their intended biological targets. Currently, comprehensive and reproducible protocols for immobilizing type I and type III collagen and a reproducible pipeline for performing in vitro binding Eu(III) TRF experiments are lacking. To overcome these limitations, reproducible methods to self-assemble and immobilize type I and type III collagen and generate stable gels and films, respectively, with the sufficient concentration of collagen required for in vitro binding assays, were developed. An optimized protocol for Eu(III) TRF of highly inert Eu(III)-DO3Aam-based complexes is presented. Finally, an optimized in vitro microplate Eu(III) TRF assay to measure the K d of Eu(III)-labeled ligands toward immobilized type I and type III collagen is demonstrated (Figure 1).

PROTOCOL:

NOTE: All product information used for this work is presented in the Table of Materials.

1.

Collagen immobilization 1.2.5. Transfer the microplates to the incubator for 1 h at 37 °C, and then transfer the microplates to the bench, and allow them 1 h to dry before using them in further binding experiments.

NOTE

Assessment of the stability of the immobilized collagen gels/films

2.1. Incubation with PBS for 1 h NOTE: During the binding experiment, incubate the immobilized collagen with the compound of interest. It is important to investigate the stability of the resulting collagen gel or film. To do this, measure the stability of three conditions: no wash = measures the immobilized collagen directly after incubation; washing = measures the immobilized collagen after washing the plate twice with 100 µL of PBS; and 1 h PBS mimic & wash = measures the immobilized collagen acter incubating for 1 h with PBS followed by two rounds of washes with PBS. Below, the PBS incubation method is explained.

NOTE: When comparing the binding properties of one compound toward different target

proteins that immobilize at different concentrations, the latter will need to be considered when calculating the amount of compound bound to the target (i.e., bound compound/protein).

4.4.2.2.

Divide the concentration of the bound compound by the concentration of the protein immobilized in the well.

NOTE: For this calculation, use the concentration of immobilized collagen that was calculated after the wells were incubated with PBS for 1 h (so-called PBS mimic experiment; section 2.1 above). This is to account for potential losses of collagen during the incubation step and washing step so that those losses will not contribute to the final fluorescence signal.

4.4.2.3.

Plot the data using a scatter plot that has the concentrations of the compound on the x-axis (µM) and the bound compound/protein on the y-axis.

REPRESENTATIVE RESULTS:

Assessing the stability and concentration of type I and type III collagen immobilized in gels/films

The quantification of the collagen concentration immobilized per well was carried out using three different conditions: a) in wells without washing with PBS after immobilizing the proteins (no wash); b) in wells with a wash step (twice with PBS) after immobilization to remove any uncoated protein; c) in wells after incubation with PBS for 1 h (PBS mimic experiment). The PBS incubation mimicking step was performed to understand the stability of the collagen gel/film following incubation with a solution containing the candidate compound during the actual binding assay. Quantifying how much collagen is immobilized per well is crucial for accurately calculating and comparing the bound fraction and dissociation constant (K d ) of a compound toward different targets, especially when those targets have different coating efficiencies. Erreur ! Source du renvoi introuvable. shows a representative stability assay for type III collagen, from which it is evident that the amount of collagen that remained immobilized on the wells decreased when washing and/or incubation steps were included. The results demonstrate that a greater loss of collagen from the gel/film occurs during the washing step and less loss occurs during the incubation step.

However, washing is a crucial step during an in vitro binding assay to ensure that the unbound compound is removed. For this reason, it is important that the amount of collagen that remains coated within the well, and not the amount of collagen initially immobilized, is calculated and used for the normalization of the results of the in vitro binding assay. The optimized conditions that resulted in the reproducible immobilization of collagen and the corresponding amount of collagen that was immobilized after following the washing and incubation steps are reported in Erreur ! Source du renvoi introuvable.. 3), a normalization step was implemented to account for the amount of immobilized collagen per well. To achieve that, the standard curve obtained from the total compound in solution (Figure 8B) was used to convert the bound fluorescence readings into concentration. These values were then normalized by dividing the bound fluorescence readings by the concentration of collagen coated onto the wells to obtain the plot shown in Figure 8C, in which the bound protein data for type I and III collagen could be presented on the same plot. It must be noted that although the K d values were not altered by the normalization, in this particular example, the results could be plotted and directly compared using the same graph. The fractional occupancy results, shown in Figure 8D, demonstrated that 71% of the collagen was occupied by the compound at the highest concentration of 10 µM. Table 1: Summary of the main differences between the intrinsic characteristics of type I and type III collagen. Adopted from Eryilmaz et al. [START_REF] Eryilmaz | In vitro analysis of the co-assembly of type-I and type-III collagen[END_REF] . collagen fibrillogenesis in vitro [START_REF] Eryilmaz | In vitro analysis of the co-assembly of type-I and type-III collagen[END_REF][START_REF] Caravan | Collagen-targeted MRI contrast agent for molecular imaging of fibrosis[END_REF] . Specifically, the reproducible coating of type I and III collagen was achieved by optimizing critical conditions, including the type of microplates, the pH, the temperature, and the ionic concentration of the buffer. Given that collagens self-assemble with increasing pH values and temperature, the neutralization and plating steps for immobilizing collagen must be completed on ice and in the cold room. This ensures that the nucleation rate of collagen, the first step of fibrillogenesis, does not start before the collagen comes into contact with the well but rather occurs within the well after the temperature is increased during the incubation step. By quantifying the amount of protein in each well, it was found that type I collagen coated reproducibly and stably using nonbinding specific microplates that were coated with a non-ionic hydrophilic surface. In contrast, type III collagen, showed reproducible and stable coating using tissue culture polystyrene-microplates that were exposed to plasma gas, making the microplate more hydrophilic. Immobilization protocols for type III collagen are less explored compared with those available for type I collagen, and, therefore, this protocol is unique. Although the immobilization protocols used for type I and type III collagen follow similar steps, there are crucial differences in the duration and temperature of the incubation due to the different intrinsic characteristics of the two collagen types. It is known that type III collagen has a faster nucleation rate and a slower growth rate than type I collagen, and these differences were considered when establishing this protocol (Table 1) [START_REF] Eryilmaz | In vitro analysis of the co-assembly of type-I and type-III collagen[END_REF] . For this reason, type III collagen requires incubation at 37 °C for only 2 h, whereas type I collagen is incubated for 18-20 h.

