Sébastien Gouin

Dr Arlette Darfeuille-Michaud

Dr Nicolas Barnich

Dr Béatrice Burdin

Dr Sabine Favre-Bonté

Isabella Polo

Mallaouia Bengoua

Pierre Alcouffe

Marion Colella

Dr Agnès Crepet

Fernande Boisson

Cécile Chamignon

Annick Waton

Dr Patrick Goetinck

Dr Senbin Chen

Dr Jing Chen

Dr Huagui Zhang

Wenyong Zhang

Biao Zhang

Dr Jing Yang

Dr Marco Delgado

Ali Ghadban

Olivier Gribelin

Ludovic Sallet

Dr François Ganachaud

Dr Michael Malkoch

Dr Julien Nicolas

Hong Tian

Hesheng Yan

This PhD work focuses on the preparation of glycopolymers bearing pendent heptyl mannose groups and the evaluation of the capability of such multivalent ligands to inhibit bacterial adhesion to human cells.

Aiming at understanding the impact of various structural parameters on glycopolymer/ E coli interactions (AIEC LF82 et UTI 89 strains of E. coli), a series of linear and star-shaped glycopolymers with tunable molecular weight, mannoside density and microstructure (block copolymers, gradient copolymers, random copolymers) has been constructed. The association of the glycopolymers with FimH adhesin, a lectin which possesses a mannose-specific receptor site and is responsible for recognition and binding to host cells, was first confirmed by static and dynamic light scattering experiments. The propensity of the glycopolymers to prevent attachment of E. coli (AIEC LF82 involved in Crohn's disease) to intestinal epithelial cells (T84 cells) was further investigated through adhesion assays. It was shown that under in vitro conditions, the addition of 10 nM or 100 nM of glycopolymer on a mannose unit basis (in pre-incubation and post-incubation respectively) decreases by half the bacterial adhesion to intestinal epithelial cells. The anti-adhesive effect of these multivalent ligands was further confirmed in ex vivo conditions for colonic loops of transgenic CEABAC10 mice (Crohn's disease model mouse).

Finally we took advantage of the nanoprecipitation process to generate glyconanocapsules with oily core. The employed strategy allowed for preparing well-defined nanocapsules bearing groups of interest (tags, ligands) or metal particles within the shell and loaded with active molecules in the core in one step.

1

Résumé

Ce travail de thèse est consacré à la préparation de glycopolymères porteurs de groupements pendants mannoside d'heptyle et à l'évaluation de la capacité de ces ligands multivalents à inhiber la fixation bactérienne sur les cellules humaines.

Nous avons synthétisé, par polymérisation radicalaire contrôlée, une série de glycopolymères linéaires ou en étoile présentant des masses molaires, des densités en mannoside et des microstructures modulables dans le but d'évaluer l'influence de ces paramètres sur les processus d'interactions avec diverses souches de bactéries E coli (AIEC LF82 et UTI 89). Nous avons tout d'abord mis en évidence par diffusion dynamique et statique de la lumière, la formation d'agrégats entre ces glycopolymères et FimH, la lectine à l'origine de la fixation de souches de bactéries E coli, traduisant des interactions fortes entre les motifs mannosides et les sites de reconnaissance au mannose de la lectine. Nous avons ensuite évalué l'aptitude de ces ligands multivalents à bloquer l'adhésion bactérienne d'AIEC LF82 (impliquée dans la maladie de Crohn) sur des cellules épithéliales intestinales T84. Il a été démontré en conditions in vitro que l'ajout de 10 nM ou 100 nM d'unités mannoside (respectivement en pré-ou post-incubation) réduit de moitié l'adhésion des bactéries sur les cellules épithéliales. L'effet anti-adhésif de ces glycopolymères a été confirmé par des tests ex vivo réalisés sur des intestins isolés de souris transgéniques CEABAC10.

Enfin, nous avons exploité la technique de nanoprécipitation pour l'élaboration de nanocapsules de glycopolymères à coeur huileux. Le procédé développé permet la synthèse de nanocapsules de dimensions contrôlées, porteuses de groupements fonctionnels (fluorophores, ligands) ou de particules métalliques et l'encapsulation de molécules actives à coeur en une seule étape.

Abbreviations

General introduction

In the history of humanity, bacteria have been responsible for a wide number of diseases and are considered as the cause of numerous deaths. Over the last century, water purification, vaccination and the systematic use of antibiotics have considerably reduced the risk of these illnesses. Nowadays, the traditional antibiotic treatments become less and less efficient since bacteria have acquired resistance. This problem is so important and global that the World Health Organization (WHO) declared in 2014 that "this serious threat is no longer a prediction for the future, it is happening right now in every region of the world and has the potential to affect anyone, of any age, in any country. Antibiotic resistance-when bacteria change so antibiotics no longer work in people who need them to treat infections-is now a major threat to public health." 1 Because of this emergency, several lines of research are currently explored. One of them consists in blocking the adhesion between the bacterial pathogens and the host cells or the tissues which leads to the infection and then to the disease. Adhesion is governed by multivalent interactions between bacterial surface lectins and carbohydrates groups on the cells surface. For instance Escherichia coli (E. coli) bacteria, which are associated with numerous infections, express adhesive hair-like organelles with mannose-specific receptor to promote adhesion and infection of tissues. Monosaccharides such as mannose derivatives have been widely described as antagonists that competitively interact with bacterial lectins to inhibit or disrupt the bacterial adhesion towards host cells. 2 Their use has gradually emerged as valuable alternative to antibiotics. To enhance their efficiency, carbohydrate moieties can also be tethered along a polymer backbone resulting in functional glycopolymer. 3 In such a way, the activity of a glycopolymer will depend on its architecture, the degree of polymerization, the density of carbohydrate groups… Therefore, the generation of glycopolymer with well-defined architecture is of crucial importance in order to precisely determine the relationship between the structure of the glycopolymer and its anti-adhesive properties.

Controlled radical polymerizations (CRP) are ideal methods to control the polymerization process and design polymers having narrow distribution and complex architectures such as block copolymers, comb like, stars, star blocks, dumbbell, dendrigrafts and so on… The living character of CRP also limits the ability of growing chains to terminate and leads to an accurate control of chain end composition.

Reversible Addition-Fragmentation chain Transfer (RAFT) is one of the most effective CRP methods and is very attractive since it allows the polymerization of a wide range of monomers including glycomonomers. In addition, the lability of the thiocarbonylthio end group paves the way to post-functionalizations. 4 For all these reasons we have chosen to design well-defined multivalent glycopolymers via RAFT polymerization of n-heptyl α-D-mannose-based monomers and to deeply investigate their efficiency as lectin FimH antagonists for anti-inflammatory treatment of adherent-invasive E.Coli (AIEC) infection.

To further explore the potential of such glycomaterials, we have subsequently explored the propensity of our tailor-made glycopolymers to form glyconanocapsules via the Ouzo process. Indeed, as core-shell structures can protect actives against degradation and elimination, nanocapsules can be advantageously applied as nano-carriers for various molecules or (bio)macromolecules (fluorescent probes, drugs, DNA, RNA…). 5 With carbohydrates moieties present at the surface of glyconanocapsules one can favor specific interactions with targeted cells or tissues and enhance the cellular uptake.

The present manuscript is organized in five chapters. First (chapter 1) a literature review describes the different routes to get carbohydrate-functionalized nanocapsules from polysaccharides and glycopolymers. Note that we have chosen to investigate the latter subject because interactions between glycopolymers and lectins have been profusely reviewed over the last decade. 3,6 The synthesis and characterization of n-heptyl α-D-mannose-based glycopolymers are described in chapter 2. Based on RAFT polymerization of mannosylated monomers the preparation of linear/star-shaped glycopolymers and of a glyco-copolymer library (block, gradient, statistical, post-modified) is reported.

In the chapter 3, the application of glycopolymers against bacterial adhesion is explored, the evaluation of anti-adhesive properties being evaluated from molecular level to in vitro and ex vivo.

As underlined in chapter 1, the Ouzo technique is a simple route to generate nanocapsules through nanoprecipitation. This feature will be investigated in chapter 4 with the glycopolymers described in chapter 2.

Finally all the experimental parts are provided in chapter 5, including characterization methods, synthetic procedures, bio-assays and preparation of nanocapsules.

Chapter 1 Preparation of Carbohydrate-based nanocapsules

Introduction

Over the past decades, polymer capsules have attracted significant attention owing to their capability to mask a smell, or a taste, to protect an active molecule from degradation or elimination and to release it in a controlled manner. For all these reasons, polymer capsules which can be constituted of biopolymers or of synthetic polymers are commonly used in numerous applications in adhesives, paintings, food and flavor industry, pharmaceutical industry, cosmetics and so on.

Synthetic capsules can be likened to vesicular systems in which an encapsulated product is confined in a cavity consisting of an inner liquid core surrounded by a polymer membrane 1 . Synthetic capsules display a core-shell structure 2 (Scheme 1.1).

Both core and shell can be loaded with (macro)molecules to deliver and the shell can also be functionalized with probes or ligands 3 . The polymer(s) composing the capsules can be chosen so that the capsules exhibit a stimuli-responsive character (pH, temperature, light, redox…) in order to facilitate or trigger the release of the sequestrated active molecules. [4][5] The shell of the capsules can be made of synthetic or natural polymers. The inner part of the capsules can contain encapsulated colloids, solids, liquids or gas. According to the techniques of preparation, the reservoir of capsules can be either lipophilic or hydrophilic and depending on the particle size, the prepared capsules can be classified into microcapsules or nanocapsules.

Microcapsules

Capsules with diameters ranging from one to one thousand micrometers are called microcapsules. Microcapsules possess a high surface area and a large inner volume which facilitates the loading of molecules of interest (dyes, catalysts, drugs, ink...).

Microcapsules are thus utilized as microcarriers 6 , microsensors 7 or microreactors 8 .

Two major advantages of synthetic microcapsules over nanocapsules are that 1)

microcapsules are relatively fragile and can thus be easily broken by friction, a very desirable feature for cosmetic, flavor, food or textile applications and 2) that a large number of simple and cheap routes of microcapsules have been established for decades. The most common methodologies affording microcapsules are listed below. [9][10] (1) Coacervation. Coacervation is a phase separation process due to partial desolvation of (fully solvated) macromolecules upon addition of salt, oppositely charged polymer, non-solvent, pH or temperature change. The solution separates into two liquid phases, a polymer-rich and polymer-lean phase. The use of coacervation to build polymer microcapsules was first developed in the fifties for carbonless copy paper applications. The general procedure consists in dispersing (for instance) oils or drugs intended to form the core of the capsules into a solution containing the polymer. Coacervation results in the deposition of the polymer at the surface of dispersed particles and in the formation of a membrane which is finally hardened through chemical or thermal treatments.

(2) Spray-drying. Spray drying is a mechanical technique developed in the thirties for microencapsulation. It is notably a popular route for encapsulation of molecules of hydrophobic nature, such as oils and flavors. These molecules that further constitute the core of the capsules are first dispersed in an aqueous concentrated solution of shell precursor (generally polysaccharides or proteins). The resulting oil-in-water emulsion is then atomized into a spray of droplets in a hot chamber. The evaporation of the solvent leads to the formation of microcapsules.

(3) Pan coating. Pan coating is widely applied in the pharmaceutical industry for manufacturing coated particles or tablets. Generally, solid particles are mixed with a coating material. As temperature increases, the coating material is melted to enclose the core particles. Afterwards, microcapsules are solidified by cooling. Alternatively, the coating material can be gradually added to core particles which are tumbling in a vessel.

(4) Centrifugal extrusion. The core and coating materials, which are immiscible with one another, are co-extruded through a spinning two-fluid nozzle. Owing to Rayleigh instability, after clearing the nozzle the jet splits into tiny round droplets of core material engulfed with the coating material. The shell can be directly hardened in flight or after the formation of the capsules through treatment in a hardening bath.

This technique is ideally suitable for preparation of microcapsules with a diameter ranging from 250 to 2000 micrometer.

(5) Air-suspension coating. In this process, the core material is first dispersed (as particles) by an upward-moving (and usually heated) air stream and coated while suspended. This method is widely applied in pharmaceutical, food and cosmetic industry.

(6) Solvent evaporation. To apply this method, a coating polymer solution which is immiscible with liquid manufacturing vehicle (LMV) is first prepared. The core materials are either dissolved or dispersed in the polymer solution. Then the resulting solution is added into LMV with agitation. The polymer is deposited on the core materials to form microcapsules after solvent evaporation. This technique is widely used in pharmaceutical industry. Microcapsules can be built through emulsion, suspension, dispersion polymerizations (using the osmotic swelling technique or hydrocarbon encapsulation) or interfacial polymerizations.

Whereas microcapsules are widely used for industrial purposes, the scope of nanocapsules is presently limited by the absence of cheap and straightforward routes to these classes of objects.

Nanocapsules

The size of nanocapsules typically ranges from 10 nm to 500 nm. Owing to their peculiar dimensions, nanocapsules have raised great interest over the last two decades in biomedical applications, especially in the field of nanomedicine. [11][12][13][14] Nanocapsules allow for improving the efficacy of drugs or biomolecules (proteins, nucleic acids) while reducing side effects and toxicity issues. Nanocarriers are indeed capable to protect sequestrated active molecules, to control their release and, in the meantime, to overcome biological barriers, to target specific sites (for release) and to enable bio-imaging through incorporation of appropriate tags (Scheme 1.2). [15][16][17] Scheme 1.2: Engineered capsules for drug delivery applications.

The size distribution of nanocarriers is a highly important parameter for drug delivery applications, especially for intravenous applications: 1) to avoid recognition by macrophages of the reticuloendothelial system (RES) and thus rapid clearance from the bloodstream, the size of the nanocarriers should be as small as possible to limit the adsorption of plasma proteins (opsonins); 2) to take advantage of the Enhanced Permeation and Retention effect (EPR) in cancer therapy or treatments of various inflammatory diseases, the size of the nanocarriers should be under 200 nm and preferentially below 100 nm.

In quest for smart and sophisticated nano-objects, considerable attention has been lately paid to the generation of nanocapsules decorated with a myriad of functions ensuring selective targeting, bio-imaging and so on. In this context, the fabrication of nanocapsules bearing carbohydrate moieties is very attractive. Together with DNA and proteins, sugar moieties are indeed key molecules in living organisms. Sugars play a significant role in cellular communication, biological recognition events, 18 immune function 19 and signal transduction. 20 To enhance bioactivity (and eventually biocompatibility), glycopolymers, polysaccharides and molecular carbohydrate derivatives are then commonly used to prepare functional glyconanocapsules.

Routes to carbohydrate-functionalized nanocapsules

As nanocapsules are attractive materials for drug delivery applications, numerous methodologies have been developed over the last two decades. In this chapter, we will describe the most common routes to this category of nanocarriers. More precisely, this chapter will be dedicated to the physical and chemical routes (Scheme 1.3) to glycopolymer and polysaccharide based nanocapsules. [21][22][23][24][25][26] Construction of glyconanocapsules exclusively based on molecular carbohydrate derivatives (liposomes, cyclodextrine…) will not be reported.

Scheme 1.3: Routes to carbohydrate-functionalized nanocapsules.

Emulsification routes to carbohydrate-functionalized nanocapsules

Emulsification routes are classical methods to prepare nanocapsules. The principle of the most common methods, that is to say, the emulsion diffusion technique, the double emulsification technique, the emulsion coacervation technique, the polymer coating technique and the mini-emulsion technique will be described and their application in the field of glyconanocapsules will be discussed below.

Emulsion diffusion technique

In the emulsion diffusion process, the polymer-dissolved oil/water system needs to be emulsified via vigorous agitation. Nanocapsules are then generated by subsequent addition of aqueous solution into the emulsion which causes the diffusion of the solvent into the external phase. The formation of nanocapsules is induced by polymer precipitation and interfacial deposition during the diffusion of solvent. The size distribution of nanocapsules prepared through this method is generally ranging from 150 nm to 200 nm. This method has been extensively applied to prepare PCL or PLA-based polymeric nanocapsules. Up to now, no carbohydrate-based nanocapsules have been reported based on this technique.

Double emulsification technique

Double emulsification, also called "emulsion of emulsion", is a process which consists in the preparation of either water-oil-water emulsion or oil-water-oil emulsion. To realize the double emulsion process, both hydrophobic and hydrophilic surfactants are needed and should be used in this two-step emulsification process. The formation of nanocapsules induced by double emulsification is also associated with nanoprecipitation and emulsion-diffusion methods.

Holban and co-workers prepared double cross-linked chitosan nanocapsules by double emulsification (Table 1.1, entry 1). 27 In a preliminary phase, an o/w/o emulsion was prepared by two-step emulsification. A toluene solution of Span80 was first co-emulsified with an aqueous solution of chitosan-gelatin (1/1, w/w) containing Tween80 (low-molecular weight chitosan, 75-85% DA). The resulting emulsion was then added to a second Span80 toluene solution (final o/w/o conditions, 1/3/17, volume ratio). After a first cross-linking step (induced by addition of sodium sulphate), the solution was emulsified by mechanical agitation. Chitosan containing nanocapsules were finally obtained after cross-linking with glutaraldehyde. The size of nanocapsules (ranging from 100 nm to 700 nm) was controlled by the molar ratio between amino groups of chitosan and gelatin and sodium sulfate. Increasing the content of sodium sulfate made the nanocapsules shrink owing to ionic cross-linking.

The resulting nanocapsules were capable to load anti-tumor drug L-phenylalanine derivatives with efficiency up to 79%. In comparison with free drug treatment, the encapsulation improved the anti-tumor activity against implanted Guerin's carcinoma in vivo.

In 2013, Wu and coworkers reported on alginate/chitosan coated nanocapsules which could be used as insulin delivery system (Table 1.1, entry 2). 28 A w/o/w double emulsion was prepared by a two-step process; a w/o emulsion was first generated by emulsification of an insulin aqueous solution/Labrafac, Span 80 with phospholipid oily phase. This emulsion was then poured into an alginate aqueous solution and a second emulsification was carried out (by stirring) to generate a w/o/w emulsion. After sequentially adding calcium chloride and chitosan (low-molecular weight, 75-85% DA), alginate-coated nanocapsules were finally obtained with a diameter of around 490 nm. The release of insulin was proven to be controlled by the acidity of the solution. For instance in simulated gastric conditions (pH 1-3), only 20 % of insulin could be released out of the alginate/chitosan shell at pH 2.5 after 30 min (37°C), while more than 85% and no leakage at all were observed at pH 1.0 and 3.0, respectively. As revealed by in vivo studies, these nanocapsules were also shown to effectively prolong the hypoglycemic effect after oral administration.

Emulsion coacervation technique

In the emulsion coacervation process, emulsion is generated through o/w emulsification by mechanical stirring or ultrasound. The polymer then precipitates from the continuous solution to form a coacervate layer on the emulsion template.

This polymer layer then generates the walls of the nanocapsules after cross-linking reactions. The crucial point of this technique is the formation of the coacervate layer which is triggered by the addition of salt, oppositely charged polymer or non-solvent, or by pH or temperature change. Up to now, sodium alginate and gelatin have been commonly used for the generation of nanocapsules by the emulsion coacervation technique.

In 2008, Lertsutthiwong's group reported on the preparation of turmeric oil loaded alginate nanocapsules by emulsion coacervation technique (Table 1.1, entry 3). [29][30] Practically, a solution of turmeric oil in acetone or ethanol was added into an alginate (800~120 kg/mol) aqueous solution. After o/w emulsification by sonication, cross-linked nanocapsules were directly generated by addition of calcium chloride and solvent removal. The size of capsules obtained from the ethanol/water system (diameter around 260 nm) was smaller than capsules obtained from the acetone/water system (diameter around 370 nm). An increase of oil concentration or oil/alginate mass ratio caused an increase of nanocapsules' size. Alternatively, the alginate coacervate layer was cross-linked by sequential addition of calcium chloride and chitosan (41 or 72 kg/mol). However, involvement of chitosan led to the formation of larger nanocapsules (above 500 nm).

Grebinsan et al. developed glutamine derivatives loaded alginate-based nanocapsules (Table 1.1, entry 4). 31 Starting from L-glutamine, formyl and acetyl groups grafted N-(p-aminobenzoyl)-L-glutamines were synthesized in five steps. The glutamine derivatives (constituting the core material) were dissolved in a mixture of ethanol, benzyl alcohol and DMSO, which was then poured into an alginate aqueous solution. After emulsification by sonication, glutamine derivatives loaded alginate-based nanocapsules (size about 400 nm) were obtained after chitosan-induced cross-linking reaction and solvent evaporation. According to in vitro anti-tumor studies, no enhancement of activity could be observed with these nanocarriers in comparison with free drugs.

Jana and coworkers recently prepared testosterone-loaded nanocapsules using the emulsion-coacervation technique (Table 1.1, entry 5). 32 Testosterone (a steroidal male hormone) dissolved in hexane was added into an alginate aqueous solution.

Nanocapsules with diameter of 34 nm were directly obtained after emulsification (first stirring and then sonication) and calcium-induced cross-linking. These nanocapsules were capable to load 30% of testosterone during the preparation and enhanced its bioavailability in Female Sprague Dawley rats.

Polymer coating technique

The polymer coating technique consists in depositing a layer of polymer on preformed nanoparticles surface. This route thus relies on a preliminary emulsification step leading to the formation of nanodroplets and a subsequent polymer deposition step at the oil/water interface. In contrast with the emulsion-coacervation technique, the adsorption of the polymer chains is generally triggered by solvent evaporation.

In 1997, Alonso's group took advantage of this approach to generate polysaccharide-based nanocapsules. Poly-ɛ-caprolactone (PCL, Mw = 40 kg/mol),

Miglyol 840 and lecithin were dissolved in acetone (Table 1.1, entry 6). After pouring the organic solution into aqueous solution of chitosan (molecular weight > 100 kg/mol), chitosan-coated PCL nanocapsules (size ranging from 300 to 500 nm) were generated by an interfacial deposition technique after evaporation of acetone (One-stage procedure). These positively charged nanocapsules were capable to encapsulate indomethacin (an anti-inflammatory drug) and enhance the corneal penetration. It was demonstrated that 85% of the drug diffuses out of the nanocapsules within 2h. [33][34][35] More recently (2006), the same group reported on the preparation of Miglyol-812 filled chitosan nanocapsules (Table 1.1, entry 7). [36][37] A miglyol nano-emulsion was first prepared by solvent shifting and further incubated in an aqueous solution of chitosan (MW < 10 kg/mol) for one hour to afford nanocapsules (diameter around 200 nm). The chitosan nanocapsules displayed a preferential association with HT29-M6 cells. Salmon Calcitonin, a polypeptide hormone, was encapsulated with an efficiency of 60% and released over a period of 6h. In 2013, the same authors post-modified the nanocapsules with hepatitis B surface antigen (HB) through electrostatic attraction (CS/HB = 1/0.025, w/w) resulting in a high antigen association efficiency (70%). 38 Imiquimod (a TLR7 receptor agonist)

was also incorporated into the oily core of the nanocapsules to elicit a specific T1-mediated immune response. Chitosan was also modified with MeO-PEG-COOH (5088 g/mol) and biotin-PEG-COOH (4060 g/mol) by EDC catalyzed coupling amidation. An analogous procedure was then used to afford PEG functionalized and biotin-PEG functionalized chitosan based nanocapsules (diameters around 250 and 200 nm, respectively). The presence of PEG (DS = 0.003) enhanced the stability, the biocompatibility and the cellular uptake capability of nanocapsules. The biotin-PEG functionalized nanocapsules were further functionalized with monoclonal antibody anti-TMEFF-2 through a convenient two-stepwise avidin-biotin interaction (Figure 1.1). The loading and delivery of docetaxel (DCX) was subsequently investigated. In comparison to free drug, increased efficacy (tumor weight reduced from 1.8 g to 0.8-1.0 g) of encapsulated DCX was shown from in vitro cell studies due to the efficient internalization of nanocapsules, but no enhancement of efficacy was observed from in vivo studies on lung cancer xenograft model. [39][40] Using a similar methodology, the same group also reported on squalene-loaded PG-nanocapsules (PG, polyglucosamine, highly deacetylated chitosan, 276 kg/mol) (Table 1.1, entry 8). 41 Specifically, squalene (core material) was dissolved with lecithin in 2-propanol. A similar route was also used by Ortega-Vinuesa and coworkers to investigate the influence of molecular weight (MW) and of the acetylation degree (DA) of chitosan on capsules stability (Table 1.1, entry 9). 42 An acetone/ethanol solution of miglyol 812 and lecithin was added into aqueous solution of low MW (11 kg/mol, DA ranging from 1.4 to 51%) or high MW (~112 kg/mol, DA ranging from 1.6 to 56%) chitosan to give birth to chitosan-based nanocapsules (diameters ranging from 150 to 185 nm) after solvent evaporation. Interestingly, nanocapsules made of chitosan with intermediate MW and high DA (85%) displayed higher cell uptake capability and stability than epikuron or pluronic ® F68 based liposomes. [43][44] Low molecular weight chitosan was capable to deposit at the interface of soybean oil nanoemulsion (generated by solvent evaporation) to afford nanocapsules with a size of 145 nm (Table 1.1, entry 10). 45 In view of developing materials for chemotherapy of leishmaniasis, amphotericin B (AmB, prototype leishmanicidal drug) was then loaded with a good steric stability in vitro. As revealed by in vitro and in vivo studies, the encapsulated drug exhibited an enhanced antileishmanial property compared with free drug.

