J ' Aimerais

Patrick Tabeling

Aurore Stéphane Ta

Benjamin Chollet

Loïc D'eramo

Jessica Delavoipière

Hubert Geisler

Antoine Chateauminois

Costantino Creton

Dominique Hourdet

Emilie Verneuil

Hélène Montès

Laurence Talini

Nadège Pantoustier

Armand Hakopian

Freddy Martin

Gilles Garnaud

Mohamed Hanafi

Pierre Christine

Guillaume Schelcher

Guillaume Laffite

Olivier Lesage

Thitima Limpanichpakdee

Mengxing Li

Laure Bluteau

Ludovic Fiege

Guillaume Votte Ma

Corentin Trégouët

Davide Colombo

Éric Lintingre

Hui Guo

Jennifer Macron

Marc Yonger

Natacha Goutay

Pascaline Hayoun

Paul Elzière

Pierre Millereau

Rémi Deleurence

Robin Masurel

Xavier Caliès

Charles Celle

Guillaume Barrand

Jingwen Chatté

Menghua Zhao

Romain Zhao

Dubourget

Saint- Martin Tom

Sans

Alice Boursier

Anne-Charlotte Le Gulluche

Antoine Fleury

Artem Kovalenko

Cécile Mussault

Francisco Cedano

Gabriel Sanoja

Gaëlle Rondepierre

Hiroto Ozaki

Julie Godefroid

Es Julien

Juliette Sayed

Mehdi Slootman

Mélanie Vahdati

Miléna Arangalage

Paul Lama

Pierre Fourton

Raphaëlle Gelineau

Robert Kulis

Valentine Gurney

Yinjun Hervio

Olivier Chen… J'exprime Ma

Colombani

Antoine Véron

Anne-Lise Launay

Pierre Launay

Valentin Aubin-Boivin

Lilian Mesnage

Justine Mesnage

Anne-Marie Langlois

Nicolas Launay

Céline Launay

Je voudrais remercier du fond du coeur les permanents pour leurs précieux conseils

Je voudrais remercier les organismes et les personnes qui m'ont aidé pendant ma thèse.

Tout d'abord, j'aimerais remercier le ministère des Sciences et de la Technologie de Thaïlande (MOST) pour son support financier, sans lequel je n'aurais pas pu réaliser ma thèse en France.

Je souhaiterais exprimer ma gratitude à Yvette Tran pour sa confiance, ses conseils précieux, sa disponibilité, sa patience … J'ai appris beaucoup de choses sur les polymères à l'interface avec son soutien. Yvette est un bon modèle d'encadrant et d'enseignant et j'espère être à mon tour un bon maître de conférences en Thaïlande.

วิ ทยานิ พนธ์ นี ้ สู งค่ า กว่ าจะส าเร็ จมา เหนื ่ อยแท้

Synthetic polymer hydrogels are chemical or physical polymer networks which can contain high water content. They have attracted many attentions due to their biocompatibility and the development for many applications in biomaterials, biotechnologies... Another attractive feature is hydrogels with stimuli-responsive properties as they can operate as switchable systems in response to small change of external parameters such as temperature.

The responsiveness allows to control reversibly the swelling/collapse transition of hydrogels, which makes them suitable for a wide range of applications, from sensors to actuators.

With hydrogel thin films, the advantages of hydrogel architecture with reduced size can be combined. Actually, macroscopic hydrogels show a long response time because the swelling/collapse transition is a diffusion-limited process. Hydrogel thin films of microscopic size can provide fast response. The response time T is expressed as T = L 2 /D, where L is the smallest dimension of the hydrogel and D is the collective diffusion constant of the hydrogel network. It turns out that smaller size hydrogels leads to faster responses. Moreover, the architecture of polymer network enables high water contents and then high amplitude change of stimuli-responsive properties.

Surface-attached hydrogel films are very attractive polymer films as they combine the advantages of being multiscale with well-controlled chemistry and their fabrication can be achieved in one pot. So far, polymer films are either coated layers, but poorly chemically controlled, or polymer brushes and LBL assemblies, for which the thickness is the limitation.

Polymer brushes are polymer chains that are attached by one of their extremities to the substrate. The thickness of polymer brushes is governed by the chain length so that it is limited to submicrometric range. In LBL assemblies, as the elementary layer is a few nanometers thick, many cyclic steps are required to build multilayers so that high thickness could be the limitation. As LBL assemblies are in general formed by physical bonds (electrostatic interactions, hydrophobic interactions, hydrogen bonds) they are susceptible to disassembly under some conditions. In contrary, surface-attached hydrogel films are chemical stable polymer layers owing to covalent cross-linking and surface-grafting. Despite these attractive features, they are still scarcely investigated. This is probably due to a lack of general and reliable strategy to synthesize hydrogel films with well-controlled chemistry.

General introduction 3

Recently, our research group has proposed a new simple and versatile approach for the synthesis of reliable and reproducible hydrogel films. We called this approach CLAG because it consists in cross-linking and grafting preformed functionalized polymers using click chemistry. The synthesis of preformed reactive chains avoids the difficulty of working under controlled atmosphere as oxygen inhibits radical polymerization, this sensitivity being enhanced for films with the high surface-to-volume ratio. The thiol-ene click reaction chosen for its versatility can be activated by thermal heating as well as by UV irradiation. Preformed ene-functionalized polymers and dithiol molecules cross-linkers are coated on thiol-modified solid substrates, then simultaneously cross-linked and grafted to the surface by thiol-ene reaction. Another advantage is that sophisticated chemistry is not required as enefunctionalized polymers can be quite easily synthesized by free radical polymerization (the control of dispersity is not necessary). The coating technique such as spin-coating and dipcoating allows the formation of hydrogel films on a wide range of thickness, from a few nanometers to several micrometers.

We have been working on surface-attached hydrogel thin films with thermoresponsive properties using poly(N-isopropylacrylamide) PNIPAM (Mengxing Li's PhD).

The temperature-responsive properties of PNIPAM hydrogels were determined by measuring the swelling ratio between thickness in air and thickness in water at various temperatures.

PNIPAM hydrogel films show LCST behavior with a transition temperature about 32°C. The swelling-to-collapse change obtained is about three-fold. Actually, the swelling ratio depends on the crosslinks density of polymer networks and it can be controlled by synthesizing different ratios of ene-reactive groups in preformed polymers. For example, 2mol% of alkenes in ene-functionalized PNIAPM chains, the swelling ratio of 4 is obtained in swollen state.

Our group has also developed new hydrogel films with adjustable architectures: multilayer hydrogel films, interpenetrating networks and nanocomposite hydrogel films.

Micro-patterned hydrogel thin films can be also produced by photolithography using UV irradiation to activate the thiol-ene reaction (Benjamin Chollet's PhD). This concept enables the preparation of hydrogel actuators for microfluidics. We have a collaborative project with Microfluidic MEMS Nanostructures (MMN) laboratory in Institute Pierre-Gilles de Gennes (IPGG) and Patrick Tabeling. The objective is to introduce a new actuation technology, using responsive hydrogel films embedded inside a microfluidic channel as a valve or functional gate that allows to open and close the channel for fluid flows. The proof-of-concepts were demonstrated with micro-valving and micro-caging functionalities. Now, we intend to exploit the full potential of these responsive hydrogel actuators to develop microfluidic devices for biotechnological applications. In collaboration with the IPGG-MMN laboratory, particularly with Hubert Geisler's PhD project, we aim to develop hydrogel actuators with various temperature-responsive properties dedicated to specific applications. This is the context in which this dissertation is placed. We aim to design surfaceattached hydrogel films with different LCST/UCST (Lower/Upper Critical Solution Temperature) temperature-responsive properties. We have to propose other thermo-sensitive polymers than PNIPAM. The idea is to generalize and optimize the CLAG strategy for a set of polymers with acrylate and acrylamide. Among these polymers, poly(PEGMA) family is interesting as the LCST can be adjusted with the number of PEG units. Poly(acrylamide) derivatives can show either LCST or UCST behavior. Polyzwitterions such as polysulfobetaines advantageously combine both UCST and bio-fouling properties.

This manuscript is separated into five chapters. The chapters 2, 3 and 4 are presented in an "article" template.

The chapter 1 is devoted to the state of art and the objectives. An overview of hydrogels and polymers with different stimuli-responsive properties is described. The polymer hydrogels at interface are reported with layer-by-layer assemblies and polymer brushes. After a description of the context, the objectives of this dissertation are set out.

The chapter 2 is dedicated to the synthesis of surface-attached polymer hydrogel films with various temperature-responsive properties. The different strategies for the generalization and optimization of the synthesis of ene-functionalized polymers are reported, together with their characteristics. The preparation of surface-attached hydrogel films and the determination of temperature-responsive properties are also shown;

In chapter 3, we investigate the effect of PEG units on the LCST behavior of poly[poly(ethylene glycol methacrylate ether)] by studying poly(PEGMA) with 2, 3 and 4-5

General introduction 5 PEG units. The effects of confinement and stress on the lateral swelling are also observed.

Finally, the transition temperatures found for hydrogel films and that obtained in bulk are compared and discussed.

In chapter 4, we develop poly(acrylamide) derivatives hydrogel films with various LCST and UCST temperature-responsive, properties. We determine their temperature phase transitions in different aqueous solutions (pure water, NaCl added salt and phosphate buffer saline physiological solution). We also show that the transition temperature can be controlled by the strategy of synthesis of ene-functionalized polymers.

In chapter 5, the polysulfobetaines hydrogel films are synthesized and investigated in pure water as well as in physiological solutions to determine their UCST, which is of crucial importance as actuators in biology applications. The preliminary results obtained help to understand the role of free counterions in UCST properties of polyzwitterions.

In conclusion, the main contributions of the PhD research are highlighted and some future perspectives are discussed.

Chapter 1. Polymer hydrogel films: generalities and objectives of the thesis Chapter 1. Polymer hydrogel films: generalities and objectives of the thesis

Polymer hydrogels

Synthetic polymer hydrogels have been widely studied over the last decades due to their ability to contain water over 90% (by weight) without dissolving and a degree of flexibility very similar to natural tissue. Hydrogels are a three-dimensional physically or covalently cross-link of hydrophilic polymer chains bearing hydrophilic functions, for example -OH, -NH 2 , -COOH, -CONH 2 , -CONH-, SO 3 H... [1][2] The presence of hydrophilic groups renders hydrogels highly biocompatible for a wide range of applications such as drug delivery, food additives, hygienic products, pharmaceuticals, biosensors and biomedical applications. [2][3][4][5][6][7][8][9][10][11] The ability of hydrogels to absorb and retain aqueous media comes from hydrophilic groups in polymer pendant chains, while their dissolution resistance increases with the crosslinks density between polymer chains. Many materials, both naturally occurring and synthetic, fit the definition of hydrogels. The natural polymer hydrogels are for example, crosslinked dextrans and collagen. The synthetic hydrogels can be obtained from poly(acrylamide), poly(methacrylic acid), poly(ethylene oxide) etc.

Several methods have been reported for the preparation of hydrogels 1,2,11 such as cross-linking polymerization, graft polymerization, network formation of water-soluble polymer, radiation cross-linking... The chemical classical way is simultaneous polymerization and crosslinking. The chemical crosslinking provides permanent hydrogel networks, while the physical crosslinking is a transient junction, consisting of polymer chains entanglements or physical interactions such as ionic interactions, hydrogen bonds or hydrophobic interactions.

Physical hydrogels can be reversible depending on the competition of interactions between chains and the environment, while chemical hydrogels are irreversible (definitely stronger interaction than that with the environment).

We are interested in hydrogels based on chemically crosslinked polymer chains for their stability and durability (Figure 1).

Chapter 1. Polymer hydrogel films: generalities and objectives of the thesis 10 Figure 1. Schematic of molecular structure of a hydrogel.

Stimuli-responsive polymers

Hydrogel based polymer networks have been designed and tailored to lead to new goals for different applications. Stimuli-responsive hydrogels also called "smart" hydrogels are very attractive for their ability to respond to environmental parameters and change network structure, mechanical strength and permeability, Several synthetic polymers possess the stimuli-responsive properties and can respond to the environment (temperature, pH, light, electric/magnetic field or other) by changing the physical or chemical properties [12][13][14][15][16][17] . These polymers are utilized to fabricate hydrogels. The control of the response is based on absorption or expulsion of water of hydrogel upon receiving different external parameters. The volume is changed reversibly and observed as a transition in the swelling degree of the polymer networks in aqueous solution, inducing changes on various properties of hydrogels such as permeability, interfacial tension, adhesion, etc. 18 Therefore, with a slight external condition, the smart hydrogels can respond with excellent change of properties.

The phase transitions in thermo-responsive hydrogels are affected to the changing in the volume of solvent and type of solvent due to the solubility ability of polymer chains.

Thermo-sensitive polymers can be classified into two categories 19 : Lower Critical Solution Temperature (LCST) or Upper Critical Solution Temperature (UCST) (Figure 2). In LCST behavior, the phase separation between polymer and water occurs at high temperature (above the LCST). At low temperature (below the LCST), polymer is soluble in water at any concentration. At high temperature (above the LCST), polymer and water are not "compatible" at any concentration, resulting in phase separation, with one phase rich in water and the other phase rich in polymer. The temperature of the phase separation depends on the concentration of polymer in water, the lowest value corresponding to the LCST. For UCST behaviour, it is the inverse. The phase separation occurs at low temperature (below the UCST). The most well-known and studied LCST polymers is poly(N-isopropylacrylamide) or PNIPAM. [20][21][22][23] It is probably due to the proximity of its LCST (around 32°C) close to the ambient and physiological temperatures and the low dependence of the LCST with the polymer concentration (the LCST is constant on a wide range of polymer concentration).

PNIPAM is soluble in water at room temperature. Below its LCST, the solvation of hydrophilic (-NH) and hydrophobic (C=O) functions in polymer chains is favored due to the formation of hydrogen bonds with water molecules. At temperature above the LCST, the affinity between solvent and polymers decreases due of the presence of -CH 3 groups, the interpolymeric hydrogen bondings being preferred with then a phase separation. It is the same for poly(N-isopropylmethacrylamide) 22,23 or PNIPMAM which shows comparable chemical structure as PNIPAM. or poly(PEGMA 300 ) with 4-5 PEG units have LCST values equal to 26°C, 35°C and 64°C respectively. 24,25 Figure 3. The structure of LCST polymers based on poly(acrylamide) derivatives (top) and poly(PAGMA)s with different PEG units (bottom).

For UCST polymers, water is expulsed below their UCST and absorbed above their UCST. For non-ionic polymers such as poly(methacrylamide) 26 or PMA, the hydrogen bonds between pendant chains favoured at ambient temperature, are likely the origin of the phase separation and the globule formation. With heating, these hydrogen bonds are weakened and the polymers become soluble in aqueous solution. 19,27 For polyzwitterions such as 29 or PSPE, the solubility in water could be due to the presence of positive and negative charges in pendant chains. Our vision is that at low temperature (below the UCST), there is the complexation between positive and negative charges of the monomer units; the polymers are not soluble in water. For sufficiently high temperature (above the UCST), the complexation between zwitterionic chains are not favored, as a result, the polymers become compatible with water. The other polymers we investigate are poly(N,N-dimethylacrylamide) or PDMA and poly(2-acrylamido-2-methylpropane sulfonic acid) or PAMPS for their molecular structure close to that of cited polymers. These polymers display no temperature-responsive properties.

PDMA is an acrylamide derivative polymer with no LCST or UCST properties. However, we have shown that LCST and UCST behaviors can be observed when PDMA is coupled to

PSPE PSPP O O N SO 3 n O HN N SO 3 n O H 2 N n PMA PDMA O N n PAMPS O HN SO 3 Na n Chapter 1.
Polymer hydrogel films: generalities and objectives of the thesis 14 poly(acrylic acid), the same being also obtained with poly(acrylamide). 30 PAMPS is a polyelectrolyte with negative charge and can respond to electrical field. 15 In this work, we will especially focus on LCST-type and UCST-type polymers, which characteristics are summarized in Table 1. Poly(N-Isopropylacrylamide) PNIPAM LCST 30-34°C [20][21][22][23] Poly(N-Isopropylmethacrylamide) PNIPMAM LCST 34-40°C

Name

Polymer hydrogels at interface

Surfaces that provide interesting properties and performances have attracted great attention due to their wide applications in biomedicine, chemical engineering and so on. The synthesis of different polymers at interfaces is developed. We present two most useful of them; layer-by-layer assembly and polymer brushes.

Layer-by-layer (LbL) assembly is a versatile technique to tailor thin films on soft and solid substrates. The majority of films are prepared by the assembly of alternating polyelectrolyte layers oppositely on a surface with inevitably wash steps in between. The LbL technique was firstly reported by R.K. Iler 32 in 1966, who obtained multilayer of colloidal particles on a glass surface. Afterwards, Gero Decher 33,[33][34][35] has shown several routes to fabricate thin films from anionic and cationic amphiphiles and polyelectrolytes by electrostatic interaction. The LbL films can also be prepared by using hydrogen bonding interaction 36,37 and covalent bonding. 38,[START_REF] Kohli | Applying Polymer Chemistry to Interfaces: Layer-by-Layer and Spontaneous Growth of Covalently Bound Multilayers[END_REF] Covalently bound films offer the advantage of higher stability due to the chemical crosslinked polymer networks.

Nowadays, layer-by-layer assembly is one of the facile methods to prepare the hydrogel films on solid substrates because of its simplicity and versatility for surface modification. Much attention has been paid for the electro-responsiveness polyelectrolyte gel.