In vitro Eu(III) TRF binding assay to characterize the binding of Eu(III)-DO3AAm-CBP toward type I and type III collagen

FIGURE AND TABLE LEGENDS:

The coating efficiency-how much of each collagen was coated onto the wells-was also studied, and the stability of the coating under different protocols, especially after washing and after incubating with PBS for 1 h, was investigated. The stability of the coated protein is crucial for the reproducible screening of the binding properties of a compound toward the target and the accurate calculation of the K d and FO values. This is a step that is rarely performed, and binding protocols typically only quantify the concentration of the immobilized protein at the start of the experiment (after immobilization but prior to the incubation) [START_REF] Caravan | Collagen-targeted MRI contrast agent for molecular imaging of fibrosis[END_REF] . However, because both the coating efficiency and stability of the coated protein may vary between different proteins, it is important to determine how much of the initial protein remains stably coated within the well at the end of the binding experiment and, therefore, contributes toward the final measurement. Knowing that there is a reproducible amount of protein coated on the plates is important when studying the interactions of these proteins with binding molecules, as this contributes directly to the fluorescence reading from the Eu(III)-labeled compound. Normalizing the bound fluorescence measurements with the amount of protein immobilized in the well (bound protein) does not affect the K d or FO per se. However, such normalization leads to the rescaling of the data plotted on the y-axis of the binding curve, enabling the direct comparison of the binding profile of the same compound toward different targets with different immobilization efficiencies.

In the analysis, it is also important to account for the non-specific binding of the compound to the experimental matrix (i.e., the plastic plate) rather than assuming that the readings acquired from the coated wells solely represent compound bound to collagen. This was accounted for by subtracting the non-specific binding of the compound to the plate (uncoated wells) from the specific reading, represented as "bound fluorescence" (collagencoated wells), under the same TRF measurement conditions.

Eu(III) TRF-based methods have been widely used for in vitro binding studies [START_REF] Phinikaridou | Tropoelastin: A novel marker for plaque progression and instability[END_REF][START_REF] Guzaeva | Protein A used in DELFIA for the determination of specific antibodies[END_REF][START_REF] Nasiri | Polymerase assays for lead discovery: An overall review of methodologies and approaches[END_REF] . It is a flexible assay that can be used in multiple applications, such as immune assays, cell cytotoxicity studies, enzyme assays, and binding studies, as demonstrated here and in previous work [START_REF] Phinikaridou | Tropoelastin: A novel marker for plaque progression and instability[END_REF][START_REF] Capuana | Imaging of dysfunctional elastogenesis in atherosclerosis using an improved gadolinium-based tetrameric MRI probe targeted to tropoelastin[END_REF] . It is also highly sensitive and takes advantage of time-resolved fluorescence properties to eliminate any non-specific background fluorescence signals that may be observed in conventional direct readings [START_REF] De Silva | Optimization of timeresolved fluorescence assay for detection of europium-tetraazacyclododecyltetraacetic acidlabeled ligand-receptor interactions[END_REF] . In standard DELFIA applications, users are able to use the standard enhancement solution to efficiently extract Eu(III) from its complexes. However, the decomplexation of Eu(III) from Eu(III)-DO3AAm-like complexes is difficult because of the very high kinetic inertness of such complexes, which means that decomplexation will be far from being complete and reproducible. A protocol to achieve Eu(III) decomplexation from such complexes has been proposed by De Silva et al. [START_REF] De Silva | Optimization of timeresolved fluorescence assay for detection of europium-tetraazacyclododecyltetraacetic acidlabeled ligand-receptor interactions[END_REF] . Such a protocol involves the incubation of the Eu(III)-DO3AAm complex with a very acidic solution (AS, 2 M HCl) for 2 h, followed by the addition of a neutralizing solution (NS, 2 M NaOH) to bring the pH back to neutrality. The enhancement solution is then added to activate the Eu(III) and to adjust the pH to be in the optimal range (3.0-3.5) for TRF readings. However, it was found that small volume inaccuracies in the amounts of acid and neutralizing solutions added to microwells could lead to significant fluctuations in the final pH, which could affect the intensity of the Eu(III) TRF signal. Thus, the protocol was modified by adding a strong glycine buffer to the NaOH solution to obtain the buffering solution (2 M NaOH and 2 M glycine). After treatment with the acid solution to extract Eu(III), the pH is adjusted directly to 3.2 in a very consistent and reproducible manner by the addition of a calibrated amount of the buffering solution. Finally, the enhancement solution can be added for Eu(III) recoordination and self-association into TRF active micelles.

A limitation of the Eu(III) TRF assay is that the maximum concentration of the Eu(III)-labeled compound that can be used in the assay is 10-15 μM because, at higher concentrations, the extraction of Eu(III) and the formation of micelles are no longer linear. Another method that has been used to measure K d values is by using compounds labeled with other lanthanides, such as Gd(III). In this approach, the Gd(III) concentration is measured in the supernatant collected after the incubation of the Gd(III)-labeled compound with the target using ICP-MS.

The ICP-MS of the supernatant collected after the incubation of the compound provides an indirect measurement of the bound fraction remaining in the well by subtracting the amount of Gd(III) in the supernatant from the "total gadolinium" concentration that was initially added in the well. Despite the indirect measurement of the "bound fraction", this approach yields comparable binding affinities to those measured with high-performance liquid chromatography (HPLC) [START_REF] Caravan | Collagen-targeted MRI contrast agent for molecular imaging of fibrosis[END_REF] . The ICP-MS method is useful in cases where the K d may be high, meaning concentrations of the compound higher than the 10-15 μM limit of the DELFIA assay are required in order to reach the plateau (saturation) of the binding curve and, thus, accurately calculate the K d . Another technique to study the interactions between molecules is SPR [START_REF] Drescher | Surface plasmon resonance (SPR) analysis of binding interactions of proteins in inner-ear sensory epithelia[END_REF] . This is an optical technique based on the immobilization of a target protein on a gold film and the analysis of the binding of another protein/molecule in a mobile state. The advantage of SPR is that it measures the interactions directly with no need for any fluorophore or lanthanide complexation to measure the binding response. However, the limitations of this technique include the complexity of optimizing the immobilization process (which can compromise the binding of the epitope), the difficulty of determining non-specific binding, and the cost [START_REF] Murali | Applications of surface plasmon resonance and biolayer interferometry for virus-ligand binding[END_REF] .

This study presents an optimized protocol that enables the reproducible immobilization of type I and type III collagen, a detailed methodology for performing Eu(III) TRF assays to study compound-collagen interactions, and a clear workflow for analyzing and interpreting the results.

: 1 1. 1 . 1 . 1 . 1 . 2 . 1 . 1 . 3 . 2 1. 1 . 5 .

