Christophe Sinturel
email: christophe.sinturel@univ-orleans.fr

Marylène Vayer

Frédéric Mahut

Franck Bonnier

Igor Chourpa

Emilie Munnier

Influence of PLGA nanoparticles on the deposition of model water-soluble biocompatible polymers by dip coating

Keywords: colloids, water-soluble biocompatible polymer, nanocomposite thin films, dip coating, AFM

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Influence of PLGA nanoparticles on the deposition of model water-soluble biocompatible polymers by dip coating

ranging from 1 to 10 % wt./wt. in PVA or PVP matrices were prepared. The presence of nanoparticles on the well-established process of thin film deposition was examined, as well as the influence of the deposition regime on the nanoparticle concentration in the deposited coating. We demonstrate that the presence of colloidal dispersion of PLGA nanoparticles in water solution of PVA and PVP does not modify the process of film deposition by dip coating. A typical "V" shaped curve was observed, with two well-known deposition regimes: capillary and draining modes respectively obtained at low and high withdrawal speeds. Due to crystallization at low withdrawal speed (favored by slow evaporation of the solvent) it was not possible to identify individual PLGA nanoparticles by AFM in the case of the PVA matrice. Amorphous PVP nanocomposite films were successfully prepared by dip coating, and allowed us to identify individual PLGA nanoparticles with AFM. Because of the prevalence of an evaporation-driven phenomenon at low withdrawal speed, incorporation of NPs was observed over the whole range of withdrawal speeds, showing original behavior compared to recent studies relying on a pure Landau-Levich regime (i.e. non-evaporative systems). Our results indicate that the nanoparticles were not equally retrieved from the solution in the capillary and the draining regime. This suggests that the balance between the viscous drag and the interfacial effects depends on the deposition mode and calls for a more detailed analysis of the physical processes involved in both regimes.

Introduction

In nanocomposite materials, the small volume of nanoscale fillers induces specific behavior in comparison to their macro-scale counterparts, such as an extremely high surface-to-volume ratio or improved chemophysical responsiveness. Given that the intrinsic properties of the nanofillers in the coating differ strongly from the bulk due to confinement effects arising from the limited amount of matter, the elaboration of nanocomposites has attracted a great deal of interest over the last decades. [START_REF] Kao | Toward functional nanocomposites: taking the best of nanoparticles, polymers, and small molecules[END_REF][START_REF] Kumar | Winey 50th Anniversary Perspective : Are Polymer Nanocomposites Practical for Applications?[END_REF] More specifically, nanocomposite materials in the form of films or coatings have found outstanding applications in packaging, [START_REF] Assis | Active biodegradable cassava starch films incorporated lycopene nanocapsules[END_REF][START_REF] Rhim | Ha Bio-nanocomposites for food packaging applications[END_REF] sensors [START_REF] Bochenkov | Sensor activity of thin polymer films containing lead nanoparticles[END_REF] , and membranes [START_REF] Khin | A review on nanomaterials for environmental remediation[END_REF][START_REF] Aroon | Performance studies of mixed matrix membranes for gas separation: A review[END_REF] thanks to improved mechanical [START_REF] Toyoda | Dispersion of carbon nanofibers modified with polymer colloids to enhance mechanical properties of PVA nanocomposite film[END_REF] , dielectric, [START_REF] Dai | Guo Improved dielectric and energy storage properties of poly(vinyl alcohol) nanocomposites by strengthening interfacial hydrogen-bonding interaction[END_REF] antibacterial, [START_REF] Guod | Polymer/nanosilver composite coatings for antibacterial applications[END_REF] antifouling, [START_REF] Selim | Huang Superhydrophobic coating of silicone/β-MnO2 nanorod composite for marine antifouling[END_REF] permeability [START_REF] Garusinghe | Batchelor Nanocellulose-montmorillonite composites of low water vapour permeability[END_REF] or optical properties. [START_REF] Gu | Cao Optical storage arrays: a perspective for future big data storage[END_REF] Drug delivery from nanocomposite coating has also emerged as a strategy to improve the integration of biomedical materials in the body and increase their efficiency through local drug release. 14,[START_REF] Westedt | Kissel Paclitaxel releasing films consisting of poly(vinyl alcohol)-graft-poly(lactide-co-glycolide) and their potential as biodegradable stent coatings[END_REF][START_REF] Dowdy | Histone deacetylase inhibitors and paclitaxel cause synergistic effects on apoptosis and microtubule stabilization in papillary serous endometrial cancer cells[END_REF] In this field, innovation is high.

Triggerable drug delivery using nanocomposite coatings has for example recently emerged. [START_REF] Vannozzi | Ricotti Nanocomposite thin films for triggerable drug delivery[END_REF] In this case, the inclusion of nanofillers renders matrices responsive to external stimuli, increasing their tunability and responsiveness in terms of drug release.