The multilayer polyelectrolyte films are very promising for their pH-and electric-sensitivity, for example, poly(acrylic acid), poly(2-acrylamido-2-methylpropane sulfonic acid), poly[(2dimethylamino)ethyl methacrylate]. The swelling properties of polyelectrolyte networks depend on their physicochemical properties and on the composition of the environmental solution. [START_REF] Sung | Swelling Properties of Hyaluronic Acid Ester Membranes[END_REF] Since the polyelectrolytes hydrogels are in solutions, the conformational changes can take place due to the ionic interaction between the polymeric ions, or between the polymeric ions and counter ions in the solvent. For weak polyelectrolyte hydrogels, the swelling degree depends on the pH of the solution, but for strong polyelectrolyte, the dissociation is completed for a wide pH range which renders them become pH-independent.

The swelling equilibrium can be modified by varying the backbone hydrophilicity of the polymer. [START_REF] Beltran | Swelling Equilibria for Weakly Ionizable, Temperature-Sensitive Hydrogels[END_REF] However, the LbL films are susceptible to disassembly under some conditions because they are not chemical crosslinked polymer networks. In order to ensure their stability, the films have to be prepared by chemically crosslinked polymer networks and grafted to the surface. As the assembling of layers has to be first ensured by physical interactions before post chemical crosslinking, the swelling ability of LbL is more limited in comparison with hydrogel films synthesized in one pot.

Polymer brushes [START_REF] Advincula | Polymer Brushes[END_REF]43 are polymers chains attached by one end on a surface. The density of attachment points is high so that the chains are obliged to stretch away from the surface in the direction normal to avoid overfilling incompressible space. This situation is different from the mushroom regime in which the grafting density is low. In the mushroom regime, the chains are free to adopt a random-walk configuration like the polymer in solution in diluted regime.

The physisoption [START_REF] Advincula | Polymer Brushes[END_REF][START_REF] Parsonage | Adsorption of Poly (2-Vinylpyridine)-Poly (Styrene) Block Copolymers from Toluene Solutions[END_REF] or the physical adsorption of homopolymer or copolymer on the surface is a route to prepare polymer brushes. However, the adsorbed layers are unstable and can be removed when they are involved in some conditions where the interactions between the polymers and the environment are stronger than the physical adsorption.

Another way to prepare polymer brushes is to form a covalent (chemically) attachment to the surface by "grating from" or "grafting onto" methods. [START_REF] Tran | Determination of the Structure of Polyelectrolyte Brushes[END_REF][START_REF] Ionov | Inverse and Reversible Switching Gradient Surfaces from Mixed Polyelectrolyte Brushes[END_REF] The "grafting from" technique, the polymers brushes are obtained by using a surfaceinitiated polymerization (SIP) with an initiator. The appropriate initiator has to be selected and the surface-initiated controlled radical polymerization allows the control the molecular weights with narrow distributions, for example, surface-initiated Atom Transfer Radical Polymerization (ATRP) and Reversible Addition-Fragmentation chain Transfer (RAFT) polymerization. [START_REF] Sanjuan | Synthesis and Swelling Behavior of PH-Responsive Polybase Brushes[END_REF][START_REF] Sanjuan | Synthesis of Random Polyampholyte Brushes by Atom Transfer Radical Polymerization[END_REF][START_REF] Slim | Microelectrochemical Patterning of Surfaces with Polymer Brushes[END_REF][START_REF] Sanjuan | Stimuli-Responsive Interfaces Using Random Polyampholyte Brushes[END_REF] In addition to its versatility, the "grafting from" approach has the advantage to provide polymer brushes with high grafting density. For the "grafting onto" method, preformed polymers with a selected chemical end-functionalized group that reacts with the surface are used. This approach is straightforward because there is no need of "sophisticated chemistry". It is definitely not as versatile as "grafting from" approach even though many preformed polymers are commercialized. It also needs the use of little amount of polymer to prepare polymer brushes. The "grafting onto" approach advantageously enables the fabrication of mixed brushes, Figure 6. Schematics of grafting from and grating onto techniques. Data from reference 51

Context and objectives of the dissertation

Context of the dissertation

For a few years now, in our group, we have been investigating surface-attached hydrogel thin films with temperature-responsive properties. The CLAG (Cross-Linking And Grafting) strategy was performed by Mengxing Li to prepare PNIPAM hydrogel films on silicon wafers with a very good reproducibility [START_REF] Li | Submicrometric Films of Surface-Attached Polymer Network with Temperature-Responsive Properties[END_REF] . The CLAG approach consists in crosslinking and surface-grafting preformed reactive chains by click chemistry. Thiol-ene click reaction was chosen for its versatility. The thiol-ene reaction can be activated by (soft) thermal heating and also by UV irradiation (even without photoinitiators). Another advantage is that many dithiol molecules are commercially available to suit the polymer chemistry. Enefunctionalized polymers can be simply synthesized by free radical polymerization with no need of sophisticated chemistry. The synthesis of preformed reactive chains avoids the difficulty of working under controlled atmosphere to avoid oxygen which inhibits radical polymerization. Even if thiol-ene is a radical reaction, the sensitivity to oxygen is much more reduced because it only involves the cross-linking and the surface-grafting and not the formation of whole chains.

Preformed ene-functionalized polymers and dithiol crosslinkers are coated on thiolmodified solid substrates before being simultaneously cross-linked and surface-grafted by thiol-ene reaction (Figure 7). To use this approach, the ene-functionalized PNIPAM was a main key and had to be synthesized. Free radical polymerization with acrylic acid was Chapter 1. Polymer hydrogel films: generalities and objectives of the thesis 18 selected using redox couple in water and followed by peptide reaction with allylamine to obtain well-defined ene-functionalized polymers. It has been shown that hydrogel films are obtained with a wide range of thickness, from a few nanometers to several micrometers using spin-coating technique. The film thickness depends on many factors such as the volatility of the solvent, the final angular velocity and the viscosity of the polymer solution. In particular, films with different thickness are obtained by changing the viscosity with the variation of polymer molecular weight and polymer concentration (Figure 8). As expected, the film thickness increases with the viscosity of polymer solution, and so with polymer concentration or molecular weight. The results are shown here for PNIPAM layers with various molecular weights but the same are obtained for PAA films. In particular, we observed that the film thickness is similar for PNIPAM and PAA using spinning polymer solutions with same concentration, same molecular weight and suitable solvent.

The temperature-responsive properties of PNIPAM hydrogel films were determined by measuring the swelling ratio with the temperature, the swelling ratio being defined as the swollen thickness in water to the dry thickness in air, as shown by Figure 9. The swelling ratio is independent of the thickness in the whole range from 100 nm to a few micrometers.

The data shown were obtained with hydrogel films of supposedly same cross-links density.

This assumption seems consistent since the synthesis of hydrogel films is performed with the same reactive copolymer (2%) and the same excess of dithioerythritol cross-linkers (30 times the ene-reactive groups). As experimental conditions used are the same except the thickness, hydrogel films can be supposed to be chemically the same.

The measure of the thickness in the swollen state is also a way to control the synthesis of hydrogel films since it provides the cross-links density (even indirectly). The weaker the cross-links density, the more the hydrogel films swell. Here, we found a swelling ratio of 4 folds with 2% ene-reactive polymer. Our value of swelling ratio is comparable to the values found by Toomey et al. [START_REF] Toomey | Swelling Behavior of Thin, Surface-Attached Polymer Networks[END_REF] If the swelling ratio is 4 for temperature below the LCST, the value of 1.5 is found for temperatures above the LCST. It means that collapsed PNIPAM hydrogel films are not free of water but indeed contains a water volume fraction of roughly 30% (which corresponds to a ratio of 2 molecules of water for 1 molecule of PNIPAM unit). The same water content was also found for PNIPAM collapsed brushes, meaning that the collapsed state does not depend on the architecture of PNIPAM film, network or brush. But it is only decided by polymer-solvent interaction (or  Flory parameter). Thiol-ene click chemistry used in our CLAG approach has also the major advantage to be activated by UV irradiation (with deep UV, there is no need of added initiator The proofs of concept with micro-valving and micro-caging functionalities using hydrogel actuators were reported in an article which is recently accepted for publication in Nature group journal. The example of micro-valving is given below. To fabricate a microfluidic valve, we integrate a patch of gel in a micro-channel. In order to seal the valve, the hydrogel patch length slightly exceeds the channel width. After closing, the excess hydrogel is squeezed by the material used for the device. This construction prevents the presence of dead volumes and the advent of leaks. Figure 11 represents the evolution of the flow-rate with the temperature in a system including a valve. One sees that the transition occurs around the LCST, and the front is steep. We aim to take advantage of the full potential of these responsive hydrogel actuators to develop microfluidic devices for biotechnological applications. Biology applications using responsive hydrogels-based technology are numerous. Digital amplification is an example. In our case, thousands of cages can be integrated on a single system, so that high throughput screening or high sensitivity analyses of samples are feasible, with direct readout. Single-cell manipulation, stimulated by a growing recognition of the important role of genomic heterogeneities in biological systems, provides another example of applications of our technology. Microbiology is another field where temperature-responsive hydrogels with wellcontrolled chemistry offer potential advantages. In this case, the permeability of the gel could be harnessed to deliver therapeutic molecules to cells trapped in cages to screen antibiotics. Also, most importantly, these applications could be developed under interesting cost conditions, since the materials are cheap and actuation only requires low voltage sources.

The development of temperature-responsive hydrogel actuators for biotechnology is achieved in close collaboration with IPGG-MMN laboratory, with Hubert Geisler's PhD project. In this context, it is indispensable to design surface-attached temperature-responsive hydrogels with various LCST and UCST properties (with various transition temperatures).

Beyond microfluidic applications, responsive hydrogel thin films are also promising mouldable materials for sensors and actuators in other domains such as optics.

Objectives of the dissertation

Inspiring from Mengxing Li and Benjamin Chollet's PhD works and the needs from our collaboration with IPGG-MMN laboratory, we are interested in LCST and UCST hydrogel thin films with various transition temperatures. The hydrogel layers have to be chemical polymer networks and covalently grafted to the solid substrates to ensure their stability and durability.

i.

What are the optimized conditions to synthesize hydrogel thin films for polymers with a wide range of thickness and with desired LCST and UCST properties?

A sample and versatile strategy has been developed to prepare surface-attached hydrogel thin films and can be easily adjusted to produce hydrogels with LCST or UCST properties. We choose to fabricate hydrogel films by chemically cross-linking preformed linear ene-functionalized polymers that we have to prepare before. This method promotes a better control of the synthesis of hydrogels grafted to the surface.

In general, the radical polymerization should be performed with controlled atmosphere because oxygen inhibits the reaction. The polymerization without rigorous caution would affect the formation of hydrogel thin films disastrously. We also optimized the approach for the synthesis of hydrogel thin films with different chemical natures and different responsiveness by using click chemistry which allows the synthesis without controlled atmosphere and without the addition of the initiator.

The click chemistry chosen is thiol-ene chemistry which allows the fabrication of surface-attached hydrogel thin films by coating the preformed ene-functionalized polymers and dithiol molecules as cross-linkers on thiol-modified substrates. The thiol-ene reaction permits the cross-linking of polymer chains and their anchoring to the surface. The thiol-ene reaction is a radical reaction; consequently, it should be carried out with controlled atmosphere and can be activated without initiator by temperature or UV-irradiation (at 250 nm). The addition of initiator is needed when using UV-irradiation at 350 nm. Besides, thiol modification surface or silanization is judicious because the glass or silicon substrates can generally be operated with thiol and in particular gold surfaces can be more easily functionalized with dithiol molecules. Moreover, thiol molecules for surface modification and dithiol molecules for cross-linking are widely commercialised. Also, the coating techniques are selected for producing hydrogel films and permit to obtain the range of thickness from a few nanometers to several micrometers which allow their characterization using ellipsometry.

Different polymers are chosen in this study (c.f. Table 1) to answer the objectives ii to iv. The synthesis of ene-functionalized polymers is carried out in different solvents. We present two strategies: the one-step strategy which is the copolymerization between the desired monomer with allyl methacrylate in organic solvents or co-solvents and two-step strategy which is the copolymerization between the desired monomer with acrylic acid and then the post-modification (peptide coupling) by using allylamine. The copolymerization selected is a free radical polymerization because there is no need to control the polydispersity of polymer chains.

ii The synthesis of ene-functionalized poly(acrylamide) derivatives for the preparation of surface-attached hydrogel films was achieved using both one-step strategy and two-steps strategy. The question whether the synthesis strategy of ene-functionalized polymers has an effect on the transition temperature is raised. We also report how to manipulate poly(acrylamide) derivatives in order to obtain hydrogel thin films with various responses to temperature: non temperature-responsive, LCST, and UCST properties.

iv.

How are the changes of UCST properties of poly(sulfobetaines) hydrogel films under salt conditions with anti-polyelectrolyte effect?

We also investigate poly(sulfobetaines) hydrogel films which are very promising for biology applications due to their advantageous combination of both UCST and anti-fouling properties. The surface-attached polymer films are characterized to determine their UCST in water and their behavior in phosphate-buffered in solutions. As reported in literature reviews, the presence of salt can cancel the UCST phase transition of polyzwitterions with an anti-electrolyte effect. What are the salt concentrations in comparison to polymer concentrations to cancel the UCST phase transition? Is the high polymer concentration in hydrogel architectures enough to keep the UCST behaviour despite the presence of salt in physiological conditions?

The answers to these questions will also be helpful to understand the role of free counterions in the UCST behaviour of polyzwitterions.

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Introduction

Polymers assemblies at the interface have drawn considerable attention in various research fields since they can provide multiscale and multifunctional polymer films. 1,2 Polymer brushes 3,4 and layer-by-layer 5,6 (LbL) assemblies are so far the most investigated during the last two decades. However, they display some drawbacks with limited high thicknesses. Polymer brushes have their thickness is decided by the chains length or the degree of polymerization so that the thickness is limited to submicrometer range. [7][8][9][10] In LbL assemblies many cyclic steps are required to build multilayers as the elementary layer is a few nanometers thick and they are susceptible to disassembly under some conditions.

Surface-attached hydrogel films which are 3D chemical polymer networks are real alternative to polymer brushes and LbL assemblies as polymer films with the advantages of one-pot synthesis and large range of thickness. Hydrogels [11][12][13] are chemically stable due to covalent crosslinks and attachment to the substrate. Hydrophilic polymer networks films become very attractive for their high biocompatibility and ability to absorb large amounts of water. Reducing the size of the hydrogel provides a material with fast response while maintaining the mechanical properties of hydrogels. [14][15][16][17] The surface-attached hydrogel films are promising materials since they associate the both properties of hydrogels and films.

However, they are still scarcely investigated. This is probably due to a lack of general and reliable strategy to synthesize hydrogel films with well-controlled chemistry.

We propose a simple and versatile strategy for the synthesis of reliable and reproducible surface-attached hydrogel films. This approach enables a fine control of the chemistry and in particular the crosslinks density which determines the swelling ratio or the water content. This point is crucial for the development of responsive hydrogel films with high amplitude change. Our CLAG strategy consists in cross-linking and surface-grafting preformed functionalized chains using thiol-ene click chemistry. 18,19 The CLAG approach allows a better control than a "grafting from"-type approach which would have been based on the polymerization of the whole layer of monomers. This latter approach is much more difficult to control due to the sensitivity of radical polymerization to oxygen which is enhanced for films (high surface-to-volume ratio). Here, ene-functionalized polymers are Chapter 2. Facile synthesis of surface-attached hydrogel thin films by cross-linking and grafting CLAG approach using click chemistry 37 coated (by spin-coating or dip-coating) in the presence of dithiol cross-linkers on thiolmodified substrates, the thiol-ene click reaction allowing simultaneous cross-linking and surface-grafting. The combination of coating (which solves the problem of limited thickness)

and fine control of chemistry (using preformed polymer) is quite innovative to enable the formation of polymer films with large range of thickness (from a few nanometers to several micrometers) and with well-controlled chemistry. [15][16][17] The CLAG strategy is versatile since it is applicable for all polymers with various targeted properties: stimuli-responsive, glassy/rubbery, neutral polymer/polyelectrolyte. Moreover, thiol molecules for surface modification and dithiol molecules for cross-linking are widely commercialized. It is straightforward since the free radical polymerization does not require sophisticated chemistry.

The ene-reactive polymers with (multiple) stimuli-responsive properties can be easily preformed by using suitable monomers.

In this article, we show the synthesis of surface-attached hydrogel films for various synthetic polymers with acrylate and acrylamide: poly[poly(oligoethylene glycol methacrylate)] or poly(PEGMA), poly(acrylamide) derivatives and ionic polymers. These polymers can show temperature-responsive properties with Lower/Upper Critical Solution Temperature LCST/UCST phase transition 20 , which is one of the research interests of our group. Poly(PEGMA) are attractive polymers for the combination of biocompatibility and LCST responsive properties. 21,22 With poly(acrylamide) derivatives, we demonstrate that a small change in the chemistry structure enables to have either LCST or UCST temperatureresponsive properties. [23][24][25][26][27] Poly(sulfobetaines) are polyzwitterions which have the advantages to combine anti-fouling and UCST responsive properties. [28][29][30] Experimental Section

Materials and reagents

Di(ethylene glycol) methyl ether methacrylate (DEGMA, 95%, Sigma), tri(ethylene glycol) monoethyl ether monomerthacry-late (TEGMA, Polysciences), poly(ethylene glycol) methyl ether methacrylate (PEGMA 300 , average Mn = 300 g.mol -1 , Sigma) and allyl methacrylate (AMA, 97%, Alfa Aesar) monomers were purified prior to use by passing through the basic alumina column to remove inhibitors.