 111112113215 Ensure each well in the microplate used during the binding assay has adjacent wells free to avoid cross-fluorescence. Carry out this part of the protocol on ice because collagen self-assembles at rising temperatures and pH levels. Perform this procedure in a tissue culture hood and under sterile conditions because the microplates are subsequently incubated in a tissue culture (TC) incubator. 1.1. Immobilization of type I collagen on the 96-well microplates (Figure 2: Workflow to immobilize type I collagen) Day Prepare a silicone tray with ice. Place the vial containing type I collagen, the cold 10x phosphate-buffered saline (PBS), and the microplates on ice, and spray everything with 70% ethanol. Place the material under the TC hood. Neutralize the collagen using equal volumes of type I collagen and 10x PBS (pH 7.4). Invert the solution a few times, ensuring that no bubbles form. 1.1.4. Add 100 µL of the neutralized collagen to every other well of the microplate, and incubate at 37 °C for 18-20 h to evaporate the collagen to dryness. Day Wash the microplates with 100 µL of 1x PBS, pH 7.4, twice to remove any unbound collagen. 1.1.6. Transfer the microplates into the incubator at 37° C for another 2 h to dry before using them for further binding experiments. 1.2. Immobilization of collagen type III on the 96-well microplates (

Figure 3 ) 1 1. 2 . 1 . 1 . 2 . 3 . 2 1. 2 . 4 .

 3121123224 Day Prepare a silicone tray with ice. Place the vial containing type III collagen, the cold 10x PBS, and the microplates on ice, and spray everything with 70% ethanol. Place the material under the TC hood.1.2.2. Neutralize the collagen using equal volumes of type III collagen and 10x PBS (pH 7.4). Add 70 µL of the neutralized collagen to every other well of the microplate, and incubate at 37 °C for 2 h by placing the microplate under the tissue culture hood to evaporate the collagen to dryness. Day Wash the microplates with 70 µL of 1x PBS, pH 7.4, twice to remove any unbound collagen.

3 .Figure 4 ). 3 . 1 .

 3431 Figure 4).

1 . 3 . 4 . 4 . 5 ) 4 . 4 . 1 .Equation 1 :

 134454411 Quantification of the concentration of collagen immobilized on the wells 4.1.1. Obtain the equation of the calibration curve of the A 560 (AU) versus the concentration (µg/mL) of the 11 standard solutions. 4.1.1.1.Use the absorbance readings acquired from the wells containing the collagen standards. 4.1.1.2. Tabulate the average values from the duplicate wells, and plot the mean absorbance against the known protein (collagen) concentrations (µg/mL) to obtain the equation for the standard curve. 4.1.2. Use the absorbance values to calculate the mass (µg) and concentration (M) of immobilized collagen. 4.1.2.1. Compute the average absorbance values across the three wells that contained immobilized collagen, and record the standard deviation. 4.1.2.2. Use the standard curve equation obtained from the collagen standard curve (step 2.2.6) to convert the absorbance measured from the collagen-coated wells into concertation. From this, calculate the concentration of collagen that was immobilized within the experimental wells in µg/mL. 4.1.2.3. Convert the concentration calculated in step 4.1.2.2 (µg/mL) first to grams/liter and, based on the molecular weight of the collagen, into molar (M).4.1.2.4. Finally, calculate the mass of the collagen immobilized in each well by dividing the concentration by the volume of collagen added to the well (100 µL for type I collagen and 70 µL for type III collagen). 4.2. Calculation of the dissociation constant (K d ) ( 4.Figure Extract the fluorescence readings. 4.4.1.1. Export the fluorescence readings from the plate reader to a spreadsheet. NOTE: In binding assays, it is important to account for the potential non-specific binding of a compound to the plastic surface of the plates 4.4.1.2. Calculate the mean values of triplicate measurements from each compound concentration for the three different plates: the specific binding readings from the coated wells (Plate A), the non-specific binding from the uncoated wells (Plate B) and the total Eu(III)-DO3AAm-CBP in solution in the uncoated wells (Plate C). 4.4.1.3. Determine the fluorescence values for the bound compound by subtracting the fluorescence readings of the uncoated wells (Plate B) from that of the coated wells (Plate A). Determining the bound fluorescence 17 : 4.4.1.4. Generate a calibration curve using the readings from the Eu(III)-labeled compound in solution (Plate C). Plot the fluorescence readings obtained against the concentration of the Eu(III)-labeled compound. Perform a linear regression fit. 4.4.2. Convert the fluorescence readings to concentrations. 4.4.2.1. Convert the readings of the bound fluorescence (step 4.2.3) into concentration using the standard fluorescence curve from the data generated using the compound concentrations in solution (step 4.2.1.4).

A

  representative analysis and results obtained from an in vitro Eu(III) TRF experiment to investigate the affinity of the Eu(III)-DO3AAm-CBP compound toward collagen type I and type III are shown in Erreur ! Source du renvoi introuvable.. The bound fluorescence readings and corresponding K d values of the candidate collagen binding compound are shown in Figure 8A. The results show that the candidate compound bound non-specifically to type I collagen (as the curve does not reach saturation), and, therefore, no K d value was reported. In contrast, the candidate compound was shown to bind to type III collagen with a K d value of 4.2 μM ± 0.7 μM. As the coating efficiencies of type I and III collagen are different (as shown in Table

Figure 1 :

 1 Figure 1: Overview of the methodology used to study compound-protein binding interactions using Eu(III) TRF. Created with BioRender.com.

Figure 2 :

 2 Figure 2: Workflow to immobilize type I collagen. Created with BioRender.com.

Figure 3 :

 3 Figure 3: Workflow to immobilize type III collagen. Created with BioRender.com.

Figure 4 :

 4 Figure 4: Chemical structure of the Eu(III)-DOTA-monoamide derivative carrying a collagen-binding peptide (CBP) used for the in vitro binding studies.

Figure 5 :

 5 Figure 5: Overview of the data analysis pipeline to calculate the dissociation constant following the in vitro binding assay. Created with BioRender.com.

Figure 6 :

 6 Figure 6: Fitting models used in the binding assays to obtain the K d Values. Created with BioRender.com.

Figure 7 :

 7 Figure 7: Amount of immobilized type III collagen in three experimental conditions. No wash = measuring the immobilized collagen directly after incubation; wash = washing the plate twice with 100 µL of PBS; 1 h PBS mimic & wash = incubation for 1 h with PBS followed by two rounds of washes with PBS to study the stability of the film before using the microplates for further in vitro binding experiments (n = 4 represented as the mean from duplicate readings and two independent experiments; error bars = standard deviation)

Figure 8 :

 8 Figure 8: In vitro binding analysis pipeline. (A,B). In vitro Eu(III) TRF binding curves using Eu(III)-DO3AAm-CBP and immobilized type I and type III collagen, respectively. (C) Total fluorescence readings of the Eu(III)-DO3AAm-CBP in solution. (D) Calculation of the bound compound/protein by rearranging the equation from Figure 8C to convert the fluorescent values into concentration and then dividing by the concentration of the immobilized protein in the wells. (E) The fractional occupancy analysis of the candidate compound against type III collagen gave an FO of 71 %. The results reported are the mean values of triplicate readings within the same plate and from two independent plate readings (error bars = standard deviation).