Tzanov and coworkers prepared cationic polysaccharide nanocapsules coated with chitosan and cellulose (Table 1.1, entry 11). 46 Thiolated chitosan (TC) was prepared by a coupling reaction between 2-iminothiolane and chitosan (~15kg/mol).

Ethylenediamine functionalized cellulose (AC) was synthesized by substitution of tosylated cellulose with ethylenediamine. Oil-loaded nanocapsules were finally obtained through a sonication-induced emulsification of an aqueous solution of polysaccharide (TC or AC) with sunflower oil. The diameters of nanocapsules prepared from TC or AC were 268 and 247 nm, respectively. These cationic polysaccharide nanocapsules were shown to disrupt bacterial membranes and were no cytotoxic to human cells.

Chitosan (150 kg/mol) was also functionalized with mannose by reductive amination (DS = 0.06). 47 A tripolyphosphate (TPP) nanoemulsion with rifampicin (anti-tubercular drug) as core material was generated by stirring of the initial DMSO/water solution and subsequently poured into an aqueous solution of mannose-conjugated chitosan to afford nanocapsules with a size of 215 nm. The encapsulation efficiency was about 39%, and more than 90% of loaded drug was released after 5h. Cellular uptake was 2.3 times higher in the presence of anchored mannose units (Table 1.1, entry 12).

Besides chitosan, Alonso and coworkers prepared miglyol 812-filled hyaluronic acid (HA, 29 or 160 kg/mol) nanocapsules through a one-stage procedure or through polymer coating on preset nanoemulsion (two stage procedure) (Table 1.1, entry 13). 48 The size of the HA nanocapsules prepared by the two stage procedure (~250 nm)

was bigger than that from the one stage procedure (~180 nm). The HA nanocapsules were capable to load DCX in miglyol and enhance efficacy of DCX in vitro.

Noteworthy, the HA nanocapsules were stable at 37°C for 90 days. Nanocapsules made of bovine serum albumin-Dextran (BSA-Dextran) conjugate and soybean oil (as core material) were used to load sensitizer/annihilator (s/a) couples for maintaining the mobility of chromophores and avoiding the quenching, displaying an intense photoluminescence emission (Table 1.1, entry 15). 51 The nanocapsules Kansal and coworkers reported alginate-containing nanocapsules by combining polymer coating and LbL strategy (Table 1.1, entry 16). [52][53] Nano-emulsions were prepared by stirring-induced emulsification using soybean oil in o/w system. The resulting droplets were coated with protamine sulfate. Sodium alginate and protamine sulfate were then sequentially deposited on the capsules to finally generate multilayered nanocapsules (size of around 340 nm). Doxorubicin (DOX) was encapsulated with efficacy above 80%. The anti-leishmanial efficacy of (encapsulated ) DOX was proven to be enhanced in vitro.

Mini-emulsion technique

Miniemulsions are kinetically stable oil-in-water dispersions generated by high shear devices such as ultrasonicators. Nanoparticles or nanocapsules can be generated using miniemulsion conditions. In a typical mini-emulsion procedure, water, oil, hydrophobic agents and stabilizers are co-emulsified resulting in the formation of nanodroplets with size ranging from 50 to 500 nm (Scheme 1.4). 55 This technique has been successfully applied to radical, ionic, or metal-catalyzed polymerizations, enzymatic or chemical polycondensation and polyaddition reactions. Polymerization normally occurs in the dispersed phase or at the interface of droplets resulting in the formation of a polymeric membrane. Thanks to its potential application in pharmaceutical and cosmetic fields, numerous examples of nanocapsules prepared under miniemulsion conditions have been reported over the last twenty years.

Scheme 1.4. The principle of mini-emulsion. 55 Chitosan, a biocompatible polysaccharide has been proposed by Landfester and co-workers as candidate for constructing glyconanocapsule membranes via interfacial polymerization under miniemulsion conditions.

In 2002, her group reported the preparation of chitosan-containing nanocapsules using the miniemulsion technique (Table 1.2, entry 1). 56 In this seminal work, a solution of toluene containing a diepoxide as cross-linker (Epikote E828), Jeffamine D2000 as cosurfactant and various diamines (1,12-diaminododecane, 4,4'-diaminodicyclohexylmethane, 4,4'-diaminobibenzyl) were dispersed in an acidic aqueous solution of chitosan. Ultrasonication of the emulsion at 0°C afforded a stable mini-emulsion. Cross-linking via epoxy-amine reations was initiated by increasing the temperature of the medium up to 68°C, leading to the formation of polymeric nanocapsules with diameter ranging from 130 to 280 nm.

Zhu and co-workers described a route to chitosan-based nanocapsules loaded with felodipine, a hydrophobic anti-hypertensive drug (Table 1.2, entry 2). 57 Capsules were built using an interfacial free radical polymerization approach involving 3). 58 The generated chitosan nanocapsules (size of 60~390 nm) were used to encapsulate anti-cancer drug (DOX). Thanks to the presence of pH-sensitive cross-linkers, the release of DOX was easily mediated by adjustment of pH of the solution (Table 1.2, entry 3). 59 The shell of the capsules was fabricated by interfacial reaction of chitosan (2.5 kg/mol) with carboxylic acid end-functionalized Pluronic F127

N
PEO 100 -PPO 65 -PEO 100 (12.6 kg/mol) under mini-emulsion conditions. Owing to the thermo-responsible character of Pluronic F127, the size of nanocapsules evolved from 37 nm at 37°C to 240 nm at 4°C, thus modifying the permeability of capsules' shell. Therefore, encapsulation and release of small molecules drugs (ethidium bromide, Mw = 394.3 g/mol) were easily controlled by shifting the temperature. The biocompatible pluronic F127-chitosan nanocapsules were identified by the authors as potential candidates for drug delivery applications in cancer treatments (Figure 1.4). In 2007, Landfester reported the preparation of hydrophilic compound-loaded nanocapsules by applying an inverse miniemulsion process. [61][62][63] 64 and of silver particles 65 . Recently, the same group described another route to dextran nanocapsules with aqueous core through inverse miniemulsion process. [66][67] After emulsification, acrylated dextran (40 kg/mol, DS between 0.13 and 0.55) was cross-linked by interfacial olefin metathesis with phenyl-di(undec-10-en-1-yl)-phosphate. The generated nanocapsules (diameter ~ 280 nm) were shown to be biocompatible (Table 1.2 entry 7).

Using a similar process, the Landfester's group also generated hydroxyethyl starch (HES)-based nanocapsules (Table 1.2, entry 8). 68 After emulsification in a water/cyclohexane system, HES (200 kg/mol) was cross-linked with TDI at the water/cyclohexane interface to generate water-loaded nanocapsules with a diameter of 275 nm (Figure 1.6). After covalent coupling with folic acid (FA), the functional nanocapsules were specifically uptaken by HeLa cells. The HES containing nanocapsules were also decorated with mannose moieties. 69 The targeting effect of these mannosylated HES nanocapsules was proven by in vitro experiments. The same group also described the preparation of enzyme-responsive hyaluronic acid-based nanocapsules using the same approach (Table 1.2, entry 9). 70 After emulsification, hyaluronic acid (140 kg/mol) was cross-linked by TDI to form water-loading nanocapsules with a diameter of 350 nm. The nanocapsules were then used to load polyhexanide, an anti-bacterial agent. The release of polyhexanide was triggered by addition of enzyme hyaluronidase.

Glyconanocapsules were also generated from an A 2 +B 2 interfacial step growth polymerization under oil/water miniemulsion conditions (Table 1.2, entry 10). 71 The word polymersome was first coined by Discher in 1999 and refers to polymers capable to mimic the self-assembly of liposomes. 72 Polymersomes or polymeric vesicles are nano-or micro-sized polymeric capsules with a bilayered membrane comprising of amphiphilic block copolymers. 73 The amphiphilic membrane structure of polymersomes is very similar to liposomes, but liposomes are built from the assembly of phospholipids with molecular weight typically less than 1 kg/mol, while polymersomes are prepared by self-assembly of amphiphilic block copolymers with molecular weight up to several hundreds thousands g/mol. This provides superior physical and chemical stability to polymersomes over liposomes.

Owing to energetic repulsion effects between the (chemically different) macromolecular segments, amphiphilic block copolymers self-assemble in solution.

The length of the blocks, the degree of repulsion and the nature of the solvent drive the self-assembly of the block copolymers into a myriad of morphologies such as spherical micelles, rods and vesicles (polymersomes). In general, the formation of polymersomes is observed when the fraction of the hydrophilic segment is less than 25%. Solvent displacement, rehydration and injection techniques have been applied

for generating polymersomes with size ranging from 50 to 800 nm.

In the next section we provide an overview on the routes to carbohydrate-fuctionalized polymersomes relying on the self-assembly of amphiphilic carbohydrate-based block copolymers.

Amphiphilic block copolymers with a glycopolymer segment

In 1999, the group of Fu-Mian Li reported the first example of glyco-polymersomes through self-assembly of amphiphilic block copolymers (Table 1.3, entry 1). 74 Lecommandoux and coworkers synthesized polypeptide block copolymers (PBLG-b-PGG) through sequential ring-opening polymerization of benzyl-L-glutamate and propargylglycine (PG) N-carboxyanhydrides with different segments ratios (PBLG 20 -b-PGG n , n = 5~32). 91 The glycosylated block copolymers were further obtained by post-polymerization modifications with azide-functionalized galactosides through CuAAC ligation (Figure 1.12). The block copolymers self-assembled through solvent shifting process into morphologies ranging from worm-like micelles (addition of water in DMSO) to polymersomes (addition of DMSO in water) depending on polymer composition and on the solvent shifting process. Stable vesicles (R h = 32 nm)

were obtained from PBLG 20 -b-PGG 25 by adding water into DMF solution of copolymer.

The vesicles were shown to specifically bind Con A lectin (Table 1.3, entry 15). 92 The self-assembly of the block copolymers in water led to the generation of temperature-responsive glycovesicles (20°C, 250 nm for P1 and 500 nm for P2). At 37°C (above the LCST of PDEGMA), the size of the vesicles decreased to 182 nm (P1) and 300 nm (P2), owing to the collapse of the PDEGMA segments (

Polysaccharide-based polymersomes

In view of generating nanocapsules for biomedical applications, polysaccharides which are natural polymers are ideal candidates due to their large availability, biocompatibility and biodegradability.

Dextran

The group of Gnanou designed dextran-based vesicles by self-assembly of amphiphilic dextran-block-PS (Table 1.3 entry 21). [97][98] The anomeric extremity of a commercial dextran (M n = 6600 g/mol) was firstly converted into an ATRP initiating site by reductive amination. The resulting macroinitiator was then engaged into the were obtained upon displacement of the organic solvent (THF or DMSO-THF) by water. Finally, the size and the shape of the assembled nano-objects were frozen by cross-linking the dextran shell through reaction of hydroxyl groups with divinyl sulfone.

In Finally, CPT loaded vesicles were proven to be effective in killing fibroblasts. and showed an enhanced efficacy against armyworm larvae compared to the free drug. was then mixed with a cationic surfactant cetyltrimethylammonium bromide (CTAB, 3×10 -4 M) in water. This mixture spontaneously self-assembled into vesicles with a hydrodynamic diameter of 200 nm. As evidenced by DLS and TEM, the formation of vesicles was favoured when longer alkyl chains were incorporated on the chitosan backbone.

In addition to the straightforward assembly of modified chitosans in solution, some research groups investigated the co-self-assembly of lipids and amphiphilic chitosan into hybrid nanocapsules. = 50 kg/mol) was functionalized with methoxyl PEG (mPEG, 2 kg/mol) or folic acid (FA) through a carbodiimide-induced coupling reaction to obtain mPEG-OQLCS and FA-OQLCS, respectively. OQLCS, mPEG-OQLCS and FA-OQLCS were then mixed with cholesterol (weight ratio, 1/1/1/1) in chloroform. These mixtures gave rise to FA-functionalized chitosan nanocapsules (size of 163 nm) by solvent evaporation.

The release behavior of these nano-objects was mediated by tuning the ratio between cholesterol and OQLCS, and a higher permeability was observed for high weight ratio of cholesterol (OQLCS/cholesterol = 0.5/1). Due to the presence of FA moieties, these nanocapsules exhibited specific uptake capability for MCF-7 cells.

Mishra and coworkers also prepared chitosan/lipid nanocapsules using amphiphilic chitosan and cholesterol (Table 1.3, entry 33). 111 Glycol chitosan (90 kg/mol, DA~82.7%) was first modified with stearic acid (SA) to generate amphiphilic chitosan (DS = 0.08). The resulting polymer was then mixed with cholesterol at a ratio of 4/1 (chitosan/cholesterol) by stirring and further vortexing. Through solvent shifting (addition of ethanol/DMA into an aqueous solution), the mixture was shown to self-assemble into vesicles (D h ~ 240 nm). Amphotericin B (AmB) was subsequently encapsulated into these vesicles during the self-assembly process with an efficiency of 26%. The vesicles exhibited high plasma stability and released 66% of AmB within 24h under sink condition. The loaded AmB showed an enhanced efficacy against leishmania in vivo compared to the free drug.

Other Polysaccharides

Besheer through solvent shifting (addition of warm water to DMSO at 55°C). The obtained polymersomes were then used to load (efficiency close to 50%) and release doxorubicin (about 55% of released DOX after 2 days). In addition, it was shown that the hyaluronan-containing carriers display high colloidal stability and specifically bind CD44 glycoprotein receptors.

Wu et al. reported the synthesis of amphiphilic β-1,3-glucan ester using a two-step process. 116 The polysaccharide (200 kg/mol) was first reacted with formic acid to generate a pyridine-soluble glucan and further esterified with acetic or hexanoic anhydrates to yield β-1,3-glucan acetate (DS = 0.92~2.46, BGA) and hexanoate (DS = 1.87, BGH). Upon solvent displacement (acetone solution dialyzed against water), the resulting polymer chains self-organized into nanocapsules or solid nanoparticles (with diameters ranging from 132 to 487 nm). Vesicles (D h = 490 nm) were only observed from the self-assembly of BGH (Table 1.3, entry 36).

Polymerization-Induced Self-assembly (PISA)

In comparison with the traditional multi-step strategy which consists in the preparation of block copolymers, their purification and their final self-assembly, PISA has emerged as a powerful approach to prepare solutions of well-defined nano-objects with high solid contents in one step. The PISA process which can be applied to emulsion or

dispersion

Micelle core removal strategy

Apart from polymersomes, micelles obtained via self-assembly of amphiphilic block copolymers can also be used to generate hollow structures through selective removal of the core. Elimination of the core can then be achieved by enzymatic degradation, ozonolysis, hydrolysis or aminolysis.

Even though her group has not described the fabrication of glyconanocapsules, the preparation of hollow nanospheres via core degradation method was first introduced by Wooley and co-workers.

Template strategy

The template strategy relies on preformed nano-sized templates (colloid, polymer, silica, polymer particles…) on which polymer chains are covalently grafted or adsorbed. Glyconanocapsules can be conveniently generated through grafting The main advantage of this strategy is that thickness and morphology of particles can be precisely controlled through carefully selection of the materials. Nano-or microcapsules are typically obtained in two steps (Scheme 1.6): 1) sequential deposition of polymer chains at the surface of the template; 2) removal of the core.

The multi-layered capsules prepared by L-b-L combine the different properties of the assembled polymers in one system, highly reinforcing its potential for applications. Kren et al. reported on the hyaluronan-coated nanocapsules for delivering genes to liver sinusoidal endothelial cells and hepatocytes (Table 1.4, entry 4). 131 The hyaluronan chains were coated on the positively charged DNA-PEI complexes through dispersion atomization process to generate hyaluronan-coated nanocapsules with a size of about 20 nm. These nanocapsules were proven to stably transfer sleeping beauty transposon into liver sinusoidal endothelial cells of mice, displaying a very interesting therapeutic application.

(

2) Multi-layered nanocapsules by L-b-L strategy

In 2004, Akashi and co-workers prepared multilayered hollow nanocapsules through L-b-L strategy from silica nanoparticles (Table 1.4, entry 5). 132 Chitosan as first layer (650 kg/mol) and dextran sulfate (500 kg/mol) were sequentially deposited on the surface of the nanoparticles (size of 330 nm) to form four layers. Polysaccharide multi-layered nanocapsules were finally generated with a diameter of about 300 nm after core removal. The same group also reported pH-responsive hollow nanocapsules using the same template (Table 1.4, entry 6, Figure 1.22). 133 The multilayered walls of the capsules were constructed from sequential deposition of chitosan (600 kg/mol) and poly(γ-glutamic acid) (γ-PGA, 1000 kg/mol), and cross-linking reactions between chitosan and γ-PGA chains (activated by carbodiimide reagent). After core removal, the size of the nanocapsules was stable (roughly 250 nm) between pH 4.0 to 10.0, and significantly increased (about 500 nm) at pH 1.0 due to electrostatic repulsion between the ammonium groups of chitosan.

Modification of the pH also allowed for tuning the release behavior of the nanocapsules. Poly(allylamine hydrochloride) (PAH, 70 kg/mol) and dextran sulfate (DS, 500 kg/mol) were sequentially deposited on silica particle templates (size of 500 nm) by Raichur and co-workers to form multilayered nanoparticles. These steps were followed by the deposition of silver nanoparticles (formation from reduction of silver nitrate during the deposition) (Table 1.4, Entry 7). 134 After core removal via HF/ NH 4 F treatment, the authors demonstrated that it was possible to encapsulate FITC-dextran in the silver nanoparticles modified nanocapsules (D h about 500 nm, thickness of 30 nm) through a permeation process. FITC-dextran was released through sonication owing to partial destruction of the polymer shell.

Silica nanoparticles (D h = 220 nm) were also coated by chitosan and negative charged heparin through L-b-L deposition (Table 1.4, Entry 8). 135 Hollow nanocapsules of 200 nm were then generated after elimination of the silica core (0.2 M HF + 0.8 M NH 4 F).

DOX was loaded in the core of the resulting glyconanocapsules with an efficiency of 89%. Interestingly, the loading and the release of DOX were controlled by adjusting the pH of the solution (64% for pH 4.8 and 77% for pH 7.4).

Li and co-workers reported insulin-loaded chitosan nanocapsules using a L-b-L process from silica nanoparticles (size of 260 nm) (Table 1.4, Entry 9). 136 Chitosan (50 kg/mol) was first modified with L-cysteine to obtain thiol-functionalized chitosan.

Poly(L-aspartic acid) (20.4 kg/mol) was reacted with L-cysteine to generate thiol-functionalized poly(L-aspartic acid). During the deposition process, the concomitant formation of disulfide bonds from coupling of thiol groups ensured the cross-linking of the polymer shell (size of 365 nm and thickness of 40 nm). Hollow nanocapsules were finally obtained after degradation of the silica core. The resulting biocompatible nanocapsules were capable to sequestrate insulin with a loading efficiency of around 13%. The release behavior of the nanocarriers was influenced by glutathione (GSH) which cleaves the disulfide bonds.

Chakravortty and co-workers reported chitosan-dextran nanocapsules as potential drug delivery system against pathogen Salmonella (Table 149 After removal of DDAB (triggered by the introduction of nonionic surfactant Triton X-100), nanocapsules with a diameter of about 500 nm were obtained. The same group also described the synthesis of dextran-loaded multi-layered nanocapsules from liposome templates. [150][151] In a preliminary step unilamellar liposomes (D h of 80 nm) were prepared using DPPC and DDAB (molar ratio at 6.5: 152

Peng and coworkers prepared paclitaxel-loaded chitosan-coated nanocapsules (Table 1.4, Entry 25). 154 Liposomes (size of 108 nm) were prepared using soya lecithin, cholesterol and stearyl amine through the thin film hydration technique.

Polyacrylic acid (PAA) and chitosan (50 kg/mol) were then used to build double layered capsules (D h = 215 nm). Paclitaxel was encapsulated during the preparation of the liposomes with an efficiency of 71%. Due to the presence of two polyelectrolyte layers, the release of paclitaxel was sustained with a lag time of 3h compared to uncoated liposomes.

Modification of preset capsules.

In addition to the template strategy described above, carbohydrate functionalized nanocapsules can also be prepared through post-modification of preformed nanocapsules. In general, the sugar moieties are introduced on the preset capsules through chemical coupling reaction or physical post-insertion of polysaccharide chains. In contrast to other strategies, the shell of preset capsules is then only partially decorated with carbohydrates (Scheme 1.7).

Scheme 1.7. General process of modification of preset capsules.

( 1.4, Entry 28). In comparison to LNC and LNC-LD, the positively charged LNC-LC showed enhanced uptake by HEK293 cells. Interestingly, post-insertion of polysaccharide chains did not influence the bio-distribution of the nanocapsules in mice. 157 Tan and co-workers prepared amphiphilic N-palmitoyl chitosan (ChP) from modification of water-soluble chitosan (roughly 30 kg/mol) with palmitoyl chloride. 158 Oleic acid based liposomes (size of 163 nm) were prepared by precise dissolution in a borate buffer and chitosan chains were then anchored on the liposomes. The size of the chitosan-functionalized nanocapsules decreased with the amount of chitosan and the smallest liposomes (size of 130 nm) were obtained when 15% of chitosan was introduced (Table 1.4, Entry 29). The authors pointed out that the presence of chitosan on the outer shell improved the integrity and rigidity of the nano-objects.

(2) Grafting strategy

Peer and coworkers described the preparation of hyaluronan-grafted nanocapsules (Table 1.4, Entry 30). [159][160] Liposomes were first prepared from the self-organization of 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE) and 1,2-dilauroyl-sn-glycero-3-glycerol (DLPG) (molar ratio of DLPE/DLPG is 9/1).

Hyaluronan chains (HA, 750 kg/mol) were covalently grafted onto the surface of the liposomes through carbodiimide induced coupling reactions. HA-grafted capsules (diameter of 100 nm) were loaded with anti-cancer drug, i.e. mitomycin C and paclitaxel (efficiency approaching 100%). HA-grafted nanocarriers were proven to enhance the efficacy of drugs and to ensure efficient targeting of CD44 cell surface receptors.

Carboxymethyl chitosan tethered lipid vesicles were reported by Banerjee and co-workers. 161 Amino-functionalized liposomes were first prepared using DPPC and 162 The grafting of the polysaccharide was performed through maleimide/thiol coupling chemistry. The resulting nanocapsules (D h = 354 and 590 nm) did not induce immunogenic response after oral administration to rats and were proven to improve the permeation of liposomes in ex vivo studies.

Nanoprecipitation

Nanoprecipitation 178 , also called as "ouzo effect" or "solvent shifting process", is a simple technique for preparation of colloids with narrow size distribution avoiding the utilization of ultra-sonication, other high shearing force or surfactants. Since no pre-emulsion is required, this solvent shifting method is more straightforward and simpler than the emulsification approaches.