Chapter 2. Facile synthesis of surface-attached hydrogel thin films by cross-linking and grafting CLAG approach using click chemistry 38 N-isopropylacrylamide (NIPAM, 97%), N-isopropylmethacryla-mide (NIPMAM, 97%), methacrylamide (MA, 98%) and N,N-dimethlyacrylamide (DMA, 99%), and acrylic acid (AA, 99%) monomers were purchased from Sigma and used without further purification.

Synthesis of ene-functionalized polymers in organic solvent

The ene-functionalized poly(PEGMA 300 ) was synthesized by free radical polymerization of PEGMA 300 and AMA using AIBN as thermal radical initiator. PEGMA 300 (24.00 g, 80.00 mmol), AMA (0.2060 g, 1.63 mmol) and AIBN (0.0201 g, 0.12 mmol) were mixed in 100 g of toluene. Then the solution was deoxygenated by nitrogen bubbling for 1 h and the reactor was put in pre-heated oil bath at 70°C. The reaction was carried out at 70°C under nitrogen for 24 h. The final solution was solubilized in minimum acetone in order to obtain a good fluid then it was precipitated in diethyl ether. The obtained precipitate was washed 3 times with diethyl ether then it was solubilized in acetone again. The solution was evaporated until all acetone were removed.

The same process was used for the synthesis of ene-functionalized poly(TEGMA), ene-functionalized poly(DEGMA), ene-functionalized PNIPAM and ene-functionalized PNIPMAM. For PNIPMAM, the solvent of reaction was dimethylformamide (DMF) instead of toluene. The solvent for the precipitation was the same of poly(PEGMA 300 ) except poly(DEGMA) and poly-(TEGMA), for which pentane was used instead of diethyl ether.

Synthesis of ene-functionalized polymers in co-solvent with water

For the ene-functionalized PSPP polymer, the process is as follows. To a two-neck flask, SPP (8.80 g, 30.00 mmol), AMA (0.20 g, 1.58 mmol), V50 (21.50 mg, 0.08 mmol), and then 100 mL of co-solvent (1:2 THF/water) were added. After that, the solution was deoxygenated by nitrogen bubbling for 1 h. The polymerization medium was allowed to proceed under refluxed condition at 70°C under nitrogen for 24 h. In order to purify the polymer at low polymer concentration to avoid having important swelling, 100 mL of water was added into the polymer solution and it was dialyzed against pure water for four days at 70°C, the polymer was acquired by freeze-drying the resulting solution. For ene-functionalized PSPE and enefunctionalized PAMPS polymers, the same conditions as the synthesis of ene-functionalized Chapter 2. Facile synthesis of surface-attached hydrogel thin films by cross-linking and grafting CLAG approach using click chemistry 40 PSPP were used. For ene-functionalized PMA, the solvent used for the polymerization was formamide/H 2 O (1:2) instead of THF/H 2 O (1:2).

Synthesis of ene-functionalized polymers in water in two steps

The detail of ene-functionalized PNIPAM synthesis in water was described in our previous article.The synthesis is performed in two steps. Briefly, the synthesis of P(NIPAMco-AA) copolymer was carried out by free radical polymerization using (NH 4 ) 2 S 2 O 8 / Na 2 S 2 O 5 as redox couple initiator in water at room temperature under nitrogen atmosphere for 24 h.

Then, the functionalization of P(NIPAM-co-AA) copolymer was realized by peptide coupling using N-hydroxysuccinimide (NHS) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC). Allylamine was added to the medium and the reaction was allowed to proceed in water for 16 h at room temperature. The final solution was purified by dialysis in 0.1 M NaCl solution for 4 days to remove the excess of EDC and NHS and then in pure water for 4 days to remove all salts. In the end, the modified polymer was recovered by freezedrying.

For ene-functionalized PNIPMAM, the solvent of reaction was H 2 0/acetonitrile (1/1 v/v) because of low solubility of NIPMAM monomer in water. Ene-functionalized PDMA was synthesized in the same condition as ene-functionalized PNIPAM and the medium could even being heated at 60°C because PDMA is not a thermo-sensitive polymer and has no LCST properties in water.

Gel Permeation Chromatography

The molecular weight and dispersity (Đ) of polymers were determined by gel permeation chromatography (GPC). Two type of GPC were used depending on the polymer.

The first GPC equipped with OHpak SB-806M HQ columns using H 2 O with 0.2 M NaNO 3 as eluent at 35°C, at a flow rate of 0.7 ml.min -1 was used for poly(PEGMA 300 ), PSPP, PSPE, PAMPS and PDMA. For PNIPAM and PNIPMAM synthesized in water by two-steps strategy, the measure was carried out at 28°C.

Chapter 2. Facile synthesis of surface-attached hydrogel thin films by cross-linking and grafting CLAG approach using click chemistry 41

The second GPC using THF as eluent was performed with GMHxl columns at 40°C, at a flow rate of 0.7 ml.min -1 . It was used for poly(DEGMA), poly(TEGMA), PSPP and PSPE, and also PNIPAM and PNIPMAM synthesized in organic solvent (one-step strategy). was used for poly(TEGMA), poly(PEGMA 300 ) and PNIPAM synthesized in organic solvent (one-step strategy). Poly(DEGMA) and PNIPMAM synthesized in organic solvent (one-step strategy) were dissolved in deuterated dimethyl sulfoxide (DMSO-D 6 ). 1 H NMR spectra of ene-functionalized poly(PEGMA 300 ), ene-functionalized PSPP and ene-functionalized PNIPAM are provided in the main article and for the other ene-functionalized polymers, their 1 H NMR spectra are provided in Supporting Information.

Ellipsometry

The thickness of hydrogel films was measured using spectroscopic ellipsometer UVISEL (Horriba Jobin Yvon) with the wavelength ranging from 260 to 850 nm (in air) and from 320 to 850 nm (in water). The refractive index used for the silicon wafer is 3.875. The measures at silicon-air interface were realized using the model with two layers. The first layer contains silica and silane (n = 1.46) which the thickness was determined before anchoring the hydrogel film. The polymer hydrogel is the second layer. For poly(PEGMA 300 ), poly(TEGMA) and poly(DEGMA), the value of refractive index used is 1.48. For PNIPAM, PNIPMAM, PSPP, PSPE, PMA and PAMPS, it is 1.50. In situ measurements in water of polymer hydrogel film were carried out using a liquid cell which is equipped with thin glass walls fixed perpendicularly to the light path with the incidence angle at 60°. The liquid cell is temperature-controllable with a regulation within ±0.1°C. We used a single model layer for polymer hydrogel film with thickness h w (in water), h a (in air) and a constant refractive index of water (1.33) and polymer (1.48 for poly(PEGMA 300 ), poly(TEGMA) and poly(DEGMA) or 1.50 for PNIPAM, PNIPMAM, PSPP, PSPE, PMA and PAMPS ). As the attachment of polymer network hydrogel is covalent bond, we should find the same polymer amount at the Chapter 2. Facile synthesis of surface-attached hydrogel thin films by cross-linking and grafting CLAG approach using click chemistry 42 surface in air and in water. Then the polymer refractive index in water (n w ) can be expressed by, n w = (n p -1.33)× +1.33 and h a = h w × which the volume of fraction of polymer in water and n p is the refractive index of polymer. The thickness in water h w, in air (h a ) and the refractive index of polymer in water (n w ) are deduced from the fitting of experimental data.

To ensure that the fittings give reliable results, the two above equations should provide the same value of .

FTIR-ATR spectroscopy

For Fourier transform-IR attenuated-total-reflection (FTIR-ATR) spectroscopy measurements, silicon substrates are used as infrared wavelength guides so that they are cut in special shape (70 mm × 10 mm × 1.5 mm trapezoidal crystal with an angle of 45°). The FTIR-ATR spectra are obtained using a Magna IR 550 (Nicolet) apparatus with a mercurycadmium-telluride (MCT) detector cooled with liquid nitrogen. The spectra are recorded with a resolution of 2 cm -1 and a 256-scan accumulation.

Results and discussion

Strategy of surface-attached hydrogel films: cross-linking and grafting CLAG

Surface-attached polymer network films are prepared by cross-linking and grafting (CLAG) method through thiol-ene click reaction. The preformed ene-functionalized polymers are coated on the thiol-modified substrates with dithiol crosslinkers (Scheme 1) to be simultaneously cross-linked and surface-grafted. The straightforward and versatile CLAG strategy allows the fabrication of chemically stable polymer hydrogel films with reliability and reproducibility.

The simplicity of the CLAG approach is mainly due those ene-reactive polymers can be fabricated by conventional radical polymerization. Actually, sophisticated radical polymerization techniques such as controlled radical polymerization (ATRP, RAFT, NMP)

Chapter 2. Facile synthesis of surface-attached hydrogel thin films by cross-linking and grafting CLAG approach using click chemistry 43 are not required because there is no need to control the dispersity of polymer to prepare the polymer networks.

The preformed polymers can be coated onto the substrates by several techniques such as dip-coating, spray-coating or spin-coating, depending on the thickness range targeted. The spin coating technique is chosen here to obtain hydrogel films with thickness widely ranging from a few nanometers to several micrometers. Spin-coating has also the advantage to necessitate a little polymer solution. For example, only 0.2 ml polymer solution is needed to cover a surface of 1 cm 2 (about 20 mg of polymer for 10 wt%) The solvent for spin-coating must be a good solvent for both ene-reactive polymers and crosslinkers. reaction allows simultaneously the cross-linking of polymer chains and their grafting on the substrate.

Synthesis of ene-functionalized polymers in organic solvent

Free radical copolymerization (with AMA) is chosen for the synthesis of enefunctionalized polymers as this polymerization technique is a straightforward process which does not require catalysis, transfer agents, etc. This strategy is a one-step synthesis allowing achieving ene-reactive polymers with different stimuli-responsive properties because there are different commercial monomers available. Also, we can select the initiators and solvents upon the type of monomer (hydrophilic, hydrophobic, ionic, etc.). Two ways are proposed for polymer synthesis in this paper. The first strategy is in one step. We used V50 as a thermal initiator in co-solvent (organic solvent/H 2 O) for the synthesis of PSPP, PSPE, PAMS and PMA ene-functionalized polymers (Scheme 2). Water is necessary to solubilize the hydrophilic monomers and the organic solvent enables to dissolve AMA. The synthesis of poly(PEGMA 300 ), poly(TEGMA) and poly(DEGMA) was carried out in an organic solvent (here toluene) with AIBN as initiator

As AMA monomer contains two reactive double bonds, they could play as crosslinkers or they could lead to a cyclization to five-or six-carbons lactone formation 31 . The radicals are preferably generated close to methacrylic group due to their stability preventing from mesomeric effect. The synthetic route likely leads to linear copolymers without crosslinking. From 1 H NMR spectra, the polydispersity of synthesized polymers is around 2 to 4 and the ene-reactive ratios are around 1 to 7 mol%., These characteristics are consistent with the formation of linear copolymers.

Actually, René Nagelsdiek and co-workers 32 have studied the copolymerization of AMA and styrene by Atom Transfer Radical Polymerization (ATRP) without any cross-linking process observed.

However, in our studies the gelation (cross-linking) was observed for the synthesis between 90 mol% PEGMA 300 and 10 mol% AMA at 50 wt% monomers concentration in toluene. The other gel formations were also remarked when copolymerized between 90 mol% DEGMA and 10 mol% AMA in dimethylformamide (DMF) at 20 wt% monomer concentration.

Chapter 2. Facile synthesis of surface-attached hydrogel thin films by cross-linking and grafting CLAG approach using click chemistry 45 Scheme 2. General strategy of radical polymerization of methacrylate or acrylamide derivatives and allyl methacrylate in organic solvent or co-solvent with water.

Table 1 shows the characteristics of ene-functionalized polymers investigated.

For poly(DEGMA), poly(TEGMA) and poly(PEGMA 300 ) polymers synthesized in toluene using AIBN as thermal initiators, the molecular weights obtained are almost the same due to their similar chemical structures. The values of dispersity between 2.3 and 3.4 are absolutely consistent with the free radical polymerization method used. The ratio of AMA found is slightly lower than the expected ratio showing that the reactivity of AMA monomers is weaker than that of PEGMA 300 monomers for the formation of polymer chains.

For PMA, PAMPS, PSPE and PSPP synthesized in co-solvent with water using V50 as thermal initiators, the molecular weight obtained increases for polyelectrolytes and polyzwitterions due to repulsive electrostatic interactions. The values of dispersity are between 2.0 and 3.7 as expected for free radical polymerization. As for the poly(PEGMA) family; the ratio of AMA found is lower than the expected ratio showing that the reactivity of AMA monomers is weaker than that of MA, AMPS SPE and SPP monomers.

For PNIPAM and PDMA and PNIPMAM polymers synthesized in water, the molecular weight is controlled by the amount of redox couple as initiator 33 . The ene-groups obtained in this strategy in two steps are due to the post-modification with allylamine by amidification reaction 34 .

The ratio of ene-groups is decisive for further synthesis of hydrogel thin films by surface-grafting and cross-linking of ene-reactive chains through thiol-ene click reaction.

C O R 1 R 2 C O R 1 R 2 + C O O n m C O O n m I , N 2 , 24H
70°C, solvent M Chapter 2. Facile synthesis of surface-attached hydrogel thin films by cross-linking and grafting CLAG approach using click chemistry 46

Actually, it has been shown that the ratio of ene-groups has to be at least 1mol% to allow the formation of hydrogel thin films. Below 1mol% of ene-groups ratio, the formation of hydrogel films cannot occur due to low grafting density and cross-linking, the polymer chains being not sufficiently grafted and cross-linked to the chemical stability of the polymer networks on the surface. On the other hand, highly cross-linked hydrogels (with ene-groups ratio above 5mol%) would not swell a lot, containing little water. Toomey et al 14 The ene-groups ratio is calculated by 2 methods which are provided in detail in the Supporting Information. The first method is used for the ene-functionalized polymers synthesized by one-step strategy. Ene groups ratio of poly(PEGMA 300 ) is found 1.2% (figure 2a). The second method is applied when the proton of polymers appeared at same chemical shifts such as PNIPAM and PNIPMAM synthesized by two-steps strategy. The ene groups of PNIPAM (figure 3) is determined at 2.7%.

Synthesis of ene-functionalized polymers in water

Water is very attractive solvent for chemical reaction. The synthesis of enefunctionalized polymers is achieved in two steps. First, the copolymers containing acrylic acid are synthesized by free radical polymerization followed by post-modification using peptide coupling reaction (Scheme 3).

This process is successful for the synthesis of ene-functionalized neutral polymers (acrylamide polymers such as PNIPAM, PNIPMAM and PDMA) and weak polyelectrolytes under acid form (acrylic acid polymers such as PAA and PMAA). The synthesis of PNIPAM was reported in detail in our previous publications. [15][16][17] The first stage is the free radical copolymerization of NIPAM and acrylic acid using ammonium persulfate/sodium metabisulfite redox couple as initiator. The advantage of this free radical polymerization technique is its relative control: the molecular weight of polymer chains is ruled by the concentration of the reducing agent as shown by Bokias et al. 33 The second step is the enefunctionalization of P(NIPAM-co-AA) by grafting allylamine in the presence of EDC/NHS couple, EDC being the dehydration agent and NHS the addition agent used to increase yields and decrease side reactions. 34 Both radical polymerization and amide formation are advantageously performed in water as all the chemicals used are water-soluble. They are carried out at ambient temperature below the LCST of PNIPAM.

The ene-functionalized polymer can be then easily purified by dialysis against water and recovered by freeze-drying to be stored and characterized by Size Exclusion Chromatography and NMR spectroscopy. It should be also noted that for ene-functionalized PAA, only the second step is required as PAA homopolymer can be easily and cheaply purchased.

Chapter 2. Facile synthesis of surface-attached hydrogel thin films by cross-linking and grafting CLAG approach using click chemistry 50

From 1 H NMR spectra such as ene-functionalized poly(NIPAM-co-AA) that shown in figure 3, the ratio of ene-functionalization is calculated. A ratio about 2% was found. A disadvantage of the strategy is the presence of acrylic acid units which are not modified by allylamine. Actually, the ratio of residuals acrylic acid is generally less than 5% and does not change dramatically the temperature-responsive properties of hydrogel films.

If ene-functionalized acrylamide polymers such as PDMA, PNIPMAM and PNIPAM were successfully synthesized in water using the two-steps strategy, it was not the same for ene-functionalized PMA. The first step of copolymerization is not a problem. The difficulty is the modification by amidification. The primary amine character of PMA might disturb the coupling reaction. The side reactions that may occur during coupling reaction using EDC/NHS: NH 2 of PMA will attack on the carbonyl function of acrylic acid which is strongly favored by the presence of the leaving group and lead to obtain six-membered ring. 35 The synthesis of ene-functionalized poly(PEGMA) in water was complex due to the formation of hydrogel favoured by physical hydrogen bonding. The approach in organic solvent was then preferred as shown in the previous part. Chapter 2. Facile synthesis of surface-attached hydrogel thin films by cross-linking and grafting CLAG approach using click chemistry 51

The strategy of synthesis of ene-functionalized polymers in water was also unsuccessful with ionic polymers, PSPE, PSPP polyzwitterions and PAMPS polyelectrolyte.

Actually, there is no problem to synthesize the copolymers containing ionic units and acrylic acid. The difficulty is the second step of amide formation with EDC/NHS coupling. The presence of ionic units probably interferes with EDC/NHS coupling mechanism.

The solution to this problem could be the substitution of acrylic acid units by carboxylic acid units with longer spacers and with more functionalities. However, we showed that the replacement by itaconic acid and butenoic acid units had no major improvement. And there is a risk that hydrophilic properties of polymer networks are modified with the use of carboxylic acid with longer spacer than butenoic acid. Finally, it seems that enefunctionalized strong polyelectrolytes (PAMPS) and polyzwitterions (PSPP, PSPE) are not able to be synthesized in water with this two-steps approach. For these ionic polymers, the one-step strategy in water/organic solvent mixture using V50 as thermal initiators is more suitable.