Among the various methods used to deposit surface layers, dip coating is a very simple way for coating objects, even with more complex shapes than flat surfaces, such as fibers [START_REF] Quéré | Fluid coating on a fiber[END_REF][START_REF] Kwon | High Luminance Fiber-Based Polymer Light-Emitting Devices by a Dip-Coating Method[END_REF][START_REF] Tang | Dip-coating for fibrous materials: mechanism, methods and applications[END_REF] that could find valuable applications in the field of biomedical implants [START_REF] Javadi | Surface engineering of titanium-based implants using electrospraying and dip coating methods[END_REF] . It consists in dipping the substrate into a solution containing the material to be deposited, and withdrawing the substrate at a constant speed. The process can be automated and can therefore be easily implemented in industrial processes, on any type of materials or objects. 22 Depending on the withdrawal speed, the nature of the solvent, and the concentration of the solvent vapors above the reservoir, the thickness of the coating can be finely tuned. Dip coating was first studied in the case of a non-evaporative liquid in the pioneering work of Landau and Levich [START_REF] Evich | ragging of a liquid by a moving plate[END_REF] . It has been extended to sol-gel processes, [START_REF] Faustini | Preparation of Sol-Gel Films by Dip-Coating in Extreme Conditions[END_REF][START_REF] Brinker | Sol-Gel Thin Film Formation[END_REF] and polymer solutions, [START_REF] Roland | Solvent Influence on Thickness, Composition, and Morphology Variation with Dip-Coating Rate in Supramolecular PS-b-P4VP Thin Films[END_REF][START_REF] Vital | Sinturel Morphology control in thin films of PS:PLA homopolymer blends by dip-coating deposition[END_REF] where, as the solvent evaporates, it has been demonstrated that deposition occurs in two specific regimes, namely the capillary and the draining regimes (for low and high withdrawal speeds respectively). The physical processes of film formation in these two regimes are different (convective and Landau-Levich deposition respectively), 22 leading to a typical "V" shaped thickness evolution. [START_REF] Roland | Morphology, Thickness, and Composition Evolution in Supramolecular Block Copolymer Films over a Wide Range of Dip-Coating Rates[END_REF] By extension, the preparation of nanocomposite coatings using dip coating can be easily carried out if the polymer solution contains a colloidal dispersion of nanoparticles (NPs). Polymer and nanoparticles are both retrieved with the solution by the moving substrate, leaving a nanocomposite film after drying. One important question at this stage is whether the presence of nanoparticles can modify the well-established process of thin film deposition and whether NP concentration in the deposited coating can be influenced by the withdrawal speed. In a similar mode of deposition, it has been indeed shown that the deposition of colloidal dispersion in the evaporative regime was strongly influenced by the vapor mass transfer during drying, leading to a deviation of the withdrawal speed dependence of the thickness. [START_REF] Loussert | Role of vapor mass transfer in flow coating of colloidal dispersions in the evaporative regime Langmuir[END_REF] In addition, it is known that enrichment in nanoparticles can occur at the receding meniscus, similarly to the "coffee ring effect", [START_REF] Deegan | A Witten Contact line deposits in an evaporating drop[END_REF][START_REF] Deegan | Capillary flow as the cause of ring stains from dried liquid drops[END_REF] leading to a modification of the NP concentration in the dried film, in comparison to the solution. [START_REF] Huang | One-Step Patterning of Aligned Nanowire Arrays by Programmed Dip Coating[END_REF][START_REF] Ghosh | Spontaneous Pattern Formation by Dip Coating of Colloidal Suspensions on Homogeneous Surfaces Langmuir[END_REF] On the contrary, it has been recently shown that a selection of nanoparticules can occur at a low withdrawal speed, [START_REF] Dincau | Capillary Sorting of Particles by Dip Coating[END_REF] eventually leading to a situation where only the liquid is entrained, the particles being trapped in the meniscus and left in the bath (capillary filtering) [START_REF] Sauret | Dressaire Capillary filtering of particles during dip coating[END_REF] .

A literature survey shows that despite an abundant number of studies on the use of dip-coating for the deposition of polymer solutions on the one hand and colloidal suspensions of NPs on the other hand, the deposition of more complex systems combining polymer and NPs is much less well documented. Although some studies describe the preparation of nanocomposites by dip-coating, [START_REF] Patil | Investigations on silver/polyaniline electrodes for electrochemical supercapacitors[END_REF][START_REF] Sugumaran | Transparent nano composite PVA-TiO2 and PMMA-TiO2 thin films: Optical and dielectric properties[END_REF][START_REF] Catauro | Bollino Characterization and biological properties of TiO2/PCL hybrid layers prepared via sol-gel dip coating for surface modification of titanium implants[END_REF] no systematic analysis of the deposition process (withdrawal speed, NP concentration, polymer type, etc.) of a polymer solution containing a colloidal suspension of nanoparticles has been reported to our knowledge. In the present work, we focused on two model biocompatible water-soluble polymers, namely polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP) for which we evaluated the effect of the presence of poly(lacticco-glycolic acid) (PLGA) nanoparticles (a typical carrier for drug encapsulation [START_REF] Galeska | Controlled release of dexamethasone from PLGA microspheres embedded within polyacid-containing PVA hydrogels[END_REF][START_REF] Tada | Sridhar Chitosan Film Containing Poly(D,L-Lactic-Co-Glycolic Acid) Nanoparticles: A Platform for Localized Dual-Drug Release[END_REF][START_REF] Santos | Mijangos Manufacture and characterization of chitosan/PLGA nanoparticles nanocomposite buccal films[END_REF] ) at moderate concentrations (up to 10%) on the deposition mechanism.

Material and methods

Material

Two polymer matrices (Polyvinyl alcohol (PVA) M w : 50 kg.mol -1 , hydrolysed 87 -89% from Aldrich, and Polyvinylpyrrolidone (PVP) M w average: 58 kg.mol -1 from Alfa-Aesar) were used. Distilled water was used as solvent to solubilize these polymers. Poly (lacticcoglycolic acid) 50:50, mol wt 30,000-60,000, and dimethyl carbonate were purchased from Sigma-Aldrich. Water was purified with a Milli-Q system (Millipore, France).

PLGA nanoparticle preparation and purification

PLGA nanoparticles were prepared by the single emulsion-solvent evaporation process. [START_REF] Tewes | Chourpa Comparative study of doxorubicinloaded poly(lactide-co-glycolide) nanoparticles prepared by single and double emulsion methods[END_REF] To prepare the organic phase, 400 mg of PLGA were dissolved in 4 mL of dimethyl carbonate.

The aqueous phase was a PVA solution at 4% wt./wt. at 4°C. The aqueous phase was placed in a cooling Rosett cell in an ice bath. The organic phase was injected in the aqueous phase under ultrasound (20 kHz; Vibra-cell ultrasonic processor, Sonics & Materials, USA). Sonication was continued during 2 minutes. Then, the dimethyl carbonate was evaporated under vacuum at 40°C, 50 mbar for 30 minutes (Welch ILMVEC, USA). Samples were then centrifuged (15000xg, 20 min) and the supernatant was discarded. The pellets were dispersed in 10 mL of ultrapure water and centrifugation was repeated to purify the sample from the excess of surfactant. The pellet was finally dispersed in 10 mL of ultrapure water and stored at 4°C. The NP concentration in the final suspension was 13 mg.mL -1 .