In nanoprecipitation, a hydrophobic solute (oil, dye, polymer) is dissolved in an organic solvent which is fully miscible with water. Upon addition of water, the solution becomes a non-solvent of the solute which comes out of the solution. Under appropriate conditions, this process spontaneously generates metastable dispersion of nanometer-scale droplets or particles without using surfactants. This process is simple, cheap and applicable to numerous solutes and solvents.

The group of Lecommandoux reported the preparation of glycopolymersomes using the nanoprecipitation technique (see previous sections). 99,115,143 However, in these studies, the formation of hollow nano-objects is mostly relying on the features of the amphiphilic carbohydrate-based block copolymers (blocks length, nature of the blocks…) which drive their self-assembly in solution. In other words, the formation of the nanocapsules is not strictly due to the solvent shifting process. From that point of view, to our knowledge, the elaboration of glyconanocapsules (exclusively) induced by the solvent shifting process has not been reported yet.

Conclusion

Due to the key role of saccharides in biological recognition events, considerable efforts have been devoted to the preparation of tailor-made glycomaterials. In view of generating drug delivery devices not only capable to carry and release therapeutic agents in a controlled manner but also to target specific sites, significant attention has been paid over the last decades on the development of synthetic glyconanocapsules.

In this Chapter, we have shown that the generation of such polymer nanocapsules remains a considerable challenge to (physical) chemists.

Chapter 2 RAFT Preparation of well-defined Heptyl mannose-based Glycopolymers

Introduction

Adhesion of bacterial pathogens to host cells and tissues, a prerequisite for a majority of infection diseases, is governed by interactions between bacteria surface lectins and complementary carbohydrates displayed at the host cell periphery. To promote adhesion and infection of tissues through multivalent interactions, bacteria such as Escherichia coli (E. coli) express hundreds of adhesive proteinaceous hair-like organelles, type 1 fimbriae, on their cell surface. These type 1 fimbriae are cylindrical rod-shaped extensions on the E. coli cell wall constituted of a major pilin subunit, FimA, and followed by a short, flexible tip fibrillum composed of minor pilins FimF, FimG and the adhesin FimH. The latter is exposed at the extremity of the fimbriae and its lectin domain possesses a mannose-specific receptor site responsible for recognition and binding to host cells. 1 An overgrowth of E. coli is associated with infections, i.e. urinary tract and bladder infections, inflammatory bowel diseases, sepsis and meningitis, which usually require antibiotic treatments. As bacterial resistance to antibiotics is becoming a serious threat to public health, the anti-adhesive therapy, which lies in the use of drugs capable of preventing bacterial adhesion step with no bactericidal effect, is progressively emerging as a valuable alternative. 2 Because carbohydrate-protein interactions are intrinsically weak (K d typically in the mM to M range), the applicability of this approach requires to develop compounds with high affinity towards bacterial lectins so that adhesion (and thus infections) can be prevented at reasonably low concentrations. In this context, extensive research has been carried out on the design of synthetic molecular inhibitors displaying structures that optimize interactions with the lectin sugar-binding site(s) to outmatch interactions with natural ligands. The feasibility of this approach has been demonstrated for E. coli. In their pionnering work, Sharon and co-workers showed that mannosides bearing aromatic aglycons are more efficient FimH inhibitors than the methyl mannoside reference. 3 X-ray structure investigations on FimH lectin co-crystallized with butyl -D-mannoside highlighted that in addition to the hydroxyl groups of D-mannose which establish hydrogen bonds with the amino acids located at the bottom of the FimH binding pocket, the butyl group develops strong hydrophobic interactions at the entrance (of the binding pocket), the so-called tyrosine gate (Tyr48, Ile52 and Tyr137). 4 Optimization of the alkyl chain length led to the discovery of n-heptyl -D-mannose (HM), a nanomolar FimH antagonist (binding affinity of 5 nM recorded by SPR). 4 These findings greatly impacted the development of FimH antagonists paving the way to the discovery of other potent compounds with biphenyl, [5][6][7] indolinyl phenyl, 8 or squarate aglycones 9 for the potential treatment of E.

coli-induced urinary tract infections. Recently, it was also showed that thiazolylaminomannosides, 10 a new family of FimH antagonists, could extend the anti-adhesive concept to the treatment of specific inflammatory bowel diseases such as Crohn's disease (CD), in which adherent-invasive Escherichia coli (AIEC) can play a key role in the inflammation process. [11][12] Targeting the cause of the inflammation is a promising alternative to the current marketed CD drugs essentially based on lowering tumor necrosis factor (TNF) levels. 13 Another conventional route for the design of high-affinity ligands consists in the incorporation of several epitopes on a common scaffold (multivalent molecule, dendrimer or polymer chain). [14][15][16][17][18][19][20][21][22][23] Such derivatives can display noticeable enhancement in activity compared to their monovalent counterparts on a per mole of epitope basis on a wide range of lectins. This trend is often referred to as the "cluster glycoside" effect. 24 Multivalent FimH antagonists have been reported by several research groups. In seminal works, Lindhorst 25 and Roy [26][27] developed multivalent FimH inhibitors from dendrimer scaffolds whereas Gouin and Bouckaert recently introduced HM ligands on functionalized hydrophilic cyclodextrin-based scaffolds and observed substantial cluster effects in vitro [28][29] and in vivo. 30 In this chapter, we will describe the preparation of macromolecular anti-adhesives of type 1 piliated E. coli through the association of a judicious ligand selection with the presentation of multiple ligands on a polymeric scaffold. More precisely, we will firstly focus on the design and synthesis of model macromolecular inhibitors of type-1 fimbriae bacterial adhesion based on the incorporation of multiple copies of n-heptyl -D-mannose (HM), one of the strongest molecular binder of the monovalent lectin domain of FimH, as pendent motifs, to build series of multivalent HM-based polymers with various chain lengths and topologies for assessment of the influence of HM valency and spatial presentation of the epitopes. In addition, in order to investigate the influence of HM density and of the relative position of HM groups on the polymer chains, the synthesis of series of well-defined glyco(co)polymers with tunable compositions and microstructures will be described in this chapter. Owing to its nanomolar affinity toward FimH lectin (K d = 5 nM as measured by SPR) 4 , heptyl mannoside (HM) is a promising building block for constructing potent anti-adhesive glycopolymers. To design a HM-based glycomonomer (HMM), a multi-step synthetic strategy was developed (7 steps, Scheme 2.1). To build the spacer between the carbohydrate and the group to polymerize, 7-bromoheptan-1-ol

Synthesis of mannosylated monomers 2.2.1 Synthesis of N-[7-(-D-mannopyranosyloxy

(1) was first generated from 1,7-heptanediol and HBr. 31 To further probe the influence of the spacing between the mannose and the polymer backbone on anti-adhesive properties of the glycopolymers, a second glycomonomer,

Synthesis of N-[2-(-D-mannopyranosyloxy)ethyl] methacrylamide (EMM)

N-[2-(-D-mannopyranosyloxy

)ethyl] methacrylamide (EMM), having a short ethyl linker between the mannose and the methacrylamide group was prepared. The synthesis of EMM was directly adapted from the procedure described for the preparation of HMM (Scheme 2.2). Briefly, 2-bromoethanol was first reacted with α-peracetyl-mannose through glycosylation and a methacrylamide group was introduced through sequential azidation, hydrogenation and methacrylation steps.

The NMR and ESI spectra of intermediate derivatives are given in Chapter 5. The 1 H and 13 C NMR spectra of this second glycomonomer after deprotection are given in

Design of multifunctional RAFT agents for the preparation of star-shaped glycopolymers

In the quest for the generation of highly potent macromolecular FimH antagonists based on HM, the influence of several parameters such as the degree of polymerization and polymer topology (linear versus 3-arm and 8-arm glycopolymers) was investigated. To generate a series of well-defined macromolecules, we decided to employ a controlled/living radical technique, the reversible addition-fragmentation chain transfer polymerization (RAFT) which has proven to be extremely valuable for the construction of glycopolymers. [32][33][34][35] Whereas precisely defined linear glycopolymers can be straightforwardly generated by polymerizing HMM in the presence of commercially available chain transfer agents (such as 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid, CPADB), the preparation of well-defined star-shaped glycopolymers requires the design of multifunctional RAFT agents. With this goal in mind, we synthesized two original RAFT agents bearing three or eight dithiobenzoate mediating groups (see Scheme 2.3) where the core of the multifunctional molecule constitutes the leaving group (R group) of the RAFT agent (see Chapter 5 for details on the principle of RAFT polymerization). In the presence of such RAFT agents, the arms grow directly from the multifunctional core.

Practically, the multifunctional RAFT agents were synthesized in two steps (Scheme

2.3).

The carboxyl group of CPADB was first activated through reaction with N-hydroxysuccinimide (NHS) to favor amidification over aminolysis of the dithiobenzoate. 36 The resulting RAFT agent was then added in slight excess (typically 1.1 eq. per amine) to a solution containing the polyamines, tris(2-aminoethyl)amine or Octa Ammonium POSS to afford trifunctional and octafunctional RAFT agents (14 and 15). 1 H and 13 C NMR spectra of multi-functional RAFT agents after purification by column chromatography (see details in experimental part) are given in Figures 2.5 For CPADB based octafunctional RAFT agent, the peaks at 7.37, 7.54 and 7.88 ppm in 1 H NMR and 222.9 ppm in 13 C NMR confirms the presence of dithiobenzoate groups on the POSS cage. The peaks due to the POSS end groups are observed between 0.52-0.78, 2.24-2.75 and 3.22-3.45 ppm. The formation of amide linkages between the CPADB units and the POSS cage is confirmed by the presence of a peak at 6.56 ppm in 1 H NMR. The comparison of the integration of peaks c and h (Fig Polymerization rates were comparable in the presence of CPADB and 14 and significantly lower in the presence of 15 as mediating agent. Importantly, homopolymerizations proceeded with pseudo-first-order kinetics whatever the nature of the RAFT agent suggesting that the concentration of propagating species is constant during the whole process. The glycopolymers were subsequently studied by dynamic light scattering analysis (DLS) in pure water (at 1mg/mL). With the exception of L55 and L69, no aggregation was observed in these conditions. A single population was observed for the glycopolymers and the hydrodynamic diameters typically comprised of values between 4.2 nm for the shortest linear glycopolymer (L31) to 9.1 nm for the longest 8-arm star (O269). (Figure 2.12).

Synthesis of glycopolymer PEMM

In addition to this library of PHMM glycopolymers, a mannosylated glycopolymer with a short ethyl linker, poly(N-[2-(-D-mannopyranosyl-oxy)ethyl] methacrylamide)

(PEMM, M n = 53 600 kg.mol -1 , DP n = 183, and Ð = 1.17, see Table 2.1) was also prepared by RAFT polymerization in the presence of CPADB (see Scheme 2.5) in order to further evaluate the influence of the alkyl spacer on the interactions within the sugar-binding pocket of the FimH adhesin and in fine on inhibition of bacterial adhesion.

Scheme 2.5: Preparation of PEMM.

Chain-end modification of glycopolymers

In view of using the PHMM glycopolymers for biomedical applications in vitro and in vivo conditions, toxicity issues needed to be addressed. As discussed by Bulmus and co-workers, end-capping of glycopolymers (or other polymers) with dithiobenzoate moiety results in significant cytotoxicity. [37][38] To overcome these problems, the dithiobenzoate functionality at the ϖ-chain was quantitatively removed through aminolysis (addition of an excess of primary amine) and the resulting thiol was

Elaboration of library of PHMM/PGMA glyco(co)polymers with tunable compositions and microstructures

In order to investigate the influence of carbohydrate density and of the relative position of the carbohydrates on the polymer backbone, we subsequently focused on the RAFT preparation of copolymers based on HMM and glycidyl methacrylate (GMA) (Scheme 2.7). HMM was evidently selected to promote interactions with FimH adhesion and GMA was chosen to allow for post-modification of the glycopolymers through ring opening with various amines to gain insight into the influence of pendent groups adjacent to the carbohydrates on anti-adhesive properties.

Scheme 2.7: General approach for the preparation of HMM-based glyco-copolymers.

In order to get a clear picture of the composition and monomer sequence distribution of glycopolymers generated in statistical copolymerization, the reactivity ratios of HMM and GMA in radical polymerization were first evaluated. Polymerizations were carried out in DMSO in the presence of CPADB as mediating agent. The reactivity ratios of this copolymerization system were estimated utilizing two different methods:

the Jaacks method 39 and the usual Kelen-Tüdos method [40][41] (experimental details given in Chapter 5). Analogous values of reactivity ratios were obtained for both methods. The reactivity ratio of HMM and the reactivity ratio of GMA were determined to be 0.14/0.13 and 2.82/2.77 respectively (Figure 2.14 A and B). These disparate reactivity ratios indicate that GMA is consumed at a significantly higher rate than HMM and that the monomer feed and copolymer composition progressively drift with conversion (increase of f HMM and F HMM ). As a consequence, when the batch copolymerization of HMM and GMA is not carried out at the azeotropic composition, the microstructure of the resulting chains evolves from the α-end to the ϖ-end of the chain to become more and more rich in HMM (gradient microstructure). In order to prepare glycopolymers with tunable compositions and microstructures, 3 different protocols of (co)polymerization were thus explored: i) sequential polymerizations of HMM and GMA leading to block copolymers ii) batch copolymerizations (as described above) spontaneously leading to gradient copolymers and iii) semi-batch copolymerizations where GMA is continuously added to the monomer feed to obtain a more uniform distribution of GMA units within the chains. For each methodology, three different GMA/HMM molar compositions (1/2, 1/1, 2/1) and a constant global degree of polymerization of 200 were targeted. Determined from SEC analysis in THF and PS calibration (after acetylation); d : Ð from SEC analysis in THF and PS calibration (after acetylation); e : concentration of GMA in the initial batch; f : concentration of GMA in the pump-controlled syringe.

RAFT Preparation of PGMA-b-PHMM block copolymers

RAFT Preparation of PGMA-grad-PHMM copolymers through batch copolymerization

Considering the high difference in reactivity between GMA and HMM (polymerization of GMA is much faster than that of HMM), copolymers with a gradient profile composition should be generated through batch copolymerization due to progressive increase of the molar fraction of HMM in the monomer mixture (Scheme 2.9). Scheme 2.9. Construction of gradient glyco-copolymers. Consistent with the values of r HMM and r GMA , consumption of GMA was significant faster than that of HMM resulting in an increase of F HMM with conversion (herein, F HMM means the average HMM molar fraction in the copolymer), see Figure 2.18B. After purification, the composition and the number average molecular weight of the gradient copolymer were evaluated by 1 H NMR (Figure 2.19). The final composition of the copolymer (33.6% of GMA and 66.4% of HMM) is in good agreement with the initial composition in monomers and experiment molecular weights are close to the theoretical values calculated from the overall conversion in monomers (see Table 2

RAFT

RAFT Preparation of PGMA-stat-PHMM copolymers through semi-batch copolymerization

The procedure for semi-batch copolymerization as shown in Scheme 2.10 was adapted from a paper of Billon and co-workers. 42 Scheme 2.10 RAFT Preparation of PGMA-stat-PHMM copolymers using a semi-batch procedure

The preparation of P(GMA 63 -stat-HMM 134 ) through semi-batch copolymerization is described below to illustrate the general procedure. In brief, one third of the GMA (to be engaged in the polymerization) was initially mixed with HMM, CPADB and ACPA in anhydrous DMSO. Copolymerization was carried out at 70 °C (see Entry 4 in Table 2.2). After 100 min of copolymerization, 92% of GMA was consumed as well as 38%

of HMM (at this stage, the copolymer is then composed of 76% of HMM) (Figure In addition to the evolution of the copolymer composition over conversion which supports the synthesis of glyco-copolymers with different microstructures using a batch or a semi-batch copolymerization procedure (PGMA-grad-PHMM vs PGMA-stat-PHMM), we attempted to gain further insight into the chain microstructure through NMR spectroscopy. 1

Post-modification of the copolymers with various amines

Scheme 2.11. Post-modification of GMA containing copolymers.

The reactive pendant epoxy groups were subsequently engaged in post-polymerization modifications [43][44] to introduce various functional groups along the copolymer backbone through ring opening with primary amines and to further The modified glycopolymers were subsequently studied by dynamic light scattering analysis (DLS) in pure water (concentration at 1 mg/mL). The results are given in Table 2.3. After modification, several copolymers, especially those modified with taurine or (2-aminoethyl)trimethylammonium and having a low HMM content tend to form large aggregates in water possibly due to unwanted cross-linking side reactions between epoxy rings or straightforward self-assembly of the copolymers in solution.

The other copolymers are observed as unimers (Entries 6EA, 6AA, 7EA, 8EA……) or as small aggregates (Entries 1AA, 1TR, 3EA, 4AA……). : determined from DLS analysis in pure water (1mg/mL); b :not determined due to aggregation.

Conclusion

Aiming at generating glycopolymers with anti-adhesive properties, we have designed and prepared series of linear and star-shaped multi-valent homoglycopolymers by RAFT polymerization based on heptyl mannoside (HM), a nanomolar FimH antagonist.

In order to investigate the influence of carbohydrate density and of the relative position of the carbohydrates on the polymer backbone, the glyco(co)polymers composed of HMM and GMA have been prepared with tunable compositions and microstructures. Neutral, positively charged and negatively charged modified copolymers have then been prepared through a epoxy ring opening procedure.

Glycopolymer/FimH interactions and anti-adhesive properties of the glycopolymers described in this chapter are presented in Chapter 3.

Chapter 3

Glycopolymers for Anti-adhesive Treatment of E.

coli-Induced Inflammatory Diseases

Introduction

In the previous chapter, we described the preparation of precisely defined glycopolymers having multiple copies of n-heptyl -D-mannose (HM), as pendent motifs. Through the association of a judicious ligand selection with the presentation of multiple ligands on a polymeric scaffold, we expect to generate highly potent macromolecular inhibitors of type-1 fimbriae bacterial adhesion. In the present Chapter, we will thus evaluate the capability of precisely defined HM-based glycopolymers to efficiently bind to FimH lectin domains of E. coli strains (UTI 89 and AIEC LF82 clinically isolated from patients with urinary tract infections and Crohn's disease respectively). Practically, we will first investigate FimH adhesin/glycopolymers interactions through dynamic and static light scattering analyses. We will then evaluate the ability of the glycopolymers to associate with E. coli strains. Finally we will estimate the capability of the glycopolymers to inhibit recognition and binding of adherent-invasive E. coli with live epithelial cells or to disrupt adherent-invasive E. coli adhesion with live epithelial cells in vitro and ex vivo (Scheme 3.1).

Epithelial cells E. coli Type 1 pili

Mannosides Epithelial cells Epithelial cells Adhesion HM-based Glycopolymers

Multivalent FimH antagonists

The macromolecular FimH antagonists with different size and topology (described in Chapter 2) have been designed to assess the influence of HM valency and spatial presentation of the epitopes. We hypothesized that the polymeric antagonists would generate stronger multivalent interactions, on a valency-corrected basis, by recruiting a higher number of bacterial pili, mimicking more accurately E. coli attachment to the glycocalyx of the host cells.

Glycopolymer/FimH interactions

To gain preliminary insight into type 1 piliated E. coli bacteria/PHMM interactions, association of FimH adhesin and glycopolymer chains was investigated by dynamic light scattering (DLS). To closely apprehend the initial conditions, DLS was first carried out on FimH adhesin (83 µM) on the one hand and PHMM (L188, 15 µM) on the other hand (Figure 3.1A, blue and black curves, respectively). The D h -values, 4.2 ± 0.1 and 825 ± 33 nm are obtained for FimH adhesin and 10.7 ± 0.3 and 259 ± 2 nm for L188, corresponding to free macromolecules and pre-assembled ones. 1 The distributions shown in Figure 3.1A are mass-weighted. When converting them into number-weighted distributions, we get N small ~ 10 3 -10 5 N big , meaning that the number of free macromolecules (N small ) is in fact much larger than that of pre-assembled ones (N big ). A polymer suspension of L188 (20 µL at 15 µM) was subsequently added in a single step to a suspension of FimH adhesin (350 µL at 83 µM, [Man]/[FimH]  2). In addition to the one-shot addition procedure described above, PHMM/FimH interactions were also investigated for a stepwise addition procedure (see Figure 3.2).

Specifically, PHMM Hepes solution (L188, 15 µM) was sequentially added into FimH

Hepes solution (83 µM) to finally reach the same composition as the one obtained for the one-shot addition procedure. Again, the populations present in the solution were probed by DLS. For clarity's sake, we exclusively focus herein on L188/FimH system (however, association of FimH adhesin with PHMM chains was also confirmed for the other glycopolymers, see Chapter 5). In contrast to the one-shot addition procedure which resulted in the formation of a unique population of supramolecular cylinders, the sequential addition of glycopolymer aliquots in solution led to a far more complex picture. All along the addition of glycopolymers, three different populations were detected. A first population observed at very low R h was attributed to free/self-assembled FimH.

The dimensions of this population progressively decreased with glycopolymer content (from 2nm to 1nm) but contrary to the one-shot addition procedure, this population remained present even after complete addition of the glycopolymer. These data suggest that when the glycopolymer chains are sequentially incorporated in the FimH solution, it is not possible to quantitatively complex the adhesin with the pendent mannosides (in spite of a favorable HM/FimH ratio).

A second population with a R h that rapidly stabilizes at about 20 nm was also systematically detected. The proportion of these objects seems to increase with glycopolymer content. Finally a third population with a R h starting from 400 nm and rapidly dropping to about 250 nm is detected. This last population could be either a "reservoir" of aggregated FimH (its proportion is decreasing upon addition of glycopolymers) or FimH/L188 aggregates. In this context of multimodal distribution, no SLS analysis was performed for this experiment.

Glycopolymer/Bacteria interactions

The capability of PHMM chains to bind FimH in their natural environment, i.e. when the lectin is located to the extremity of bacteria pili, was then investigated. In this view, a bacterial suspension of type 1 piliated E. coli strain UTI89 (OD 650nm =1.3) was subjected to PHMM chains at concentrations ranging from 1 to 3 M of L188 (See and resulting in a "bacterial egg" agglomerate with approximate dimensions 98 x 51 μm.

Adhesion Tests

The propensity of the glycopolymers to prevent attachment of E. coli to host cells was further investigated through adhesion assays. Infections of intestinal epithelial T84 cells were performed with AIEC reference strain LF82 previously incubated with a series of mannosides (HMM, HM and -D-mannose) or PHMM (pre-incubation and T200). These results demonstrate that PHMM chains are not only capable to efficiently inhibit recognition events in a prophylactic approach but also to disrupt established interactions between epithelial cell receptors and FimH adhesion domains.

Ex-vivo tests

Based on the pre-and post-incubation assays data, L188 was finally selected for testing PHMM anti-adhesive properties in ex vivo conditions. More specifically, the ability of this macromolecular antagonist to prevent AIEC LF82 adhesion was tested on a model of colonic loops of transgenic CEABAC10 mice expressing CEACAM6. As observed in vitro on T84 cells, L188 actively prevents AIEC attachment to the colonic tissues (6.4

x 10 6 CFU/g of tissue for PHMM-free experiment vs 2.5 x 10 6 CFU/g of tissue in the presence of PHMM, 61% decrease of bacteria adhering to colonic mucosa).

ELLSA tests

Competitive Enzyme-Linked Lectinosorbent assays (ELLSA) were finally carried out to gain further knowledge on the association of the glycopolymers to FimH (as a function of the HM valency and topology). Briefly, the principle of this assay lies in the competition between FimH/glycopolymers and FimH/RNAseB interactions, RNAseB confirming the importance of the heptyl group between the mannosides and the polymer backbone. Taken together, these results suggest that the significant enhancement in activity over monovalent ligands (highlighted by the anti-adhesive tests) is not related to a higher intrinsic affinity of the polymer bound ligands for FimH, but to the capability of the glycopolymers to undergo multiple cross-linking interactions with living bacteria and to generate large aggregates in which only peripheral bacteria can adhere to the cells.

Preliminary results on copolymers anti-adhesive properties

Adhesion tests were also performed on the post-modified PHMM/PGMA copolymers described in the Chapter 2. In pre-incubation tests, infections of intestinal epithelial T84 cells were performed on AIEC reference strain LF82 which were previously incubated with a series of copolymers with different compositions, microstructures and pendent groups (see Table 2.2 and 2.3 in Chapter 2 for details). To establish comparisons between homopolymers and copolymers, one of the most potent homopolymers (L188) was used as control (52%). Pre-incubation assays were performed at 0.1 µM (on mannose unit basis) of FimH antagonists (Figure 3.8).