Synthesis of hydrogel thin films

Thiol-ene click chemistry was used to simultaneously cross-link polymers chains and graft them onto thiol-modified substrates. This technique is versatile and powerful to connect permanently ene-reactive polymers with dithiol cross-linkers and thiol-modified substrates.

The surface-attachment and the crosslinking can be activated by using thermal heating under vacuum (for 24 h) or under UV irradiation (without any initiator for 7h with standard UV lamp). Polymer hydrogel films are washed in organic solvent or in water (the solvent used for coating) to remove all unreacted chains and dithiol excess. They are then immersed in pure water for 24 h in order to ensure their stability. It was shown that all polymer chains are crosslinked and surface-attached as the thickness measured by ellipsometry is the same before and after washing and immersion. The film thickness depends on many factors such as the volatility of the solvent, the final angular velocity and the viscosity of the polymer solution. 36 In particular, films with different thickness are obtained by changing the viscosity with the variation of polymer molecular weight and polymer concentration.

The results are shown here for PNIPAM layers with various molecular weights but the same are obtained for PAA films. In particular, we observed that the film thickness is similar for PNIPAM and PAA using spinning polymer solutions with same concentration, same molecular weight and suitable solvent. As expected, the film thickness increases with the viscosity of polymer solution, and so with polymer concentration or molecular weight. For molecular weight of 66 kg/mol, submicrometer thicknesses can be obtained with high precision (below 5% deviation) by varying the concentration below 10 wt%. For higher molecular weight, the films have a bigger deviation of the thickness but the advantage is the large range with the formation of micrometric films. [15][16][17] For poly(PEGMA 300 ) and poly(TEGMA) hydrogel films, the variation of the thickness as function of polymer concentration is quite different from that obtained with PNIPAM hydrogel films. It is probably due to higher dispersity values for poly(PEGMA)s polymer chains.

Chapter 2. Facile synthesis of surface-attached hydrogel thin films by cross-linking and grafting CLAG approach using click chemistry 54 and PNIPAM hydrogel film after 24 hours thermal activation (blue).Data from reference 16.

The chemical cross-links density depends on the ratio of ene-functionalization and the efficiency of thiol-ene reaction. If it is quite easy to determine the ratio of enefunctionalization by 1 H NMR spectroscopy with free polymer chains, it is more problematic to characterize the ratio of thiol-ene reaction on hydrogel films. X-ray Photoelectron Spectroscopy (XPS) and IR-ATR seem to be the most suitable and usual spectroscopic methods to probe flat surfaces. However, XPS technique could only probe the superficial nanometric layer (the standard penetration depth of the technique is about 5 nm) and cannot provide the characterization of the whole hydrogel films.

Infrared spectroscopy in Attenuated Total Reflection with micrometric penetration depth was used to probe the whole films.

Si-Si bonds)

. There is some interesting information that could be extracted from 4000 -1600 cm -1 range. The absorption bands between 3100 and 2800 cm -1 are characteristics of asymmetric and symmetric stretching vibrations of CH, CH 2 and CH 3 groups. The absorbance is the same for the four spectra as expected. The loss of the peak at 2500 cm -1 attributable to dithiol cross-linkers (S-H stretch) for spectra (c) and (d) is explained by the thiol-ene reaction and the removal of dithiol in excess. It is also consistent with the high increase of the absorption band at 3300 cm -1 characteristics of O-H (due to H bonds) found in the spectrum b (because of the presence of dithioerythritol in excess), this broad and high band even hiding the peak at 3430 cm -1 characteristics of N-H amide groups. As expected, it is not possible to quantify the thiol-ene reaction for two main reasons. First, the absorption peak which is characteristics of S-C bond is at very low wave number (around 700 cm -1 ). Second, the thiol-Chapter 2. Facile synthesis of surface-attached hydrogel thin films by cross-linking and grafting CLAG approach using click chemistry 55 ene ratio is weak with a maximum of 2%, the ratio of ene groups measured by 1 H NMR being around 2%.

Actually, the measure of the thickness in the swollen state is a way to control the synthesis of hydrogel films since it (indirectly) provides the cross-links density. The weaker the cross-links density, the more the hydrogel films swell. This point will be developed in the following part.

We showed that dithiol molecules had to be added in excess in the polymer solution

Introduction

The field of stimuli-responsive polymer and polymeric materials that exhibit significant properties changes in response to external stimuli, has considerably increased in the last few decades. [1][2][3][4][5] Applications in diverse areas such as sensors and actuators, microelectromechanical systems, colloids, drug deliveries, tissue engineering, self-healing materials are developed. Responsive hydrogel films are increasingly explored being an actual alternative to polymer brushes and layer-by-layer assemblies as smart surfaces. Hydrogel thin films combine the advantages of stability with the surface attachment and the structure of polymer networks. They are then advantageously miniaturized devices with fast response times and high volume changes. 6,7 Among stimuli, the temperature is amongst the most widely investigated for biomedical applications and its easy control. Poly(N-isopropylacrylamide) has been by far the most commonly studied among temperature-responsive polymers. 8 It exhibits a Lower Critical Solution Temperature (LCST) around 32°C which is very convenient because near room temperature and also physiological conditions. We ourselves recently reported studies on the temperature-responsive properties of PNIPAM hydrogel films in a few publications. [9][10][11][12] In particular, we determined the swelling-to-collapse transition with the thickness of PNIPAM hydrogel films as a function of temperature. The amplitude deformation is remarkably high with 3-fold change or more and the transition occurs in a narrow range of temperature.

Poly(PEGMA) chains that bear short oligo ethylene glycol side chains are an emerging class of temperature-responsive polymers owing to their versatile and relevant structures. The methacrylate backbone enables radical polymerization and the PEG side chain provides water solubility and biocompatibility. The thermosensitivity is attributable to the delicate hydrophobic/hydrophilic balance between the backbone and the side chain and the LCST can be adjusted with the oligo ethylene glycol units with the advantage of keeping the same chemistry. A few excellent reviews on poly(PEGMA) polymers highlight their polymerization, their temperature-responsive properties and their design for materials. [13][14][15] In this letter, we report LCST phase transition of poly(PEGMA) hydrogel thin films with well-controlled chemistry. Three poly(PEGMA) with various PEG side chains length are Chapter 3. Lower Critical Solution Temperature phase transition of poly(PEGMA) hydrogel thin films: effect of Oligo Ethylene Glycol units 78 investigated: DEGMA bearing two oligo ethylene glycol units, TEGMA with three units and PEGMA 300 with M n = 300 g/mol. The surface-attached hydrogel thin films are synthesized using a simple and versatile "grafting onto" approach through thiol-ene click reaction. Using ellipsometry, the thickness of poly(PEGMA) hydrogel films is determined as a function of temperature, in various aqueous solutions, and in particular in phosphate saline buffer (PBS) physiological solutions. The temperature-responsive properties of poly(PEGMA) hydrogel films are also confronted to that of linear chains. These results hopefully enable to highlight the potential of materials based on poly(PEGMA) polymers such as surface-attached hydrogel films.

Chapter 3. Lower Critical Solution Temperature phase transition of poly(PEGMA) hydrogel thin films: effect of Oligo Ethylene Glycol units 79

Results and Discussion

The synthesis of surface-attached hydrogel films is schematized in Figure 1. It consists in performing ene-functionalized poly(PEGMA) chains first and then cross-linking and grafting them to thiol-modified substrates by thiol-ene reaction. This "grafting onto" approach via thiol-ene click chemistry is preferred to a "grafting from" approach in which (radical) polymerization and cross-linking take place simultaneously. The inconvenience of the "grafting from" technique is the difficulty to finely control the chemistry, and in particular the crosslinks density of the hydrogel film, which is decisive for stimuli-responsive properties.

This is mainly due to the sensitivity of radical polymerization reaction to oxygen, which is amplified by the film geometry with high surface to volume ratio.

Ene-functionalized poly(PEGMA) chains are synthesized by free radical polymerization of PEGMA and allyl methacrylate in toluene using azobisisobutyronitrile (AIBN) as thermal radical initiator. The copolymers are purified by precipitation in diethyl ether for storage and characterization by size exclusion chromatography and NMR spectroscopy. They are then coated on thiol-modified silicon substrates by spin-coating to get uniform films which thickness is decided by the polymer concentration in the solution. Here, the solvent is toluene as it solubilizes both poly(PEGMA) and 2,2′-(ethylenedioxy)diethanethiol used as cross-linkers and it allows the wetting and spreading of the polymer film on thiol-modified surface. If the spin-coating method allows the coating of polymer films with thickness ranging from a few nanometers to a few micrometers, other coating techniques such as dip-coating can also be used to get (micrometer-) thicker films.

Hydrogel films are obtained in dry state, after the cross-linking between polymer chains and the grafting on substrates owing to thiol-ene reaction which is achieved by annealing samples at 120°C under vacuum. Polymer network films are stable and durable as the chains are covalently cross-linked and surface-grafted. Actually, the dry thickness which corresponds to the polymer amount per unit area, is always the same while systematically controlled (by ellipsometry) after every use and handle such as immersion in water for the study of swelling properties. The details on the preparation of surface-attached poly(PEGMA) hydrogel films are provided in the Supporting Information. Temperature-responsive properties of poly(PEGMA) hydrogel films are shown in Figure 2. The swelling ratio, which is defined as the ratio between the swollen thickness of hydrogel films in water and the dry thickness in air, is measured at different temperatures.

Figure 2a displays temperature-responsive swelling of poly(PEGMA 300 ) hydrogel films. At low temperature, the poly(PEGMA) hydrogel film is swollen with a swelling ratio of 4.5 whereas at high temperature, hydrogel collapses with a swelling ratio of 1.5. The swellingcollapse transition shows high amplitude with three-fold change. The transition temperature is about 55°C. Moreover, the phase transition curve is the same in various aqueous solutions: pure water, phosphate saline buffer and NaCl salt solutions even at a concentration of 1 mol/L. It means that water is a good solvent for poly(PEGMA) and the quality of solvent is not altered by the presence of salt. The insensitivity of poly(PEGMA) hydrogel to the addition of salt is very important for the development of responsive hydrogels in biotechnologies.

The same insensivity to salt is observed for poly(TEGMA) hydrogel films with a transition temperature around 17°C. Note that the swelling-to-collapse transition of Figure 2c shows temperature-responsive behavior of poly(TEGMA) hydrogel films synthesized with three different dry thickness, 68 nm, 380 nm and 860 nm. The transition temperature of poly(TEGMA) hydrogel films is around 17°C and is the same for any thickness. The swelling ratio is a little different for the thinnest film. For low temperature, the value of 2.9 is found for 68 nm-thick film instead of 3.3 for the two other films. This could be explained by the effect of the surface attachment on the swelling of hydrogel films. As the grafting density is much higher than the crosslinks density, nanometric hydrogel films swell on average less than micrometric films. The same was already observed for surface-attached poly(N-isopropylacrylamide) hydrogel films. For films with dry thickness lower than 150 nm, the swelling ratio is dependent of the thickness due to the covalent grafting to the surface. For films with higher dry thickness, the swelling ratio is the same whatever the film thickness as the swelling is only determined by the crosslinks density and there is no effect of the grafting density.

The influence of the PEG chain length on the transition temperature of poly(PEGMA) hydrogel film is clearly shown in Figure 3a. The transition temperature increases with the number of oligo ethylene glycol units. It is 15°C for poly(DEGMA) with 2 units, 17°C for poly(TEGMA) with 3 units and 55°C for poly(PEGMA 300 ) with 4-5 units. This is consistent with the increasing of hydrophilic character of poly(PEGMA) with the number of PEG units.

This demonstrates that poly(PEGMA) hydrogel films can be interestingly exploited as responsive materials with a wide range of temperature transition for specific applications, in particular in biology. Temperature-dependent phase transition of mixed poly(PEGMA) hydrogel films was also investigated. The swelling ratio versus temperature of hydrogel films made with the mixture of poly(TEGMA) and poly(PEGMA 300 ) at stochiometric ratio is shown in Figure 4.

As "pure" poly(TEGMA) and poly(PEGMA 300 ) hydrogel films exhibit two very distinct transition temperatures (about 17° for poly(TEGMA) and 55°C for poly(PEGMA 300 )) and two different swelling ratios (at low temperature it is 3.3 for poly(TEGMA) and 4.5 for poly(PEGMA 300 )), this is helpful to discern the phase transition of the mixture. Two regimes can be observed. One first regime is close to the phase transition of poly(TEGMA) with a transition temperature about 17°C. The swelling ratio in the swollen state of the mixed hydrogel at low temperature is the average of the swelling ratio of the single hydrogels. The second regime is close to the phase transition of poly(PEGMA 300 ) which transition temperature is approximately 55°C. The crossover swelling ratio is between 2.0 and 2.5 within temperature range of 25°C-30°C. In this crossover zone, the hydrogel film contains poly(TEGMA) collapsed chains co-existing with poly(PEGMA 300 ) swollen chains. This could be explained by the formation of nano-aggregates in the phase separation.

Temperature-responsive properties of poly(PEGMA) copolymers hydrogel films were also shown in the same figure. Ene-functionalized poly(TEGMA-co-PEGMA 300 ) copolymer with stochiometric ratio (50/50 mol/mol) of TEGMA and PEGMA 300 were used for the formation of hydrogel films. It should be noted that the LCST-type phase transition of copolymer hydrogels is comparable to that obtained with homopolymers with two welldefined plateaus on both sides of the LCST. This behavior which is different from that observed with hydrogels made by the mixture likely indicates that there are no aggregated nano-structures in hydrogel films made by copolymers. The transition temperature found here for poly(TEGMA-co-PEGMA 300 ) copolymer hydrogel film is 37°C, which is between the transition temperature for TEGMA and PEGMA 300 homopolymer hydrogel films (17 and 55°C respectively). It turns out that the strategy of synthesis of hydrogel films with copolymers is relevant and elegant to finely adjust the transition temperature. Ene-functionalized poly(TEGMA-co-PEGMA 300 ) are synthesized by free radical polymerization of TEGMA, PEGMA 300 and ally methacrylate (AMA) in the same conditions as the synthesis of ene-functionalized poly(PEGMA 300 ). Ene-functionalized poly(DEGMA), poly(TEGMA) and poly(PEGMA 300 ) are performed by free radical polymerization using ABIN as initiator in organic solvent (toluene).

C O O C O O + C O O n m C O O n m AIBN, N 2 , 70°C
The molecular weights are almost the same for all poly(PEGMA) synthesized due to their similar chemical structures. The dispersities are 2.3-3.4 which are absolutely consistent with the free radical polymerization method used. The ratio of ene-groups is close to what we expected for further synthesis of hydrogel thin films by surface-grafting and cross-linking of ene-reactive chains through thiol-ene click reaction. Actually, it has been shown that the ratio of ene-groups has to be at least 1mol% to allow the formation of hydrogel thin films. Below 1mol% of ene-groups ratio, the formation of hydrogel films cannot occur due to low grafting density and cross-linking, the polymer chains being not sufficiently grafted and cross-linked to the chemical stability of the polymer networks on the surface.

Ene reactive groups are calculated by using 1 H NMR spectroscopy. For example, in the case of poly(PEGMA 300 ) (Figure S5), the ene-groups ratio is deduced from the ratio between the average integral of one proton (g) and 2 protons (h) of AMA and the integral of two protons of co-monomer (c). The ene-groups ratio can be calculated as follows:

1H (AMA) = [1H(g) + 2H(h)] 3 1H(co-monomer) = 2H(c) 2
Chapter 3. Lower Critical Solution Temperature phase transition of poly(PEGMA) hydrogel thin films: effect of Oligo Ethylene Glycol units 91

The ene-groups ratio is then equal to {1H(AMA) [1H(AMA)+1H(co-monomer)]} X 100

The value found for poly(PEGMA 300 ) sample is 1.2%

The same method is used to calculate the ratio of ene-groups for poly(DEGMA) and poly(TEGMA).

The ene-groups ratio of ene-functionalized poly(TEGMA-co-PEGMA 300 ) can be calculated as follows:

2H(AMA)+2H(TEGMA)+2H(PEGMA) = 35.38 2H(AMA) = 2.13 %(AMA)= {2H(AMA) ) [2H(AMA)+2H(TEGMA)+2H(PEGMA)]} X 100
The value found for poly(TEGMA-co-PEGMA 300 ) sample is 6.0%

Chapter 4. Temperature-dependent phase transition of poly(acrylamide) derivatives hydrogel thin films Introduction

Stimuli-responsive polymers also called smart polymers can respond to their environment by changing their physical and chemical properties. 1,2 Responsive properties can be obtained with various existing stimuli parameters such as pH, temperature, light, etc. [1][2][3][4][5] Stimuli-responsive hydrogel films are excellent candidates for the development of smart modulable materials with fast response. Actually, they combine the properties of hydrogel with the advantages of being biocompatible and with high water contents 6,7 and the properties of films with microscopic size which provides fast responses. [8][9][10] We develop a simple and versatile approach to synthesize reliable and reproducible surface-attached polymeric hydrogel films. This strategy allows a fine control of the chemistry and especially the crosslink density in polymer networks which determines the water content and the swelling ratio. The ability to control the crosslink density is crucial for the development of responsive hydrogel films with high amplitude change. The CLAG strategy we used consists in cross-linking and grafting onto the surface preformed reactive polymer chains using thiol-ene click chemistry. This allows the fabrication of chemically stable hydrogel films with the desired responsiveness. This process offers a better control than a "grafting from" approach thank to the utilization of preformed ene-reactive polymers. The CLAG strategy is versatile and is adaptable to various polymers and in particular polyacrylamide derivatives.