Nanoparticle characterization

The nanoparticle size obtained after deposition of the nanoparticle suspension onto a silicon wafer and evaporation of the solvent was measured by Atomic Force microscopy (AFM, Dimension Icon with ScanAsyst, Bruker, USA). A typical AFM image of deposited nanoparticles is shown in Figure 1. From a systematic analysis of the nanoparticle size performed on a 2 x 2µm AFM image, a mean size of 45 ± 10 nm was found.

Thin film preparation and characterization

Nanocomposite polymer thin films were prepared from polymer solutions in water, to which a colloidal suspension of PLGA nanoparticles was added. The PVA was solubilized in water by heating (90°C) under magnetic stirring for 2h while the PVP solution was prepared by mixing the polymer and water at room temperature, under magnetic stirring for 1h. The concentration of both PVA and PVP in water was 30 mg.mL -1 . Specific amounts of aqueous suspension of PLGA nanoparticles (13 mg.mL -1 , 1.3% wt./vol.) were added to the polymer solution (30 mg.mL -1 , 3% wt./vol.) in order get a concentration of PLGA nanoparticles in the dried film between 1 and 10 % wt./wt. The concentration of solid material in the solution after mixing the two parent solutions was therefore <3% and thus can be considered as a dilute solution.

The viscosity of the solution was measured by a DV2T LV viscometer from AMETEK Brookfield (Middleboro, USA). Thin films of polymers were prepared on a silicon substrate by dip coating (dip coater QPI-168, Qualitech Industrial Products, India). The substrate was immersed in the solution (7 mL) at 200 mm.min -1 , left in the solution for 1 min and withdrawn at a speed from 0.05 to 200 mm.min -1 depending on the samples. After complete evaporation of the water, dried nanocomposite thin films on silicon were obtained. The thickness of the films was measured by visible reflectometry (F20 Film Thickness Measurement System, Filmetrics, KLA-Tencor, USA). The thicknesses of the films were measured at more than 5 different places and the averaged thicknesses and standard deviations were calculated. For all the films, these standard deviations were less than 10%. AFM (Dimension Icon with ScanAsyst, Bruker, USA) in tapping mode was performed in order to monitor the different surface topographies depending on the nanocomposite film composition. The surface roughness R was extracted from the AFM image. R represents the amplitude of the deviations (z) of surface heights from the mean height and was estimated by means of the Root Mean Squared (RMS) roughness using the formula where n is the number of heights considered:

Results and discussion

PVA/PLGA nanocomposite thin films

Figure 2 shows the typical variation of the polymer film thickness (after drying) as a function of the withdrawal speed. The results shown here were obtained with neat PVA and blends of PVA/PLGA NP (5%), deposited by dip coating from solutions in water. In a log/log scale, the thickness versus the withdrawal speed exhibits a typical "V" shaped curve. It first linearly decreases with increasing speed, goes through a minimal thickness for a critical speed, and then linearly increases for higher values of the speed. This is consistent with the expression of the thickness (h f ) versus the withdrawal speed (U 0 ) which, in the case of a dried film formed upon solvent evaporation, can be written as follows (equation 1): [START_REF] Faustini | Preparation of Sol-Gel Films by Dip-Coating in Extreme Conditions[END_REF] (Eq 1.)

h f k i U U
where k i depends on the mass concentration of the polymer in the solution (c i in g.cm -3 ) and the density of the polymer ρ i in g.cm -3 ) by k i = c i /  i . E, L, D are the evaporation speed, the film width and the global draining constant respectively. In our case, k i was 2.5 10 -5 and L was 0.01 m.

As seen from this expression, the thickness is proportional to the inverse of the speed at low withdrawal speed and proportional to 2/3 of the withdrawal speed at high withdrawal speed.

These two stages correspond to the so-called "capillary" and "draining" regimes respectively (represented in blue and red for better clarity on Figure 2), and are clearly verified in our results with a slope in log-log scale of -1 for the low withdrawal speeds and 2/3 for the high withdrawal speeds. These two regimes result from different physical processes that dominate as a function of the withdrawal speed. [START_REF] Brinker | Dip coating[END_REF][START_REF] Berteloot | Limat Dip coating with colloids and evaporation[END_REF] In brief, the formation of higher thicknesses is favored at low speed due to a continuous capillary flow that drags the solution towards the film in formation, upon an evaporation-driven phenomenon. In contrast, higher film thicknesses are built-up above the critical speed due a direct increase in the amount of solution removed by the withdrawn substrate, resulting from the interplay between the viscous drag of the solution and surface tension (described by the Landau-Levich equation [START_REF] Evich | ragging of a liquid by a moving plate[END_REF] ). The superposition of the curves for the neat polymer and the nanocomposite thin film shows that PLGA nanoparticles do not significantly modify the deposition process of PVA film, whether in the draining or the capillary regime. From a fit of the linear parts with Eq. 1, E and D were estimated respectively at 4 10 -13 m 3 .s -1 and 3 10 -4 m 1/3 .s 2/3 for both neat PVA and the blend PVA/PLGA NP (5%). These values are in the range of the values obtained in previous work [START_REF] Vital | Sinturel Morphology control in thin films of PS:PLA homopolymer blends by dip-coating deposition[END_REF] showing similar values of D for polymer solutions loaded at 30 mg.mL -1 (the same concentration as in the present study) but higher values of E since the solutions were obtained in organic solvent having a much faster evaporation rate. Since distinct physical processes drive the formation of the films in the "capillary" and the "draining" regimes, the question that arises is whether or not the withdrawal speed affects the composition of the film (i.e. the NP concentration in the final film). From the applications standpoint mentioned in the introduction, such as drug delivery, this is a crucial aspect since it can lead to enrichment or depletion of the NPs in the film. In order to evaluate the NP concentration in the film, we carried out AFM analysis of the surface of the film. Because the NP size (45 nm) is not negligible compared to the film thickness (20 to 200 nm), we anticipated that embedded NPs could be identified by AFM. ). In contrast, the PVA/PLGA NP (5%) blend displays white spots on the surface that can be attributed to embedded nanoparticles, exhibiting higher RMS roughness (1.6 nm at 200 mm.min -1 ). From a systematic analysis of the AFM images, we found an average feature size of 51 ± 11 nm, in good agreement with the size of the NPs (see experimental part). The increased diameter of the NPs in the film can be attributed to a PVA coating of the PLGA NPs. At low withdrawal rate (Figure 3 a)) the topography is very different. Both systems (PVA and PVA/PLGA NP (5%) blend) exhibit a very rough surface with a RMS roughness respectively of 13.4 and 10.1 nm, with strong local variations of the z profile (as shown in the insert). This phenomenon can be attributed to a crystallization of the polymer, which dominates the surface roughness. In previous work, we showed that withdrawal speed has a significant impact on the crystallization of polymers, [START_REF] Vayer | Constrained crystallization of poly(L-lactic acid) in thin films prepared by dip coating[END_REF] which in turn greatly affects the surface roughness. We demonstrated that the crystallization process was favored at low withdrawal speed, where the slow solvent evaporation allows for the nucleation and growth of the crystalline domains. In contrast, the high evaporation that occurs at higher withdrawal speeds acts as a quench, and favors the formation of an amorphous phase. In the present case, it is well known that PVA is a semi-crystalline polymer, even in its atactic form (which can crystallize up to 65%). [START_REF] Peppas | Crystallization kinetics of poly(vinyl alcohol)[END_REF] This was confirmed by DSC which shows a strong exothermal signal while heating (see Figure SI-2). As a result, the surface topography is dominated by the formation of crystalline features at low withdrawal speed, preventing the observation of the particles in the nanocomposite films.