Inhibitory activities tended to slightly increase with ethanolamine content (48~58% for 1EA~3EA, 40~42% for 4EA to 6EA, 30~50% for 7EA~9EA) and to significantly decrease with the incorporation of positively charged trimethylammonium (60~100%).

No specific influence is observed for negatively charged copolymers (49~52%, 1T~3T). These results suggest that when it comes to (glyco)copolymers, the nature of the comonomer plays an important role on the anti-adhesive properties. The influence of the copolymers microstructure and HM density was far more difficult to unravel since no clear correlation between these features and the residual adhesion could be established. Post-incubation experiments were subsequently performed at a concentration of 0.1 µM (on mannose basis). Again, copolymer 7EA was the most potent inhibitor (35% vs 45% for L188). No enhancement of activity was observed for the other copolymers.

Pré-incubation à 0.1µM

L F 8 2 M O N O M E R E L 1 8 8 ( 1 7 9 ) 1 E A 2 E A 3 E A 4 E A 5 E A 6 E A 7 E A 8 E A 9 E A 1 A 2 A 3 A 6 A 1 T 2

Conclusions

In summary, in this Chapter we have evaluated the potential interest of the multivalent approach in preventing/disrupting adherent-invasive E. coli adhesion implied in gives the segment length (b ~ 7 × 0.154 nm). This calculated value is compatible with the experimental value 10.7 nm, thus showing the presence of single molecule coils. The molar mass of FimH adhesin (16.9 kg.mol -1 ) is also coherent with the experimental value 4.2 nm and data given in reference 5.

Barnich

Chapter 4

Simple but Precise Engineering of Functional

Nanocapsules through Nanoprecipitation

Introduction

The nanoprecipitation technique, is a simple means for generating narrow size distribution colloids without using ultra-sonication or high shear mechanical mixing methods. The principle of the technique lies in the supersaturation of whatever hydrophobic solute (oil, polymer, dye…) primarily dissolved in a hydrophilic solvent (e.g. acetone, THF……) when a large excess of water is added. Upon addition of water, the solution turns to be a non-solvent of the hydrophobic solute, inducing spontaneous emulsification. For certain proportions of solute, solvent and non-solvent, submicronic droplets or particles are generated (Scheme 4.1). 1 In this chapter, we wil aim at developing an undemanding method for the construction of multifunctional, oil-filled nanocapsules in a simple and straightforward batch experiment. In contrast to the general applications of nanoprecipitation in the preparation of particles. 2 we will intend to find experimental conditions allowing for the encapsulation of liquids by a polymer using the Ouzo effect. To identify a domain of composition in which narrowly dispersed core-shell nano-objects can be elaborated, we will first establish phase diagrams and will then focus on the production of nanocapsules decorated with biorelevant molecules within the shell and loaded with hydrophobic molecules in a simple one-pot procedure.

Determination of phase diagram

For preparing the carbohydrate-based membrane of the capsules, 2 water soluble linear glycopolymers (PHMM1 and PHMM2) were prepared by RAFT polymerization using agent CPADB as RAFT agent. The polymerization proceeded smoothly to yield polymers exhibiting molar masses of 24 and 102 kg mol

Cloudy point boundary of PHMM in water/acetone mixtures

As a first step towards the preparation of nanocapsules, phase diagrams for polymer/ water (solvent)/ acetone (non-solvent) and hexadecane (HD)/ acetone(solvent)/ water (non-solvent) ternary systems were determined. The cloud point boundary for the polymer/water/acetone ternary system was readily determined by titrating aqueous solutions of polymer with acetone (non solvent). It can basically be described by a straight line at a constant mass fraction of acetone (Figure 4.5). Noteworthy, the boundary is slightly shifted to lower mass fraction of solvent when the titration is performed with the polymer possessing the highest molar mass. Below this boundary, the polymer is fully dissolved in the solvent mixture whereas above it the glycopolymer chains generate large aggregates.

Ouzo domain of Hexadecane/Acetone/Water ternary system

Similar to the determination of the cloudy point boundary for PHMM/acetone/water mixtures, the binodal curve for the HD/acetone/water system was determined by titration of HD/acetone solution with water. The Ouzo limit, beyond which large droplets are formed, was determined by dynamic light scattering. Special requirements were made here to ensure reproducible measurements of the Ouzo limit: i) a non-ionic surfactant (Brij56) was added in the organic phase to stabilize the HD droplets once formed; ii) after adding the required content of water for emulsification, an extra load of water was rapidly poured to dilute the samples and avoid Ostwald The two curves were finally overlapped to obtain the Ouzo domain of HD, where monodisperse hexadecane nanodroplets can be generated in a reproducible manner without addition of surfactant.

Preparation of glyco-nanocapsules through Nanoprecipitaion

Phase diagram for nanocapsules preparation

The phase diagrams of glycopolymer/water/acetone and HD/water/acetone ternary systems were then overlapped (Figure 4.7), to identify conditions where nanocapsules with narrow size distribution can be built. A prerequisite for the generation of glyconanocapsules is to work in a domain of composition where HD will generate exclusively nanodroplets (in the Ouzo domain) and at the meantime where polymer chains preferentially stick at the surface of the nanodroplets.

After overlapping of the 2 phase diagrams, 6 regions can be distinguished (a~e). In zones a, b and c, PHMM is fully soluble forbidding the formation of a polymeric shell.

In zones d, e and f, PHMM generates aggregates. HD is fully soluble in zones c and d, demixes in zones a and c, and generates metastable emulsion in zones b and f. The domain f is the only one in which well-defined nanocapsules can be built through the conjoint precipitation of HD and glycopolymer (domain f given in green in Figure 4.7).

Practically, PHMM2 which leads to a cloudy point boundary for lower content of acetone (Figure 4.5), was preferred over PHMM1 in order to widen the domain of application. As a consequence, we decided to maintain the morphology of the nanocapsules through cross-linking of the shell.

Mass fraction of HD or glycopolymer

Capsules' stabilization using cross-linking strategy

For "freezing" the structure of capsules and simultaneously promoting their functionalization, isophorone diisocyanate (IPDI), a low water-sensitive cross-linker, was initially added to the organic solution. In were however still present. A further increase in IPDI content resulted in progressive shrinkage of the capsule membranes as a result of cross-linking reaction between PHMM chains, and the D z value of nanocapsules dropped to 200 nm and 150 nm upon the addition of 5 and 24 equivalents, respectively. The nano-objects gradually evolved to robust and perfectly spherical glyconanocapsules. Accordingly, leakage of HD was suppressed, and the polymer membranes are sufficiently robust not to be broken during the TEM process (casting/analysis).

Nanocapsules

Functionalization and loading of glyconanocapsules

To extend the scope of our procedure leading to the straightforward preparation of

Functionalization of the glyconanocapsules using biological molecules

The glyconanocapsules generated through nanoprecipitation are potential candidates for drug delivery systems. It is therefore of crucial importance to demonstrate that surface functionalization of the nanocapsules with molecules paving the way to imaging or targeting (dyes, ligands) can be undertaken. Incorporation of biotin in the course of the preparation of the nanocapsules was thus explored. In this view, an amino functionalized biotin (2 eq. per PHMM chain) was dissolved together with the glycopolymer in the aqueous solution, and the nanoprecipitation was carried out as previously described (24 equivalent per chain) to afford functionalized nanocapsules with a D z ~246 nm (PDI = 0.12). Consistent with a successful functionalization,

Functionalization of glyconanocapsule using Fluorescence probes

Fluorescence imaging is a common and practical technique for investigating and tracking of the delivery system in vitro and in vivo. Herein, a methodology for fluorescence labeling was explored to generate glyco-nanocapsules suitable for in vitro or in vivo imaging.

Encapsulation of hydrophobic probes within the core of the nanocapsules

To prepare dye-loaded nanocapsules, hydrophobic probes (e.g. pyrene) were directly added to the organic solution (HD with 2.5 % pyrene inside) prior to the nanoprecipitation procedure (24 eq. IPDI). The encapsulation of pyrene was assessed by the fluorescence emission spectrum of the resulting dispersion. The spectrum showed an ensemble of monomeric peaks (between 375 nm and 410 nm) and an additional broad unstructured band (from 425 to 550 nm) due to excited-stat-dimer

Multi-functionalization of glyconanocapsule using one-pot strategy

One pot procedure allowing for the preparation of well-defined nanocapsules and concomitant surface functionalization and loading of actives within the liquid core was then investigated. As a proof of concept, the preparation of biotinylated nanocapsules loaded with pyrene was explored. Practically, amino-functionalized biotin (2 equivalents per polymer chain) was dissolved with glycopolymer in water, and pyrene (2.5 % in HD) was added in the organic solution to perform the one-pot

Fabrication of NP coated glyconanocapsules

Besides the grafting of functions on the surface of nanocapsules and loading of hydrophobic actives within the core, the incorporation of metal nanoparticles could be of interest for biomedical application (imaging, therapy……). Preliminary experiments were carried out with commercially available dodecanethiol-functionalized gold nanoparticles (3~5 nm, AuNP) dispersed in the HD (2 wt%) and then in acetone.

Owing to their long alkyl grafts, the AuNPs were expected to be loaded within HD core during the nanoprecipitation process. This procedure afforded the incorporation of AuNP within the nanocapsules but contrary to our assumption, the AuNP were finally located in the shell of the nanocapsules and not inside the nanocapsules (Figure

4.16).

Again, the addition of nanoparticles had very limited impact on the colloidal properties of the nanocapsules. Using the same approach, nanocapsules with dodecanethiol-functionalized iron oxide nanoparticles (5 nm) within the shell were straightforwardly obtained (Figure 4.17

Investigation of the scope of the approach 4.5.1 Generation of PEMM based nanocapsules through nanoprecipitation

For emphasizing the general applicability of this method, we finally explored the preparation of nanocapsules from a different oil, miglyol 812 (more environment-friendly and biocompatible), and another synthetic glycopolymer (poly

Generation of P(HMM-co-PGMA) based nanocapsules through nanoprecipitation

As epoxy pendent groups are attractive for post-nanoprecipitation modification of the nanocapsules (with amino or thiol functionalized compounds for instance), we further attempted to build epoxy-functionalized glyconanocapsules from P(HMM-co-PGMA) copolymer having an homogeneous distribution of epoxy groups along the chains (to avoid the formation of micelles of copolymer in aqueous conditions). A new copolymer, P(HMM 206 -co-GMA 17 ) was then constructed using the semi-batch process described in Chapter 2. a) calculated from monomer conversion; b) determined from relative integration of the aromatic chain end group and polymer backbone peaks; c) determined of acetylated polymer from SEC with RI/light scattering detection in THF solution using PS calibration; d) Ð determined of acetylated polymer from SEC analysis in THF solution using PS calibration; e) determined from DLS analysis in pure water.

Determination of phase diagram

The cloudy point boundary for this glyco-copolymer in water (solvent)/acetone (nonsolvent) mixtures was first determined (see Figures 4. 19 for the establishment of phase diagrams) as previously described. Interestingly, the incorporation of around 10% mol of GMA units within the glycopolymer chains leads to a significant shift of the cloud point boundary to lower fraction of acetone (from 0.65 for PHMM to 0.58 for copolymer). By overlapping the two phase diagrams (See Scheme 4.3), one can immediately see the importance of this shift that allows for widening the tiny domain in which miglyol-filled epoxy-functionalized glyconanocapsules can be obtained.

Preparation of glyconanocapsules through nanoprecipitation

We first considered cystamine as a potential cross-linking agent in order to maintain the morphology of the objects without inducing any toxicity and at the meantime, to confer a degradable character of the nanocapsules (through reduction of the disulfide bond by glutathione or DTT). Unfortunately, under conditions of cross-linking

Post-nanoprecipitation modification of nanocapsules

After elaboration of the nanocapsules through nanoprecipitation and cross-linking of the polymeric shell, the presence of epoxy moieties of the surface of the nanocapsules was exploited to introduce new functional groups. The avidin-biotin system was investigated as a model of protein-ligand interaction with the broad application like tumor pretargeting clinical diagnostics and so on. Biotin ethylenediamine (1 eq. per polymer chain) was thus added into a mixture of capsules

A C B

Conclusions

In this chapter, we have demonstrated that the exploitation of phase diagrams of different solutes solvent-shifted by the ouzo effect enables the generation of well-defined nanocapsules by an extremely fast and straightforward method. The nanocapsules from PHMM can be shell-cross-linked, shell-functionalized, and loaded with actives at the same time in a one-pot procedure. This simplified route to functional nanocapsules is general and can be readily transposed to other systems to design new materials of practical applicability in different fields in which colloids are used. For instance, the generation of glyconanocapsules from P(HMM-co-GMA) leads to a convenient platform for post-nanoprecipitation modification, resulting in multi-functional nanocapsules. The glyconanocapsules exhibits the strong affinity towards E coli bacteria leading to the formation of huge bacteria/capsules aggregates. Dodecanethiol-functionalized gold nanoparticles (Sigma-Aldrich, particle size 3-5 nm, 2% w/v in toluene) were dispersed in hexadecane and toluene was removed under vacuum. Iron oxide (II, III) magnetic nanoparticles (Sigma Aldrich, particle size 10nm, 5mg/mL in Toluene) were dispersed in hexadecane and toluene was removed under vacuum. Amino-functionalized fluorescein (Fc) was obtained from protonated Fluorescein-PEG3-Amine TFA (Broadpharm) by adding 1.5 eq. of triethylamine (Sigma Aldrich) in water for 2 days. Amino-functionalized biotin was obtained from protonated biotin ethylenediamine hydrobromide (Sigma Aldrich) by adding 1.5 eq. of triethylamine (Sigma Aldrich). Miglyol 812 was purchased from SASOL (Germany).

Acetone, methanol, DMSO and petroleum ether were purchased from Carlo Erba.

Dialysis membranes (Mw cut-off 1000 Da) were purchased from Spectrum Laboratories, Inc. Other reactants were purchased from Sigma-Aldrich.

7-bromoheptan-1-ol (1) was prepared as described in the litterature. 1 Human intestinal epithelial cell line T84 were purchased from American Type Culture Collection (ATCC, CCL-248) and maintained in the culture medium recommended by ATCC in an atmosphere containing 5% of CO 2 at 37°C.

Intestinal epithelial cells

Human intestinal epithelial cell line T84 and human cervical adenocarcinoma cell line (Evrogen). The fragments and the pDONR221-nadB-cat were digested using BamH1.

Upon ligation, the plasmid pDONR221-nadB-cat-kat was obtained. The plasmid was transformed into E. coli LF82 and clones containing the plasmid selected on kanamycin/chloramphenicol LB-agar during an overnight incubation at 37 degrees.

Incubation during another four days at room temperature showed pinki colonies, confirming the expression of the katushka near-infra red fluoresent protein.

Enzyme-linked lectinosorbent assay

Enzyme-linked lectinosorbent assay (ELLSA) was performed using microplate reader BioTek-ELx800. RNAse B proteins (Sigma, R-7884) were used as a substrate of oligomannose glycans. For detection of FimH binding towards nature glycans were used rabbit anti-FimH polyclonal antibodies (Eurogentec) and goat anti-rabbit HRP-labeled secondary antibody (Enzo Life Sciences).

202

E. coli strain LF82 isolated from an ileal biopsy of a CD patient was used as AIEC reference strain. 4 Bacteria were grown overnight at 37 °C in Luria Bertani (LB) broth. A bacterial suspension was prepared in phosphate buffer saline (PBS) for in vitro adhesion assays or ex vivo assays on colonic mucosa from CEABAC10 mice. The protected monomer (6.1g, 11.5mmol) was finally deacetylated in methanol (250 mL), using 1M NaOMe/MeOH solution (5.1mL). The solution was stirred at room temperature for 3h. The pH of the solution was adjusted to 5, the solution was then filtrated and the solvent was removed to obtain the pure monomer (HMM) as a colorless gum. 4.1 g (98% yield).

Methods

[α] D = +11 (c= 0.4, Methanol). The protected monomer (2.1g, 4.46mmol) was finally deacetylated in methanol (87 mL), using 1M NaOMe/MeOH solution (1.8mL). The solution was stirred at room temperature for 3h. The pH of the solution was adjusted to 5, the solution was then filtrated and the solvent was removed to obtain the pure monomer (EMM) as a colorless gum. 1.2 g (92% yield).

[α] D = +17 (c= 0.

Synthesis of CPADB-based multi-functional RAFT agents

Preparation of Glcopolymer (PHMM and PEMM) RAFT polymerization of HMM

In a typical experiment (L47), a Schlenk flask (10 mL) was charged with HMM (100 mg, 2.8×10 -4 mol) dissolved in 0.

RAFT Polymerization of EMM

The polymerization procedure was adapted from the one described above for HMM.

In brief, solution polymerization of EMM (150mg) was performed in DMSO (0.

Acetylation of HM-based glycopolymers for SEC analysis in THF.

The glycopolymer (L47, 2mg, 1.16×10 -7 mol) was dissolved in a solution of anhydrous DMF (1mL) containing pyridine (4.3 mg, 2 eq. per hydroxyl group). Acetic anhydride (14.0 mg, 5 eq. per hydroxyl group) was incorporated into the solution. The solution was heated overnight at 50°C. After reaction, 150 mL of dichloromethane was added.

The solution was washed with water (2×200mL), dried by MgSO 4 and the solvent was evaporated to obtain the acetylated glycopolymer (yield 90%) as a beige paste.

Preparation of Glyco-copolymer

Determination of Reactivity ratios of HMM and GMA

Jaacks method

The Jaacks method was perfomed as high monomer feed ratio ([HMM] 0 /[GMA] 0 = 34 and 0.028, respectively). Specifically, HMM (122 mg, 3.40×10 -4 mol) and GMA (1.4 mg, 1.00×10 -4 mol) were dissolved in anhydrous DMSO (0.7 mL), CPADB (2 mg, 7.16×10 -6 mol) was engaged as RAFT agent and V70 (0.43, 1.39×10 -6 mol) was used as initiator.

Ran the polymerization at 40 °C, kinetics studies on the polymerization for each monomer via entire RAFT process were systematically conducted. Using -ln(1-Conv. HMM ) as y-axis and -ln(1-Conv. GMA ) as x-axis to make the graph, the results was treated with linear simulation, the reactivity ratio of HMM was just the slope. r GMA could be determined applying the reverse condition.

Kelen-Tüdӧs method

Kelen-Tüdӧs method needs three kinetic experiments were performed. The monomer feed ratio ([HMM] 0 /[GMA] 0 ) was used at 0.25, 1 and 2. CPADB was selected as RAFT agent using V70 as initiator ([HMM+GMA]/[CTA]/[Initiator] = 50/1/0.2). Performed the polymerization at 40 °C, kinetics studies on each copolymerization were systematically conducted. Using G/(α+F) as y-axis and F/(α+F) as x-axis to do the graph, the results was treated with the linear simulation. r GMA was the G/(α+F) as the F/(α+F) equaled to 1, and r HMM was the product of α |intercept|. α is used to distribute uniformly and symmetrically the experimental points between 0 and 1.

2 1 2 1 2 (m / m ) / F dm dm  1 2 1 2 1 2 / ( / 1) / m m G dm dm dm dm   mM a F F 

Preparation of Block copolymers

In a typical experiment, two step RAFT homopolymerization was employed. In the first polymerization, a Schlenk flask (10 mL) was charged with monomer GMA (200 mg, 14.1×10 -4 mol) dissolved in 2 mL of DMSO, V70 (1.08 mg, 3.50×10 -6 mol), and CPADB (3.92 mg, 14.1×10 -6 mol) dissolved in 1.5 mL of DMSO. After mixing, the pink solution was deoxygenated by three consecutive freeze-pump-thaw cycles. After the last cycle, the Schlenk flask was filled with nitrogen, allowed to warm to room temperature and finally immersed in an oil bath at 70 °C. After polymerization (conversion at 61%), the crude product was precipitated in an Et 2 O to remove unreacted monomer and initiator.

The resulting pink powder was dried overnight under vacuum to give 100 mg of pGMA 65 as a pale pink solid. M n SEC =11.5 kg.mol -1 , Ð = 1.31(using PS calibration in THF).

In the second step, a Schlenk flask (10 mL) was charged with monomer HMM (140 mg, 3.87×10 -4 mol dissolved in 0.5 mL of DMSO, ACPA (0.18 mg, 0.64×10 -6 mol), pGMA 65 (19 mg, 2.0×10 -6 mol) dissolved in 0.2 mL of DMSO. After mixing, the pink solution was deoxygenated by three consecutive freeze-pump-thaw cycles. After the last cycle, the Schlenk flask was filled with nitrogen, allowed to warm to room temperature and finally immersed in an oil bath at 70 °C. After polymerization (conversion at 64%), the Schlenk flask was plunged into iced water and the solution was then freeze-dried overnight. The crude product was redissolved in a minimal quantity of solvent (methanol/DMSO, v/v, 20/1) and precipitated in an acetone/petroleum ether mixture (v/v, 7/3) to remove unreacted monomer and initiator.

The resulting pink powder was dried overnight under vacuum to give 80 mg of pGMA 65 -b-pHMM 133 as a pale pink solid. M n SEC =51.0 kg.mol -1 , Ð = 1.20. (after acetylation, using PS calibration in THF).

Preparation of Gradient copolymers

In a typical experiment, a Schlenk flask (10 mL) was charged with monomer HMM (130 mg, 3.60×10 -4 mol) and GMA (22 mg, 1.54×10 -4 mol) dissolved in 0.3 mL of anhydrous DMSO, ACPA (0.26 mg, 0.93×10 -6 mol), and CPADB (0.65 mg, 2.34×10 -6 mol) dissolved in 0.1 mL of DMSO. After mixing, the pink solution was deoxygenated by three consecutive freeze-pump-thaw cycles. After the last cycle, the Schlenk flask was filled with nitrogen, allowed to warm to room temperature and finally immersed in an oil bath at 70 °C. After polymerization (conversion at 87%), the Schlenk flask was plunged into iced water and the solution was then freeze-dried overnight. The crude product was redissolved in a minimal quantity of solvent (methanol/DMSO, v/v, 20/1) and precipitated in an acetone/petroleum ether mixture (v/v, 7/3) to remove unreacted monomer and initiator. The resulting pink powder was dried overnight under vacuum to give 110 mg of p(GMA 66 -grad-HMM 130 ) as a pale pink solid. M n SEC =52.9 kg.mol -1 , Ð = 1.18. (after acetylation, using PS calibration in THF).

Preparation of Statistical copolymers

In a typical experiment, a Schlenk flask (10 mL) was charged with monomer HMM (150 mg, 4.15×10 -4 mol) and GMA (8 mg, 0.56×10 -4 mol) dissolved in 0.25 mL of water, ACPA (0.38 mg, 1.35×10 -6 mol), and CPADB (0.75 mg, 2.70×10 -6 mol) dissolved in 0.07 mL of DMSO. After mixing, the pink solution was deoxygenated by three consecutive freeze-pump-thaw cycles. After the last cycle, the Schlenk flask was filled with nitrogen, allowed to warm to room temperature and finally immersed in an oil bath at 70 °C. After 100 min, a DMSO solution (0.14 mL) of the GMA (0.84 M) was progressively added under argon by syring pump at a rate of 0.038 mL/h. After polymerization (conversion at 88%), the Schlenk flask was plunged into iced water and the solution was then freeze-dried overnight. The crude product was redissolved in a minimal quantity of solvent (methanol/DMSO, v/v, 20/1) and precipitated in an acetone/petroleum ether mixture (v/v, 7/3) to remove unreacted monomer and initiator.

The resulting pink powder was dried overnight under vacuum to give 125 mg of p(GMA 63 -stat-HMM 134 ) as a pale pink solid. M n SEC =54.8 kg.mol -1 , Ð = 1.15 (after acetylation, using PS calibration in THF).

Bio-assay for anti-adhesive of piliated E.Coli

Cytotoxicity tests

The cytotoxicity tests were performed using FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) according to manufactures' instructions and generally accepted procedures. Propidium iodide (PI) was used to counterstain necrotic cells (PI+). Apoptotic cells were detected as Annexin-V-FITC+/PI-cells.