Poly(N-isopropylacrylamide) [11][12][13][14] (PNIPAM) has been extensively investigated for its LCST responsive properties in solution. We have recently reported the LCST properties of PNIPAM hydrogel films at the interface 8 . In this article, we investigate the various water solubility properties of hydrogel films based on polyacrylamide derivatives. We show that temperature-responsive properties can be obtained with both LCST and UCST phase transition. The LCST with different transition temperatures can also be found. The ene-functionalized PNIPAM was carried out by free radical polymerization of NIPAM and AMA using AIBN as thermal initiator. NIPAM (8.3492 g, 73.8 mmol), AMA (0.4986 g, 3.88 mmol) and AIBN (0.0212 g, 0.16 mmol) were mixed in 100 g of toluene.

Then the solution was deoxygenated by nitrogen bubbling for 1 h and the reactor was set in pre-heated oil bath at 70°C. The reaction was allowed to proceed at 70°C under nitrogen for 48 h. The final solution was solubilized in acetone to obtain a good fluid, then it was precipitated in diethyl ether. The obtained precipitate was washed 3 times with diethyl ether, then it was solubilized in acetone again to recover all polymers. The solution was evaporated until all acetone were removed. The ene-functionalized PNIPMAM was synthesized under the same conditions as ene-functionalized PNIPAM, except that the solvent of reaction was dimethylformamide (DMF).

Synthesis of ene-functionalized PMA in co-solvent with water.

To a two-neck flask, V50 (10.7 mg, 0.040 mmol), AMA (1.00 g, 7.92 mmol) and MA (12.8 g, 15 mmol), then 50 mL of co-solvent (1:2 formamide/water) were added. After that, the solution was deoxygenated by nitrogen bubbling under reflux conditions for 1 h. The reaction was allowed to proceed under reflux conditions at 80°C under nitrogen for 24 h. To the final solution was added 100 ml of water to decrease the polymer concentration and then it was dialyzed against pure water for four days at 80°C, and finally the polymer was recovered by freeze-drying.

Synthesis of ene-functionalized polymers in water by two steps.

The synthesis of ene-functionalized PNIPAM, PNIPMAM and PDMA was reported in our previous articles 8 . Briefly, the synthesis of P(NIPAM-co-AA) was carried out between NIPAM 95 mol% and AA 5mol% by free radical polymerization using (NH 4 ) 2 S 2 O 8 /Na 2 S 2 O 5 as redox couple initiator in water. The reaction was allowed to proceed at room temperature under nitrogen atmosphere for 24 h. The ene-functionalization of P(NIPAM-co-AA) was performed in water at room temperature by peptide coupling using N-hydroxysuccinimide (NHS)/ N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) at pH 4.5 for 2 h. Allylamine was added to the reaction and the medium was allowed to proceed in water at Chapter 4. Temperature-dependent phase transition of poly(acrylamide) derivatives hydrogel thin films 103 pH 10 for 16 h at room temperature. In order to obtain the purified polymers, the final solution was dialyzed in 0.1 M NaCl for 4 days and in pure water for 4 days. In the end, enereactive polymers were recovered by freeze-drying. The synthesis of ene-functionalized P(NIPMAM-co-AA) and ene-functionalized of P(DMA-co-AA) is the same process as the synthesis of ene-functionalized P(NIPAM-co-AA), except that the co-solvent H2O/acetonitrile (1:1) was used for the copolymerization between NIPMAM and AA instead of water due to the low solubility of NIPMAM.

Thiol-modification of substrate.

The silicon wafers were prepared firstly by freshly cleaning using UV-ozone irradiation for 15 min. Then the wafers were kept inside the sealed reactor containing 3 vol % of 3-mercaptopropyltrimethoxysilane (95%, ABCR Gelest) in anhydrous toluene under nitrogen for 3 h. In the end, the thiol-modified substrates were obtained and rinsed in a large quantity of toluene, sonicated in toluene during 1 min and dried with nitrogen flow. We used the ellipsometer to measure the thickness of thiol-modified layer on the substrates. The thiolmodified layer was obtained around 1-2 nm with a deviation lower than 10%.

Synthesis of hydrogel thin films.

The synthesis of PNIPAM hydrogel films was described in our previous articles (reference Mengxing and Benjamin's paper). PNIPAM solution was prepared in methanol/butanol (1:1) and dithioerythritol (99%, Sigma-Aldrich) was added as cross-linker.

The ratio of cross-linker was used 30 times (in excess) of ene-reactive function in polymer chains. Then the polymer solution was deposited on thiol-modified substrate and spin-coated with the final angular velocity of 3000 rpm and the spinning time of 30 s. The polymer films obtained on silicon substrates were annealed at 120°C for 24 h under vacuum to facilitate the thiol-ene click reaction which allows the crosslinking of polymer chains and the surfacegrafting. The obtained PNIPAM hydrogel films were washed and sonicated in methanol for 30 seconds. The thiol-modified substrates were then dried with nitrogen flow to remove unreacted chemicals. Before using the samples, the polymer hydrogel films are immersed in Chapter 4. Temperature-dependent phase transition of poly(acrylamide) derivatives hydrogel thin films 104 pure water for 24 h in order to ensure the stability of polymer networks. The same conditions are applied for the preparation of PNIPMAM and PDMA hydrogel films.

PMA hydrogel films were prepared by dip-coating instead of spin-coating. PMA are carried out in water and heated at 80°C and dithioerythritol crosslinkers are added. The ratio of crosslinkers and ene-reactive groups is 40 times (in excess) to facilitate the formation of polymer networks and the grating onto solid substrates. The thiol-modified substrates are immersed in the polymer solution for 1 min and then placed on a hotplate at 80°C for 5 min before the annealing at 170°C under vacuum for 21 hours. PMA hydrogels are washed in water at 80°C in a sonicator for 1 min and dried with the nitrogen flow to remove unreacted chemicals. The hydrogel films are then soaked in pure water at 80°C for 24 hours to ensure their stability before their use.

Ellipsometry.

The measure of the thickness of hydrogel films was carried out using the spectroscopic ellipsometer UVISEL (Horriba Jobin Yvon). The range of wavelength is decided from 260 to 850 nm for the measurements in air and from 320 to 850 nm in water. The refractive index used for the silicon wafer is 3.875. The measurements at silicon-air interface were analyzed using the model with two layers. The first layer is the layer containing silica and silane (n =1.46) whose thickness was measured before coating the polymer film. The polymer hydrogel is the second layer; PNIPAM, PNIPMAM or PMA which refractive index is equal to 1.48. In situ measurements (in water) of polymer hydrogel films were carried out using a temperaturecontrollable liquid cell (with a regulation within ±0.1°C). The liquid cell was equipped with thin glass walls fixed perpendicularly to the light source and the incidence angle was fixed at 60°. The thickness of hydrogel film was measured in water (h w ) and in air (h a ). The hydrogel films synthesized are covalently attached to the substrates so that the amount of polymers at the surface has to be the same in air and in water. Therefore, the refractive index in water (n w ) can be expressed as n w = (1.48-1.33)× +1.33 and h a = h w × , where is the volume fraction of polymer in water. By fitting the experimental data, we obtained the thickness in water (h w ), in air (h a ) and the refractive index of polymer in water (n w ). The fitting is considered as reliable when the same value of was provided from the two equations above.

Chapter 4. Temperature-dependent phase transition of poly(acrylamide) derivatives hydrogel thin films 105

Results and discussion

Synthesis of surface-attached hydrogel films

Surface-attached hydrogel thin films are prepared by the cross-linking and grafting (CLAG) method through thiol-ene click reaction. The preformed ene-reactive polymers and dithiol crosslinkers are coated on thiol-modified silicon wafers in order to be simultaneously cross-linked and grafted to the surface (Scheme 1). The CLAG strategy is a straightforward and versatile technique because it allows the fabrication of chemically stable polymer hydrogel films with reliability and reproducibility. The utilization of preformed enefunctionalized polymers helps to avoid using complex chemistry during polymerization at the surface. Ene-functionalized polymers are obtained by free radical polymerization, which is a simple technique since the control of polymer disparities is not necessary.

Scheme 1 Preparation of surface-attached hydrogel films. Ene-functionalized polymers are coated on thiol-modified silicon substrates with dithiol crosslinkers. The cross-linking of polymer chains and their grafting on the substrate are performed by thiol-ene click chemistry.

Spin-coating and dip-coating are the usual deposition methods we have selected. The spin-coating technique allows the spreading of polymer films on a wide range of thickness from a few nanometers to several micrometers. It is suitable for ene-functionalized polymers which are soluble in organic solvents with low surface tension (close to thiol-modified surfaces). Dip-coating was utilized for the fabrication of PMA films because they can be fewly solubilized in organic solvents. Water is used for the solubilization of PMA but as water has high surface tension, the dewetting of polymer films can be observed with spincoating while it seems to be slowed down with the dip-coating. show a phase separation at temperature above the LCST. At high temperature, the solvatation cage is disrupted with the decrease of the affinity between water and polymers due to the presence of -CH 3 groups, the interpolymeric hydrogen bonding being preferred.

If PNIPAM LCST properties in aqueous solutions are well reported in literature, the temperature-responsive properties of PNIPAM surface-attached hydrogel films are more scarcely investigated. Here, the temperature-responsive properties of PNIPAM hydrogel films using ene-functionalized PNIPAM synthesized by copolymerization with AMA monomer in organic solvent (one-step strategy). Figure 2a displays the swelling ratio of PNIPAM hydrogel films as function of temperature. The swelling ratio is the ratio between the thickness in water (swollen state) and the thickness in air (dry state). The data of swelling ratio in various aqueous conditions are provided: pure water, phosphate buffered saline (PBS) physiological solution, at pH 2 and pH 9. The swelling-collapse transition shows high amplitude with three-fold change. At low temperature, the PNIPAM hydrogel film is swollen with a swelling ratio of 4.3 whereas at high temperature, it collapses with a swelling ratio of 1.2. The same swelling-collapse transition with the same transition temperature (about 23°C) is observed whatever the aqueous conditions.

Figure 2b shows the temperature-responsiveness of PNIPAM hydrogel films using ene-reactive PNIPAM synthesized by copolymerization with AA and then, following by the functionalization with allylamine using peptide coupling (two-steps strategy). For experimental conditions in pure water, PBS and at pH 2, we found the same swelling-collapse transition. The transition shows high amplitude with four-fold change, the swelling ratio being about 5.5 below 28°C and 1.5 for temperature above 36°C. The transition temperature of PNIPAM is determined at about 32°C. PNIPAM hydrogel films in solution of pH 9 displayed the swelling ratio of 6.7 below 30°C, and the swelling ratio was 1.5 above 36°C, which is the same ratio as that in pure water, PBS and at pH 2. The higher value of the swelling ratio in the swelling state at pH 9 (6.7 instead of 5.6) might be explained by the presence of residuals acrylic acid in the hydrogel architecture. These acrylic acid units copolymerized with NIPAM were not post-modified by allylamine. At temperature below the LCST, the polymer network containing deprotonated acrylic acid units at pH 9 is more swollen or absorbs more water in comparison with the situations in pure water, PBS solution and at pH 2.

LCST properties of poly(N-isopropylmethacrylamide) PNIPMAM hydrogel films

We also investigated temperature-responsive properties of poly(Nisopropylmethacrylamide) PNIPMAM hydrogel films. The CLAG approach was used with the ene-functionalized PNIPMAM synthesized in organic solvent by one-step strategy. The swelling ratio of PNIPMAM films as function of temperature in pure water and PBS is shown in figure 4.a. It clearly demonstrates that PNIPMAM has a LCST behavior in both pure water and PBS solutions. We found that the transition temperature of PNIPMAM hydrogels is about 33 °C. The swelling ratio is 3.3 below 30°C and 1.5 at temperature above 38°C. The transition temperature of PNIPMAM hydrogel films (33°C) is surprisingly higher than that obtained with PNIPAM hydrogel films (23°C) using ene-functionalized polymers synthesized with the same one-step strategy. Actually, as PNIPMAM contains CH 3 group on the backbone which should increases its hydrophobic character, it is expected that the transition temperature of PNIPMAM is lower than that of PNIPAM. However, this result found for hydrogel films could be due to a concentration effect so that it is helpful to compare the LCST of enefunctionalized PNIPMAM and PNIPAM chains synthesized with the same one-step strategy, as shown right afterwards. NMR spectroscopy. The similar ratio was also found for PNIPAM chains which LCST is 20°C (see Figure 3). This confirms that PNIPMAM has a higher LCST value than that of PNIPAM (synthesized in the same conditions and containing the same ratio of ene-groups). Indeed, the transition temperature which is enably measured (in water using ellipsometry standard device) should be lower than 80°C. This would require a polymer fraction between 0.25 and 0.3 (or a swelling ratio about 3.5-4). Another solution is to decrease the transition temperature of PMA hydrogel films by copolymerizing PMA with hydrophilic comonomers such as acrylamide. Indeed, it is interesting to get UCST behavior with a transition temperature around 35-40°C in order to be close to the physiological temperature, which is indispensable for biology applications. The lines are guides for the eye.

Poly(dimethylacrylamide) PDMA hydrogel films

The swelling ratio of PDMA hydrogel films in pure water as function of temperature is displayed in Figure 6. The degree of swelling is found to be between 3.25 and 3.9 for the temperature increasing from 6°C to 85°C. The linear variation of the swelling ratio clearly demonstrates that there is no swelling-collapse phase transition with temperature. The little change of swelling ratio from 3.25 to 3.9 observed on the large temperature range of 80°C

Conclusions

We showed that surface-attached hydrogel films based on poly(acrylamide)

derivatives can be synthesized with the cross-linking and grafting CLAG strategy using thiolene click chemistry. It allows the fabrication of stable synthetic hydrogel thin films covalently grafted to the surface. We generalized and optimized the synthesis of ene-functionalized acrylamide derivatives polymers. We demonstrated that various temperature-responsive properties can be obtained. If PDMA hydrogel films have non-temperature-responsiveness, PNIPAM and PNIPMAM hydrogel films show LCST-type phase transition. For PNIPAM, the transition temperature found is the effective LCST. It also has been shown that the transition temperature can be controlled by the strategy used for the synthesis of enefunctionalized polymers. As expected, the presence of acrylic acid shifts the transition temperature on a higher range. Moreover, there is no change of LCST properties of PNIPAM and PNIPMAM hydrogels with salt addition such as in phosphate buffer saline physiological conditions, which is indispensable for the use of responsive hydrogel as actuators in biology applications. ) and poly(allylamine-co-allylurea) copolymers for which the ratio of comonomers controls the transition temperature. [1][2][3][4][5] For these neutral polymers, the hydrogen bonding plays a major role in the UCST behavior. The structure of water is mainly affected by the polar groups that the dissolved polymers contain in their structure. Contrariwise, the hydrophobic groups are included in a cage of water molecules. The cage is easily broken when the polymers comprise the more hydrophobic groups, or the larger hydrophobic groups, and the phase separation into polymerpoor and polymer-rich phase is observed. The process which authorizes the dissolution of UCST polymers in aqueous solutions is mainly dipole-dipole interactions and also the hydrogen bonding between the polar groups in the polymer structures. The non-polar parts of polymer are bordered by a cage of water molecule. The water molecules prefer bonding with each other because the formation of hydrogen bonds between water molecules is stranger than the interaction between the non-polar groups and water. [1][2][3][4] The other polymers are polyzwitterions such as poly(sulfobetaines) which can advantageously combine both UCST with anti-fouling properties. These polymers contain both positive and negative charges in the same monomer unit. The UCST behaviour might be explained by the presence (or not) of free counterions complexating the monomer unit. At temperature below the UCST, the phase separation might be due to the complexation of oppositely charged groups of the monomer unit without the need of any free counterion. At higher temperature, the presence of free counterions for complexation is favoured by entropy.

Chapter 5. Polyzwitterions hydrogel thin films

With this vision, it is expected that the presence of salt in physiological conditions can disturb UCST properties of poly(sulfobetaines), which is known as anti-polyelectrolyte effect.

Actually, instead of screening electrostatic charges, the addition of added salt makes the presence of free counterions more favourable and even cancels the UCST behaviour. Upon dissolution of PNAGA in water, it was proven that the two carbonyl groups present in polymer chains could serve as hydrogen bonding acceptors, while the two amide groups play a role as hydrogen bonding donors (Figure 2). Unlike to most of the other polymers with the same hydrogen bonding interaction, PNAGA prefer exceptionally to form intra-interaction (with itself) rather than inter-interaction (with water) 9 . With raising The PNAGA copolymers with alkyl (meth) acrylates are less stable to hydrolysis than with alkyl acrylamide. In phosphate-buffered saline or PBS solution, the cloud points of poly(BA-co-NAGA) decreased from 60°C to 5°C with nine measurement cycles (Figure 3a) while the PNAGA copolymers with styrene exhibit the same UCST because styrene cannot hydrolyze but the increasing of styrene affects the sharpness of transition curve and also the decreasing of UCST (Figure 3b).

Poly(uracilacrylate) or poly(6-acryloyloxylmethyl uracil) or PAU was synthesized in the first time by BRAHME et al 11 , it was reported by Aoki et al about its thermosresponsivity using the transmittance technique and its UCST was found around 20°C. 1,10 Later, PAU was reported that the UCST phase transition is not only in distilled water but also in physiological solution. 12 Like PNAGA, in aqueous solution N-H and C=O groups behave as hydrogenbonded donors and acceptors, respectively (Figure 4a). Poly(acrylamide-co-acrylonitrile) copolymers show interesting UCST properties.