The particle amount as a function of the deposition rate is consequently not accessible. After observing this behavior, we therefore did not conduct a detailed NP concentration analysis of the surface topography and we switched to a fully amorphous system, PVP, which is now described.

PVP/PLGA nanocomposite thin films

In order to cope with the crystallization process observed in the case of the PVA films formed at low withdrawal speed, we moved to the formation of nanocomposite thin films using PVP, a fully amorphous polymer. Both the film thickness and NP concentration were studied with respect to the withdrawal speed, on a range of PLGA NP concentration spanning from 1 to 10%.

Figure 4 shows a typical variation of the polymer film thickness as a function of the withdrawal speed, for the neat PVP and nanocomposite thin film. Similarly to the PVA case, the NPs do not significantly modify the thickness of the PVP film, over the range of NP concentration explored in this study, whether in the capillary or the draining regime. All the thickness curves are superimposed onto a single master curve. As for the PVA case, the values of E (1 10 -11 m 3 .s -1 ) and D (3 10 -4 m 1/3 .s 2/3 ) were extracted from a fit of the linear parts with equation Eq. 1. The difference between the E values for PVA (4 10 -13 m 3 .s -1 ) and PVP (1 10 -11 m 3 .s -1 ) can be attributed to the better affinity of PVA compared to PVP towards water. [START_REF] Vital | Sinturel Morphology control in thin films of PS:PLA homopolymer blends by dip-coating deposition[END_REF] In contrast, the value of D does not differ for the solutions of PVA and PVP and is not affected by the NP concentration. This suggests that the viscosity of the solutions is not affected in our case by the nature of the polymer, nor the NP concentration. This was verified by measuring the viscosity of the solutions which was found to be constant (1.13 mPa.s) and relatively close to the viscosity of water (0.90 mPa.s). This is in good agreement with the fact that all solutions contained a limited amount of solid material (< 3%). This case is quite different from the work of Gans and coworkers [START_REF] Gans | Dip-coating of suspensions[END_REF] , where the solid content in solution was between 10 and 41%, with a dramatic impact on the viscosity. Figure 5 displays the surface observations of films obtained using three representative conditions of the withdrawal speed (0.5 mm.min -1 in the capillary regime, 10 mm.min -1 near the critical point and 200 mm.min -1 in the draining regime), as a function of the NP concentration. On the neat PVP film, the surface of the film remained perfectly smooth whatever the withdrawal speed, indicating that no crystallization occurred upon drying. This is consistent with the structure of PVP that exhibits an amorphous behavior, which was confirmed by DSC (see Figure SI-3). Consequently, the modification in the film topography can be solely attributed to the presence of the PLGA nanoparticles, which appear as white spots on the surface (with dimensions in good agreement with the NP size) in both the capillary and the draining regime. In this case, the systematic analysis of the AFM images gives an average feature size of 58 ± 15 nm. Again, the increased diameter of the NPs in the film can be attributed to a PVP coating.