Competitive Enzyme-Linked Lectinosorbent Assay (ELLSA)

Immunosorbent microplates (Nunc, Maxisorp) were coated with 100 µl of 5 mg/ml solution of RNAse-B in 100 mM carbonate/bicarbonate buffer pH 9.6. Plates were incubated at 4°C overnight and then washed (300 µl/well) three times with 10 mM phosphatebuffered saline (PBS) containing 0.15% Tween -20. All wells were blocked with 200 µl 3% bovine serum albumin (BSA) in 10 mM phosphatebuffered saline (PBS) containing 0.15% Tween -20 (PBST) and incubated at 37 °C 2 h. Then washed three times with PBST. Thiazole-bearing mannosides were dissolved in PBST at the concentrations indicated on graphs and added to the microwells. FimH was diluted in PBST to 0.07µM and added to each well of plate and incubated for 1h at room temperature. Wells were washed three times with PBST. Wells were incubated with 100 µl of rabbit-anti-FimH antibodies IgG (aFimH) diluted 1:5000 in PBST for 1h at room temperature. Wells were washed 3 times with PBST and incubated with 100 µl of goat-anti-rabbit HRP-labeled secondary antibody, Enzo Life Sciences (2ndAb-HRP) diluted 1:10000 in PBST for 1h at room temperature. The wells were washed three times with PBST and 100 µl of 3,3′,5,5′-Tetramethylbenzidine (TMB) were added to each well and incubated in darkness for 5 -15 min. The reaction was stopped with 100 µl/well of 1N sulfuric acid. Plate absorbance was analysed at 450 nm using microplate reader BioTek-ELx800.

Adhesion tests

Detailed procedures on experiments with Katushka UTI89+L188 and with

Caco-2 /HeLa with LF82 and L188

To evaluate the effectiveness of synthesized glycopolymers were used the model of co-incubation of Crohn-disease provoking strain LF82 of E.coli bacteria with human epithelia cells of Caco-2 and HeLa cell lines. Cells were grown on the glass. Before adding of a bacterial suspension, cells were washed three times by fresh culture medium (without antibiotic). 4 µl of bacterial suspension of type 1 piliated E. coli strain LF82 (OD=1.3) were added to cells. L188 inhibitor was added at concentration of 500 nM. After 2h of co-incubation of cells with bacteria (under the presence or absent of L188 inhibitor), samples were analyzed by fluorescent microscopy.

Adhesion assays of AIEC LF82 strain on intestinal epithelial cells T84.

The intestinal epithelial cells T84 were seeded in 48-well tissue culture plates at a density of 1.5 x 10 5 cells/well and were incubated at 37 °C for 48h. For pre-incubation protocol, AIEC LF82 bacteria were incubated for 1 h with mannoside derivatives (-D-Mannose, HM or HMM) or HM-based glycopolymers at final concentrations of 1; 0.1 and 0.01 µM (in mannose unit basis) then cells were infected with the mixture at a multiplicity of infection (MOI) of 10 bacteria per cell for 3 h. Monolayers were washed 3 times with phosphate buffer (PBS) and lysed with 1% Triton X-100 (Sigma) in deionized water. Samples were diluted and plated onto LB agar plates to determine the number of colony-forming units (CFU). For the post-incubation protocol, cells were infected with AIEC LF82 bacteria at a MOI of 10 for 3 h, then monolayers were extensively washed with PBS and inhibitory compounds were added at final concentrations of 10, 1 and 0.1 µM (in mannose unit basis) onto the cells for an additional 3 hours-period.

Ex vivo assay : Adhesion assays of AIEC LF82 strain on colonic tissues from CEABAC10 mice

Adhesion assay of AIEC LF82 bacteria were performed using colonic tissues from transgenic mice expressing the human CEACAM6 protein. 5 Briefly, 10 to 12-week-old FVB/N CEABAC10 transgenic mice were anesthetized, euthanized by cervical dislocation and colons were removed. Colons were washed twice in phosphate buffer saline (PBS) and segmented in independent loops of ≈ 0.6 cm. A volume of 100 µL containing 6×10 5 CFU of LF82 bacteria in PBS in the presence or absence of 3.4 µmol of the glycopolymer L188 were injected into the loops. Loops were incubated 4 h at 37°C in Krebs-Ringer bicarbonate buffer (Sigma) in an atmosphere containing 5% of CO 2 , and then opened and washed 4 times in PBS. Tissues were homogenized, appropriately diluted and plated onto Luria-Bertani agar plates containing ampicillin (100 µg/mL) and erythromycin (20 µg/mL) to select AIEC LF82 bacteria.

Preparation of glyconanocapsules through nanoprecipitation

Homopolymer

Phase diagrams' determination

Cloud point boundary. This equilibrium limit corresponds to the solubility limit of the polymer in acetone-water mixtures. The method consists of titrating with acetone various aqueous solutions of glycopolymer unimers (L47 or L221) until the mixtures turn milky (generation of swollen micelles). Compositions at the cloud point line are deduced from the mass of acetone added at the onset of turbidity and at a given glycopolymer initial concentration. Binodal curve. This equilibrium limit corresponds to acetone/water compositions where the HD is not soluble anymore. The method consists of titrating with water acetonic solutions of hexadecane of increased concentrations until the mixture turned milky, or even phase separates, through the generation of droplets.

"Ouzo limit". This boundary curve separates the Ouzo domain, where stable HD nanodroplets can be formed, from the high concentration region where HD phase-separates from the mixture. Special requirements were made here to ensure reproducible measurements of the Ouzo limit: i) a non-ionic surfactant (Brij56) was added in the organic phase to stabilize the HD droplets once formed; ii) just after the required content of water, an extra load of water was rapidly pourred to dilute the samples and avoid Ostwald ripening. The Ouzo limit was determined by measuring the number of counts by DLS, before and after filtrating the mixture with 1.20 μm polyethersulfone filters. Identical numbers of counts before and after filtration correspond to homogeneous emulsions of HD droplets, whereas a decrease of it indicates a loss of big, numerous HD droplets on the filter.

Preparation of PHMM-based glyconanocapsules

Typically, L221 (1mg) was dissolved in 350 mg H 2 O, whereas 2 mg HD were poured in 650 mg acetone separately. L221 aqueous solution was then poured into the acetone solution all at once, the transparent solution turned to be milky immediately.

After shaking for seconds, the solution was immediately characterized by DLS and TEM (Figure S4). To prepare cross-linked nanocapsules, various contents of IPDI were added primarily in the acetone phase prior solvent shifting. The solutions were left overnight and then characterized by DLS and TEM.

Preparation of PEMM-based nanocapsules

PEMM (0.5 mg) was dissolved in 540 mg of water, whereas 0.5 mg miglyol 812 and IPDI (0.05 mg, 24 eq.) were poured in 460 mg acetone. Polymer aqueous solution was then incorporated into the acetone solution all at once. The transparent solution turned to be milky immediately. The solution was left overnight and then characterized by DLS and TEM.

Glyconanocapsules' functionalization

Post-functionalization of glyconanocapsules using a simple amine. After 2h of cross-linking (1 eq. IPDI per PHMM chain), 0.0016mg of octylamine (1 eq. comparing to IPDI) was introduced into the milky mixture solution. After shaking for seconds, the solution was left overnight before being observed by TEM and DLS analyses.

Functionalization of glyconanocapsule using amino-functionalized biotin. L221

(1mg) and 0.007mg amino-functionalized biotin (2 eq per PHMM chain) was dissolved in 350 mg of water. 2 mg of HD and 0.06 mg IPDI (24eq per PHMM chain) were dissolved in 650 mg acetone. The L221 aqueous solution was then poured into the acetone solution to generate functionalized glyconanocapsules. The solution was left overnight. After dialysis for 3 days, 0.125 eq. avidin in 0.5 mL water was added into the glyconanocapsule solution and the evolution of the aggregated complex was analyzed by DLS. In the meantime, the dialyzed sample was measured by UV-Vis after introducing HAVA/Avidin reagent for determining the efficiency of biotin grafting reaction.

Fluorescence tags and assays

One-pot functionalization of glyconanocapsule by amino-functionalized fluorescein. L332 (1mg) and 0.015 mg amino-functionalized fluorescein (2 eq per PHMM chain) were dissolved in 350 mg of water. 2 mg of HD and 0.06 mg IPDI (24 eq per PHMM chain) were dissolved in 650 mg of acetone. The aqueous polymer solution was then poured into the acetone solution and the solution was left overnight.

After dialysis for 3 days, the glyconanocapsule solution was analyzed by fluorescence spectroscopy, DLS and TEM. No signal was observed for blank experiments with fluorescein.

Pyrene-Loading experiment. L221 (1 mg) was dissolved in 350 mg H 2 O. On the other hand, 2 mg hexadecane containing 2.5% wt pyrene and IPDI (0.06 mg, 24eq.) were added to 650 mg of acetone.The aqueous polymer solution was poured into acetone solution and the solution was left for overnight. After dialysis for 3 days, the glyconanocapsule solution was analyzed by Fluorescence spectroscopy, DLS and TEM.

One-pot preparation of pyrene-loaded biotinylated nanocapsules L221 (1 mg) and 0.007mg of amino-functionalized biotin (2 eq per PHMM chain) were dissolved in 350 mg of water. 2 mg of HD with 2.5% wt pyrene and IPDI (0.06 mg, 24eq.) were added to 650 mg of acetone. The aqueous solution was then poured into the acetone solution and the solution was left for overnight. After dialysis for 3 days, the glyconanocapsule solution was analyzed by Fluorescence spectroscopy and DLS.

AuNP-coated glyconanocapsules. L221 (1 mg) was dissolved in 350 mg of H 2 O. On the other hand, 2 mg hexadecane (with 2% wt dodecanethiol functionalized gold nanoparticles) were mixed with IPDI (0.06 mg, 24eq.) in 650 mg acetone. PHMM2 aqueous solution was poured into acetone solution and the solution turned immediately milky. The solution was left overnight and further analyzed by DLS and TEM.

Iron oxide NP-coated glyconanocapsules.

NP coated nanocapsules

L221 (1 mg) was dissolved in 350 mg of H 2 O. 2 mg of HD containing 0.6% wt of iron oxide nanoparticles and IPDI (0.06 mg, 24eq.) were added to 650 mg of acetone. The aqueous solution was poured into the acetone solution and the solution was left for overnight. The solution was further analyzed by TEM.

Copolymer

Glycopolymer/Water/Acetone system: Cloud point boundary. This equilibrium limit corresponds to the solubility limit of the polymer in acetone-water mixtures. The method consists of titrating with acetone various aqueous solutions of glycopolymer unimers until the mixtures turn milky (generation of swollen micelles). Compositions at the cloud point line are deduced from the mass of acetone added at the onset of turbidity and at a given glycopolymer initial concentration.

Miglyol/water/acetone system: Binodal curve. This equilibrium limit corresponds to acetone/water compositions where the Miglyol 812 is not soluble anymore. The method consists of titrating with water acetonic solutions of miglyol of increased concentrations until the mixture turned milky, or even phase separates, through the generation of droplets .

"Ouzo limit". This boundary curve separates the Ouzo domain, where stable Miglyol nanodroplets can be formed, from the high concentration region where Miglyol phase-separates from the mixture. Special requirements were made here to ensure reproducible measurements of the Ouzo limit: i) a non-ionic surfactant (Brij56) was added in the organic phase to stabilize the Miglyol droplets once formed; ii) just after the required content of water, an extra load of water was rapidly pourred to dilute the samples and avoid Ostwald ripening. The Ouzo limit was determined by measuring the number of counts by DLS, before and after filtrating the mixture with 1.20 μm polyethersulfone filters. Identical numbers of counts before and after filtration correspond to homogeneous emulsions of Miglyol droplets, whereas a decrease of it indicates a loss of big, numerous Miglyol droplets on the filter (Figure S3).

Preparation of glyconanocapsules

Typically, Glyco-copolymer (2.5 mg) was dissolved in 4100 mg H 2 O, whereas 5 mg miglyol was dissolved in 5900 mg acetone. The polymer aqueous solution was then poured into the acetone solution all at once, the transparent solution turned to be milky immediately. To prepare cross-linked nanocapsules, various contents of IPDI were added primarily in the acetone phase prior solvent shifting. The solutions were left overnight and then characterized by DLS and TEM.

Glyconanocapsules' functionalization

Functionalization of glyconanocapsule using amino-functionalized biotin. Glycopolymer (2.5 mg) was dissolved in 4100 mg of water. 5 mg of Miglyol and 0.34 mg IPDI (48 eq per polymer chain) were dissolved in 5900 mg acetone. The polymer aqueous solution was then poured into the acetone solution to generate glyconanocapsules. After overnight, the 0.01 mg amino-functionalized biotin (1 eq per PHMM chain) was added into solution and reacted at 40 °C for overnight. After dialysis for 3 days, 0.125 eq. avidin in 0.5 mL water was added into the glyconanocapsule solution and the evolution of the aggregated complex was analyzed by DLS. In the meantime, the dialyzed sample was measured by UV-Vis after introducing HAVA/Avidin reagent for determining the efficiency of biotin grafting reaction.

Functionalization of glyconanocapsule using amino-magnetic particle. Glycopolymer (2.5 mg) was dissolved in 4100 mg of water. 5 mg of Miglyol and 0.34 mg IPDI (48 eq per polymer chain) were dissolved in 5900 mg acetone. The polymer aqueous solution was then poured into the acetone solution to generate glyconanocapsules. After overnight, the 0.030 mL amino-turbobead was added into solution and reacted at 40 °C for overnight, and then characterized by DLS and TEM. Functionalization of glyconanocapsule using cadaverine functionalized Alexa 555 probe. Glycopolymer (2.5 mg) was dissolved in 4100 mg of water. 5 mg of Miglyol and 0.34 mg IPDI (48 eq per polymer chain) were dissolved in 5900 mg. The polymer aqueous solution was then poured into the acetone solution to generate glyconanocapsules. After overnight, the cadaverine functionalized Alexa 555 probe 0.03 mg amino-functionalized biotin (1 eq per polymer chain) was added into solution and reacted at 40 °C for overnight, and then characterized by DLS, fluorescent spectra and TEM.

Glyconanocapsules induced E.Coli aggregation.

E. Coli AIEC LF82 marked with GFP was incubated in the medium to produce a batch of bacteria solution (OD = 0.6). After evaporation of acetone, the aqueous solution of glyconanocapsules is concentrated to 33 and 66 μM. 10μL of capsules solution was added into 10μL bacteria solution with soft shaking. After 3h incubation, the solution was observed by confocal microscopy.

Kinetic study on capsules/bacteria aggregation was also performed in the similar process as above. 10μL of 66 μM capsules solution was added into 10μL bacteria solution with soft shaking. The observation of aggregates by confocal microscopy was performed after incubation for 30 min, 1 h, 2 h and 3 h.

General Conclusion

In the frame of the fight against antibiotic resistance, this PhD work was dedicated to the rational design of n-heptyl -D-mannose-based macromolecular anti-adhesives of type 1 piliated E. coli and to the evaluation of the capability of these glycomaterials to block bacterial adhesion.

In the first Chapter dedicated to the state of art in the field of carbohydrate-functionalized nanocapsules, we reviewed all the chemical and physical processes enabling to build synthetic glyconanocapsules from glycopolymers and polysaccharides. We highlighted that emulsification routes, self-assembly of amphiphilic copolymers or template strategies are routinely employed to generate such morphologies. We also pointed out that the nanoprecipitation process had not been used yet to build glyconanocapsules.

In Chapter 2, we described the preparation of well-defined linear and star-shaped glycopolymers. A first series of well-defined linear and star-shaped (3-arms and 8-arms) glycopolymers with multiple HM epitopes and different chain length (DP n from 31 to 269) was generated via RAFT polymerization. To further investigate the influence of carbohydrate density and of the relative position of the carbohydrates on the polymer backbone on the anti-adhesive properties, a series of precise-defined glyco(co)polymers with 3 different microstructures (block, gradient and statistical) and compositions (molar ratios of GMA/HMM are about 2/1, 1/1 and 1/2) was also constructed. The presence of pendent oxirane groups allowed post-modification via epoxy-amine reaction to introduce new functionalities or ionic charges on the copolymers.

In Chapter 3, we investigated the capability on the glycopolymers described in Chapter 2 to block bacterial adhesion. We explored glycopolymer/FimH and glycopolymer/bacteria interactions and showed that HM-based glycopolymers efficiently inhibit bacterial adhesion and disrupt established cell-bacteria interactions in vitro at very low concentration (0.1 µM). On a valency corrected basis, HM-based glycopolymers were proven to be respectively 10 2 and 10 6 times more potent than HM and D-mannose for their capacity to disrupt the binding of adherent-invasive E. coli to T84 intestinal epithelial cells. Anti-adhesive capacities of HM-based glycopolymers were finally demonstrated ex vivo in the colonic loop of a transgenic mouse model of CD.

In Chapter 4, we reported a simple and precise route to multi-functional glyconanocapsules constituted of anti-adhesive glycopolymers through nanoprecipitation. The phase diagrams of different solutes (hexadecane, miglyol 812 and glycopolymers) were precisely determined leading to the generation of monodisperse oil-filled and glycopolymer coated nanocapsules. The proposed route allows for elaborating the nanocapsules' walls, cross-linking and functionalizing the walls and encapsulating active compounds in a rapid one-pot procedure. Importantly, this approach to functional nanocapsules is not limited to PHMM homopolymers and can be transposed to other polymer systems to design new materials of practical applicability in different fields. For instance, miglyol-filled glyconanocapsules were conveniently built from P(HMM-co-GMA) and the presence of the oxirane groups was further exploited to carry out post-nanoprecipitation modifications with bio-relevant molecules (amino-functionalized biotin, fluorescent probes) or magnetic nanoparticles.

As PHMM polymer chains, HMM-containing glyconanocapsules were proven to interact strongly with E. coli leading to the formation of large bacteria/nanocapsules clusters.

To pursue this work dealing with the preparation of potent multivalent FimH Copolymerization of cyclic ketene acetals with HMM could thus give birth to biodegradable glycopolymers and could pave the way to the generation of biodegradable HM-based glyconanocapsules. Furthermore, it is also interesting to explore the preparation of multi-component nanocapsules through nanoprecipitation.

Taking advantage of the simple and straightforward process described in Chapter 4, polymers sharing a same domain of nanoprecipitation could be simultaneously coprecipitate to form multi-component nanocapsules. The success of this approach would extremely simplify the techniques to prepare multi-component nanocapsules, and accelerate the progress in the development of multi-functional nanocapsules.

1. 2 . 2 43 1. 2 . 2 . 1 1 . 2 . 2 . 2 1 . 2 . 2 . 3 1 . 2 . 3 64 1. 2 . 3 . 1 1 . 2 . 3 . 2 1 . 2 . 3 . 3 ( 1 ) 83 ( 2 ) 85 1. 2 . 4 86 1. 3 86 1. 4

 224322112221223123642311232123318328524863864 Formation of hollow nano-objects from amphiphilic copolymer self-assembly .................................................................................................... Polymersomes ............................................................................................................. Polymerization-Induced Self-assembly (PISA) ................................................... Micelle core removal strategy ................................................................................. Template strategy ..................................................................................... Particles-based strategy ................................................................................... Liposomes-based strategy ............................................................................... Modification of preset capsules. ..................................................................... Post-insertion ..................................................................................................... Grafting strategy ................................................................................................ Nanoprecipitation ..................................................................................... Conclusion ......................................................................................................... Reference ......................................................................................................... 88

Scheme 1 . 1 :

 11 Scheme 1.1: Different Capsular Structures 2 : (a) Liquid Core, (b) Polymer Matrix and (c) Active substance in molecular dispersion.

( 7 )

 7 Polymerization techniques. The generation of microcapsules by direct polymerization has represented a main development in the field of polymer colloids.

  The solution was then poured into an aqueous PG solution under stirring, resulting in PG nanocapsules (diameter around 240 nm) after evaporation of 2-propanol. The biodistribution of nanocapsules was preferably limited to the lymphatic circulation, to inject indium-111 labeled nanocapsules into rabbits. The γ-scintigraphy imaging analysis revealed a slow clearance of the nanocapsules from the injection site and their progressive accumulation (3.8% of the injected dose) in the popliteal lymph node over 48h.

Figure 1 . 1 .

 11 Figure 1.1. Functionalization of CS-PEG-Biotin nanocapsules with anti-TMEFF.40

(

  UCNC) encapsulated different s/a couples [PtTPBP/BDP-G (UCNC-G), PtTPBP/BDP-Y (UCNC-Y) or PdOEP/DPA (UCNC-B) to afford green, yellow or blue luminescent emissive nanocapsules (Figure 1.2). UCNC-G and UCNC-Y exhibited up conversion luminescence (UCL) emission (λ ex = 635 nm) with quantum efficiencies of 1.7% and 4.8%, respectively. The resulting nanocapsules may be applied to in vivo UCL bio-imaging of mice without skin removal.

Figure 1 . 2 .

 12 Figure 1.2. Schematic Illustration of TTA-UCL Process of the Upconversion Nanocapsules (UCNC), and Chemical Structures of Sensitizers (PdOEP and PtTPBP) and Annihilators (DPA, BDP-G, and BDP-Y).

  -maleoylchitosan (NMCS) (Chitosan, 500 kg/mol, DS = 0.23) as macromonomer. A surfactant Triton X-100 and a felodipine/chloroform mixture were dispersed in an aqueous phase containing NMCS. Polymerization of the vinyl groups at the (chloroform/water) interface initiated by K 2 S 2 O8/NaHSO 3 gave birth to nanocapsules with a diameter of 100 nm. Kinetics of drug release was in correlation with feed concentration and the DS of NMCS. The drug encapsulation efficiency was independent of feed concentration (~80%). In a similar manner, Chen et al. cross-linked N-maleoyl-modified chitosan (MCS) (Chitosan, 5 kg/mol, DS = 0.48) with 1,4-butanediol bis(3-mercapto-propionate) via UV-induced thiol-ene reaction at the oil/water interface (Figure 1.

Figure 1 . 3 .

 13 Figure 1.3. Preparation and TEM picture of modified chitosan nanocapsules.58

Figure 1 . 4 .

 14 Figure 1.4. Schematic representation of the preparation of Pluronic F127-chitosan nanocapsules by interfacial polymerization in emulsion conditions.59

Figure 1 . 5 .

 15 Figure 1.5. (a) Schematic diagram of the preparation of dextran based nanocapsules through interfacial polymerization under miniemulsion conditions. (b) TEM picture of DexMA based glyconanocapsules. (c) TEM picture of DexMA-PLMA based glyconanocapsules.60

Figure 1 . 6 .

 16 Figure 1.6.Preparation of HES nanocapsules using inverse miniemulsion process.68

  diazido-6.6'-dideoxysucrose (in the aqueous phase) and bi(propargyloxy)butane (in the miglyol phase) resulted in the formation of stable glyconanocapsules with a D h value of 200 nm (Figure 1.7).

Figure 1 . 7 .

 17 Figure 1.7. Preparation of glyconanocapsules by interfacial CuAAC step growth polymerization 71 .

  -b-poly[(2-β-Dglucopyranosyloxy)ethyl acrylate] (PS-b-PGEA) and different block lengths were designed by sequential ATRP polymerizations (PS 101 -b-PGEA 7 , PS 88 -b-PGEA 4 , PS 77 -b-PGEA 6 and PS 55 -b-PGEA 9 ). The block copolymers were then dissolved in organic solvents such as dioxane. Self-assembly of the block polymers was then induced by addition of DMF resulting in several morphologies (from vesicles to fibers) (Figure 1.8).

Figure 1 . 8 .

 18 Figure 1.8. Morphologies obtained from PS 55 -b-PEGA 9 in dioxane/DMF mixtures. 74-75 Content of dioxane (wt%): (A) 0, (B) 10, (C) 30, (D) 50, (E) 80, (F) 100.

Figure 1 . 10 .

 110 Figure 1.10. Access to various glyconano-objects through double post-polymerization

Figure 1 . 12 .

 112 Figure 1.12. Synthesis of PBLG-b-PGG block copolymers (Left) and high magnification TEM image of galactosylated polymersomes made of PBLG 20 -b-PGG 25 (Right).