Table 3 :Supplemental File 1 :

 31 Optimized conditions for the immobilization of type I and type III collagen and the quantified amount immobilized. Preparation of the acidic solution (AS) and buffering solution (BS) and calibration of the volume. DISCUSSION: This work presents a reproducible method for immobilizing type I and type III collagen. It also demonstrates a protocol for acquiring, analyzing, and interpreting in vitro Eu(III) TRF binding data to characterize the binding properties of a candidate ligand toward type I and III collagen. The protocols for immobilizing type I and type III collagen presented here were developed and optimized considering previously published work on type I and type III

  2.1.1. Add 70 μL of PBS (1x) to each of the wells coated with collagen, and incubate the microplate at room temperature for 1 h.

	2.2.4. Place the microplate on a plate shaker for 30 s. Cover the microplates, and incubate
	at 37 °C for 30 min.
	2.2.5. Remove the microplates, and allow to cool at room temperature. Measure the
	absorbance at 560 nm using a plate reader.
	2.2.6. Plot a calibration curve by plotting the A 560 (AU) versus the concentration (µg/mL) of

2.1.2. Aspirate the excess liquid from each well using a pipette, and wash with PBS (1x) twice before carrying out the protein quantification assay described below.

2.2. Quantification of the amount of immobilized collagen using a bicinchoninic acid assay (BCA) NOTE: Use the Pierce BCA Protein Assay Kit (Table of Materials) following the manufacturer's instructions. Make respective collagen standards for this assay. The concentration range for collagen I is from 0-3,000 µg/mL and for collagen III from 0-750 µg/mL. In total, make 11 standards per collagen.

2.2.1.

Prepare the total volume of working reagent (WR) needed by following the manufacturer's instructions.

2.2.2. Add 25 μL of each of the collagen standard into the microplate wells (in duplicate).

These solutions are used to draw the standard curve. 2.2.3. Add 200 μL of working reagent solution to each of the wells containing the standards and the wells coated with unknown concentrations of collagen. the 11 standard solutions, and use the calibration curve to calculate the amount of collagen.

  .2.1. Add 54 μL of acidic solution (AS) to each well, and place the plate in the incubator at 37 °C for 90 min, covering the microplates with foil to avoid evaporation. The temperature and incubation time must be carefully controlled to achieve reproducible decomplexation.3.2.2. Add 46 μL of buffering solution (BS) to each well, and gently shake the plate for 30 s.3.2.3. Add 100 μL of enhancement solution (ES), and shake the plate for 30 s.3.2.4. Wait for 30 min before reading the plate using a TRF plate reader. Use the parameters listed in Erreur ! Source du renvoi introuvable..

	3.2. Acid extraction of Europium(III) and time-resolved fluorescence (TRF) readings
	NOTE: Please refer to the supplemental information about the preparation and calibration
	of the volumes of the acidic solution (AS) and the buffering solution (BS). The volumes of the
	AS and BS required to reproducibly achieve an optimal pH were 54 μL and 46 μL,

3.1.2. Add 75 μL of each concentration of compound into the collagen-coated wells (Plate A). Perform the experiment in triplicate to calculate the amount of compound that binds to the collagen. 3.1.3. Use a second uncoated plate (Plate B), and add 75 μL of each compound to the empty wells to calculate the non-specific binding of the compound to the plate. Use triplicates for each concentration. 3.1.4. Incubate the microplates for 1 h at room temperature.

3.1.5. Using a pipette, aspirate and discard the excess solution from each well, and wash the wells with 1x PBS twice to remove excess, unbound compound. Perform this step using both the collagen-coated and uncoated microplates. 3.1.6. To a third uncoated plate (Plate C), add 10 μL of the same range of Eu(III)-DO3AAm-CBP concentrations (in duplicate). Use the fluorescence reading from the Eu(III)-DO3AAm-CBP in solution to make a calibration curve. NOTE: Do not wash or aspirate the solution from this plate. respectively, in this work. Carry out the following operation on plate A, plate B, and plate C.

3

Table 2 : Parameters for Eu(III) TRF measurements.

 2

ACKNOWLEDGMENTS:

We are grateful to the following funders for supporting this work: (1) the UK Medical Research Council (MR/N013700/1) and King's College London member of the MRC Doctoral Training Partnership in Biomedical Sciences; (2) BHF program grant RG/20/1/34802; (3) BHF Project grant PG/2019/34897; (4) King's BHF Centre for Research Excellence grant RE/18/2/34213; (5) the ANID Millennium Science Initiative Program -ICN2021_004; and (6) ANID Basal grant FB210024.

DISCLOSURES:

The authors have no conflicts of interest to disclose.


G Conzatti\

S Cavalie

F Gayet\

J Torrisani C

N Carrère

A Tourrette

• Clrima

Pnipam

Elaboration of a thermosensitive smart biomaterial: From synthesis to the ex vivo bioadhesion evaluation

Polysaccltarldes PEC

Introduction

Bioadhesion during wound dressing is an important part of bioma terial science. Indeed, bioadhesion, defined as the ability of material to adhere to a biological entity, is required to achieve an effective healing. However, a highly bioadhesive material could cause injuries and be cou nterproductive. Thus, the manipulation of such materials during surgery is a critical consideration, as it is sometimes neoessary to adjust the material position precisely when applied to soft organs.

In this context, a controlled bioadhesion would be a great way to obtain a balance between position holding and easy manipulation. Bioadhesion is a complex phenomenon that involves proteins such as fibronectin that act as an anchor for the cells. (1) The ability of a sur face to support cell adhesion is depaident on its ability to support protein adsorption. Many factors have to be considered, such as the biomaterial stiffness, surface topography and hydrophilicity. To control bioadhesion, it is necessary to control the interactions between cell adhesion proteins and the biomaterial surface, i.e. the biomaterial surface state.

Numerous studies have been focused on smart biomaterials ob tained by grafting stimuli responsive polymers, such as pH or thermo • Corresponding author. sensitive polymer s. [2,3) Poly N (isopropylacrylamide) (PNIPAM) is a widely studied thermosensitive polymer that changes its hydrophilicity around 32 •c, called its lower critical solution temperature (LCST) [START_REF] Conzatti | PNIPAM grafted surfaces through ATRP and RAFT polymerization: chemistry and bioadhesion[END_REF]5). Therefore, this is an interesting material, as the human body temperature is close to 37 •c. Once grafted, its ability to change from a passive state at room temperature to an active state at body tempera ture is depaident on chain length and graft density. Nonetheless, the difficulty in efficiently characterizing the surface state of polymer scaffolds has oriented the use of PNIPAM for bioadhesion studies to ward the surface modification of well controlled surfaces, such as glass [START_REF] Tsai | Two-dimensional patterns of poly(N -isopropylacrylamide) microgels to spatially control fibroblast adhesion and temperature-responsive detachment[END_REF] or gold [START_REF] Plunkett | PNIPAM chain collapse depends on the molecular weight and grafting density[END_REF]. These surfaces are amenable to theoretical models [START_REF] Halperin | Theoretical considerations on mechanisms of harvesting cells cultured on thermoresponsive polymer brushes[END_REF].