Unlike PVA, PVP-based nanocomposite films can be used to evaluate the influence of the deposition mechanisms on the incorporation of the NPs in the films. For that purpose, instead of a direct, time-consuming counting of NPs on the AFM images (which cannot be automated due to complex threshold adjustments), we used the film roughness as an indicator of the NP concentration in the film. As all the images are at the same z-scale, one can directly compare the different conditions. Clearly, the intermediate withdrawal speed leads to higher roughness. However, it does not mean that the number of PLGA nanoparticles is higher. One has to keep in mind that the thickness depends on the withdrawal speed. At 10 mm.min -1 , the thickness of the film is minimal (c.a. 35 nm), and is thinner that the size of the nanoparticles. This makes the nanoparticles clearly visible on the AFM images, in contrast to the thicker films where the nanoparticles are embedded in the polymer matrice, leading to a smoother appearance of the surface. This clearly indicates that the roughness of the films cannot be used to compare the NP concentration between two films of different thicknesses. However, for two films having the same thickness, but obtained in two deposition regimes, the roughness extracted from the AFM images can be used to conclude on the influence of the deposition regime on the amount of NPs in the film. Figure 6 shows the thickness dependence of the thin film roughness, in the two deposition regimes (draining in red and capillary in blue). For the neat polymer, the roughness is almost constant (0.2 nm) with the film thickness indicating that the deposition regime (draining or capillary) has no influence. For the nanocomposite films, the roughness is maximum for the smallest value of film thickness (~35 nm), and decreases for higher thickness values, eventually reaching a plateau, in a similar way for both the capillary and draining regimes. As previously mentioned, this phenomenon mostly originates from a scale difference between the nanoparticle diameter and the PVP thickness: the NPs become more deeply buried in the film as the thickness increases, resulting in the observed decrease in roughness (a plateau is reached when the thickness of the film becomes similar to or higher than the NP size, c.a. 50 nm). As previously mentioned, the roughness variation between films of different thicknesses is not an indication of the NP concentration in the dried film. However, if one considers a similar film thickness, the roughness values can be used as qualitative information to compare the concentration of nanoparticles in the dried films: a low concentration leads to low roughness, and a higher concentration to higher roughness. To gain greater insight into the influence of this property, Figure 7 shows the roughness level observed for films at 50 nm. A lower roughness value is observed in the capillary regime, indicating a depleted NP concentration in the dried film and consequently suggesting that NP entrainment is influenced by the deposition mode in this case. This was confirmed by an individual counting of the NPs on 3 different 2 x 2µm scale images (more than 300 measurements). 25 NPs.µm -2 were found in the draining regime, versus 18 NPs.µm -2 in the capillary regime, confirming a depletion of NPs in the capillary regime (to be compared to the theoretical value of 25 NPs.µm -2 calculated with nanoparticules having a diameter of 50 nm, loaded at 5 % in a film). These results can be analyzed in light of the very recent works by Dincau [START_REF] Dincau | Capillary Sorting of Particles by Dip Coating[END_REF] and Sauret [START_REF] Sauret | Dressaire Capillary filtering of particles during dip coating[END_REF] studying the deposition of particles from a liquid suspension in dip coating. As described by these authors, the NP entrainment by a moving substrate in such a situation results from the competition of two opposite forces. On the one hand, the viscous forces are responsible for retrieving the NPs out of the solution whereas on the other hand the capillary forces tend to curb the meniscus deformation, preventing the NPs from being incorporated in the film. NPs are thus incorporated in the deposited films when the viscous drag overcomes the capillary forces, which is obtained for capillary numbers greater than a critical value that depends mainly on the viscosity of the solution, the NP size and the withdrawal speed. For a given viscosity and NP size, NPs are entrained at high withdrawal speed because of viscous drag. In contrast, NPs incorporation in the film decreases at low withdrawal speed because the capillary forces dominate and may even lead to the point that no particle is found in the film, as shown by Dincau et al. [START_REF] Dincau | Capillary Sorting of Particles by Dip Coating[END_REF] and Sauret et al. [START_REF] Sauret | Dressaire Capillary filtering of particles during dip coating[END_REF] . In their situation, such a phenomenon relied on a pure Landau-Levich regime (i.e. without solvent evaporation -the particles are dispersed in non-volatile oil) over the whole range of withdrawal speed values.

In our results, NPs are embedded in the films whatever the withdrawal speed. This is because in our case, the liquid phase evaporates during deposition which leads, in the low withdrawal speed range, to an increase in thickness. This specific effect originates from a self-initiated continuous capillary flow that drags the solution towards the film in formation, in an evaporation-driven phenomenon (similar to the coffee ring effect). Consequently, the NPs are submitted to the same antagonist effects described for the draining regime (viscous vs interfacial forces) that allow for the incorporation of the NPs in the film in our case. Interestingly, our results suggest that the balance of these two effects is not equivalent in the draining and capillary regime. The underlying physical origin of this phenomenon is currently under investigation.

Conclusion

We showed that the presence of colloidal dispersion of PLGA nanoparticles in a water solution of PVA and PVP does not modify the general process of film deposition by dip coating. A typical "V" shaped curve is observed with two well-known deposition regimes: capillary and draining modes respectively obtained at low and high withdrawal speeds. For both polymer matrices, the thickness is independent of the NP concentration (up to 10%). However, a close look at the coating topographies allowed us to point out differences in the capillary and the draining regimes for both matrices. In the case of the PVA, crystallization was observed at low withdrawal speeds, which is explained by the slow evaporation of the solvent. It was not possible to identify individual PLGA nanoparticles by AFM in this case due to the high surface roughness generated by the crystallization process. In contrast, amorphous PVP nanocomposite films were successfully prepared over the whole range of withdrawal speeds, and allowed us to identify individual PLGA nanoparticles with AFM.

When the thickness is sufficiently low, the particles' presence modifies the topography on the surface and in this case, the roughness measured by AFM was correlated to the NP concentration in the film. Because of the prevalence of an evaporation-driven phenomenon at low withdrawal speed, incorporation of NPs was observed over the whole range of withdrawal speeds, in contrast to other studies relying on a pure Landau-Levich regime (i.e. non-evaporative systems). Our results indicate that the balance between the viscous drag and the interfacial effects depends on the deposition regime and call for a more detailed analysis of the physical processes involved in both regimes. In comparison to the existing literature on the dip-coating deposition of polymer solutions on the one hand and colloidal suspensions on the other hand, this work offers a new perspective. It brings new insights into the influence of NPs on the deposition of polymer solutions by dip-coating (and vice-versa), building essential knowledge dealing with phenomena occurring during the deposition. Combining these two systems allowed us to reveal new behavior, particularly concerning the incorporation of NPs at low withdrawal speed, making this work particularly relevant, relative to prior literature on the separate systems. From the application standpoint (such as the formation of nanocomposite coatings containing NPs for controlled drug delivery), this work demonstrated that it is important to consider the withdrawal speed for these two major biocompatible polymer models. In the case PVA, crystallinity will greatly depend on the process. In the case of PVP, the NP concentration in the dried film is affected by the mode of deposition.

Fig. 1 :

 1 Fig. 1: AFM image (2 x 2 µm) showing individual PLGA nanoparticles obtained from the

Fig. 2 :

 2 Fig. 2: Influence of PLGA nanoparticle concentration on the thickness of PVA films

Figure 3

 3 Figure3shows the surface topographies of the neat PVA (left) and PVA/PLGA NP (5%)

Fig. 3 :

 3 Fig. 3: Influence of PLGA nanoparticle concentration and withdrawal speed on the morphology of PVA thin films investigated by AFM. a) 0.05 mm.min -1 in the capillary

Fig. 4 :

 4 Fig. 4: Influence of PLGA nanoparticle concentration on the thickness of PVP films

Fig. 5 :

 5 Fig. 5: Influence of PLGA nanoparticle concentration on the morphology of PVP films in three representative conditions of the withdrawal speed: a) 0.5 mm.min -1 in the capillary regime b) 10 mm.min -1 near the critical point c) 200 mm.min -1 in the draining

Fig. 6 .