91 Pasparakisand

 91 Alexander constructed block copolymers from 2-glucosyloxyethyl methacrylate (PGEMA) and diethyleneglycol methacrylate (PDEGMA) using sequential ATRP (P1, M n = 11.2 g/mol, Ð = 1.34, LCST = 28°C) or RAFT (P2, M n = 15.2 g/mol, Ð = 1.11, LCST = 28°C) polymerizations.

  96 A block copolymer polystyrene-b-poly(tetra-acyl-α-1-mannosyl styrene) (PS75 -b-PManAc 25 ) was generated by RAFT polymerization in THF. The deacetylation of the PManAc block in THF triggered a self-assembly process, leading to the formation of vesicles with glyco-inside structure (R h = 42 nm, R g /R h = 0.96). The vesicles were decorated with gold nanoparticles in the core by directly adding HAuCl 4 (0.2 mM) to the vesicles solution of PS 75 -b-PMan 25 in THF. Importantly, no addition of reducing agent was required. Solvent shifting (addition of water to THF) led to the destruction of vesicles and formation of micelles (R h = 11 nm) with a glycopolymer shell and a PS core (Figure 1.13).

Figure 1 . 13 .

 113 Figure 1.13.Glyco-inside and glycol-outside morphologies reported by Jiang and co-workers.96

  controlled polymerization of styrene to afford Dextran 40 -b-PS n (n = 5, 270, 775) block copolymers. Depending on the length of the PS block, micelles (Dextran 40 -b-PS 5 , R h = 28 nm) and vesicles (Dextran 40 -b-PS 270 , R h = 64 nm or Dextran 40 -b-PS 775 , R h = 77 nm)

(

  from 110 nm to 120 nm, TEM image). Addition of esterase (under physiological conditions) within the solution resulted in the cleavage of the ester linkages between the dextran backbone and the hydrophobic tails causing a fast release of CPT or Rh-B.

Figure 1 . 14 .

 114 Figure 1.14. Dextran-based nanovesicles developed by Jayakannan and coworkers. 103

Figure 1 . 15 .

 115 Figure 1.15. Route to photo-crosslinked chitosan hollow nanocapsules. 105-106

Figure 1 . 16 .

 116 Figure 1.16. Preparation of antibacterial chitosan-based nanocapsules. 107

  functionalized with alkyne groups by reductive animation on the anomeric position with propargylamine. The amphiphilic copolymer PBLG 23 -b-hyaluronan was then generated by CuAAC between ω-alkyne-functionalized hyaluronan and α-azide-PBLG ( Mw= 5000 g/mol, Ð = 1.1). As evidenced by DLS, SANS, TEM and AFM, the copolymers self-assembled into bilayer vesicles (R h = 220 nm, PDI = 0.08)

Scheme 1 . 5 .

 15 Scheme 1.5. Principle of the polymerization-induced self-assembly (PISA) method.117

Figure 1 . 17 .

 117 Figure 1.17. (Left) Preparation of nano-objects (spheres, worms or vesicles) by polymerization induced self-assembly; (Right) TEM images of the obtained nanoobjects.118

  Amphiphilic block copolymers (Py-PCL 32 -b-PVBG 10 , Ð = 1.28) were prepared by ring-opening polymerization of ɛ-CL initiated by hydroxymethyl pyrene and -(4-vinylbenzyl)-1,2:3,4-di-O-isopropylidene-D-galactose (VBIG). The self-assembly of these block copolymers resulted in the formation of micelles with a PCL core and a glycopolymer shell (size between 20 and 90 nm) by solvent shifting (addition of water into DMF). After cross-linking of the glycopolymer shell with glutaraldehyde, removal of the polyester core through basic hydrolysis afforded hollow glyconanocapsules with size ranging from 50 to 120 nm (Figure 1.18).

Figure 1 . 18 .

 118 Figure 1.18. Synthesis of amphiphilic block glyco-copolymer (Left) and TEM image of hollow glyconanocapsules after hydrolysis (Right).119

Figure 1 . 19 .

 119 Figure 1.19. Fabrication of hollow sugar balls relying on the core removal approach. 120 (a) Hexanediol diacrylate/water/AIBN/60°C; (b) hexylamine.

  reactions and/or adsorption of glycopolymers or polysaccharides on the surface of the templates. The most common technique to build the walls of capsules is based on Layer-by-layer (L-b-L) deposition. L-b-L deposition is a methodology first introduced by G. Decher 124 based on electrostatic attraction between polycations and polyanions.

Scheme 1 . 6 .( 1 )Figure 1 . 20 .

 161120 Scheme 1.6. L-b-L assembly on colloidal templates: i) deposition of a positively charged polyelectrolyte and washing; ii) deposition of a negatively charged polyelectrolyte and washing; iii) repeat i & ii to generate multi-layered core-shell particles; iv) core removal.125

  methacrylate (CPEM) were selected as templates. Surface initiated AGET-ATRP (activator generated by electron transfer for atom transfer radical polymerization) polymerization of N-[2-(4-vinylbenzenesulfoneamido)ethyl] lactobioneamide (VBSAELA) was further performed on the surface of core particles to generate lactose-functionalized glycoparticles. Hollow nanocapsules (diameter of 640 nm) were finally obtained after dissolution of PS template in THF solution. After core removal, the size of nanocapsules was shrunk by 15%. Glycomicrocapsules were prepared applying similar strategy using micro-sized PS as templates (Figure 1.21).

Figure 1 . 21 .

 121 Figure 1.21. Glyconanocapsules fabrication through template strategy (Top) and TEM image (bottom) of preparation of glyconanocapsules and glycomicrocapsules.

  130

Figure 1 .

 1 Figure 1.22. pH-triggered release behavior of CT-γ-PGA hollow nanocapsules. 133

  Thanks to the presence of thiols along chitosane backbones, the shell was gradiently cross-linked through the formation of disulfide bonds. The gradiently disulfide cross-linked nanocapsules (before core removal, diameter of 151 nm) were obtained after the degradation of the silica core by HF/NH 4 F treatment. These biocompatible nanocapsules were capable to encapsulate BSA (66.7 kg/mol) with an efficiency up to 80% by electrostatic attraction of the negatively charged BSA on the silica particles during the deposition. The release behavior was triggered by addition of glutathione (Figure1.23).

Figure 1 . 23 .

 123 Figure 1.23. Schematic representation of pH-responsive dextran sulfate (DS)/chitosan (CS) nanocapsules through L-b-L strategy. 139

Figure 1 . 24 .

 124 Figure 1.24. Schematic diagram of preparation of glucose-responsive glyconanocapsules by LBL strategy.141

Figure 1 . 25 .

 125 Figure 1.25. Design of Virus-like Polymer Particles by deposition of glycoprotein-mimic amphiphilic Block Copolymer chains around RNA-Based Polyelectrolyte Complexes.

  3.5) by reverse-phase evaporation. The polyelectrolyte shell was then formed by L-b-L sequential deposition of chitosan and alginate resulting in multi-layered nanocapsules with a diameter of 300 nm (thickness of 20 nm). These nanocapsules were capable to load FITC-Dextran (efficiency ~ 50%) with different MW (20, 40 and 70 kg/mol). The release of FITC-Dextran was triggered by the dissolution of the (liposome) inner part of the nanocapsules. The release behavior was further controlled by adjustment of pH and by the MW of FITC-Dextran (Table 1.4, Entry 23).

Figure 1 . 26 .

 126 Figure 1.26. Alginate-Chitosan nanocapsules through L-b-L strategy. 149

Figure 1 . 27 .

 127 Figure 1.27. Preparation and decomposition of multi-layered glyconanocapsules through DNA denaturation.

Scheme 2 . 1 :

 21 Scheme 2.1: Preparation of HMM. Reagents and conditions: a) HBr, toluene (reflux); b) anhydrous NaOAc, acetic anhydride, 100 °C; c) SnCl 4 , CF 3 CO 2 Ag, CH 2 Cl 2 ; d) NaN 3 , DMF, 70 °C; e) H 2 , Pd/C, p-TsOH, EtOH; f) methacryloyl chloride, CH 2 Cl 2 , Et 3 N; g) NaOMe, MeOH.

2 . 1 .

 21 D-mannose was then fully acetylated using anhydrous NaOAc and acetic anhydride at high temperature. The resulting α-peracetyl-mannose(2) was then engaged in a glycosylation reaction to attach the heptyl chain to the sugar moiety. The crude product (3) was subsequently reacted with sodium azide and the resulting azide group of (4) was then converted in a primary amine through catalytic hydrogenation. 5 was reacted with methacryloyl chloride to incorporate the polymerizable group through the formation of a stable amide bond. N-[7-(-D-mannopyranosyloxy)heptyl] methacrylamide (HMM, compound 7 in scheme 2.1) was finally obtained after quantitative deprotection of the mannose under basic conditions. The NMR and ESI spectra of intermediate derivatives are given in Chapter 5 (experimental methods). The 1 H and 13 C NMR of HMM are given in Figure The presence of the mannoside is clearly assessed by the peaks between 3.5 and 4 ppm and the peak at 4.91 ppm corresponding to the anomeric proton. 13 C spectrum further confirms the presence of the mannoside (typical signature of the anomeric carbon of mannose at 99.7 ppm, peak l). Peaks e-i belonging to the heptyl linker are observed between 1.4 and 1.7 ppm whereas the formation of the methacrylamide group is highlighted by the presence of the peaks a-c at 1.98, 5.48 and 5.71 ppm in 1 H NMR (peaks a-c at 17.75, 120.52 and 139.44 ppm in 13 C NMR). The preparation of HMM is further confirmed by ESI-MS analysis. Experimental mass for the main population at 384.1980 m/z are in excellent agreement with the theoretical mass of (M+Na) + ([C 17 C 31 NO 7 Na] + at 384.1993 m/z) (see Figure 2.2).

Figure 2 . 1 . 1 HFigure 2 . 2 .

 21122 Figure 2.1. 1 H NMR and 13 C NMR spectra of HMM (D 2 O, r.t.).

Figure 2 . 3 .Figure 2 . 3 . 1 H

 23231 Figure 2.3. The synthesis of EMM was confirmed by NMR analyses. Again, characteristic peaks of mannose moieties were clearly distinguished, especially the ones corresponding to anomeric proton (4.82 ppm) and carbon (100.3 ppm). The incorporation of polymerizable double bond group was ascertained by the presence of peaks at 1.89, 5.42 and 5.65 ppm for 1 H NMR and 18.35, 121.69 and 139.71 ppm for 13 C NMR. ESI-MS analysis gave further evidence of the preparation of EMM with the detection of a main population at 314.1204 m/z matching with the theoretical mass for (M+Na) + ([C 12 C 21 NO 7 Na] + 314.1210 m/z) (see Figure 2.4).

Figure 2 . 4 .

 24 Figure 2.4. ESI-MS spectrum of EMM.

and 2. 7 .Scheme 2 . 3 : 1 H

 7231 Scheme 2.3: Preparation of multifunctional RAFT agents. a) DCC, NHS, Chloroform; b) 13, MeOH/THF; C) 13, DIPEA, MeOH/THF.

Figure 2 . 5 .Figure 2 . 6 .

 2526 Figure 2.5. 1 H NMR and 13 C NMR spectra of CPADB-based trifunctional RAFT agent (CDCl 3 , r.t.).

2. 7 )

 7 gives clear evidence of the generation of the CPADB-based octafunctionalized RAFT agent. ESI-MS analysis reveals one major population at 1485.2639 m/z which is attributed to (M+2H) 2+ (theoretical mass of (M+2H) 2+ , i.e. C 128 H 154 N 16 O 20 S 16 Si 8 , is 2970.5200 (see Figure 2.8).

Figure 2 . 7 .

 27 Figure 2.7. 1 H NMR and 13 C NMR spectra of CPADB-based octafunctional RAFT agent (CDCl 3 , r.t.).

Figure 2 . 8 .Scheme 2 . 4 :Figure 2 . 9 .

 282429 Figure 2.8. ESI-MS of CPADB-based octafunctional RAFT agent.

Figure 2 . 9 .

 29 Figure 2.9. Semi-logarithmic plots of monomer conversion versus time for RAFT polymerization of HMM for CPADB, 14 and 15. (Conditions of polymerization given in Table 2.1) .

Figure 2 .

 2 Figure 2.10 1 H NMR spectrum of L47 (D 2 O, r.t.).

Figure 2 . 12 .

 212 Figure 2.12. DLS of PHMM glycopolymers (see Table2.1). A) Linear glycopolymers; B) 3-arms

Scheme 2 . 6 .

 26 Scheme 2.6. Chain-end modification of glycopolymers.

Figure 2 . 13 .Figure 2 . 14 :

 213214 Figure 2.13. A) SEC trace for peracetylated L31 between before and after chain-end modification (Use of PS calibration in THF); B) UV-vis absorption spectra between before and after chain-end modification.

13 CFigure 2 . 14 .

 13214 Figure 2.14. (A) Jaacks plots of the RAFT polymerization obtained for [HMM] 0 :[GMA] 0 =34; (B) Jaacks plots of the RAFT polymerization obtained for [HMM] 0 :[GMA] 0 =0.028; (C) Kelen-Tudӧs plot for the RAFT copolymerization of HMM and GMA in DMSO at 35 °C (α=0.57).

a:

  Calculated from monomer conversion; b : Determined from relative integration of the aromatic chain end group and polymer backbone peaks; c :

Scheme 2 . 8 .Figure 2 . 15 .

 28215 Scheme 2.8. Construction of block glyco-copolymers.

Figure 2 .

 2 Figure 2.16. 1 H NMR spectra of block copolymer PGMA 65 and PGMA 65 -b-PHMM 133 (DMSO-d6,

Figure 2 . 17 .

 217 Figure 2.17. SEC traces of PGMA 65 and PGMA 65 -b-PHMM 133 after acetylation (PS calibration, THF as eluent).

  Conditions of polymerization and macromolecular characteristics of the PGMAgrad-PHMM copolymers are presented in Table2.2 (Entries 3, 7 and 11) and supplementary characterizations are given in Chapter 5. The preparation of PGMA 66 -grad-PHMM 130 is discussed below. Copolymerization of HMM and GMA ([HMM] 0 /[GMA] 0 = 2.3, [GMA] 0 = 0.39 M) was carried out in anhydrous DMSO-d6 using a Young NMR Tube for in-situ NMR monitoring of the copolymerization at 70 °C in the presence of CPADB and ACPA as initiator.

Figure 2 .

 2 18A shows the first-order kinetic plot of the batch copolymerization, which was in an agreement with the theoretical prediction (simulated from the reactivity ratios of two monomers).

Figure 2 . 18 .

 218 Figure 2.18. (A) The fraction of HMM on copolymer chain during propagation in RAFT polymerization of P(GMA 66 -grad-HMM 130 ); (B) Pseudo first-order kinetic plots for copolymerization

Figure 2 . 19 . 1 H

 2191 Figure 2.19. 1 H NMR of gradient copolymer P(GMA 66 -grad-HMM 130 ) (DMSO-d6, r.t.).

Figure 2 . 20 .

 220 Figure 2.20. SEC analysis of P(GMA 66 -grad-PHMM 130 ) after acetylation (PS calibration, THF as eluent).

2 .Figure 2 . 21 .Figure 2 . 22 . 1 H

 22212221 Figure 2.21. (A) Evolution of F HMM with overall monomer conversion for preparation of P(GMA 63 -stat-HMM 134 ); (B) Pseudo first-order kinetic plots for the copolymerization of HMM and GMA (Entry 4, Table2.2)

Figure 2 . 23 .

 223 Figure 2.23. SEC analysis of P(GMA 63 -stat-HMM 134 ), SEC was performed after acetylation. (Use of PS calibration in THF, figures for other statistic copolymers were given in annex 6)

Figure 2 .

 2 Figure 2.24. 1 H NMR spectra (in DMSO-d6 at r.t.) of PHMM and PGMA homopolymers and of statistical, gradient and block copolymers having similar HMM/GMA composition (HMM/GMA ≈

Figure 2 .

 2 Figure 2.25. 13 C NMR spectra (in DMSO-d6 at r.t.) of of PHMM and PGMA homopolymers and of

  investigate the impact of the post-modification on the anti-adhesive properties of the glyco-copolymers. Post-polymerization modifications were carried out with ethanolamine, taurine and (2-aminoethyl)trimethylammonium (see Scheme 2.11). To avoid undesired cross-linking of the chains due to the coupling of secondary amines (formed after reaction of a primary amine with an epoxy) with unreacted epoxy groups, the post-modifications were carried out in anhydrous conditions in the presence of a very large excess of amine (50 equivalents of amine per epoxy ring, glycopolymer solution added drop by drop into the solution containing the primary amine). DMPP and NIPAAM were also engaged in the reaction to simultaneously ensure effective chain end capping of the copolymers. After dialysis and freeze-drying, white fluffy powders were obtained. The success of the post-modification and the removal of dithiobenzoate were confirmed by combining 1 H NMR and 13 C NMR (Figure2.26 and 2.27, ethanolamine modified P(GMA 63 -stat-HMM 134 ) was used as an example). The disappearance of the peaks of dithiobenzoate (about 8 ppm in 1 H NMR) directly confirmed the removal of the DTB group. The disappearance of the peaks of the epoxy ring (2.6 ppm and 2.8 ppm 1 H NMR, 49 ppm in13 C NMR) and of the adjacent methylene (66 ppm in13 C NMR) gave evidence of the success of the epoxy-ring opening reactions. The generation of ethanolamine functionalized copolymer was finally proved by the appearance of a new peak at 61 ppm corresponding to NH-CH 2 -CH 2 -OH in 13 C NMR. In SEC analysis, the shift of a peak towards the higher molecular weights (M nSEC = 63.3 kg/mol, Ð = 1.19) also confirmed the success of the post-modification (Figure 2.27).

Figure 2 .

 2 Figure 2.26. 1 H NMR spectra of ethanolamine modified glyco-copolymer using P(GMA 63 -stat-HMM 134 ) as precursor.

Figure 2 . 26 .Figure 2 . 27 .

 226227 Figure 2.26. 13 C NMR spectra of ethanolamine modified glyco-copolymer using P(GMA 63 -stat-HMM 134 ) as precursor.

Scheme 3 . 1 :

 31 Scheme 3.1: Principle of HM-based glycopolymers anti-adhesive action; A: Sequestration of free bacteria in the lumen of the gut; B: Disruption of established E coli-cell interactions

Scheme 3 . 2 :

 32 Scheme 3.2: Molecular and macromolecular FimH antagonists explored in this study.

1 ]Figure 3 . 1 :

 131 Figure 3.1: A) DLS, autocorrelation function (g(2) -1) and relaxation time distribution (A) at 90°; B) DLS, inverse of the relaxation time distribution maximum (1/τ) on squared wave vector modulus (q 2 ) between 15 and 140° by step of 5° (hydrodynamic diameter (D h ) and correlation coefficient (R 2 )); C) SLS, scattering intensity (I) on wave vector modulus (q) between 14 and 143° by step of

Figure 3 . 2 :

 32 Figure 3.2: Comparison plots between one-shot and stepwise addition.

Figure 3 . 3 ).Figure 3 . 3 :

 3333 Figure 3.3). As shown in Figures 3.3B and 3.3C, PHMM acts as a powerful glue to agglutinate E. coli bacteria. Introduction of PHMM results in the formation of bacterial clusters whose size increases with the concentration of ligands (50 x 98 m after adding 3M) confirming that the glycopolymer chains induce multiple binding events with the bacteria-bound FimHs and that one glycopolymer chain is capable to establish cross-linking interactions with FimH adhesins expressed by multiple bacteria.

Figure 3 . 4 :

 34 Figure 3.4 : A) Residual adhesion of the bacterial strain AIEC LF82 to T84 cells at a concentration of 0.1 µM of inhibitors (on mannose unit basis) in pre-incubation experiments. B) Residual adhesion of the bacterial strain AIEC LF82 to T84 cells at a concentration of 1 µM of inhibitors (on mannose unit basis) in post-incubation experiments. C) Inhibitory effects of mannosides or glycopolymers (at 0.01, 0.1 and 1 µM) on AIEC LF82 adhesion levels to T84 cells in pre-incubation experiments. D) Inhibitory effects of mannosides or glycopolymers (at 0.1, 1 and 10 µM) on AIEC

Figure 3 . 5 :

 35 Figure 3.5: Residual adhesion of AIEC strain LF82 to T84 cells at a concentration of 1, 0.1 and 0.01 µM of EMM or PEMM inhibitors (on mannose unit basis) in pre-incubation experiments.Results are expressed as percentages of adherent bacteria, AIEC LF82 adhesion in absence of inhibitors was considered as 100%

L F 8 2 LFigure 3 . 6 :

 236 Figure 3.6: Inhibitory effect of glycopolymer L188 on the adhesion level of the AIEC LF82 strain to intestinal tissue from transgenic CEABAC10 mice expressing human CEACAM6. Bacteria were pre-incubated 15 min with the inhibitor at a dose of 3.4 µmol of mannose units, then a volume of 100 µL of bacteria/inhibitor mixture was injected into colonic loops (0.6 cm for 1h). Results are expressed as percentages of bacteria adherent to the colonic mucosa (means ± sem, n=5 to 8 mice). 100% corresponds to the bacterial adhesion in absence of any treatment (NT for non-treated). *: P<0.05. With that aim, LF82 bacteria (6 x 10 5 CFU) or a mix of LF82 bacteria (6 x 10 5 CFU) and L188 (3.4 µmol of mannose units) were injected into the loops and incubated for one hour. After washing steps, LF82 adhesion levels to the intestinal tissue in the presence or absence of inhibitor were estimated (See Figure 3.6). As observed in

  being a protein (coated on the immunosorbent plates) which displays a mixture of oligomannose glycans (Man 5 GlcNAc 2 , Man 7 GlcNAc 2 , and Man 8 GlcNAc 2 ) capable to strongly bind to FimH (see technical details in Chapter 5). The intensity of FimH binding to the substrate represented by the optical density of chromophore absorbance at 450 nm was measured for a dilution series of glycopolymers as well as for -D-mannose, methyl -D-mannoside and HM as molecular references to build dose-response inhibition profiles (See Figure 3.7). In contrast to the conclusions drawn from the adhesion tests, the highest level of inhibition (IC 50 = 0.11 M) was obtained for HM. Thanks to the favoured binding of FimH to pendent HM ligands, the glycopolymers also efficiently disrupt FimH/RNAseB association but no gain of affinity is observed with macromolecular inhibitors (IC 50 = 0.40-1.5 M Man) owing to the monovalent nature of the FimH binding site. The apparent affinity of the pendent HM bound to the polymeric structure is indeed lower than the one observed for the free HM reference probably due to steric hindrance.

Figure 3 . 7 :

 37 Figure 3.7: (Top left) Principle of competitive lectin ELLSA assays (Bottom and Top left) Affinity testing of glycopolymers toward oligomannose glyco-epitopes using ELLSA. a: linear glycopolymers; b: 3-arms glycopolymers; c: 8-arms glycopolymers.

Figure 3 . 8 :

 38 Figure 3.8 : (Up) Residual adhesion of the bacterial strain AIEC LF82 to T84 cells at a concentration of 0.1 µM of inhibitors (on mannose unit basis) in pre-incubation experiments; (Bottom) Residual adhesion of the bacterial strain AIEC LF82 to T84 cells at a concentration of 1 µM of inhibitors (on mannose unit basis) in post-incubation experiments. Results are expressed as percentages of adherent bacteria, AIEC LF82 adhesion in absence of inhibitors was considered as 100% (means ±SEM). *P<0.05, **P<0.01, ***P<0.001.

Crohn's disease3. 10 References 1 .