However, the development of biodegradable biomaterials that would allow controlled bioadhesion is of interest when considering implanted devices. Polysaccharides are a class of such polymers that are generally considered biocompatible. Among others, alginate and chit osan are relatively inexpensive biopolymers that are well tolerated by the immune system. [9,10) Their combination, in the form of poly electrolyte complexes (PECs), allows the production of biodegradable absorbent and resistant matrixes that are able to 1imit fluids, such as bleedings. They can also be used to disseminate or to release drugs [START_REF] Castel-Molieres | Influence of homogenization technique and blend ratio on chitosan/ alginate polyelectrolyte complex properties[END_REF][START_REF] Conzatti | Alginate/ chitosan polyelectrolyte complexes: a comparative study of the influence of the drying step on physicochemical properties[END_REF]. Previously, we reported that calcium reticulated PECs showed E-mail address: Audrey.tourrette@univ•t1se3.fr (A. Tourrette). https://doi.org/10.1016/j.colsurfb. 2018.11.084 12,000 g/mol and dried under vacuum.

The theoretical molecular weight, Mn th , of the polymer was calcu lated with Eq. 1:

= × × Mn mono CTA M M [ ] [ ] th mono CTA 0 0 (1)
where M mono represents the molecular weight of the monomer, [mono] 0 its initial concentration, % conv is the percentage of conversion of the monomer (considered as 100%) and [CTA] 0 the initial concentration of the CTA.

Characterization of the PNIPAM COOH

The molar mass of the PNIPAM was measured by size exclusion chromatography (SEC) using DMF/LiBr (10 mM, refractive index of 1.431) as solvent. Before analysis, the samples were filtered through a 10 μm nylon filter. Detection was performed by the measurement of the refractive index variation (Optilab rEX detector) combined to a light scattering analyzer (miniDawn, 690 nm). Mass determination was per formed using the ASTRA software with a dn/dc of PNIPAM in DMF equal to 0.0731 mL/g. [START_REF] Audouin | Surface-initiated RAFT polymerization of NIPAM from monolithic macroporous polyHIPE[END_REF] The functionality of PNIPAM was determined by COOH pH metric titration of the polymer (0.5 g in 20 mL H 2 O) with NaOH (0.02 M). The pH was measured using a 766 Calimatic pH meter (KCl electrode).

Surface functionalization

The grafts on the surface of the PECs were achieved through an NHS/EDC coupling. To increase the reactivity of the PECs (PEC and PEC EDA), an acidic treatment was performed by immersing the PECs in water (HCl, pH 2.5) for 1 h prior to any reaction.

Grafting of EDA on alginate/chitosan PECs

To increase the grafting density on the films, EDA was previously grafted on the desired samples (PEC EDA). Briefly, PECs (30 g) were immersed in DMSO (0.6 mL) containing NHS (100 mmol/L) and EDC (200 mmol/L) for 3 h before EDA addition (0.6 mL, final concentration of 7.2 mol/L). The mixture was left to react for 12 h. The mixture was slightly agitated on a gyratory rocker. Films were thoroughly rinsed with EtOH and DMSO to remove any unreacted species.

Grafting of PNIPAM COOH on alginate/chitosan PECs

The grafting of the PNIPAM COOH was performed both on EDA grafted PECs (PEC EDA) and on native PECs (PEC). The PNIPAM COOH (2 mmol/L) was activated for 3 h with NHS (25 mmol/L) and EDC (50 mmol/L) in DMSO (1 mL). PEC of PEC EDA (30 g) were added to the reaction mixture. The mixture was left to react for 12 h. The mixture was slightly agitated on a gyratory rocker. Films were thor oughly rinsed with EtOH and DMSO to remove any unreacted species.

Surface characterization

The surface characterization before and after modification was performed via X ray photoelectron spectroscopy (XPS), on an ES CALAB 250, Thermo Electron. The excitation was monochromatic, Al Kα ray at 1486.6 eV. The diameter of analysis was 400 μm and a charge com pensation was done through an electron beam at -2 eV. The signal of aliphatic carbons was used as reference and set to 285.0 eV.

Before analyses, samples were stored in glass containers, under vacuum to avoid contamination.

In vitro evaluation

Cytotoxicity studies were performed with hPNE (human Pancreatic Nestin positive Epithelial) cells expressing green fluorescent protein (GFP) to facilitate their visualization. The samples (5 x 5 mm) were immersed in the culture medium for 20 min before the cells were an interesting resistance toward enzymes, along with a good balance between degradation and resistance in saline environment [START_REF] Conzatti | Alginate/ chitosan polyelectrolyte complexes: a comparative study of the influence of the drying step on physicochemical properties[END_REF]. How ever, only a few studies report the development of biodegradable wound dressings with thermo controlled bioadhesion by the grafting of PNIPAM on polysaccharide substrates [START_REF] Chen | Thermo-responsive wound dressings by grafting chitosan and poly(N-isopropylacrylamide) to plasma-induced graft polymerization modified non-woven fabrics[END_REF]. To date, no studies are devoted to the exploration of a thermosensitive PEC.

Herein, the grafting of a functional PNIPAM COOH polymer in a brush structure on alginate/chitosan PEC film surfaces through NHS/ EDC coupling was studied. To control the chain length of the grafted PNIPAM, it was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization, a controlled radical polymerization, [START_REF] Conzatti | PNIPAM grafted surfaces through ATRP and RAFT polymerization: chemistry and bioadhesion[END_REF] and compared to a commercial polymer. The grafting strategy was also set to modulate the graft density of the PNIPAM brush. The re sulting films were characterized by X ray photoelectron spectroscopy (XPS) prior to characterization of cytotoxicity and thermo controlled bioadhesion with human cells (in vitro) and organs (ex vivo).