Poly(acrylamide) or PAM is one of the oldest known synthetic polymer that has never been

H 2 N O n CN m b O O n CH 2 O O n ---- CH 2 N N H O O H N N H O O H ---- a
Chapter 5. Polyzwitterions hydrogel thin films 124 reported about its UCST. Since the primary amide can be considered to be similar as the PNAGA, PAM may have a possibility to be a UCST-type polymer with the reversible hydrogen bonding. Seuring et al 11 have confirmed the UCST of PAM being below 0°C by copolymerization with a hydrophobic copolymer. Poly(acrylamide-co-acrylonitrile) exhibit a UCST in water with varying amounts of acrylonitrile from 6°C to 60°C (Figure 5). This was prepared by free-radical copolymerization between acrylamide and acrylonitrile in DMSO using AIBN as the nonionic initiator. Poly(allylamine-co-allylurea) or ureido-modified polymers was synthesized by Shimada et al using modification of poly(allylamine) 13 . Poly(allylamine) hydrochloride was reacted with potassium cyanate in order to transform the amine groups into ureido groups.

The solution 0.1 wt% was investigated in electrolyte solution (150x10-3 molar NaCl, 10x10-3 molar HEPES-NaOH buffer, pH 7.5) and shown the UCST-type phase transition. The UCST of poly(allylamine-co-allylurea) with a degree of functionalization around 91% increased with the molar mass from 25°C, 32°C to 54°C for Mw equal to 5 kg/mol, 15 kg/mol and 150 kg/mol respectively (Figure 6).

Polyzwitterions

Polyzwitterions have attracted a lot of attention over the past decade. They bear the equal numbers of positive and negative charges on the same monomeric unit and are virtually neutral. Polymers containing betaine groups are ubiquitous in organisms. In hydrated state, they are biocompatible [14][15][16][17] , lubrificating [18][19][20] and antifouling. which are polyzwitterions and comprise the anti-fouling and UCST properties (Figure 7).

The betaine association consists of the electrostatic interaction between cationic and anionic charged groups, which causes the phenomenon referring to the decreasing of transition temperature by salt, differently from polyampholytes becoming more water soluble (chain expansion), a so-called antipolyelectrolyte effect [27][28][29][30][31][32] (Figure 8). Indeed, salts disturb the electrostatic association of zwitterionic betaines by decreasing the Debye length l D = (4l B .C s ) -0.5 , where l B is the Bjerrum length in pure water (0.7 nm) and C s is the concentration of salt. The Debye length is the distance where the opposite charges sense each other. The UCST phase transition of aqueous PSPP was reported at 18°C, 21°, 26°C and 33°C for the mass molar being 40, 70, 100 and 230 kg/mol respectively 36 (Figure 10). The PSPP polymer brushes synthesized using surface initiator-modified by atom-transfer radical polymerization (ATRP) was studied by Quintana et al. 37 The PSPP film with dry thickness of 130 nm showed the swelling ratio at 1.2 in water and 2.2 in NaCl 0.7 M at room temperature.

Preparation of poly(sulfobetaines) hydrogel films

The stable and durable hydrogel thin films are needed for our studied. The hydrogel films have to be chemically cross-linked and covalently grated to the substrates. Therefore, the surface-attached hydrogel films have been developed by using "grafting onto" approach which is thiol-ene click reaction. The ene-functionalized polymers are deposited on thiol modified-substrates with dithiol molecules, and the polymers films can be obtained thanks to coating technique deposition and then using heating to activate the polymer networks formation and grating polymers films to the surface. To apply our strategy for producing hydrogel films, the ene-functionalized polymers have to be preformed.

Free radical polymerization

The one-step strategy to synthesize the ene-functionalized poly(sulfobetaines) by using free radical copolymerization was chosen because there is no need to control narrow distribution of chains.

The synthesis of ene-functionalized PSPP and PSPE was already described in Chapter 2. Briefly, the copolymerization between SPE or SPP and AMA monomers in co-solvent (THF/H 2 O: ½). AIBN is not a good candidate for this reaction why we selected V50 because of its solubility parameter. SPP monomer, AMA, and V50 and then the co-solvent, were added to a round flask. The solution was deoxygenated by nitrogen bubbling with the reflux condition for 1 h. After that, the reactor was set in a pre-oil bath at 70°C, and the reaction was Chapter 5. Polyzwitterions hydrogel thin films 129 allowed to proceed with reflux condition and under a nitrogen atmosphere for 24 h. To recover the polymer yields, we diluted the medium by adding water as the same volume as co-solvent we added in the beginning and purified the polymers by dialyzing in pure water for four days. The last solution was freeze-dried and the polymers were obtained in a solid state.

The same process was utilized for the synthesis of end-functionalized PSPE.

We have tried to synthesize ene-functionalized PSPE and PSPP polysulfobetaines in water using two-steps strategy, which might be suitable as PSPE and PSPP are very little soluble in solvents except water. It consisted in copolymerizing sulfobetaines and acrylic acid in water first and then post-modificating with allylamine. Unfortunately, we did not succeed to obtain ene-functionalized PSPP and PSPE with this two-steps strategy. The substitution of acrylic acid units by carboxylic acid units with longer spacers and with more functionalities was also envisaged. However, we showed that the replacement by itaconic acid and butenoic acid units had no major improvement. And there is a risk that hydrophilic properties of polymer networks are modified with the use of carboxylic acid with longer spacer than butenoic acid. It seems that ene-functionalized polyzwitterions (PSPP, PSPE) are not able to be synthesized in water with this two-steps approach. The peptide reaction with EDC/NHS might be disturbed by the positive and negative charges of poly(sulfobetaines),. The positive and negative charges of poly(sulfobetaines) can interact with the positive form of EDC which reduces the efficiency of EDC for carbodiimide-mediated amidation. Besides, the long pendant chains of poly(sulfobetaines) avoids the access of EDC and NHS to carboxylic acid groups. For ionic polymers, the one-step strategy in water/organic solvent mixture is more suitable.

Fabrication of hydrogel films

The spin-coating was selected for PNIPAM and poly(PEGMA) hydrogel films.

However, PSPE and PSPP are soluble in a few solvents such as formamide, formic acid and water. They are not a suitable solvent for spin-coating due to their high surface tension (58.2 mN/m for formamide, 37.58 mN/m for formic acid, and 72.8 mN/m for water at 20°C). We have to find a suitable solvent to solubilize the polymers and to spread onto the thiol-modified substrates. Also, we should avoid the dewetting of the polymer solution during the coating.

Among these solvents, water was chosen because it is an ideal solvent despite its surface tension. With water, the UCST polymer films cannot be prepared by spin-coating at room temperature to prevent from the phase separation. The dip-coating is promoted because it is quite easy to heat polymer solution and silicon wafer above their UCST. After spin-coating, the silicon substrate with deposited polymers is placed on a hot plate at 90°C to evaporate water for 5 min. The polymer films are then annealed at 170°C for 21 hours under vacuum to activate the cross-linking and surface-grafting through thiol-ene reaction. After, the polymer hydrogel films are cleaned with pure water at room temperature in the ultrasonic sonicator for 10 seconds and then in hot water at 70°C for 2 min to remove all unreacted crosslinkers and polymer chains. Finally, the polymer films are soaked in pure water at room temperature for 24 hours to ensure the stability of the grafting on the solid substrate before using.

Temperature-dependent phase transition

Poly(sulfobetaine) hydrogel films are investigated in air and in water at various temperatures in order to determine their transition temperature. The study of hydrogel films in phosphate-buffered saline or PBS solution is also helpful for biology applications.

Measure of the thickness by ellipsometry

The determination of the thickness (in air and in water) was performed using spectroscopic ellipsometry. From the measure of the two ellipsometric angles, Psi and Delta, which are the changes of the amplitude ratio and the phase shift of the polarized reflected beam, the refractive index and the thickness of the hydrogel layers are deduced.

Chapter 5. Polyzwitterions hydrogel thin films 131

The samples have to be homogenous and the thickness range should be 300-1500 nm in dry state. Actually, if the hydrogel film is outside this range, the ellipsometric data (,  are not reliable for the adjustment by theoretical models, in particular for the experiments in aqueous solutions which provide weaker signals. The model used for the fit consists of two layers between the two semi-infinite media (silicon wafer and water or air). The first layer includes silica layer and thiol self-assembled monolayer. The second layer corresponds to the hydrogel thin film.

PSPP hydrogel films are firstly measured in the air to obtain the thickness at dry state.

The result is valid when the ellipsometric data (,  are superimposed or well followed the model. Moreover, the refractive index has to be ~1.5, and the thickness has to be similar to the average thickness measured with the ellipsometer equipped with mono wavelength at various angles. Figure 12 displays reliable fit of Delta and Psi spectra of PSPP hydrogel films. When the hydrogel films are immersed in an aqueous solution, they can absorb water and become swollen. The swelling ratio can be 3-fold or more which depends on the crosslinks density. For a specimen with a thickness of 500 nm, its thickness in a swollen state will be around 1500 nm or more. The data analysis becomes more difficult to fit when the swollen thickness exceeds more than 9000 nm (limit of the resolution of the ellipsometry).

The obtained data and the models are not then correctly superimposed. The measurements have to be repeated for at least 3 times (for each temperature) to check the reliability and the reproducibility of the reliable data.

PSPP hydrogel films were investigated in pure water and PBS. The ellipsometric data at 20°C, 50°C and 80°C are shown in Figure 13. The period of the oscillations is indicative of the thickness of the hydrogel film. The more the oscillations, the thicker the film is. It appears that PSPP hydrogel films are thicker or are more swollen in PBS than in pure water. The thickness also seems to increase with temperature, which is expected for UCST hydrogel films. (e) and 80°C (f). The thickness of PSPP films is 1150 nm at dry state.

Delta and Psi data can be quite properly fitted for PSPP hydrogel films in pure water.

For data in PBS, the gap between the raw data and the fit can be observed. This gap is increasing with the thickness of films. It can be explained by the simple model used for the fit. Actually, the density profile of the hydrogel layer should be soft instead of a homogeneous layer supposed in the model. But the periods of oscillations, which correspond to the thickness of the hydrogel film, were always cautiously fitted. The average thickness obtained is then reliable. The swelling ratio of PSPP hydrogel films as a function of temperature is shown in Figure 14. The swelling ratio is defined as the ratio between the thickness in water and that in air. UCST behaviour is clearly observed with a transition temperature about 50°C. The UCST properties are found both in pure water and in PBS.

In pure water, the swelling ratio is from 2 (between 20 and 40°C) to 4 (between 60 and 80°C). The swelling of hydrogel at high temperature (> UCST) is mainly ruled by the architecture and then depends strongly on the crosslinks density. In the collapsed state at low temperature (< UCST), it is more governed by polymer-water interaction than by the architecture. It should be noticed that the swelling ratio of 2 in the collapsed state is higher than for neutral hydrogel films such as poly(PEGMA) and poly(NIPAM) for which the value of 1.5 was found. It might indicate that the collapsed neutral polymer hydrogels are more hydrophobic than the collapsed polyzwitterions. If Manning condensation is considered, the monomer concentration in PSPP hydrogel films could remain higher than the salt concentration. For example, with a ratio of one third condensed counterions giving a monomer concentration of 0.2 mol/L, it is still higher than salt concentration.

135

The swelling ratio of PSPP hydrogel films in PBS solutions shows higher values than in pure water. It goes from about 5.3 (between 20 and 40°C) to 8 (between 60 and 80°C). The higher swelling behavior in PBS salt added solution is consistent with the complexation of the PSPP by free counterions. However, PSPP hydrogel films still show UCST properties even with salt addition while usually it is shown in literature that UCST behavior of polyzwitterions is cancelled with monovalent salt addition.

It could be explained by an effect of concentration. Actually, the concentration of PSPP in polymer networks is higher than the concentration of salt in PBS. PBS solution contains 0.154 mol/L of NaCl (9 g/L) and the monomer concentration in PSPP hydrogel films is 0.60 M. The monomer molar concentration can be calculated as follows:

where  is the volumetric mass of PSPP (g/mL), M is molar mass of SPP monomer (M = 292.39 g/mol) and  the volume fraction of polymer ( in hydrogel film).

This effect of concentration is consistent with the change of UCST behaviour observed in literature for polyzwitterions in diluted regime. For example, Peter A. Woodfiled and coworkers 38 They observed the decrease of UCST from 59.4°C to 7°C with the concentration of NaCl from 3.0 mM to 75.8 mM respectively. For their studies, the concentration of PSPP was equal to 16 mM, which explains the effect of salt on the UCST behavior. In our case, PSPP hydrogel films show higher monomer concentration than that of added salt in physiological conditions. The phase diagram concentration-temperature of PSPP in water is shown in Figure 15.

It was obtained using linear PSPP homopolymers with Mw equal to 199 kg/mol and 322 kg/mol. The UCST phase transition of PSPP is found at 33°C and 65°C for Mw = 199 kg/mol and 322 kg/mol respectively. The UCST for PSPP with Mw 199 kg/mol is in agreement with that observed by C. Durand-Gasselin and co-workers 36 . The increase of UCST with molecular weight is also consistent with the literature. We also found that there is no phase transition of PSPP when Mw is lower than 12 kg/mol. It should be also noticed that the variation of the transition temperature with the polymer concentration is more significant for polymers of higher molecular weight.

The transition temperature obtained for PSPP hydrogel films with the transition temperature at ~50 ±2°C. The volume fraction of polymer calculated in hydrogel films is 0.25 15. We can observe that the transition temperature of hydrogel films is higher than that of linear polymer chains, which is not surprising.

Beyond that, it should be interesting to determine the phase diagram of PSPP hydrogel films. It means that PSPP hydrogel films with various concentrations of polymer or various swelling ratios have to be prepared. It can be achieved with ene-functionalized PSPP with various ratios of ene-reactive groups or by modulating the thiol-ene reaction yield (various amounts of dithiol crosslinkers available, various temperatures for the activation of thiol-ene reaction…). However, the range of polymer concentrations is limited for surface-attached hydrogel films. We have shown that the swelling ratio reached is limited to values below 10 (or corresponding polymer volume fraction above 0.1). It is not possible to obtain hydrogel films with very high swelling ratios as they are submitted to strong mechanical stresses due to the swelling which lead to the degrafting of the hydrogel film from the solid substrate. We are also investigating UCST properties of PSPE hydrogel films. But unfortunately, we did not have enough time to obtain stable PSPE hydrogel films to determine the swelling ratio with temperature. We have just determined the phase diagram of free PSPE homopolymers in water (with Mw equal to 460 kg/mol) as reported in figure 16. The UCST UCST properties can also be achieved with poly(sulfobetaines) surface-attached hydrogel films. We showed that PSPP show a UCST behavior with a transition temperature at about 50°C in pure water and at about 55°C in phosphate buffer saline. The swelling of PSPP hydrogel films is higher in aqueous solution with added salt, in agreement with an antipolyelectrolyte effect. The UCST properties observed even in the presence of salt is probably due to a concentration effect. The concentration of added salt used (0.15 M) remains lower than the concentration of polymer in hydrogel architecture. This shows that polysulfobetaine hydrogel films can be suitably used as actuators in salt physiological conditions. These preliminary results obtained with surface-attached hydrogel films based on PSPP poly(sulfobetaines) are very encouraging and open many fundamental questions. What is the role of free counterions complexating the polyzwitterion chains on UCST behavior? What is the role of free counterions on biofouling or anti-adsorption properties of poly(sulfobetaines)?

We are also investigating are acrylamide-acrylonitrile copolymers which are known as UCST neutral polymers. They are helpfully insensitive to salt addition so that their UCST properties are the same in physiological conditions. Another advantage is the facile control of transition temperature with the variation of acrylamide-acrylonitrile ratio.

Besides, the shift of the transition temperature could be also achieved by copolymerizing with hydrophobic/hydrophilic comonomers. The transition temperature could General conclusion 148 be then ajusted for any LCST/UCST polymer hydrogel, poly(PEGMA)s, poly(acrylamide) derivatives and poly(sulfobetaines).

Beyond the use of temperature-responsive hydrogels as micro-actuators, we are developing other applications such as optical sensors and actuators. Actually, the submicrometric range of hydrogel films thickness is suitable for optics. The large amplitude change of the thickness with temperature enables high spectroscopic shifts which could be a real breakthrough for advanced optics.

Figure 2 .

 2 Figure 2. A phase diagram showing the UCST (red) and LCST (black) points for a thermoresponsive polymer. Data from reference 19.

Chapter 1 .

 1 Polymer hydrogel films: generalities and objectives of the thesis 12 For poly[poly(ethylene glycol) methyl ether methacrylate] poly(PEGMA), their solubility below their LCST are due to the hydrogen bonding between water molecule and the hydrophilic (-CH 2 -CH 2 -O) x and hydrophobic (C=O) groups in polymer structures. The LCST of poly(PEGMA) increases with the numbers of ethylene oxide units in pendant chains. It has been demonstrated that poly[di(ethylene glycol) methyl ether methacrylate] or poly(DEGMA) with 2 PEG units, poly[tri(ethylene glycol) methyl ether methacrylate] or poly(TEGMA) with 3 PEG units and poly[poly(ethylene glycol) methyl ether methacrylate]

n 1 .

 1 polysulfobetaines, for example poly([3-(methacryloylamino)-propyl]dimethyl(3-sulfopropyl)ammonium hydroxide) 19,28 or PSPP, poly([2-(methacryloyloxy)-ethyl]dimethyl-(3-Chapter Polymer hydrogel films: generalities and objectives of the thesis 13 sulfopropyl)ammonium hydroxide)

Figure 4 .

 4 Figure 4. Chemical structure of UCST polymers based on poly(sulfobetaine)s and poly(methacrylamide).

Figure 5 .