 101 inflammation. Investigation of AIEC strain LF82 adhesion to T84 cells in the presence of molecular and macromolecular antagonists highlighted the remarkable anti-adhesive capacities of HM-based glycopolymers in vitro. Compared to the monovalent HM reference, HM-based glycopolymers were more than 100-fold more potent (valency corrected basis) in preventing/disrupting AIEC attachment to intestinal epithelial cells. The anti-adhesive effect was further evidenced ex vivo, in the colon of a transgenic mouse model of CD. As evidenced by the lower IC 50 's observed for macromolecular inhibitors in ELLSA assays performed with recombinant FimH, the enhancement in activity is not due to a greater intrinsic affinity for FimH targets but rather to crosslinking interactions and aggregation of the bacteria as suggested by DLS and fluorescence microscopy analyses on HM-based glycopolymers and FimH and bacteria/glycopolymer mixtures. These disparities between ELLSA and adhesion assays also indicate that in the quest for highly efficient pathogen anti-adhesives, classically performed carbohydrate-lectin interactions studies such as ELLSA, ITC or SPR, should be interpreted with caution when they do not account for pathogen aggregation that significantly impact the cell-adhesive properties. Importantly, the in vitro and ex vivo results reported here illustrate the strong potential of the multivalent concept to prevent/disrupt AIEC attachment to the gut and may constitute an important step towards an anti-adhesive treatment of E. coli-induced inflammatory diseases. To validate this last point, the most promising glycopolymers (L188) are currently tested on model mice in a laboratory of INSERM Clermont-Ferrand (Team of Dr Nicolas Barnich/Pr Arlette Darfeuille-Michaud) in in vivo conditions. The diameter of gyration (D g ) of 68.4 kg.mol -1 glycopolymer (DP n = 188) in Θ-solvent conditions is crudely given, taking into account the polymer pending groups, by:

Scheme 4 . 1 .

 41 Scheme 4.1. Schematics of the liquid-liquid nucleation process of Ouzo effect 1 .

10 (Figure 4 .

 104 -1 and Ð values below 1.4A). Dynamic light scattering was performed to evaluate the hydrodynamic diameter of individual chains (below 10 nm) in pure water solution (Figure 4.4B).

Figure. 4 . 4 .

 44 Figure. 4.4. (A) SEC traces of glycopolymers in NaNO 3 /NaOH aqueous solution (pH =7); (B) Hydrodynamic diameters as measured by DLS in pure water (1mg/mL).

Figure 4 . 5 .

 45 Figure 4.5. Phase diagram of PHMM1 (red) and PHMM2 (black) in water/acetone mixtures. The content of water is deduced from the two other weight fractions.

  ripening. The Ouzo limit was determined by measuring the number of counts by DLS, before and after filtrating the mixture with 1.20 μm polyethersulfone filters. Identical numbers of counts corresponded to homogeneous emulsions of HD droplets (Ouzo conditions), whereas a decrease of it indicates a loss of large HD droplets on the filter (out of Ouzo conditions) (Figure 4.6).

Figure 4 . 6 .

 46 Figure 4.6. Phase diagram of HD shifting in acetone/water mixture, showing the binary curve (in blue) and the Ouzo limit (in red).

Figure 4 . 7 .

 47 Figure 4.7. Overlapped phase diagrams of PHMM and HD in acetone/water mixtures. PHMM is soluble in a, b, c, and micellizes in d, e and f; HD is fully dissolved in c and d, generate metastable emulsions in b and f, and demixes in a and e. The domain f in which well-defined nanocapsules

  are built through conjoint precipitation of HD and glycopolymer is shown in green. ( ) HD molecule, ( ) HD droplet, ( ) swollen polymer chain, ( ) polymer coil, ( ) core-shell HD/glycopolymer capsule.To prepare the glyconanocapsules, the content of acetone should be between 65 and 70 wt%, with a final weight fraction of HD around 0.2 wt%. In a preliminary test, PHMM2 (1mg) was dissolved in 350 mg of water, whereas 2 mg of HD were dissolved in 650 mg acetone separately. Aqueous solution of PHMM2 was then poured into the acetone solution all at once, and the transparent solution turned to be milky immediately. Dynamic light scattering (DLS) of the resulting dispersion highlighted the presence of a single population with diameter (D z ) of 135 nm and a polydispersity index of 0.11 (Figure4.8A). However, after evaporation of the acetone, the population corresponding the nanocapsules totally disappeared and a new one corresponding to individual chains of PHMM2 (Diameter ~10nm) appeared. Besides, the nanocapsules (in water/acetone mixture) do not survive to TEM analysis (Figure4.8B). HD leaked out of the droplets and dark dots of precipitated glycopolymers were observed (similar morphologies were observed when aqueous solution of PHMM2 were directly casted).

Figure 4 . 8 .

 48 Figure 4.8. (A) DLS of non-crosslinked HD-filled glyconanocapsules (right, black curve) obtained by dissolving PHMM2 (1mg) in water (0.35g), dissolving HD (2mg) in acetone (0.65g) and pouring

Figure 4 . 9

 49 are given the TEM pictures of a series of nano-objects of increasing complexity. The first attempt of shell cross-linking with 0.5 equivalent of IPDI per chain failed at efficiently stabilizing the formed glyconanocapsules. (see Figure 4.8B and 4.9B). Upon the addition of 1 equivalent of IPDI per chain, the D z value of the capsules increased to 248 nm (PDI = 0.07), and the formation of a thin, fragile and deformable membrane was observed by TEM at the surface of the droplets (Figure 4.9). HD domains and glycopolymer nanoparticles (from self-assembly of the free unreacted chains during sample casting)

Figure 4 . 9 .

 49 Figure 4.9. (A) DLS of glyconanocapsules cross-linked with 1 (black), 5 (green) and 24 (red)

Figure 4 . 10 .

 410 Figure 4.10. Morphologies observed for different HD/Acetone/water compositions after solvent shifting and IPDI polymer cross-linking.

Scheme 4 . 2 .

 42 Scheme 4.2. One-pot cross-linking and functionalization of the polymer shell.

4. 4 . 1 Figure 4 Figure 4 . 11 .

 414411 Figure 4.11. (A) Size distribution of IPDI-cross-linked nanocapsules and octylamine-functionalized nanocapsules in water (B) TEM picture of the octylamine-functionalized glyconanocapsules.

biotinylated nanocapsules were proven to disrupt 4 'Figure 4 . 12 .

 4412 Figure 4.12. (A) Size distribution of biotin-functionalized glyconanocapsules in water before and after conjugation to avidin; (B) UV-absorption spectra of biotinylated and non-biotinylated nanocapsule. HABA test results were calculated using absorbances measured at 500nm.

Fluorophores were attached to 20 F l u o r e s c e n t l y l a b e l l e d g l y c o n a n o c a p s u l e s F r e e F l u o r e s c eFigure 4 . 13 .

 20413 Figure 4.13. (A) Calibration curve of amino-functionalized fluorescein (Fc) in water. (B) Fluorescence emission spectra of fluorescein-labelled glyconanocapsules (λ ex =450 nm). (C) TEM picture of fluorescein-labelled glyconanocapsules. (D) Size distribution of Fc-functionalized glyconanocapsules in water.

(Figure 4 Figure 4 . 14 .

 4414 Figure 4.14. (A) Calibration curve of pyrene in hexadecane (λ ex = 335 nm). (B) Fluorescence emission spectra of Pyrene-loading glyconanocapsules. The straight line shows the loaded pyrene glyconanocapsules with concentration at 0.32 ppm, the dash line corresponds to the reference (free pyrene at same concentration in hexadecane). The presence of a clear excimer peak (from 460 nm to 550 nm) for the pyrene-loaded sample confirms the hydrophobic environnement of pyrene molecules embedded in HD oil. (C) TEM picture of pyrene-loaded glyconanocapsules. (D) Size distribution of pyrene-loaded glyconanocapsules in water.

Figure 4 . 15 .

 415 Figure 4.15. (A) Fluorescence emission spectra of pyrene-loading glyconanocapsules. (B) Size distribution of biotin-functionalized glyconanocapsules in water before and after conjugation to avidin.

Figure 4 . 16 .

 416 Figure 4.16. (A) DLS and (B) TEM picture of AuNP-coated glyconanocapsules obtained using a one-pot procedure .

Figure 4 . 17 .

 417 Figure 4.17. TEM picture of iron oxide NP-coated glyconanocapsules obtained using a one-pot procedure.

Figure 4 . 18 .

 418 Figure 4.18. A) Phase diagrams of miglyol/acetone/water. (B) Overlapped phase diagrams of PEMM and miglyol/acetone/water. (C) Size distribution of IPDI-cross-linked PEMM based nanocapsules in water (D) TEM picture of the resulting glyconanocapsules.

D

Figure 4 . 19 .

 419 Figure 4.19. (A) Phase diagram of copolymer in water/acetone mixtures. The content of water is deduced from the two other weight fractions. (B) Phase diagram of Miglyol shifting in acetone/water mixture, showing the binary curve and the Ouzo limit.

Scheme 4 . 3 .

 43 Scheme 4.3. Overlapped phase diagrams of Copolymer and Miglyol 812 in acetone/water mixtures.

(Figure 4 .

 4 Figure 4.20 could not be obtained in a reproducible manner. As a consequence, this route was finally given up and IPDI was further used as a cross-linking agent.

Figure 4 . 20 .

 420 Figure 4.20. TEM pictures of glyconanocapsules cross-linked 9 equivalent cystamine.

Figure 4 . 21 .

 421 Figure 4.21. (A) DLS of glyconanocapsules cross-linked with 24 (green) and 48 (red) equivalents of IPDI (per polymer chain). (B) and (C) Selected TEM pictures of glyconanocapsules cross-linked with 24 and 48 eq. of IPDI.

  was heated overnight at 40°C. The surface decoration does not impact the dimensions of the nano-objects (D z = 111 nm). After dialysis, the biotinylated nanocapsules were mixed with an aqueous solution of avidin (0.25 eq per chain), resulting in the formation of very large aggregation of nanocapsules owing to strong interaction between avidin and grafted biotin (Figure4.22A). Accordingly, the characteristic UV based of pre-established HABA/Avidin complexes at 500 nm in water (see Figure4.22B) rapidly dropped when modified capsules was added thanks to the strong affinity of avidin for biotin moieties.

Figure 4 . 22 .Figure 4 27 BFigure 4 . 23 .Figure 4 . 24 . 4 . 5 . 6

 422427423424456 Figure 4.22. (A) Size distribution of biotinylated nanocapsules in water before and after conjugation to avidin. (B) UV-absorption spectra of biotinylated and non-biotinylated nanocapsules. HABA test results were calculated using absorbances measured at 500nm. (C) TEM picture of the biotin-functionalized glyconanocapsules.

Figure 4 . 25 .

 425 Figure 4.25. Confocal fluorescence microscopy pictures of (A) E. Coli AIEC LF82 bacteria. (B) Glyconanocapsules (33 μM)-bacteria aggregates. (C) Glyconanocapsules (66 μM)-bacteria aggregates.

Figure 4 . 26 .

 426 Figure 4.26. Kinetic study of aggregation between glyconanocapsules and bacteria (A) 30 min, (B) 1h, (C) 2h and (D) 3h.

  cyanovaleric acid) (ACPA), isophorone diisocyanate (IPDI), hexadecane, water (HPLC), deuterium oxide (D 2 O), avidin from egg white, and pyrene were purchased

  HeLa were purchased from American Type Culture Collection (ATCC, CCL-248; ATCC, CCL-2) and maintained in the culture medium recommended by ATCC in an atmosphere containing 5% of CO 2 at 37°C. Human colorectal adenocarcinoma cell line Caco-2 was obtained from the Cell Culture Collection of the Department of Structural and Functional Glycobiology, Faculty of Biology, University of Science and Technology of Lille 1. Cells were cultured in EMEM medium supplemented with 20% fetal bovine serum (FBS) and penicillin/streptomycin.Construction of fluorescent strain UTI89 nadB::cat-katThe strain LF82 nadB::cat-kat was constructed with the recombination system reported by Datsenko and Wanner 2 except that the linear fragments used were long homologous recombinant sequences amplified from pDONR221-nadB-catkat derived from pDONR221-nadB3 . Briefly, the recombinant plasmid pDONR221-nadB-cat was constructed first by amplification of the chloramphenical gene from pKD32 using the primers EcatF GCAATGAATTCTAGAGAATAGGAACTTCGGAAT and EBcatR (ATTGCGAATTCGGATCCAAGTATAGGAACTTCGGCGCGCCTA), enzymatic digestion by EcoRI and ligation into pDONR221-nadB. The katushka gene kat were amplified with primers KatF, containing the BamHI site and em7 promoter sequences (CGCGGATCCTGTTGACAATTAATCATCGGCATAGTATATCGGCATAGTATAATA CGACAAGGTGAGGAACTAAACCATGGTGGGTGAGGATAGCGTGCTGA) and KatR (CGCG GATCCGCATTAGCTGTGCCCCAGTTT) from pTurboFP-635C

1 : 1 ,

 11 Scheme 5.1: Preparation of HMM. Reagents and conditions: a) HBr, toluene (reflux); b) 1, SnCl 4 , CF 3 CO 2 Ag, CH 2 Cl 2; c) NaN 3 , DMF; d) H 2 , Pd/C, p-TsOH, EtOH; e) methacryloyl chloride, CH 2 Cl 2 , Et 3 N; f) NaOMe, MeOH.

  35 mL of water, 4,4'-azobis (4-cyanovaleric acid) (ACPA, 0.52 mg, 1.85×10 -6 mol), and 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid (1.56 mg, 5.55×10 -6 mg) dissolved in 0.35 mL of DMSO. After mixing, the pink solution was deoxygenated by three consecutive freeze-pump-thaw cycles. After the last cycle, the Schlenk flask was filled with nitrogen, allowed to warm to room temperature and finally immersed in an oil bath at 70 °C. After polymerization (conversion at 84%), the Schlenk flask was plunged into iced water and the solution was then freeze-dried overnight. The crude product was redissolved in a minimal quantity of methanol and precipitated in an acetone/petroleum ether mixture (Volume ratio at 7/3) to remove unreacted monomer and initiator. The resulting pink powder was dried overnight under vacuum to give 60 mg L47 as a pale pink solid. Polymerizations of HMM in the presence of CPADB-based trifunctional RAFT agent were carried out in similar conditions. Polymerizations of HMM in the presence of CPADB-based octafunctional RAFT agent were carried out in pure DMSO using AIBN as initiator owing to the poor solubility of the RAFT agent in water. Kinetics of polymerization and SEC traces of the resulting star-shaped glycopolymers are given in Figures S1 and S2

  5mL) and mediated by 4-cyano-4-(phenylcarbonothioylthio) pentanoic acid using 4,4'-azobis (4-cyanovaleric acid) as initiator ([M] 0 /[CTA]/[ACPA]=250/1/0.3). The solution was deoxygenated by three consecutive freeze-pump-thaw cycles and the Schlenk flask was finally filled with nitrogen, allowed to warm to room temperature and finally immersed in an oil bath at 70 °C. After polymerization (73% of conversion), the Schlenk flask was plunged into iced water and the solution was then freeze-dried overnight. The crude product was redissolved in a minimal quantity of DMSO and precipitated in an acetone/petroleum ether mixture (Volume ratio at 7/3) to remove unreacted monomer and initiator. The powder was dried overnight under vacuum to give 90 mg of PEMM as a pale pink solid. PEMM (M w = 96 kg.mol-1, Ð = 1.17) was characterized by SEC with RI/LS detection in NaNO 3 /NaOH aqueous solution (pH =7).Removal of the dithiobenzoate chain ends. HM-based glycopolymer (L47, 40mg, 2.32×10 -6 mol) was dissolved in a 1mL of DMSO (1mL). The solution was deoxygenated by three consecutive freeze-pump-thaw cycles. Ethanolamine (1.4 mg 10 eq) was added in solution and the pink color immediately disappeared. NIPAAm (13.2 mg, 50eq) and dimethylphenylphosphine (0.3 mg 1eq) were then incorporated and the solution was stirred overnight at room temperature. Water (2 x 100 mL) and dichloromethane (200 mL) were added to the solution. The aqueous phase was collected, dialyzed against deionized water for 2 days and freeze-dried to afford a fluffy white powder (36 mg, 90%).

  antagonists, it would be of particular interest to synthesize a new generation of glycomonomers based on thiazolylaminomannosides.1 Indeed, our close collaborators, Sébastien G. Gouin, Julie Bouckaert and Arlette Darfeuille-Michaud recently demonstrated that this class of monovalent FimH antagonists leads to a 100 times increase in inhibitory potency compared to HM. It would also be valuable to explore other routes to multivalent inhibitors. Instead of recoursing to controlled radical polymerization techniques, the antagonists could be directly attached onto biodegradable reactive polysaccharide backbones. As the HM-based glycopolymers have shown good results in vitro and ex vivo, it would be interesting to study more in details their anti-adhesive properties in in vivo conditions In order to extend the scope of HM-based nanocapsules in drug delivery applications, we should first solve the problem of biodegradability of the present polymer shell. To overcome this issue, we propose to incorporate biodegradable groups into the glycopolymer backbones. For instance, it has been shown by Julien Nicolas and co-workers that controlled radical copolymerization of cyclic ketene acetals with common monomers such methacrylates or styrene affords the preparation of well-defined macromolecules with cleavable ester links within the chain.2

Annex Annex 1 .Annex 2 .Annex 3 .Annex 4 . 247 Annex 6 .Annex 7 .Figure A1. 1 HFigure A2. 1 HFigure A3. 1 HFigure A4. 1 HFigure A5. 1 HFigure A6. 1 HFigure A7. 1 H

 1234247671111111 Figure A1. 1 H NMR and 13 C NMR of azido-functionalized mannoside (4, CDCl 3 )

Chapter 1 Preparation of Carbohydrate-based nanocapsules 1.1 Introduction

  ....................................................................................................... 20 1.2 Routes to carbohydrate-functionalized nanocapsules .................................. 24 Emulsion diffusion technique ................................................................................. Double emulsification technique ............................................................................ Emulsion coacervation technique ..........................................................................

	1.2.1 Emulsification routes to carbohydrate-functionalized nanocapsules . 24
	1.2.1.1 1.2.1.2 1.2.1.3

1.2.1.4 Polymer coating technique ...................................................................................... 1.2.1.5 Mini-emulsion technique ..........................................................................................

Table 1 . 1 :

 11 Emulsification routes to carbohydrate-functionalized nanocapsules.

	Entry	Routes	Polymer	Solution	Emulsification Method	Cross-linker	Dimension (nm)	Ref.
	1	Double emulsification	Chitosan, Gelatin	Toluene/water/Toluene	Mechanical agitation	Sodium glutaraldehyde sulphate,	100~700	27
	2	Double emulsification	Alginate	Water/Phospholopid oil/Water	Stirring	Calcium chitosan chloride,	488	28
	3	Emulsion coacervation	Alginate	Acetone or ethanol/Water	Sonication	chloride Calcium	373 (acetone) 263 (ethanol),	29-30
	4	Emulsion coacervation	Alginate	Ethanol, benzyl alcohol or DMSO/Water	Sonication	Chitosan	400	31
	5	Emulsion coacervation	Alginate	Hexane/Water	Stirring, sonication	Calcium chloride	34	32
	6	Polymer coating	Oligochitosan, PECL	Acetone/Water	Stirring		300~500	33-35
	7	Polymer coating	Oligochitosan	Acetone/Water	Solvent displacement		200	36-37
	8	Polymer coating	Polyglucosamine	2-propanol/Water	Stirring		242	41
	9	Polymer coating	Chitosan	Acetone-ethanol/Water	Solvent displacement		148~185	42
	10	Polymer coating	Chitosan	Ethanol/Water	Solvent evaporation		145

Table

Table 1 . 2 :

 12 Formation of carbohydrate-functionalized nanocapsules using mini-emulsion conditions.

	Entry	Interfacial Reactions	Polymer	Monomer	Solution	Emulsion method	Cross-linker	Dimension (nm)	Ref.
		Cross-linking	Diamine, Chitosan		Toluene/Water	Ultrasonication	diepoxide	130~280	56
		Cross-linking	N-maleoylchitosan		Chloroform/Water	Mechanical agitation		100	57
		Cross-linking	N-maleoylchitosan		Chloroform/Water	Ultrasonication	1,4-butanediol bis(3-mercapto-propionate)	57~388	58
		Cross-linking	Chitosan		1,4-dioxane/Water Ultrasonication	Pluronic F127 PEO 100 -PPO 65 -PEO 100	37~240	59
		Cross-linking	N,N-diethylaminoethyl dextran, polylactide macromonomer		Chloroform/Water	Mechanical agitation		233	60
								324
		Cross-linking	Dextran or Starch		Water/Clyclohexane	Sonication	Tolylene diisocyanate	524 (dextran),	61-63
								(starch)
		Cross-linking	Acrylated dextran		Water/Clyclohexane	Sonication	Phenyl-di(undec-10-en-1-yl) phosphate	280	66-67
		Cross-linking	Hydroxyethyl starch		Water/Clyclohexane	Sonication	Tolylene diisocyanate	275	68
		Cross-linking	Hyaluronic acid		Water/Clyclohexane	Sonication	Tolylene diisocyanate	350	70
				6,6'-diazido-
	10	Polymerization		6,6'-dideoxysucrose, bio(propargyloxy)	Miglyol/water	Sonication		200
				butane

2 Formation of hollow nano-objects from amphiphilic copolymer self-assembly 1.2.2.1 Polymersomes

Table 1 .3, entry 2). 76

 1

	-77 These block copolymers

Table 1 .3, entry 5). 81

 1

	Poly(macromonomer)s (PMMs) bearing various carbohydrate
	residues (galactose, glucose, ribose and xylose) were prepared by repeating ring
	opening metathesis polymerization (ROMP). Depending on terminal functionalization
	by norbornene, the PMMs were capable of conducting further polymerization. After
	coupling with polyethylene glycol (PEG), amphiphilic block copolymers (PMMs-b-PEG)
	with different segments ratios were prepared (Figure 1.9). Nano-objects exhibiting
	different morphologies were generated after self-assembly of these block copolymers
	in aqueous solution. Polymersomes with a hydrodynamic diameter of 230 nm were
	prepared from PMMs 20 -b-PEG 110 . One year later, Xiao et al. synthesized an
	aldehyde-functionalized	glycomonomer,	1,2,3,4-di-isopropylidene-
	6-O-(2'-formyl-4'-vinylphenyl)-D-galactopyranose (IVDG), which was polymerized by
	RAFT polymerization (M n = 13 kg/mol, Ð = 1.09). After deprotection of the sugar
	moieties, glycopolymersomes with R h ranging from 80 to 205 nm were generated
	through solubilization in water. The resulting vesicles were further used to immobilize
	BSA proteins thanks to the presence of aldehyde groups on the shell (

Table 1 .3, entry 6). 82 Figure 1.9. Synthetic

 1 route to PMMs-b-PEG.81

	In	2008,	Dong's	group	prepared	4-arm	star-shaped
	poly(ɛ-caprolactone)-b-glycopolymer block copolymers (SPCL-b-PGAMA and
	SPCL-b-PLAMA) by combining ring opening polymerization (ROP) of ɛ-caprolactone
	using pentaerythritol as initiator and subsequent chain-extension ATRP
	polymerization	of	gluconamidoethylmethacrylate		(GAMA)	or
	lactobionamidoethylmethacrylate (LAMA). 83-84 By varying the length of the
	glycopolymer segments, the produced 4-arm glyco-copolymers (SPCL-b-PGAMA and
	SPCL-b-PLAMA) spontaneously self-assembled in aqueous solution to form vesicles,
	micelles or worm-like nano-objects (

Table 1 .3, entry 7 and 8). Vesicles

 1

	were only

Table 1 .3, entry 9).

 1

	Utilizing

Table 1 .3, entry 10).

 1

	Stenzel's group reported block copolymers having pendent glucosamine and uridine
	moieties. 87 The monomers, 5'-O-methacryloyl uridine (MAU) and 2-methacrylamido
	glucopyranose (MAG), were first prepared by chemo-enzymatic reaction and
	sequentially polymerized through RAFT polymerization. Self-assembly of the block
	copolymers by solvent displacement process (DMA solution dialyzed against water)
	generated nano-objects with morphologies evolving from micelles to worm-like

Table 1 .3, entry 11).