Materials and methods

Materials

Sodium alginate, chitosan, calcium chloride, acetic acid, N Isopropylacrylamide (NIPAM), poly (N Isopropylacrylamide) (PNIPAM, Mw = 5000 g.mol -1 ), N Hydroxysuccinimide (NHS), N (3 Dimethylaminopropyl) N' ethylcarbodiimide hydrochloride (EDC), ethylene diamine (EDA), Tetrahydrofuran (THF), dimethylsulfoxide (DMSO), Methyl 2 (dodecylthiocarbonothioylthio) 2 methylpropio nate (CTA) and 4.4′ Azobis(4 cyanovalericacid) (radical initiator, ACVA), as well as all solvents, were provided by Sigma Aldrich.

The alginate and chitosan were characterized as previously re ported. [START_REF] Conzatti | Alginate/ chitosan polyelectrolyte complexes: a comparative study of the influence of the drying step on physicochemical properties[END_REF] The G/M unit ratio of alginate was estimated using a water suppressive NMR method to M/G = 2.7 [START_REF] Vilén | Application of diffusion-edited NMR spectroscopy for selective suppression of water signal in the determination of monomer composition in alginates[END_REF]. Viscosimetry determined its molecular weight to be 340 kDa, and α and κ parameters to be 1.13 and 6.9 × 10 -4 mL.g 1, respectively. [START_REF] Martinsen | Comparison of different methods for determination of molecular weight and molecular weight distribution of alginates[END_REF] Using the Shigemasa method, the degree of acetylation (DA) of the chitosan was estimated to be 23% [START_REF] Shigemasa | Evaluation of different absorbance ratios from infrared spectroscopy for analyzing the degree of deacetylation in chitin[END_REF] with Mw = 1 300 kDa (α = 0.93, κ = 1.81 × 10 -1 ml. g 1). [START_REF] Kasaai | Calculation of Mark-Houwink-Sakurada (MHS) equation viscometric constants for chitosan in any solvent-temperature system using experimental reported viscometric constants data[END_REF] NIPAM was purified by hexane recrystallization prior to use. Other products were used in the as received state.

Alginate/chitosan PECs elaboration

The procedure for the PEC elaboration was based on our previous studies with minor modifications. [START_REF] Castel-Molieres | Influence of homogenization technique and blend ratio on chitosan/ alginate polyelectrolyte complex properties[END_REF][START_REF] Conzatti | Alginate/ chitosan polyelectrolyte complexes: a comparative study of the influence of the drying step on physicochemical properties[END_REF] The sodium alginate was dissolved in water at a concentration of 1.5% w/v and mechanically stirred overnight (500 rpm). In parallel, the chitosan was dissolved in acidic water (acetic acid, 0.34% v/v) at a concentration of 1.5% w/v and mechanically stirred overnight (500 rpm). After dissolution, 50 g of the alginate solution and 30 g of the chitosan solution were mixed to obtain a mass of 63/37 alginate/chitosan. The mixture was homo genized with an Ultra Turrax ® (11 000 rpm) for 10 min to form the PEC. The mixture was poured in a 24 well culture plate (0.2 mL/well) and dried at 50 °C for 2 h. The films were reticulated with a CaCl 2 solution (0.5 mL, 1% m/V) for 30 min, washed with distilled water and dried for another 2 h to obtain thin semitransparent films.

Synthesis of PNIPAM COOH

Functional PNIPAM COOH was synthesized through a RAFT poly merization in THF. The monomer (NIPAM) was added to a balloon containing the CTA dissolved in THF. The reactor was closed with septa and argon was bubbled in the reaction mixture for 30 min to remove dissolved oxygen. The initiator was added into the mixture and the reaction was maintained for 12 h. After the polymerization, the polymer was precipitated in ether, filtered and washed with ether. This step was repeated three times to remove the ether soluble monomer. The precipitate was dialyzed in water for 3 days with a cut off of

= - × T Q Q Q (%) (37) (27) (27) 100 (2)
where Q( 27) is the number of adherent cells at 27 °C and Q(37) is the number of adherent cells at 37 °C.

Ex vivo adhesion evaluation

Porcine organs (liver and pancreas) were furnished from a local slaughterhouse, where they were excised directly after slaughtering, kept in physiological solution at 4 °C and used within the day. Before the measurement, samples of fresh organ external part were excised and immersed in a physiological solution at 25 °C or 40 °C. The samples were fixed on a 10 mm diameter probe and contact was made with the organ surface for 5 min with an application force of 500 g. The bio material was separated from the surface of the organ (0.5 mm/s). The maximal force required for this separation was measured with a TA.XTplus texturometer equipped with a 5 kg captor. The associated constraint corresponded to the adhesive force. The experiment was conducted on three samples of each type.

Results and discussion

Alginate and chitosan display reactive sites with R COOH carboxylic acids and R NH 2 amines, respectively. A PNIPAM end functionalized with a carboxylic acid can be grafted on the amino functions of the chitosan via an amide formation (Fig. 1, step 1). The "grafting to" strategy proposed here consisted of an increase in the number of re active sites at the PECs surface through the grafting of a diamine on the carboxylic acids of the alginate (Fig. 1, step 2a). This resulted in an amination of the alginate and the covalent binding of the PNIPAM COOH to both diamine and amines of the chitosan (Fig. 1, step 2b). As a result, a higher grafting density of the PNIPAM was expected. In ad dition, the control of the molecular weight of the PNIPAM could modulate the chain length of the grafted brush.

PNIPAM COOH synthesis

To graft PNIPAM onto alginate/chitosan PEC films, the synthesis of a functionalized polymer was needed. The RAFT polymerization allows the production a functional polymer with well controlled chain lengths.

[ 19 21]. The COOH functionality mainly resulted from the R group re initiation of the reaction (see supporting information, S1).

Table 1 reports the experimental conditions. As observed for PNI N, the polymerization of 2 g of NIPAM without CTA generated a high molecular weight polymer (M n = 100 000 g/mol) and polydispersity index Ð equal to 2.17, which is demonstrative of a non controlled polymerization. As the control agent was added (M n,th of 25 000 g/ mol), a control over the polymerization was observed with a sig nificative diminution of the M n (PNI 1, 26 000 g/mol) and Ð (1.19). The obtained molecular weight was close to the theoretical mass calculated with Equation 1, which is consistent with a RAFT mechanism. However, this polymerization was performed at a low content of NIPAM (2 g) and to use the synthesized polymer for surface grafting, it was necessary to master the polymerization of a higher content of polymer. Therefore, 15 g of NIPAM was RAFT polymerized using the same conditions (PNI 2). The obtained polymer showed a well defined size with a M n of 24 000 g/mol and Ð equal to 1.09. The scaling up of this polymerization did not affect the efficiency of the RAFT mechanism.

For the following surface modification, it was necessary to control the functionality of the used polymer. Titrations were performed on PNI 2. The COOH/PNI ratio of 1.0 obtained for PNI 2 is consistent with its low Ð, confirming the absence of significant recombination of the RAFT polymerization. To compare to a smaller PNIPAM, a commercial PNIPAM COOH was studied and showed a M n of 6 000 g/mol, Ð of 1.56 and a functionality COOH/PNI of 1.4. Both polymers were suitable for PEC surface grafting through their carboxylic functionality.