 6 Fig. 6. Variation of the film roughness in the capillary regime (a) and the draining

Fig. 7 .

 7 Fig. 7. Variation of the film roughness at 50 nm in the capillary and draining regimes,

D. Grosso, How to exploit the full potential of the dip-coating process to better control film formation, J. Mater. Chem., 0 , , 70 -70 8 https://doi.org/10.1039/C1JM12837J

Acknowledgements

We thank the Région Centre-Val de Loire for funding (MISTIC project ARD 2020 Cosmétosciences 2017-00118114 and Fonds Européen de Développement Régional 2017-EX003256).


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Plasma induced acceleration and selectivity in strain-promoted azide-alkyne cycloadditions

Introduction

Bioorthogonal reactions can be described as chemical reactions occurring in a biological environment without interfering with any of its processes or materials. Initially coined by Carolyn R. Bertozzi in the early 2000s, bioorthogonal chemistry has since benefited from many improvements in terms of reactions selectivity and kinetics thanks to the development of more soluble and stable reagents with reduced toxicities. [1][2][3][4][5][START_REF] Gordon | Chemoselective and Bioorthogonal Ligation Reactions[END_REF][START_REF] Lang | [END_REF][8][9] Strain-promoted azidealkyne cycloaddition (SPAAC) is a striking example of such an achievement: reported for the first time in the context of bioorthogonal chemistry in 2004, it is now considered as an indispensable reaction of the chemical biology toolbox, due to the stability of the resulting triazole products, along with its simplicity of operation, low toxicity and fast kinetic rates. [START_REF] Gordon | Chemoselective and Bioorthogonal Ligation Reactions[END_REF] The latter characteristic is a crucial parameter to take into account for in vivo applications, as the reaction has to be rapid in order to occur at low concentrations, before reactants are metabolized or excreted. Major efforts have thus been dedicated to optimizing SPAAC kinetic rates via the development of more reactive alkyne through the modulation of stereoelectronic parameters. [10][11][12][13][14] In this race for the ideal bioorthogonal reagent, the effect of the surrounding biological environment in which the reaction takes place has been somehow overlooked. Even though it is well known that solvent effects strongly influence cycloaddition rates, 15 most studies have been conducted in organic-aqueous solvent mixtures, those systems being often considered as representative of the more complex biological media. Interestingly however, the group of Carolyn R. Bertozzi reported the unexpected reactivity of several alkynes probes when using SPAAC to label azidefunctionalized glycans in living mice. [START_REF] Chang | Proc. Natl. Acad. Sci[END_REF] Despite comparable labelling efficiencies in vitro, in coherence with similar second-order rate constants of the parent cyclooctynes in model reactions, [START_REF] Agard | [END_REF][18][19] several alkyne probes gave markedly different results when tested in vivo, suggesting that the chemical structure of the probe itself was a key parameter of its in vivo reactivity. In the same vein, Robillard and coworkers described the use of SPAAC reactions for the modification of azide-containing antibodies in live mice and showed that serum proteinsand especially albuminhad a negative impact on the reaction kinetics, due to their strong interactions with alkynes, making the latter less available to react with the antibodies' azide groups. 20 Altogether, these results support the hypothesis that the outcome of bioorthogonal reactions in vivo cannot be predicted solely based on the parent alkyne's reactivity in model reactions conducted in simple solvent systems. As more complex parameters may play a substantial role, new in vitro models are thus needed to mimic this environment in order to decipher the underlying rules governing the fate of bioorthogonal reactions in complex media.

Results and discussion

Building on a previous work in which we reported the in vivo neutralization of the azido derivative 1 of the anticoagulant warfarine by a SPAAC reaction with bicyclononyne (BCN) 3, 15 we started by evaluating human plasma as a surrogate for in vivo models. It became quickly apparent that rate constants were extremely similar between in vivo and plasma media, and in sharp contrasts with those measured with the model azide 2 in both methanol and phosphate buffered saline (PBS) solutions (Figure 1 andS2). While such strong discrepancies underlined a major influence of the medium on SPAAC kinetics, the fact that we were comparing two different systems (1 → 4 in plasma and in mice vs 2 → 5 in PBS and methanol) prevented us from drawing this definitive conclusion.

In order to probe the influence of plasma on the outcome of SPAAC reactions, we decided to perform a systematic study by measuring the cross reactivity of pairs of alkyne / azide under standardized conditions and determining their corresponding second order kinetic rate constants. We designed a coherent series of BCN derivatives, all synthesized from the simple BCN alcohol 6: one bearing an apolar dioxole group ( 7) and three equipped with polar moieties, displaying either a permanent charge at physiological pH (8 and 9) or possessing a short and neutral oligoethyleneglycol (OEG) chain (3). Each of these BCN probes was paired with a counterpart azide equipped with similar functional groups (2, 10-12). These moieties were chosen with the aim of modulating the biophysical properties of the reagents without affecting their intrinsic chemical reactivity. Each pair was then reacted in equimolar conditions at a concentration of 0.1 mM in human plasma. This standard concentration was chosen since it was consistent with the maximum plasmatic concentration of PEG 3 measured after sub-cutaneous injection (52 mg.kg -1 ) in mice. 15 In methanol, the concentration had to be increased to 5 mM in order to start seeing product formation within the two hours timeframe of a classical experiment.