 5 Figure 5. Chemical structure of other polymers investigated with no responsive properties: PDMA and PAMS.

Figure 7 .

 7 Figure 7. Schematic of hydrogel films synthesis: ene-reactive polymer is spin-coated on thiol-modified substrate with dithiol cross-linkers. Thiol-ene click reaction allows simultaneous polymer chains crosslinking and surface attachment. Data from reference 52.

Figure 8 .

 8 Figure 8. Dry thickness of PNIPAM hydrogel films as a function of polymer concentration in the spin-coating solution for various molecular weights (66 kg/mol: red markers, 254 kg/mol: blue markers, 681 kg/mol: green markers; the solid lines being guides for the eye). Data from reference 52.

Chapter 1 . 20 Figure 9 .

 1209 Figure 9. Swelling ratio of PNIPAM hydrogel film as a function of temperature. The data are shown for polymers with various molecular weight used for the synthesis of the hydrogel films. Blue data: 66 kg/mol (the dry thickness of the film is 93 nm), red data: 405 kg/mol (80 nm), green data: 681 kg/mol (83 nm); solid lines are guides for the eye. Data from reference 52.

Figure 10 .

 10 Figure 10. Different architectures of hydrogel films; multilayers hydrogel films (left), interpenetrating networks (middle) and nanocomposite hydrogel films (right).

Figure 11 .

 11 Figure 11. Normalized flow in a microchannel integrating PNIPAM hydrogel valve as function of temperature. The results are shown here for 100 m-wide valve but the same is obtained for 50 m-wide valve. Data from reference 56

[ 3 -Figure 1

 31 Figure 1 Molecular structures of monomers investigated: oligo ethylene glycol methacrylates, acrylamide derivatives and sulfobetaine zwitterions and sulfonate-based monomers.

1 H NMR spectroscopy 1 H

 1 NMR spectroscopy was recorded on a 400 MHz Bruker spectrometer. The solvents used were deuterium oxide (D 2 O) for PSPP, PSPE, PAMPS, PMA, PDMA and PNIPAM and PNIPMAM synthesized in water (two-steps strategy). The deuterated chloroform (CDCl 3 )

Figure 2a 1 H

 1 Figure 2a 1 H NMR spectrum of ene-functionalized poly-(PEGMA300) in CDCl 3 synthesized by free radical polymerization with AMA in toluene.

Figure

  Figure 2a. displays the 1 H NMR spectrum of ene-functionalized poly(PEGMA 300 ) in CDCl 3 and the spectrum can be interpreted as follows:

Scheme 3 .

 3 Scheme 3. Synthesis of ene-functionalized polymers in water using two steps. The first step consists in free radical copolymerization reaction between NIPAM and AA in water (a) and the second step is the ene-modification of poly(NIPAM-co-AA) by amidification reaction with allylamine (b).

  -co-AA) Allylamine Ene-functionalized poly(NIPAM-co-AA) a b

Figure 3 . 1 H

 31 Figure 3. 1 H NMR spectra of ene-functionalized poly(NIPAM-co-AA) in D 2 O synthesized by free radical polymerization with acrylic acid and followed by peptide coupling reaction in H 2 O.

Figure 4 .Chapter 2 .

 42 Figure 4. demonstrates that hydrogel films synthesized by CLAG approach are obtained on a wide range of thickness. Using spin-coating, the thickness can range from a few nanometers

Figure 5 .

 5 Figure 5. ATR spectra FTIR-ATR spectra of PNIPAM films. The absorption spectra are obtained with PNIPAM films at the surface of silicon prisms (waveguides): spin-coated film containing PNIPAM (red), spin-coated film containing PNIPAM and dithioerythritol (green)

Figure 5

 5 shows FTIR-ATR spectra of PNIPAM films. Are compared four absorption spectra: spin-coated film containing only PNIPAM, spin-coated film containing PNIPAM and dithioerythritol cross-linkers, PNIPAM hydrogel film after grafting and cross-linking of chains by thiol-ene reaction by thermal activation. As silicon substrates are used as (infrared) waveguides, it is difficult to quantitatively analyze the wave number range below 1600 cm -1 due to the high absorbance of the substrates (O-H, Si-O,

  for spin-coating (typically 15 times of dierythritol molecules to ene-functionalities). It is probably not only due to the thiol-ene reaction efficiency to cross-link polymer chains in their melting state. The excess of dithiol molecules is likely necessary for the formation of oligomers to facilitate the junction between polymer chains (with the formation of S-S disulfure bonds from less stable S-H thiol functions). The oligomers formed should have flexible length to adjust to the connection between chains, which is indispensable to build the network from polymer melt.Actually, molecules with fixed length are not able to efficiently cross-link functionalized polymer chains in the melting state. An alternative chemistry to thiol-ene click reaction was tested. The esterification reaction was envisaged since it is suitable with polymers (acrylate and acrylamide families) and the surfaces (silica) used, although this chemistry is not appropriate for the fabrication of patterned hydrogels as the reaction cannot be activated by UV irradiation contrarily to thiol-ene reaction (as explained in Part 3.3). The esterification reaction is obtained with the condensation of the carboxylic acid and alcohol groups in dry conditions to inhibit the inverse hydrolysis reaction, which fits our experimental conditions (heating under vacuum). The copolymers containing acrylic acid units (which are easily synthesized by radical polymerization in one step) and the substrates functionalized by epoxy groups (as (3-glycidyloxypropyl) trimethoxysilane monolayers described in Part 2.1.2 on polymer brushes) are adequate. Epoxide bifunctional molecules (commercially available) are needed for the cross-linking of polymer chains.Poly(ethylene glycol) diglycidyl ether molecules with two different length (M n = 194 g/mol and M n = 6 000 g/mol) were tested for the synthesis of hydrogel films. Unfortunately, Chapter 2. Facile synthesis of surface-attached hydrogel thin films by cross-linking and grafting CLAG approach using click chemistry 56 only nanometric films were obtained (from spin-coated hundred nanometers-thick layers) which means that polymer monolayers are covalently grafted but chains are not (or very lowly) cross-linked. It demonstrates that cross-linker molecules with fixed length cannot adjust to the junction between functionalized chains.

Figure 6 .

 6 Figure 6. Swelling ratio of poly(PEGMA 300 ) (blue) and PSPP (green) hydrogel films as a function of temperature, the solid lines being guides for the eye. 1 and 2 correspond to hydrogel films in swollen state and collapsed state respectively.

Chapter 2 .Chapter 2 .Figure 7 .Figure S1. 1 H

 2271 Figure 7. Cloud point temperature of poly(PEGMA) (blue) and PSPP (green) in aqueous solutions as a function of polymer volume fraction. The arrows indicate the volume fraction of polymer in hydrogel films. The swelling ratio of poly(PEGMA) film of 4.5 means a volume fraction of polymer equal to 0.22. The swelling ratio of PSPP films is 4 which correspond to a volume fraction of polymer being 0.25. In the region 1, the mixture of polymer and water is miscible while the region 2 corresponds to a phase separation with one phase rich in polymer and the other phase poor in polymer.

1 HChapter 2 .Figure S2. 1 HChapter 2 . 26 ( 2 . 38 ( 2 .Chapter 2 . 2 .

 121226238222 Figure S2. 1 H NMR spectrum of ene-functionalized poly(TEGMA) in CDCl 3 synthesized by free radical polymerization with AMA.

2 . 1 . 2 Two-steps strategy 1 . 2 . 1 2 . 1 . 2 . 2 3 .

 21212121223 Figure S8.1 H NMR spectra of ene-functionalized PDMA synthesized by free radical polymerization with AA and then followed by peptide reaction with allylamine.

Chapter 3 .Figure 1 .

 31 Figure 1. Schematic of synthesis of surface-attached hydrogel film. Ene-functionalized poly(PEGMA) is spin-coated on thiol-modified silicon substrate with 2,2′-(ethylenedioxy)diethanethiol as crosslinkers. The oligo ethylene glycol units number, p, under investigation is 2 (for DEGMA), 3 (for TEGMA) and 4-5 (for PEGMA 300 with Mn = 300 g/mol). Thiol-ene click reaction allows simultaneously the chemical cross-linking of polymer chains and the covalent attachment to the surface.

Figure 2 .Chapter 3 .

 23 Figure 2. (a) and (b) Swelling ratio of poly (PEGMA 300 ) and poly(TEGMA) hydrogel films as a function of temperature in various aqueous solutions: pure water (blue), NaCl solution at 0.15 mol/L (red), NaCl solution at 1 mol/L (violet) and phosphate-buffered saline (green).The cartoons show hydrogel films in swollen and collapsed states. Dry thickness, 350 nm for poly(PEGMA 300 ) and 380 nm for poly(TEGMA). (c) Swelling ratio of poly(TEGMA)

Figure 3 .Chapter 3 .( 7 .

 337 Figure 3. (a) Swelling ratio of poly(DEGMA) (red), poly(TEGMA) (green) and poly(PEGMA 300 ) (blue) hydrogel films as a function of temperature, the solid lines being guides for the eye. The swelling ratio of poly(DEGMA) and poly(TEGMA) is normalized in relation to poly(PEGMA 300 ). (b) Cloud point temperature of poly(DEGMA) (red), poly(TEGMA) (green) and poly(PEGMA 300 ) (blue) solutions as a function of polymer volume fraction. The arrows indicate the volume fraction of polymer in poly(PEGMA) hydrogel films. The swelling ratio of poly(PEGMA 300 ) hydrogel film of 4.5 means a volume fraction of polymer equal to 0.22. For poly(TEGMA) and poly(DEGMA) hydrogel films, the swelling ratio is 3.3 and 2 respectively, which give 0.3 and 0.5 as volume fraction. For figure (a), 1 and 2 correspond to hydrogel films in swollen state and collapsed state respectively. For figure (b), polymer/water mixture is miscible in 1 and 2 corresponds to a phase separation with one phase rich in polymer and the other phase poor in polymer.

Figure 4 .

 4 Figure 4. Swelling ratio of mixed poly(TEGMA)/poly(PEGMA 300 ) hydrogel film (violet) and poly(TEGMA-co-PEGMA 300 ) (red). The mixed poly-(TEGMA)/poly(PEGMA 300 ) hydrogel film is made by a mixture of poly(TEGMA) and poly(PEGMA 300 ) with stochiometric ratio (50/50 mol/mol). Poly(TEGMA-co-PEGMA 300 ) is produced by using copolymer TEGMA and PEGMA 300 synthesized with stochiometric ratio (50/50 mol/mol). The temperaturedependent phase transition of "pure" poly(TEGMA) (green) and poly(PEGMA 300 ) (blue) hydrogel films is also provided for comparison.

Chapter 3 .

 3 Lower Critical Solution Temperature phase transition of poly(PEGMA) hydrogel thin films: effect of Oligo Ethylene Glycol units 85

Chapter 3 . 1 . 1 . 1 Figure S1 .

 3111S1 Figure S1. General strategy of the synthesis of ene-functionalized poly(PEGMA) polymers by free radical polymerization in organic solvent. Poly(DEGMA) contains two PEG units (p = 2)with R 1 corresponding to CH 3 . For poly(TEGMA), p =3 and R 1 = CH 3 and for poly(PEGMA 300 ) p = 4-5 (with Mn = 300 g/mol) and R 1 = CH 2 -CH 3 .

Chapter 3 . 2 . 45 (Chapter 3 . 2 . 2 Figure S4. 1 HChapter 3 . 2 . 3 Figure S5. 1 HChapter 3 . 2 . 4 Figure S6. 1 HChapter 3 .

 32453221323132413 Figure S3. 1 H NMR spectrum of ene-functionalized poly(DEGMA) in DMSO-D 6 synthesized by free radical polymerization with allyl methacrylate (AMA).

Chapter 4 .

 4 Temperature-dependent phase transition of poly(acrylamide) derivatives hydrogel thin (NIPAM, 97%), N-isopropylmethacrylamide (NIPMAM, 97%), methacrylamide (MA, 98%), N,N-dimethlyacrylamide (DMA, 99%), and acrylic acid (AA, 99%) monomers, 2,2′-azobis(2-methylpropionitrile) (AIBN, 98%) and 2,2′-azobis(2methylpropionamidine) dihydrochloride (V50, 97%) initiators and allylamine (98%) were purchased form Sigma and used without further purification. Allyl methacrylate (AMA, 97%) monomer was purchased from Alfa Aesar and purified prior to use by passing through the basic alumina column to remove inhibitors.

Figure 1 . 2 AllylamineChapter 4 .

 124 Figure 1. Molecular structures of investigated acrylamide derivative monomers (top) and comonomer allowing the ene-functionalization for one-step (AMA) and two-steps (AA and allylamine) strategies (bottom).

Chapter 4 .

 4 Temperature-dependent phase transition of poly(acrylamide) derivatives hydrogel thin films 106LCST properties of poly(N-isopropylacrylamide) PNIPAM hydrogel filmsPNIPAM is the most well-known temperature-responsive polymer with a Lower Critical Solution Temperature (LCST) around 32°C. PNIPAM LCST properties have been extensively investigated with in particular a recent review.1,[8][9][10] PNIPAM is water-soluble below the LCST due to the solvation of hydrophilic (-NH) and (C=O) functions in polymer chains favored by the formation of hydrogen bonds with water molecules. In contrast, they

Chapter 4 . 107 Figure 2 .

 41072 Figure 2. Swelling ratio of PNIPAM hydrogel films as a function of temperature. Enefunctionalized PNIPAM are synthesized by one-step (a) and by two-steps in water (b). The phase transition is investigated in various aqueous solutions: pure water (blue), phosphate saline buffer (green), at pH 2 (red) and pH 9 (violet). The cartoons show hydrogel films in swollen and collapsed states, the lines are guides for the eye.

Chapter 4 . 108 Figure 3 .Chapter 4 .

 410834 Figure 3. Transition temperature of ene-functionalized PNIPAM synthesized by two-steps strategy (containing AA in polymer chains, blue) and ene-functionalized PNIPAM synthesized by one-step strategy (containing AMA, violet) as a function of volume fraction of polymer (ɸ) studied in pure water. Polymer/water mixture is miscible in 1 and 2 corresponds to a phase separation with one phase rich in polymer and the other phase poor in polymer.

Figure 4 .

 4 Figure 4. a. Swelling ratio of PNIPMAM hydrogel films as a function of temperature. Enefunctionalized PNIPMAM are synthesized in one-step with AMA in toluene. The phase transition is investigated in various aqueous solutions: pure water (blue), phosphate saline buffer (green). The cartoons show hydrogel films in swollen and collapsed states, the lines are guides for the eye. b. Transition temperature of ene-functionalized PNIPMAM synthesized by one-step strategy (containing AMA in polymer chains as a function of volume fraction of polymer (ɸ) studied in pure water. Polymer/water mixture is miscible in 1 and 2 and corresponds to a phase separation with one phase rich in polymer and the other phase poor in polymer.

Chapter 4 .Chapter 4 .

 44 Figure 5. a. Swelling ratio of PMA hydrogel films as a function of temperature in pure water. b. Transition temperature of PMA as a function of polymer volume fraction (ɸ).Polymer/water mixture is miscible in 1 and 2 and corresponds to a phase separation with one phase rich in polymer and the other phase poor in polymer, the lines are guides for the eye.

Figure 6 .

 6 Figure 6. Swelling ratio of PDMA hydrogel film as a function of temperature in pure water.

Chapter 4 .

 4 Temperature-dependent phase transition of poly(acrylamide) derivatives hydrogel thin films 113 could be explained by the slight dependence of  Flory parameter with temperature. However, as already mentioned, the incorporation of hydrophilic dimethylacrylamide units in PNIPAM, PNIPMAM and PMA poly(acrylamide) derivatives hydrogel films should be relevant to control UCST and LCST properties with fine adjustment of transition temperature. Chapter 4. Temperature-dependent phase transition of poly(acrylamide) derivatives hydrogel thin films 114

3. 3

 3 Comparison between interface/bulk ......................................................................... 3.4 Outlook ..................................................................................................................... 4. References ................................................................................................................... Chapter 5. Polyzwitterions hydrogel thin films 120 Some polymers can have a phase separation in water with decreasing temperature. The phase separation occurs below their UCST. Only a few synthetic polymers with UCST properties are known, for example, poly(N-acryloylglycinamide) (PNAGA) and polyzwitterions such as poly(sulfobetaines). The other examples are poly(acrylamide-coacrylonitrile

  acryloylglycinamide) or PNAGA is one of the most studied UCST polymer which was first time synthesized by Hass and Schuler in 19645 . They reported that the polymer exhibited UCST behavior in aqueous solution like gelatine. The transition temperature of PNAGA depends on molecular weight and concentration like many UCSTtype polymers. In 2010, Sering et al6 have shown that PNAGA has a temperature-responsive property because of the hydrogen bonding between the polymer side groups. They showed the cloud points from 0.03 to 5.4 wt% and gel melting points until 14 wt% (Figure1). The cloud point is shifted to lower temperature when the amount of sodium thiocyanate is increased, which disturbs hydrogen bonds[6][7][8] .

Figure 1 .

 1 Figure 1. UCST and gel melting temperature of PNAGA as a function of the concentration in deionized water. The heating rate was 1 K/min. Data from reference 6.

Chapter 5 .

 5 Polyzwitterions hydrogel thin films 122 temperature, the polymer/polymer hydrogen bonds are broken and PNAGA displays a UCST behavior.

Figure 2 .Figure 3 a

 23 Figure 2. Schematic representation of hydrogen bonds between PNAGA side chains, NH and C=O being donors and acceptors respectively.

Figure 4 a

 4 Figure 4 a. Schematic representation of hydrogen bonds between poly(uracilacrylate) and b. the chemical structure of poly(acrylamide-co-acrylonitrile).

Figure 5 .