Surface functionalization

To provide thermosensitivity to the PEC surfaces, the PNIPAM COOH, commercially obtained or obtained from RAFT synthesis, was grafted onto alginate/chitosan matrix through NHS/EDC process. To this purpose, after their elaboration, PECs were acidified to improve the reactivity of functional groups. Two strategies were tested to modulate the graft density (Fig. 2).

All the reactions were conducted in DMSO, a non swelling solvent for PECs, to limit internal grafting and alginate/chitosan cross reaction. Surface characterization was performed using XPS (Fig. 3). First, the grafting of EDA (PEC EDA) was revealed by the increase of the overall nitrogen content, which increased from 4.4% to 7.4% compared to non treated PECs (PEC). This nitrogen was non protonated, which was consistent with the formation of amide linkage between the amines of EDA and the carboxylic acids of PEC surfaces. At the same time, the CO, CN increased from 27.7% for PEC to 36.7% for PEC EDA, which could confirm the presence of EDA on the surface. The XPS signal of PNIPAM was characterized by a strong contribution of aliphatic carbons (CC, CH, 73.5%) as approximately 10% of nitrogen, which was exclusively in the form of amides hence without protonation (see supporting in formation, S2). Without EDA, PEC 6 K exhibited a slight increase of aliphatic carbons to 31.8% as compared to 23.2% for PEC. The nitrogen content increased to 8.6% and was mainly non protonated (amine/ amide), which is consistent with the presence of amides. The decrease of oxygen content, combined with the increase of their proportion in C = O form, was also attributed to the presence of PNIPAM. All of these results confirmed the presence of PNIPAM over the PEC surfaces. The same measurements were acquired for PEC 24 K: an increase of CC, CH seeded to release from the materials any potential reagent remnants. After 20 min, the medium was aspirated and the cells were poured in the culture wells at a density of 10 3 cells per well (V = 1 mL). Control experiments, without material, were performed in parallel. Cultures were incubated at 37 °C for 4 days. The materials were gently removed, and the cells were removed from their culture plate by the action of 1 mL of trypsin EDTA. A sample of 100 μL was withdrawn and put in an isotonic solution to perform counting with a Beckman Coulter Z1 par ticle counter, which was set to count cells with a diameter greater than 10 μm. The control experiment was considered to be 100% and the percentage of proliferation of the wells containing samples were cal culated as the ratio of the number of cells in the sample, N sample /N control x 100. The experiment was separately repeated three times for each type of sample with freshly prepared biomaterials. Two parallel samples were evaluated for each experiment. The mean was calculated with the higher value of each separate experiment.

Bioadhesion studies were performed with hPNE cells. The samples were elaborated directly on glass slats to ensure the complete recovery of culture wells. Once at the bottom of the wells, the samples were covered with culture medium (1 mL) for 20 min to allow swelling and to evacuate any toxic compounds adsorbed on the surface. The medium was aspirated and the hPNE cells were gently seeded on the PECs (60,000 cells/well, 1 mL). Cultures were incubated at 37 °C or 27 °C. After 8 h, the medium was aspirated, the PECs were transferred to clean culture wells and rinsed twice with PBS to eliminate non adherent cells. In the case of cultures carried out at 37 °C, the flushing PBS was heated at 37 °C before being used. Sample surfaces was observed with a Zeiss Axio Vert A 1 inverted fluorescence microscope with a wavelength of excitation of 493 nm (emission at 517 nm). The number of cells on the surface was counted by image processing using ImageJ. The experiment was separately repeated three times for each type of sample with freshly prepared biomaterials. Two parallel samples were evaluated for each experiment. The mean was calculated with the higher value of each separate experiment. The thermosensitivity, T, was calculated using Eq. 2:

(33.8%) and overall nitrogen (8.1%), the majority of which was in a neutral form and a diminution of oxygen rates (18.6%), mainly in C = O form. It appeared that the increase of the chain length of the PNIPAM COOH was not detrimental to its graft efficiency on the algi nate/chitosan PEC surfaces.

Concerning the grafting of PNIPAM COOH on PEC EDA, the grafting of a small acrylamide of 6000 g/mol was efficient, with the same evo lution of the characteristic signals of PNIPAM compared to PEC EDA 6 K. However, in the case of the grafting of higher molecular PNIPAM COOH (PEC EDA 24 K), it was not possible to confirm the presence of PNIPAM over the surface, as aliphatic carbons diminished and the protonated amines (NH 3 + ) increased. A possible surface passivation due to an EDA diamine bridging over the surface could obstruct the surface, and the higher molecular weight PNIPAM could react less ef fectively with the hindered reactive COOH, explaining this grafting limitation. Also to mention, the possible alginate/chitosan cross linking, while not evaluated, could limit the grafting efficiency on all the biomaterial surfaces.

In brief, the grafting of PNIPAM COOH was efficiently performed, leading to various surfaces by modulating the graft density and the chain length of the PNIPAM. As no PNIPAM was observed for PEC EDA 24 K, this sample was not further studied. Cytotoxicity testing was performed by comparing the average number of hPNE cells after four days in a culture medium containing biomaterial samples to a control experiment. The results were expressed as percentage compared to control (100%), as shown in Fig. 4, Right panel. It appeared that all cell viability and cell proliferation were greater than 70%, which is the considered limit by the ISO 10993 5 to suspect a cytotoxic effect. Therefore, no sample showed cytotoxicity whether EDA was used, or not, and whether the PNIPAM was synthe sized or from a commercial source.

In vitro bioadhesion

Bioadhesion was evaluated on all the samples to determine whether a thermosensitive effect was caused by the PNIPAM. As shown in the literature, bioadhesion on the PNIPAM brush structure is dependent on the chain length and the graft density on its substrate, as these para meters play a role in the adsorption of the proteins involved in cell bioadhesion. [START_REF] Conzatti | PNIPAM grafted surfaces through ATRP and RAFT polymerization: chemistry and bioadhesion[END_REF][START_REF] Xue | Protein adsorption on poly(N-isopropylacrylamide) brushes: dependence on grafting density and chain collapse[END_REF] Bioadhesion is a process that occurs in the first hours after cell seeding [START_REF] Anselme | Cell/Material interfaces: influence of surface chemistry and surface topography on cell adhesion[END_REF]. For this reason, and to avoid proliferation influence, the cell cultures were maintained only for 8 h. After this time, non adherent cells were removed. Adherent cells were quantified by fluorescence image treatment. On PEC, cells tended to aggregate in clusters. The thermosensitivity was calculated as the difference between adherent cells at 27 °C and at 37 °C using Equation 2 (Fig. 4, Left panel) (don't we read from left to right? why did you put the Right panel before the Left panel in the description of your results?). The negative thermosensitivity for non modified PEC ( 64%) was indicative of the number of adherent cells at 37 °C compared to 27 °C, which decreased by approximately two thirds.