In each case, 1,7dihydroxynaphthalene was used as an internal standard in order to improve the precision of our analytical method, and the reactions were monitored by HPLC at various time points, following both azide disappearance and triazole product formation (see SI). As all BCN and azide compounds evaluated in these experiments were perfectly stable in plasma and since the expected triazole was the only product formed in all reactions, the second-order kinetic constant of the different SPAAC reactions was calculated by plotting the evolution of the inverse of concentration of the azide component as a function of time according to the following equation:

1/[A] = 1/[A]0 +kt where [A]0 is the initial molar concentration of the azide reactant; [A], the molar concentration of the azide reactant at a given time; t, the time in seconds; and k, the secondorder kinetic constant expressed in M -1 .s -1 (see ESI for details about calculations). We found that SPAAC kinetics were quite slow in methanol (≤ 0.57 M -1 s -1 ) and with little variations between each BCN / azide pair evaluated (σ ~ 0.17 M -1 s -1 ), clearly suggesting that the reactants structure had little to no influence on the outcome of SPAAC reactions in the organic solvent (Figure 2). In plasma however, reactions were found to be greatly accelerated compared to methanol, up to a 70-fold with second-order kinetic constant values culminating at ~19 M - 1 s -1 . In addition, striking differences in reactivity were found between the different reactant pairs depending on their structure and skeleton (σ ~ 4.73 M -1 s -1 ). One notable exception concerned carboxylate-bearing azide 12, whose reactivity was barely impacted by either the BCN reagent or the medium composition. At this stage, it is important to reiterate that reactions were fifty times more concentrated in methanol than in plasma (5 mM vs 0.1 mM), and differences in conversion rates between the two media were logically even more pronounced when reactions were conducted at 0.1 mM in both cases (see ESI). Lastly, comparison between kinetics in PBS and methanol showed that while reactions between azide 11 and all BCN probes were accelerated in watera feature previously reported in the literature -, this was to a much lesser extent than in plasma, highlighting that the aqueous nature of the reaction medium cannot be the sole explanation for the effects observed (see ESI). Taken altogether, these data demonstrate that the reactivity of a strained alkyne or an azide in plasma can be strongly modulated by the chemical structure of the moieties appended to it. Consequently, by performing SPAAC in plasma instead of classical solvent systems, it should be possible in theory to discriminate between two azides or two BCN and attain unprecedented selectivity. We thus selected BCN-azide pairs 3-11 and 3-12, whose corresponding SPAAC showed similar kinetic rates in methanol (0.21 M -1 .s -1 and 0.11 M -1 .s -1 , respectively) but substantial differences in plasma (2.39 M -1 .s -1 and 0.19 M - 1 .s -1 ) and performed competition experiments. As expected, both azides were found to be equally reactive at 0.1 mM in methanol, leading to an equimolar mixture of triazoles 13 and 14 (Figure 3,A). As anticipated also at this concentration and in this solvent, less than 10% conversion was observed after 60 minutes. In plasma however, azide 11 predominantly and quickly reacted, leading to ~40% conversion to the expected triazole product 13 after 60 minutes, while azide 12 did not participate in any visible reaction (blue line and red line, respectively; Figure 3,B). This illustrates that selectivity between two nearly identical SPAAC can be attained just by switching from classical solvent to plasma.

To evaluate the potential applications of such behaviour in the context of in vivo chemistry, where SPAAC are usually conducted with an excess of strained alkyne, the concentration of BCN 3 was increased to 0.5 mM while keeping the same concentrations in azides 11 and 12 (i.e. 0.1 mM), now making them the limiting reactants. As expected, faster reactions were observed in both methanol and plasma (Figure 3, C and D). Despite this acceleration, the same trend as before was still observed. No differences between 11 and 12 were noticed in methanol at 0.5 mM and poor conversion was detected for both substrates, while an even better level of selectivity between azides 11 and 12 was found in plasma: while carboxylate derivative 12 gave less than 20% conversion to triazole 14, the ammonium derivative 11 gave almost complete conversion to 13 after 60 minutes.

To understand better the underlying effects at stake in these reactions, we conducted plasma protein-binding assays to determine the fraction of free azides and BCNs in plasma. Utilizing the equilibrium dialysis method (see ESI for detailed procedures), which had previously allowed us to determine the percentage of warfarin azide 1 bound to plasma proteins to be 94%, 15 we found sharp variations between our set of compounds (Figure 4). Binding values of azides were shown to range from 15% for compound 2 to 98% for 1. A comparable variation was also observed for BCN, going from 26% for both 3 and 9, to almost 99% for compound 7. This plasma-protein binding affinity did not seem to be directly correlated to the functional groups equipping the SPAAC reagents: primary alcoholcontaining azide 2 and BCN 6 gave completely opposite values (15% and 91%, respectively), similar to what was observed with OEG-functionalized BCN 3 (26%) and azide 10 (78.5%). When comparing binding values and second order kinetic constants in an attempt to find a correlation between the two sets of values, we observed that the most bound azides (i.e. 1, 10 and 11) systematically gave the highest SPAAC kinetics, while the less bound azides 2 and 12 led to mediocre rate constants. In addition, for a given azide, SPAAC kinetics appeared to be closely related to and modulated by the percentage of proteins-bound BCNs, with compound 7 giving always the best results. These findings relate well to the previously mentioned study from the Robillard group, who also reported that serum proteins were strongly interacting with alkynes. 20 While in the latter case this proved to be detrimental to SPAAC reactions with azide groups borne by bulky immunoconjugates, a similar effect might be beneficial for reactions between compounds of lower molecular weights. Even though other factors might also be at stake, such as the compounds hydrophobicity or flexibility, the binding of reagents to surrounding proteins seems to have a major influence on the acceleration of SPAAC in plasma. Albeit counterintuitive, local increase of concentration at the surface of a plasma protein could facilitate interactions between azides and BCN in such dilute medium and result in the acceleration of SPAAC. validate this hypothesis, we reacted our previously described model system -BCN 3 and azide 1 -15 in PBS in the presence of increasing amounts of human serum albumin (HSA, see ESI for more details), the most abundant protein in human plasma. 21 Gratifyingly, the second-order kinetic constant determined at plasma-like concentrations of HSA in PBS (i.e. 50 g.L -1 ) was found to be in agreement with that measured in human plasma (1.66 M -1 .s -1 vs 1.55 M -1 .s -1 , respectively). While this observation tends to support the hypothesis that protein binding is responsible for the acceleration of SPAAC in human plasma, we also found that the highest rate acceleration was observed in relatively diluted HSA solutions (3.33 M -1 .s -1 at 2.5 mg.mL -1 of HSA in PBS). This shows that plasma proteins and their concentrations exert a strong influence on the kinetics of SPAAC. Understanding better the factors governing this peculiar behaviour and the role of these "participating spectators" in biological media could open up new directions in chemical biology.