 5 Figure 5. Turbidity cooling curves of poly(acrylamide-co-acrylonitrile)s with different copolymer compositions. The numbers state the acrylonitrile content in mol % (error 1.0-1.4 mol %). Data from reference 10.

Figure 6 .

 6 Figure 6. a The chemical structure of poly(allylamine-co-allylurea). b Transmittance curves of PAUs (1 mg/mL) in 10 mM HEPES-NaOH (pH 7.5) containing 150 mM NaCl. PAUs dissolved in pure water were diluted into the cells using the buffer solutions. Transmittances at 500 nm of the polymer solution were measured at scanning rate of 1 °C/min from 70°C to 5°C. Data from reference 13.

Chapter 5 .Figure 7

 57 Figure 7. a. Schematic representation of electrostatic bonds between zwitterionic groups in poly[[3-(methacryloylamino)propyl] dimethyl (3-sulfopropyl) ammonium hydroxide] (PSPP) and b. poly[[2-(methacryloyloxy) ethyl] dimethyl-(3-sulfopropyl) ammonium hydroxide] (PSPE).

Figure 8 .-.

 8 Figure 8. a Schematic of the polyelectrolyte effect and b the anti-polyelectrolyte effect. Data from reference 32.

Figure 9 .Figure 10 .

 910 Figure 9. Effect of salt concentration on PSPE (with Mw equal to 410 kg/mol) on phase behavior, ; H2O, ∆; 0.05% NaCl (0.085M), ; 0.1% NaCl (0.17M), ; 0.3% NaCl (0.51M). Data from reference 34.

Figure 11 .

 11 Figure 11. Molecular structure of monomers: acrylic acid, butenoic acid and itaconic acid.

Chapter 5 .

 5 Polyzwitterions hydrogel thin films 130

Figure 12 .

 12 Figure 12. Delta and psi (ellipsometric data) of PSPP hydrogels as a function of wavelength investigated at dry state with thickness of 1150 nm.

Chapter 5 .Figure 13

 513 Figure 13 Delta and psi (ellipsometric data) of PSPP hydrogels as a function of wavelength investigated in H 2 O at 20°C (a), 50°C (b) and 80°C (c) and PBS solution at 20°C (d), 50°C

Figure 14 .

 14 Figure 14. Swelling ratio of PSPP hydrogel films as a function of temperature in pure water (bleu) and in phosphate-buffered saline or PBS (green). The cartoons show hydrogel films in swollen and collapsed states. The thickness at dry state is 1170 nm and the lines are guides for the eye.

Chapter 5 .

 5 Polyzwitterions hydrogel thin films

  have synthesized poly{[[3-(methacry-loylamino)propyl] dimethyl(3-sulfopropyl) ammonium hydroxide]-co-benzylacrylamide) or poly(SPP-co-Benz) with the obtained ratio of 0.47 mol% SPP and 0.53 mol% Benz with Mw equal to 27.2 kg/mol and the copolymer exhibits UCST at 63.6°C for the polymer concentration of 10g/L ( 0.034 mol/L or 1 wt%).

Chapter 5 . 3 . 3 Figure 15 .

 53315 Figure 15. Cloud point temperature of homopolymer PSPP with Mw = 199 kg/mol and Ip = 1.7 (violet) and with Mw = 322 kg/mol and Ip = 2.0 (blue). Polymer/water mixture is miscible in 1 and 2 corresponds to a phase separation with one phase rich in polymer and the other phase poor in polymer. The arrow indicates the volume fraction of PSPP hydrogels films and lines are guides for the eye. The picture of simple showing 1 and 2 upon heating and cooling are presented on the right.

Chapter 5 .

 5 Polyzwitterions hydrogel thin films 137 as indicated by the arrow in Figure

Figure 16 .

 16 Figure 16. Cloud point temperature homopolymer PSPE with Mw = 460 kg/mol and Ip = 2.8 as a function of volume fraction of polymer. Polymer/water mixture is miscible in 1 and 2 corresponds to a phase separation with one phase rich in polymer and the other phase poor in polymer. The line is guide for the eye.

Chapter 5 .3. 4 Outlook

 54 Polyzwitterions hydrogel thin films 138 of PSPE is 90°C while the UCST of PSPP with 322 kg/mol is 65°C. The difference of UCST found here can be explained by the molecular weight as the chemical structure of PSPE and PSPP are quite similar.These preliminary results obtained with surface-attached hydrogel films based on PSPP poly(sulfobetaine) are very encouraging. The thickness of PSPP hydrogel films is measured in aqueous solutions at various temperatures. We show that PSPP hydrogel films have UCST properties with a transition temperature about 55°C in pure water. UCST behavior is also observed in phosphate buffer conditions, which is promising for the exploitation of PSPP hydrogel actuators for biology applications in microfluidics. The conservation of UCST properties of polyzwitterion hydrogel films in phosphate buffer solution could be explained by a concentration effect. The concentration of monovalent added salts is weaker than the polymer concentration in hydrogel films. This work is continuing to understand the role of free counterions complexating the polyzwitterion chains. Systematic studies are planned by using surface-attached hydrogel films with various swelling ratios, NaCl and KCl added salts at various concentrations for example. The swelling behavior of hydrogel films will be investigated at different temperatures using ellipsometry. But we are also interested in quantitative characterizations of free counterions by ATR-IR spectroscopy and neutron reflectivity. Sulfonate and deuterated ammonium counterions (instead of NaCl and KCl) in poly(sulfobetaine) hydrogel films can be detected and quantified by ATR-IR and neutron reflectivity. Indeed, the architecture of surface-attached hydrogel films is suitable to determine the role of free counterions as it concentrates enough monomers and counterions to be detected by infrared and neutron techniques.steps strategy is applied for the synthesis of polymers in water. The desired monomer is copolymerized with acrylic acid (AA) which is post-modified by allylamine.Poly(PEGMA) hydrogel films with various numbers of PEG units on the pendant chains were investigated: poly[di(ethylene glycol)methyl ether methacrylate] poly(DEGMA) with 2 PEG units, poly[tri(ethylene glycol)methyl ether methacrylate] poly(TEGMA) with 3 PEG units and poly[poly(ethylene glycol)methyl ether methacrylate] with Mw equal to 300 g/mol or poly(PEGMA 300 ). Poly(PEGMA) hydrogel films show LCST behavior in pure water with transition temperatures in the range between 15°C and 60°C . The transition temperature increases with the number of PEG units, which is consistent with the increase of hydrophilic character of PEG units. The effect of added salt (phosphate buffer saline PBS and 0.15M and 1M NaCl) on the LCST properties of hydrogel films is also investigated, which is important for the use of responsive hydrogels for biology. The same LCST behavior is observed for poly(PEGMA) hydrogel films with the same transition temperature and same swelling ratio, indicating that poly(PEGMA)-water interaction is not modified by the presence of salt. As a result, various LCST can be finely adjusted with poly(PEGMA) hydrogels by changing the PEG units in pendant chains or by copolymerizing two poly(PEGMA) with different PEG units.We have showed how to manipulate poly(acrylamide) derivatives to target either LCST or UCST properties. Poly(N-Isopropylacrylamide) PNIPAM and poly(N-Isopropylmethacrylamide) PNIPMAM hydrogel films display LCST-type behavior in aqueous solutions. We showed that the transition temperature could depend on the way enefunctionalized PNIPAM was synthesized. The one-step strategy gives PNIPAM gels with LCST at ~23°C, while with two-steps strategy, the PNIPAM films show LCST at ~32°C. The difference of LCST may come from different copolymers compositions. In the case of onestep strategy, there are PNIPAM and AMA (hydrophobic group) and for two-steps strategy there are PNIPAM, AA (hydrophilic group) and modified AA (hydrophobic group). The hydrophobic groups probably decrease the LCST while the hydrophilic functions should increase the LCST. PNIPMAM hydrogel films (which ene-functionalized polymers were synthesized in one-step copolymerization) exhibit a LCST at ~33°C in aqueous solutions. The transition temperature of PNIPMAM hydrogel films (33°C) is surprisingly higher than that of General conclusion 147 PNIPAM (23°C) while the more hydrophobic character of PNIPMAM polymer is expected to increase the LCST.We have also succeeded in synthesizing UCST-type hydrogel films based on acrylymide polymers with poly(methacrylamide) PMA. Ene-functionalized PMA was synthesized in one-step copolymerization with AMA in co-solvent with water. PMA phase diagram of free homopolymers (with Mw equal to 228 kg/mol) in water displays the UCST at 90°C. This high UCST explained the difficulty of observing the phase transition of PMA hydrogel films (the measure of the thickness by ellipsometry is very complex for temperatures above 85°C). Actually, it could be interesting to decrease the UCST of PMA hydrogel films around 35-40°C to be close to human body temperature for biology applications. This could be achieved by copolymerizing methacrylamide with another acrylamide hydrophilic comonomer.

Table of Contents Chapter 1. Polymer hydrogel films: generalities and objectives of the thesis

  .................... 1. Polymer hydrogels ........................................................................................................... 2. Stimuli-responsive polymers ......................................................................................... 3. Polymer hydrogels at interface ...................................................................................... 4. Context and objectives of the dissertation..................................................................... 4.1 Context of the dissertation ..........................................................................................

4.2 Objectives of the dissertation ..................................................................................... References ............................................................................................................................

Table

new actuation technology, where responsive hydrogel embedded inside

	). A powerful
	consequence is the facile fabrication of micro-patterned hydrogel thin films by
	photolithography, which is indispensable for the preparation of hydrogel actuators in
	microfluidic devices. The collaboration with Patrick Tabeling (MMN-IPGG) enables the
	invention of a a
	microfluidic channel can operate as a valve (or functional gate) which reversibly opens and
	closes the channel for fluid flows.
	In our approach,

hydrogel microactuators are synthesized ex situ, i

	.e. grafting and
	patterning responsive hydrogel films onto substrates before the system is sealed to
	microstructured material. The ex situ technology is much better controlled than the in situ
	one: the fabrication of the surface-attached patterned hydrogels can be checked step by step
	and the optimization of the hydrogel film properties can be achieved efficiently. The ex situ
	strategy allows a rigorous fabrication process, reproducible and controllable, two clearly
	needed conditions for developing applications of the technology. The power of our ex situ
	technology is the

combination of both top-down and bottom-up approaches. The

	Chapter 1. Polymer hydrogel films: generalities and objectives of the thesis
	approach enables molecules and macromolecules assembling with the fine control of
	chemical properties.
	top-down
	approach with photolithography is usually applied in the microfabrication. The bottom-up
	22

Chapter 2. Facile synthesis of surface- attached hydrogel thin films by cross- linking and grafting CLAG approach using click chemistry

	54) Chollet, B.; Li, M.; Martwong, E.; Bresson, B.; Fretigny, C.; Tabeling, P.; Tran, Y.
	Multiscale Surface-Attached Hydrogel Thin Films with Tailored Architecture. ACS Appl.
	Mater. Interfaces 2016, 8 (18), 11729-11738.
	(55) Chollet, B.; D'Eramo, L.; Martwong, E.; Li, M.; Macron, J.; Mai, T. Q.; Tabeling, P.;
	Tran, Y. Tailoring Patterns of Surface-Attached Multiresponsive Polymer Networks. ACS
	Appl. Mater. Interfaces 2016, 8 (37), 24870-24879.
	(56) Chollet, B. Hydrogels greffés stimulables comme actionneurs microfluidiques. PhD
	thesis, 2015, Université Pierre Marie Curie, Paris

Table of Contents Chapter 2. Facile synthesis of surface-attached hydrogel thin films by cross-linking and grafting CLAG approach using click chemistry

  ................................................................. Introduction .......................................................................................................................... Experimental Section ........................................................................................................... Results and discussion .......................................................................................................... Strategy of surface-attached hydrogel films: cross-linking and grafting CLAG ............. Synthesis of ene-functionalized polymers in organic solvent .......................................... Synthesis of ene-functionalized polymers in water .......................................................... Synthesis of hydrogel thin films....................................................................................... Conclusions .......................................................................................................................... References ............................................................................................................................

	Supporting information ........................................................................................................

Table 1 .

 1 have shown that the swelling amplitude is not so that different for reactive groups ratio equal to 5mol% or 10mol%, the variation being not significant above 5mol%. It results that the ene-groups ratios targeted are around 2mol%, for which the hydrogel films obtained have responsive properties with quite high amplitude deformation. Characteristics of ene-functionalized polymers: polymerization conditions with the thermal initiator and the solvent used, molecular weight Mw and dispersity Đ and the ratio of ene-groups expected and obtained.

	Polymer	Initiator Solvent	Mw (kg/mol)	Đ	Ene groups Expected/ Obtained
	Poly(DEGMA)	AIBN	Toluene	933	2.3	10 / 7.0 ± 1
	Poly(TEGMA)	AIBN	Toluene	1007	3.4	5 / 6.2 ± 1
	Poly(PEGMA 300 )	AIBN	Toluene	983	2.8	2 / 1.2 ± 1
	PNIPAM	AIBN	Toluene	869	2.5	5 / 5.5 ± 1
	PNIPMAM	AIBN	DMF	72	2.0	5 / 5.3 ± 1
	PSPP	V50	THF/H 2 O	510	3.0	5 / 1.7 ± 1
	PSPE	V50	THF/H 2 O	484	3.7	5 / 2.2 ± 1
	PAMPS	V50	THF/H 2 O	101	2.0	5 / 1.5 ± 1
	PMA	V50	FM/H 2 O	29	1.4	5 / 1.9 ± 1
	PNIPAM	Redox	H 2 O	337	2.2	5 / 2.7 ± 1
	PDMA	Redox	H 2 O	320	2.2	5 / 1.9 ± 1
	PNIPMAM	Redox	H 2 O/ACN	45	1.4	5 / 1.9 ± 1

Table of Contents Chapter 5. Polyzwitterions hydrogel thin films

  ................................................................. 1. UCST polymers: a brief state of the art. ...................................................................... 1.1. Neutral polymers .................................................................................................. 1.2. Polyzwitterions .................................................................................................... 2. Preparation of poly(sulfobetaines) hydrogel films ...................................................... 2.1. Free radical polymerization ................................................................................. 2.2. Fabrication of hydrogel films ............................................................................... 3. Temperature-dependent phase transition .................................................................... 3.1 Measure of the thickness by ellipsometry ................................................................ 3.2 Swelling ratio of hydrogel films ...............................................................................

Remerciements

Table of Contents

Abstract

Surface-attached hydrogel films with well-controlled chemistry are a very promising alternative to polymer brushes and layer-by-layer assemblies as stable and durable polymer coatings. Hydrogel films can also respond to stimuli with high volume change and very short time. We report the temperature-responsive properties of poly(PEGMA) hydrogel thin films which combine the advantages of biocompatibility with PEG side chains and the adjustment of the transition temperature with the oligo ethylene glycol units. We use a simple, versatile and well-controlled grafting onto approach through thiol-ene click reaction to synthesize surface-attached poly(PEGMA) hydrogel films. We show that the transition temperature ranges from 15°C and 20°C for poly(DEGMA) and poly(TEGMA) to 55°C for poly(PEGMA300) hydrogel films. This could be also finely adjusted by using copolymers or mixing homopolymers. Moreover, the LCST properties, swelling-to-collapse amplitude and transition temperature, is insensitive to salt, such as in phosphate saline buffer, which is important for biosciences.

Chapter 3. Lower Critical Solution Temperature phase transition of poly(PEGMA) hydrogel thin films: effect of Oligo Ethylene Glycol units

General conclusion

General conclusion 145

Surface-attached hydrogel films with LCST/UCST (Lower/Upper Critical Solution Temperature) temperature-responsive properties have been successfully designed. They are chemical polymer networks covalently attached to solid substrates. We have developed a versatile and straightforward approach to prepare reliable and reproducible hydrogel thin films. Our approach consists in coating ene-functionalized polymers and dithiol molecule crosslinkers on thiol-modified substrates, the thiol-ene click reaction allowing simultaneous cross-linking between polymer chains and grafting on the surface. Using this CLAG (Cross-Linking And Grafting) strategy, chemically stable hydrogel films can be synthesized with a wide range of thickness and with the desired temperature-responsiveness. The LCST/UCST properties are determined by measuring the thickness of surface-attached hydrogel films as function of temperature using ellipsometry.

The conditions of the synthesis of ene-reactive polymers are essential in the CLAG approach with thiol-ene click chemistry. Actually, the amplitude of responsiveness and the swelling ratio of hydrogel films is governed by the crosslinks density. The less the hydrogel is crosslinked the more it swells. As the crosslinks density of hydrogel films is determined by the thiol-ene reaction, it depends on the ratio of ene-reactive groups, the annealing time and temperature. We showed that the amount of ene-function less than 1 mol% in polymer chains cannot allow the fabrication of hydrogel films. Lowly crosslinked hydrogels are fragile and cannot support high mechanical stress due to swelling. Besides, the ratio of ene-groups more than 10 mol% in polymers chains provide hydrogel films with very low swelling ratio (less than 1.5). Highly crosslinked hydrogels are not suitable for the determination of transition temperature. Beyond, they cannot be interestingly exploited as actuators with high amplitude responsiveness. Actually, we showed that the ratio of ene-functionalized polymers should be between 2 mol% and 5 mol%.

The synthesis of ene-reactive polymers was generalized and optimized. Three polymer families were investigated using either one-step or two-steps strategy: poly[poly(ethylene glycol) methyl ether methacrylate] or poly(PEGMA), poly(acrylamide) derivatives and poly(zwitterions). On the one hand, the one-step strategy consists of the synthesis of the enefunctionalized polymer using free radical copolymerization of the desired monomer with allyl methacrylate (AMA) in organic solvent or co-solvent with water. On the other hand, two-