It is risky to explain this phenomenon, but it could be that a lower temperature diminished the cluster formation kinetics (see supporting information, S3), while these clusters were more prone to detach off the surface during the washing steps. For the PNIPAM grafted biomaterials (PEC 6 K, PEC EDA 6 K and PEC 24 K), the thermosensitivity was re versed and became positive, meaning that a better bioadhesion was achieved by the hPNE cells at 37 C rather than at 27 C due to the presence of PNIPAM. The gain of bio adhesiveness seemed to be higher when the graft density was increased by the use of EDA, with the number of cells multiplied by up to 9.5 (PEC EDA 5 K, 840%) above the LCST of PNIPAM. However, no statistical difference was observed. The size of the grafted PNIPAM (6 000 g/mol or 24 000 g/mol) did not show to affect the bioadhesion thermosensitivity, which would result of the suppression of primary adsorption bioadhesion proteins. [START_REF] Xue | Protein adsorption on poly(N-isopropylacrylamide) brushes: dependence on grafting density and chain collapse[END_REF] These results confirmed the potential of obtaining smart responsive biopoly mers.

Ex vivo evaluation

Adherence between these surfaces and porcine organs was eval uated. Ex vivo experiments allowed a first approximation of the beha vior of a developed biomaterial before further in vivo assays. Modified and unmodified PEC bioadhesion was tested using pancreas and liver freshly extracted from pigs (Fig. 5). The non thermosensitive PEC samples adhered two times harder on the pancreas than on the liver, which emphasized the importance of the considered organ itself on the bioadhesion. Specifically, the nature of the tissue (cells, extra cellular matrix), its roughness or softness are parameters that can affect bioadhesive properties.

Data obtained from the pancreas were ambiguous. Whereas no statistical difference was observed between the two temperatures, for all the other samples, only PEC 6 K was affected by the passage of the LCST. It had a strong decrease in the adhesive force, from 16 kPa at 25 °C to 6 kPa at 40 °C. Therefore, long PNIPAM chains or high graft densities tended to limit the control over bioadhesion.

When in contact with the porcine liver, non modified biomaterials (PEC) did not show any variation between the measured adhesive force at 25 °C and 40 °C (4 kPa). The PEC 6 K, PEC EDA 6 K and PEC 24 K, however, exhibited a significant difference between the temperatures below and above the LCST of PNIPAM. Indeed, at 40 °C, the bioadhe sion between these PNIPAM grafted samples was lower, reflecting poorer interactions between PNIPAM chains and the liver surface above the LCST.

The PNIPAM surfaces on retinal implants were reported to exhibit similar magnitude of adhesive force (25 kPa). [START_REF] Tunc | Reversible thermosensitive glue for retinal implants[END_REF] Nonetheless, it was difficult to determine whether the observed phenomenon was due to physical or biological interactions, so as to extract some trends con cerning the impact of the length of the grafted PNIPAM or its graft density, as it was also dependent of the studied organ. Nonetheless, although the thermosensitivity of the surfaces tended to favor interac tions at low temperatures, the observed adherence at warm tempera tures is in the range of those considered as safe [START_REF] Waring | Skin adhesion properties of three dressings used for acute wounds[END_REF]. The PNIPAM in teractions were the result of a balance between intra/interchain and water molecules. The influence of a highly polar substrate, composed of alginate and chitosan, could, however, play a critical role on this bal ance.

Conclusions

Functional PNIPAM was efficiently synthesized via RAFT poly merization for comparison with a shorter commercial PNIPAM to study the influence of the chain length on bioadhesion. Two different stra tegies were used to graft these polymers on alginate/chitosan PECs through NHS/EDC coupling. The PNIPAM was directly grafted or used in combination with EDA to increase the graft density. Studies of the chemical of the surface through XPS revealed the presence of PNIPAM for PEC 6 K, PEC EDA 6 K and PEC 24 K, without being able to confirm whether the EDA allowed a higher graft density or not. However, the combination of EDA with a high molecular weight PNIPAM did not allow its efficient grafting.

No cytotoxicity was found for all samples, and it was shown that the PNIPAM grafted surfaces exhibited a higher bioadhesion toward cells above the LCST of PNIPAM, whatever the chain length or graft density. Thermosensitivity was also observed using porcine organs. A higher chain length or graft density did not allow a controlled bioadhesion with pancreas above the LCST, whereas it did not strongly affect the thermosensitivity with the liver.

Further work has to be considered to better understand the inter actions between the grafted samples and the surface of organs, taking in consideration possible interactions between the PNIPAM chains and

Fig. 1 .

 1 Fig. 1. Grafting strategies to control the chain length and graft density of PNIPAM on PEC substrates.

Fig. 2 .

 2 Fig. 2. Grafting of PNIPAM-COOH onto alginate/chitosan PECs.

3. 3 .

 3 Toward surface adhesion 3.3.1. Cytotoxicity

Fig. 3 .

 3 Fig. 3. Atomic compositions determined by high resolution XPS.

Fig. 4 .

 4 Fig. 4. (Right panel) Number of cells after 4 days in presence of a biomaterial versus a control without any biomaterial. (Left panel) Thermosensitivity, calculated with Eq. 2, of bioadhesions as measured after 8 h of culture at 27 C and 37 C.

Table 1

 1 Experimental conditions for the polymerization of PNIPAM and the resulting polymer characteristics.

	Sample	Volume	m M0	a)	[M 0 ]/[I 0 ]/[CTA 0 ]	T	M n,th b	M n	c	Ð	pKa	COOH/PNI
		(mL)	(g)		(mol/L)	(°C)	(g/mol)
	PNI-N	5	2		1400/1/-	70	-	100 000	2,17	nd	nd
	PNI-1	5	2		1400/1/7	70	25 000	26 000	1,19	nd	nd
	PNI-2	38	15		1400/1/7	70	25 000	24 000	1,09	3.9	1.0
	Commercial	-	-		-	-	-	6 000	1.56	4.0	1.3

a monomer mass. b theoretical molar mass of PNIPAM calculated through Eq. 1. c determined with SEC.

Acknowledgment

Authors would like to thank the French National Research Agency for its financial support (ANR 14 CE17 0002 01, FP BioPrev project).

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2018.11.084.

Elaboration of a thermosensitive smart biomaterial: from the synthesis to the ex vivo bioadhesion evaluation