Figure 1 .

 1 Figure 1. Differences in kinetics between strain-promoted azide-alkyne cycloadditions run either in classical solvent systems or in plasma. The green dots represent the conversion observed in-vivo.

Figure 2 .

 2 Figure 2. Influence of azides and cyclooctynes structures on the rate of SPAAC reactions; (a) structures of the different BCN and azide derivatives utilized in the study; (b) representation of the different second-order kinetic constants measured in plasma (100 µM) and in methanol (5 mM) for the SPAAC reactions between different BCN-azide pairs. These values represent the average of measurements made in triplicates (n = 3).

Figure 4 .Figure 3 .

 43 Figure 4. Plasma proteins-binding values for all BCN and azide compounds evaluated (curves) plotted together with the previously determined second order kinetic constants of SPAAC reactions in plasma (histograms). Measurements were done in triplicates (n = 3).

Acknowledgements

The authors would like to thank the "Agence Nationale de la Recherche" for financial support (DW, ED).

Conclusions

In conclusion, we showed the rate enhancement of various SPAAC reactions conducted in plasma compared to methanol and PBS. Functional groups borne by different BCN and azide moieties were found to have a profound impact on the reactions' kinetic constants. We took advantage of this effect to successfully conduct competition experiments, where only one of two azide compounds reacted with a given BCN. To the best of our knowledge, this is the first report of an in situ selectivity in plasma between two identical bioorthogonal reactions, only caused by the chemical structure of the reactants. Exploring interactions between biological medium and reactive partners of SPAAC reaction and including this medium as an influential reaction partner should be taken into account when developing or optimizing bioorthogonal reactions.

Such phenomena open unexpected perspectives in the development of effective orthogonal pairs based on a single reactive core and expand the in vivo chemistry toolbox while alleviating the need for the production of new families of strained alkynes.

Author Contributions

DW took care of investigation, methodology, validation, visualisation and writing the original draft of this manuscript. ED took care of methodology and validation MR and SU took care of data curation & early work and investigation. MM and JS took care of methodology. WK took care of conceptualisation, funding acquisition, resources, supervision and reviewing & editing the final manuscript. GC took care of funding acquisition, project administration, supervision, visualisation, writing the original draft and reviewing & editing the final manuscript. AW took care of conceptualisation, funding acquisition, project administration, resources, supervision and reviewing & editing the final manuscript.

Conflicts of interest

There are no conflicts to declare.


Nicolas Bredeche
email: bredeche@lri.frlouis.hugues@wanadoo.fr

Louis Hugues

Inférence Apprentissage

Evolutionary Robotics: Incremental Learning of Sequential Behavior

Keywords:

I. INTRODUCTION

Evolutionary Robotics offers an efficient and easy-to-use framework for automatically building behaviors for an autonomous robots. However, a major drawback of this approach relies in the difficulty to define the fitness function (i.e. the learning setup) in order to get satisfying results. As a consequence, many works have been limited to simple problems such as wall-avoidance or target-following [START_REF] Nolfi | Evolutionary Robotics[END_REF].

On the one hand, recent works addressed this issue either by decomposing the learning task or by endowing the agent with such capabilities that should make the goal easier to achieve [START_REF] Nolfi | Evolutionary Robotics[END_REF][START_REF] Godzik | Evolving Symbolic Controllers[END_REF]. On the other hand, literature in Evolutionary Approach shows that modifying the very nature of genetic operators and/or fitness during the course of evolution may lead to better results for complex problems [START_REF] Lohn | A Comparison of Dynamic Fitness Schedules for Evolutionary Design of Amplifiers[END_REF].

Fig. 1. The task: a robot wanders through the environment and has to perform a given behavior in order to access resources (i.e. the yellow/red balls). The behavior to perform is a sequence of "key" movements to "open" the resources. The more complex the behavior needed to access a resource, the greater the reward (one key is enough for the yellow balls while four keys are needed to "eat" the red ball). The goal is to learn the complete sequence that makes it possible to access any resources (the agent accesses simpler resources first ; performing the whole sequence gives access to any resource).

EXPERIMENTS

In the scope of this short paper, we are interested in the reformulation of a straightforward complex fitness function into more subtle versions using different approaches. The original task we are interested in is described in fig. 1 * . Fig. 2 shows the results of our experiment. The task is successful if an individual's genotype exactly matches a reference genotype (i.e. the complete sequence of keys to be found). A genotype is defined as N 4-uplets of Boolean * A robot performs a basic random wandering behavior and automatically performs its access sequence anytime a food item is found. In the following, results are concerned only with the learning of this access sequence.

attributes. Every 4-uplets is a 'key' of the sequence and N is the length of the sequence (i.e. the task complexity). A sequence is evaluated from the first key, key by key, until a key is found incorrect or the end is reached. We defined four fitnesses as follow:

1. brute fitness : reward 1 if exact match, otherwise 0 (i.e. only the most difficult to access food items can be found in the environment); 2. static incremental : each accepted key is rewarded; 3. dynamic elitism : reward only the best individual(s); 4. self-tuned : each individual accepts (or not) reward for a given key (evaluation from start until error). On the one hand, the brute fitness unsurprisingly leads to a greater search space. On the other hand, the self-tuned fitness does not perform that well -looking at the genotypes, one can see that it actually behaves like a static incremental fitness with tuning that is unfortunately disrupted by the genetic operators (there is no bias toward a relevant selftuning). Regarding the static incremental and dynamic fitnesses, there is no real difference as the two of them rely on the selective pressure of the genetic algorithm even though dynamic elitism should be more aggressive regarding this very pressure.

Fig. 2. Comparing learning speed using four different fitnesses used on a more or less difficult task (i.e. sequence length up to 16 keys (i.e. 64 bits)). The xaxis shows the sequence length the agent has to learn in order to be able to get the best reward for this task (genotype length is fixed according to the sequence length). The y-axis shows the number of generations before the optimal behavior is learnt. Results with more than 1000 gen. are not shown.