 1

	Polymersomes having a hydrodynamic diameter around 35 nm were obtained by
	self-assembly of PMAG 59 -b-PMAU 36 . The polymersomes were shown to display an
	outer shell constituted of PMAG and a core built from PMAU segments. Owing to the
	establishment of H-bonding interactions between adenosine and PMAU, these
	materials were thought to be potential therapeutic carriers for ASONs (antisense
	oligonucleotides). More recently, the same group prepared random copolymers from
	N-isopropylacrylamide (NIPAAM)/ N-homocysteine thiolactone acrylamide (TiaAm) by
	RAFT copolymerization. After polymerization, the pendent thiolactone groups were
	then opened with various amines (n-propylamine, n-hexylamine and n-dodecylamine)
	to release thiol moieties which were finally reacted in situ with
	2-bromoethyl-2′,3′,4′,6′-tetra-O-acetyl-α-D-mannopyranoside	(Figure	1.10).

Employing a solvent displacement strategy, these double modified copolymers gave birth to micelles (ring opening with n-propylamine), large compound micelles (ring opening with n-dodecylamine) or vesicles at 100-600 nm (ring opening with n-hexylamine) which could specifically bind ConA lectin (

Table 1 .3, entry 12). 88

 1

11, Table 1.3, entry 13). Figure 1.11. Fluorescent

  vesicles formed from self-assembly of P3HT 25 -b-PMAGP 97 .

89

Du et al. studied the synthesis and self-assembly of glycosamide-grafted glycopolysiloxanes (

Table 1 .3, entry 14). 90 The authors first prepared aminopropyl

 1

	functional polysiloxanes (APFPS) by ring opening polymerization of
	octamethylcyclotetrasiloxane	(D4)	in	the	presence	of
	3-aminopropyldiethoxymethylsilane (APDEMS) and tetramethylammonium hydroxide
	(TMAH, catalyst). The resulting polymers were further reacted with glyconolactone to
	give glycopolysiloxanes. Investigation on the self-assembly behavior of the

Table 1 .3, entry 16). The

 1

	vesicles were capable to

Table 1 .3, entry 17).

 1

	Decrease of pH to 4.68, resulted in an increase in size to 130 nm. The vesicles were
	finally used to load anti-cancer drug Dox (efficiency up to 70%) and release it (40.1%
	in 6h at pH 4.68) by adjusting the pH value.
	Photosensitive glyconanocapsules	were designed by Das and coworkers. 94
	Poly(spiropyran	methacrylate)-b-poly(3-O-4-vinylbezoyl-D-glucopyranose)
	(PSP 8 -b-PBG 32 ) block copolymers were synthesized in two steps by ATRP
	polymerization (

Table 1 .3, entry 18). The

 1

	prepared copolymers self-assembled to

Table 1 .3, entry 19). Owing to

 1

	the formation of dynamic covalent bonds between BA and the pendent sugars
	moieties in basic conditions (pH = 9), the authors prepared "graft copolymer like"
	structures. Thanks to the thermo-sensitive character of the PNIPAAM block (~32°C),
	the PNIPAAM-BA/glycopolymers (2/1) mixtures were shown to self-assemble at 33°C
	to give birth to uniform vesicles (R h = 62 nm and 68 nm, PDI = 0.09 and 0.10 for
	V-PGal and V-PGlc, respectively). DLS experiments highlighted that sugar-coated
	vesicles were capable to specifically interact with appropriate lectin (PNA and ECA for
	PGal and none for PGlc). The same group also reported the preparation of
	glyco-vesicles with a sugar core and a PS shell (

Table 1 .3 entry 20).

 1

Table 1 .3 entry 22). 99

 1 2009, Schatz et al. designed polysaccharide and polypeptide based nano-objects (

	An alkyne-functionalized dextran (M n = 6600 g/mol, Ð = 1.35)
	was first synthesized by reductive amination with propargylamine, while an
	azide-functionalized PBLG (DP = 59) was obtained by ring opening polymerization of
	γ-benzyl L-glutamate N-carboxylic anhydride (BLGNCA). The coupling reaction
	between the two polymers applying CuAAC in DMF generated amphiphilic block
	copolymers Dextran-b-PBLG, which gave rise to thick-membrane vesicles (R h = 45
	nm, PDI = 0.2, thickness = 20 nm) through solvent shifting (addition of DMSO into
	water).
	Borsali and coworkers reported the preparation of thermo-responsive vesicles based
	on maltoheptaose (

Table 1 .3 entry 23). 100

 1

	Alkyne-functionalized maltoheptaose (Mal)

Table 1.3 entry 25). 102 After partial esterification of

	dextran (70 kg/mol) with octadecanol-carbamate imidazole, octadecanol modified
	dextrans (OMD) with different composition ratios (DS = 0.03, 0.05, 0.09 and 0.12)
	were generated. OMD17 (DS = 0.05) self-assembled into vesicles (R h = 96 nm, R g /R h
	= 1.56, 10% v/v initial water in DMF-water) through solvent displacement (addition of
	water into polymers/DMF/water solution). The size of the OMD17 polymersomes was
	tuned (R h from 31 to 96 nm) by increasing the initial water/DMF ratio (initial water
	content, from 10% to 40%, v/v).
	Jayakannan and coworkers developed original vesicular carriers from dextran
	derivatives (

Table 1 .3 entry 26). 103

 1

	Dextran (6 kg/mol) was initially partially

Table 1 .3 entry 27). 104

 1

	Carboxymethyl-hexanoyl chitosans (CHC) with

.15, Table 1.3, entry 28). 105

	-106 Chitosan (560 kg/mol) was modified by
	chloroacetic acid and azidobenzaldehyde through a two-step reaction. Self-assembly
	of modified chitosan chains in water afforded nanocapsules with a diameter of 150 nm
	at neutral pH. Due to the photo-sensitive character of the azidobenzyl groups,
	cross-linked nanocapsules (with hydrodynamic diameter around 120 nm) were finally
	obtained after UV exposition (15 min, λ = 253 nm). The nanocapsules were finally
	loaded with a pesticide (methomyl) during the assembly process (efficiency up to 90%)

Table 1 .3, entry 30). 108 Chitosan (200 kg/mol) was modified with alkylaldehyde (butyraldehyde or octaldehyde) in methanol

 1

	to generate amphiphilic chitosan (DS, 0.26~0.27). The modified chitosan (5 mg/mL)

Table 1 .3, entry 31). 109

 1

	Li and coworkers described chitosan/lipid cationic liposomes for gene delivery

Chang and coworkers also prepared chitosan/cholesterol based nano-objects (Table

1

.3, entry 32).

110

Octadecyl-quaternized lysine modified chitosan (OQLCS, M n (chitosan)

  112 Amphiphilic HES was generated by esterification of HES(70, 200, 450 kg/mol) with lauric, palmitic and stearic acids (average DS, 0.29, 0.44 and 0.17). Fatty acid modified HES was then dissolved into a water/THF (3/1, v/v) solution. Mixture of micelles (D

et al. prepared hydroxyethyl starch (HES)-containing micelles and vesicles through self-assembly process (Table 1.3, entry 34). h ~ 20-30 nm) and vesicles (D h ~ 250~350 nm) mixtures were obtained after THF evaporation. In 2009, hyaluronan-based polymersomes 113-115 were reported by Upadhyay et al. (Table 1.3, entry 35). The hyaluronan ( Mw= 5140 g/mol, Ð = 1.41) was primarily

17, Table 1.3, entry 37).

Table 1 . 3 .

 13 Preparation of carbohydrate-functionalized nanocapsules by self-assembly.

	Entry	Classification	Polymer	Protocol of Self-Assembly	Solvent	Size (nm)	Ref.
	1	Polymersomes	Polystyrene-b-poly[(2-β-D-glucopyranosyloxy)ethyl acrylate]	Solvent shifting Dioxane/DMF		74-75
	2	Polymersomes	Poly(L-alanine)-b-poly(2-acryloyloxyethyllactoside)-b-poly(L-alanine)	CMC	H 2 O		76-77
	3	Polymersomes	2,3,4,6-tetra-O-acetyl-β-D-1-thioglucopyranose modified 1,2-polybutadiene-block-polystyrene	CMC	THF	250 (R h )	78-79
	4	Polymersomes	2,3,4,6-tetra-O-acetyl-1-thio-β-D-glucopyranose modified 1,2-polybutadieneblock-poly(ethylene oxide)	CMC	H 2 O	280 (R h )	80
	5	Polymersomes	Poly(macromonomer)s-b-polyethylene glycol bearing galactose, glucose, ribose and xylose	Solvent shifting	THF/H 2 O	231	81
	6	Polymersomes	Poly[1,2,3,4-di-isopropylidene-6-O-(2'-formyl-4'-vinylphenyl)-D-galactopyranose]	Solvent shifting	DMSO/H 2 O	80~205 (R h )	82
	7	Polymersomes	Poly(ɛ-caprolactone)-b-poly(lactobionamidoethylmethacrylate)	Solvent shifting	DMF/H 2 O	432	83
	8	Polymersomes	poly(ɛ-caprolactone)-b-poly(gluconamidoethylmethacrylate)	Solvent shifting	DMF/H 2 O	250	84
	9	Polymersomes	Poly(D-gluconamidoethyl methacrylate)-PPR-poly(D-gluconamidoethyl methacrylate)	Solvent shifting	DMF/H 2 O	81	85
		Polymersomes	Poly(γ-benzyl L-glutamate)-b-poly(D-gluconamidoethyl methacrylate)	Solvent shifting	DMF/H 2 O	274	86
		Polymersomes	Poly(5'-O-methacryloyl uridine)-b-poly(2-methacrylamido glucopyranose)	Solvent shifting	DMA/H 2 O	35	87
		Polymersomes	PNIPAAM-co-glycopolymer bearing mannoside and n-hexylamine	Solvent shifting	MeOH/H 2 O	100-600	88
		Polymersomes	Poly(3-hexylthiophene)-b-poly(3-O-methacryloyl-D-galactopyranose)	Solvent shifting	THF/H 2 O	89 (R h )	89
		Polymersomes	Glucosamide-grafted amphiphilic glycopolysiloxanes	CMC	H 2 O	300~600	90
		Polymersomes	PBLG-b-PGG glycopeptides block copolymer	Solvent shifting	DMF/H 2 O	32 (R h )	91
		Polymersomes	Poly(2-glucosyloxyethyl methacrylate)-b-poly(diethyleneglycol methacrylate)	T	H 2 O	251, 500 (20°C)	92
		Polymersomes	Poly(L-lysine)-graft-hexanoyl-lactobionolactone	Solvent shifting	MeOH/H 2 O	110 (R h , pH 7.4)
				Solvent shifting	DMF/H 2 O	366

93 Polymersomes Poly(spiropyran methacrylate)-b-poly(3-O-4-vinylbezoyl-D -glucopyranose) 11 -stat-Phe 10 )-g-Cs] x -stat-Cs 2x -stat-ECs 120 CMC: Critical Micelle Concentration. T: Temperature.

Table 1 .4, entry

 1

2).

An original route was reported by

Taniguchi et al. who developed glyconanocapsules and glycomicrocapsules by combining polymerization and core removal strategies (Table 1.4, entry 3). 130 Nano-sized particles (diameter of 440 nm) prepared by miniemulsion polymerization of styrene (St) and 2-chloropropionyloxyethyl

  Li and co-workers deposited dextran sulfate (500 kg/mol) and chitosan (50 kg/mol) on SiO 2 -NH 3 + nanoparticles (size of 166 nm). 138 Hollow nanocapsules were obtained after removal of core particles by HF/ NH 4 F treatment (D h of 217 nm before core

1.4, Entry 10).

137

Silica particles were coated by chitosan (150 kg/mol) and dextran sulphate (500 kg/mol) to generate four layered nanoparticles. After silica dissolution, the biocompatible nanocapsules (diameter of 180 ± 20 nm) were capable to encapsulate ciprofloxacin and ceftriaxone (drugs for Salmonella infection) with 78% of efficiency. The release of drugs was influenced by the pH of solution (70% after 48h at pH 7.4; 51% after 48h at pH 4.8). These drug-loaded nanocapsules showed effective clearance ability of Salmonella infection in vitro and in vivo. removal, Table

1

.4, entry 11). The template was further modified with tosyl-β-cyclodextrin to afford β-cyclodextrin conjugated silica nanoparticles (D h of 100 nm, PDI = 0.319), and chitosan was functionalized with cysteine through amidation to afford cysteine-conjugated chitosan (CH-SH, targeting modification of 5, 10, 20 and 40%).

139

Dextran sulfate and CH-SH were then sequentially deposited on β-cyclodextrin conjugated silica template by L-b-L deposition (Table

1

.4, entry 12).

Table 1 . 4 :

 14 Template strategy to prepare carbohydrate-functionalized nanocapsules.

	Entries	Template	Template Dimension (nm)	Polymer	Deposition Manner	Cross-linker	Layers	Core Removal	Capsules (nm) Dimension	Ref.
	1	P(S-co-MAA) particles	200	Chitosan	Electrostatic interaction	Glutaraledehyde Monolayer	THF	190~250	126-127
	2	SC/MC Silica particles	268, 382, 574	Chitosan	Electrostatic interaction	Glutaric dialdehyde	Monolayer	HF	440 220, 270,	128-129
	3	P(St-CPEM) particles	440	P(VBSAELA)	Surface polymerization		Monolayer	THF	640	130
	4	DNA-PEI complexes		Hyaluronan	Electrostatic interaction		Monolayer		20	131
	5	Silica particles	330	Chitosan, Dextran sulfate	Electrostatic interaction		Multilayer	HF	300	132
	6	Silica particles	330	Chitosan, γ-PGA	Electrostatic interaction		Multilayer	HF	250	133
	7	Silica particles	500	PAH, Dextran sulfate	Electrostatic interaction		Multilayer HF/NH 4 F	500	134
	8	Silica particles	220	Chitosan, Heparin	Electrostatic interaction		Multilayer HF/NH 4 F	200	135
	9	Silica particles	260	Cysteine-Chitosan, Cysteine-PASA	Electrostatic interaction		Multilayer	HF	365 (before removal) core	136
	10	Silica particles		Chitosan, Dextran sulphate	Electrostatic interaction		Multilayer	HF	180

Cette thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2015ISAL0011/these.pdf © [X. Yan], [2015], INSA de Lyon, tous droits réservés

(1) Monolayer capsules

Park and coworkers reported Vitamin C loaded chitosan (4 kg/mol)-coated nanoliposomes.

144

Liposomes were first prepared through self-assembly using phosphatidylcholine and cholesterol and further coated with chitosan. The resulting nanocapsules (D h = 96 nm) were capable to load vitamin C with a maximal efficiency up to 96.5%, while the payload was about 46.8% (Table

1

.

4, Entry 17). In

	the injection method. After deposition of chitosan chains, the nanocapsules (D h = 182
	nm) were used to encapsulate fluorescein (efficiency ~ 94%). The release of
	fluorescein was slowed down by the incorporation of the chitosan layer (56% vs
	65.5% for the uncoated liposomes after 24h). Due to the presence of folate groups,

order to increase bioavailability of the nanocarriers, Yang et al. introduced folate groups onto chitosan-coated liposomes. Folate groups were incorporated on chitosan chains (10kg/mol, DS is not given) using carbodiimide chemistry (Table

1

.4, Entry 18).

145

Liposome templates were then prepared from phosphatidylcholine (PC) using the nanocapsules were easily uptaken by MCF-7 cells.

Besides chitosan, pectin with different molecular weight (76, 110 and 96 kg/mol, LM, HM and AM pectin) was also introduced by Klemetsrud and coworkers to investigate the interaction between pectin-coated liposomes and mucin (

Table 1 .4, Entry 19). 146

 1

	Liposomes were firstly prepared using dipalmitoyl phosphatidylcholine (DPPC) and
	dipalmitoyl trimethylammoniumpropane (DPTAP) through the lipid film hydration
	method. Due to the mucoadhesive properties of pectin, these coated liposomes
	(0.02% of coated pectin, diameter of 229, 302 and 418 nm for LM, AM and HM pectin
	based capsules, respectively) were shown to interact with mucin to form aggregates
	(diameter of 312, 446 and 582 nm for aggregates of LM, AM and HM-based
	capsules/mucin, respectively).

Borsali and Soldi decorated PS-b-PAA based polymersomes with chitosan (37CS and 5CS, molecular weight of 37 kg/mol and 5 kg/mol) for generating finasteride-loaded nanocarriers (finasteride is a drug for benign prostatic hyperplasia and male pattern baldness). 147 PS 139 -b-PAA 17 and PS 404 -b-PAA 63 were first employed to construct polymersomes (C3 and C7, diameter of 414 and 180 nm, respectively) through a co-solvent self-assembly process. The chitosan-coated polymersomes (C3/37CS, C3/5CS, C7/37CS and C7/5CS, hydrodynamic diameter of 712, 404, 363 and 183 nm, respectively) were capable to encapsulate finasteride and enhance the permeation of the drug in ex vivo studies. Due to the presence of positively charged chitosan, the coated polymersomes exhibited strong interaction with skin components compared to uncoated ones (Table

1

.

4, Entry 20). (2) Multilayer capsules through Layer-by-layer strategy

  In 2010, Ge and coworkers prepared alginate-chitosan containing liposomes (

Table 1 .

 1

	4, Entry 21). Unilamellar liposomes were first generated from DPPC,
	N-dioctadecyldimethylammonium bromide (DDAB) and cholesterol applying
	evaporation and post-extrusion techniques (the solution was finally filtrated by 100 or
	200 nm polycarbonate filters). Nanocapsules (four layers, D

h = 414 nm) were then obtained by successive deposition of alginate and chitosan (10 kg/mol) on the cationic liposomes through L-b-L process. The nanocapsules were stable in SDS aqueous solution and hydrophilic solution (1/10 water/ethanol, v/v).

148

Using a similar L-b-L process, Cuomo and coworkers constructed alginate/chitosan based nanocapsules from cationic liposomes (diameter of 220 nm) of DDAB (

Table 1 .4, Entry 22, Figure 1.26).

 1

  As evidenced by NMR and TEM, dextran functionalized nanocapsules (diameter of 73 nm) were finally obtained through insertion of modified dextran chains on the preformed nanocapsules at room temperature or 60 °C for 24h

	(mass incorporation, 5% at room temperature and 10% at 60°C:).
	In a same approach, lipid nanocapsules (LNC, diameter of 49 nm) were also
	prepared by Benoit and co-workers in order to generate chitosan/lipid nanocapsules.
	Lipochitosan chains (LC, D acyl = 1.3) obtained by modification of chitosan (CS, 4~6
	kg/mol) with stearic anhydride, were then inserted within the shell of LNC (through
	incubation at 37 °C for 24h) to generate chitosan-functionalized nanocapsules

) Post-insertion In 2008, Richard et al. designed dextran-containing lipid nanocapsules through post-insertion process (

Table 1.4, Entry 26).

155

Lipid nanocapsules (diameter of 59 nm) were prepared using commercial lipids (solutol, labrafac and phospholipon) through phase inversion method. Amphiphilic dextran was generated from modification of Dextran (40 kg/mol) with amine terminal-functionalized Brij 58P by reductive animation.

(LNC-LC, size from 50 to 62 nm). LNC-LC nanocapsules were post-modified with chitosan and dextran sulphate (6.5~10 kg/mol) by L-b-L deposition to form multi-layered nanocapsules (diameter of 200 nm). Fondaparinux sodium (FP, a heparin-like pentasaccharide) was also incorporated within the shell in the course of the wall construction (Table

1

.4, Entry 27).

156

More recently, the same group reported a similar post-insertion route to elaborate polysaccharide/lipid nanocapsules using hydroxylamine modified dextran (lipodextran, LD, 40 kg/mol) (Table

4, Entry 31).

	Chitosan-thioglycolic acid (77 and 150 kg/mol) was grafted onto DPPC/
	1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohe
	xane-carboxamide] liposomes (D h = 201 nm) prepared through thin film rehydration
	method (

1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) applying the thin-film hydration method. Carboxymethyl chitosan (7000 kg/mol) was then grafted at the surface of liposomes through amidation.The resulting nanocapsules (with a size from 200 to 300 nm) allowed for the encapsulation of paclitaxel with an efficiency up to 83.8%. In vivo efficacy in subcutaneous model of B16F10 underlined significant improvement of tumour growth inhibition and survival with orally administered paclitaxel-loaded chitosan-decorated vesicles (vs lipid vesicles) (Table

1

.

Table 1 .4, Entry 32).

 1

Table 2 . 1 .

 21 Library of linear and star homoglycopolymers : Determined from relative integration of the aromatic chain end group and polymer backbone peaks; f : Determined from SEC with RI/light scattering detection in NaNO 3 /NaOH aqueous solution (pH =7); g : not determined due to aggregation in NaNO 3 /NaOH aqueous solution; h : Determined from SEC analysis in THF and PS calibration (after acetylation);

	Ligand [M]/[CTA]/[I]	[M] (mM)	M n th (kg.mol -1 ) d	M n NMR (kg.mol -1 ) e	M w (kg.mol -1 ) f	M n SEC (kg.mol -1 ) h	e DP n	j (nm) D h
	L31 a	32/1/0.2 k	0.45	8.7	11.5	15.0 (1.04)	14.0 (1.06) i	31	4.2
	L47 a	50/1/0.33 k	0.45	15.6	17.2	24.0 (1.03)	17.9 (1.07) i	47	5.0
	L55 a	74/1/0.33 k	0.45	17.8	20.2	26.4 (1.06)	18.4 (1.06) i	55	5.2
	L69 a	94/1/0.5 k	0.45	23.5	25.3	29.2 (1.07)	21.2 (1.08) i	69	5.9
	L106 a	123/1/0.5 k	0.45	34.2	38.6	45.5 (1.06)	25.5 (1.09) i	106	6.5
	L188 a	300/1/0.5 k	0.90	64.5	68.4	n.d g	30.2 (1.15) i	188	7.9
	L 221 a	500/1/0.2 k	1.13	80.1	81.2	102.5 (1.02)		221	8.9
	T51 b	74/1/0.33 k	0.45	14.4	19.6	n.d g	20.4 (1.12) i	51	5.9
	T74 b	99/1/0.5 k	0.45	22.8	28.0	n.d g	22.9 (1.13) i	74	6.4
	T93 b	123/1/0.5 k	0.45	30.7	34.8	n.d g	27.6 (1.12) i	93	6.5
	T200 b	300/1/0.5 k	0.90	72.1	73.2	n.d g	32.1 (1.13) i	200	8.0
	O74 c	123/1/1 k	0.45	20.3	29.7	n.d g	17.2 (1.10) i	74	6.4

a : Linear homopolymers; b : 3-arm star homopolymers; c : 8-arm star homopolymers; d : Calculated from monomer conversion; e i : Ð from SEC analysis in THF and PS calibration (after acetylation); j : determined from DLS analysis in pure water; k : Polymerization carried at 70°C; l : Polymerization carried out at 80°C. C Figure 2.11. Evalution of the GPC traces for the synthetic acetylated glycopolymers. A) Linear glycopolymer; B) 3-arms glycopolymer; C) 8-arms glycopolymer. (Use of PS calibration in THF)

Table 2

 2

.1). A) Linear glycopolymers; B) 3-arms glycopolymers; C) 8-arms glycopolymers. The volume weighted sizes were obtained from measurements performed with 1 mg/mL glycopolymer aqueous solution.

Table 2 . 2 .

 22 Library of glyco-copolymers

	Entries	Polymer	T (°C)	[M]/[CTA]/[I]	[HMM] 0 (M)	[GMA] 0 (M)	[GMA] sp f (M)	Solvent	Conv. (%)	Mn th (g.mol -1 ) a	Mn NMR (g.mol -1 ) b	c (g.mol -1 ) Mn SEC	Ð d
	1	PGMA 65	40	100/1/0.25		0.40		Toluene	61	8900	9500		1.31
	2	PGMA 65 -b-PHMM 133	70	200/1/0.33	0.55			DMSO	64	55100	57500		1.20
	3	P(GMA 66 -grad-HMM 130 )	70	220/1/0.4	0.90	0.39		DMSO	87	55400	56600		1.18
	4	P(GMA 63 -stat-HMM 134 )	70	220/1/0.5	1.23	0.17 e	0.84	DMSO	88	56700	57600		1.15
	5	PGMA 100	40	150/1/0.25		0.40		Toluene	61	13300	14500		1.25
	6	PGMA 100 -b-PHMM 107	70	140/1/0.33	0.73			DMSO	68	50300	53900		1.19
	7	P(GMA 103 -grad-HMM 108 ) 70	220/1/0.5	0.90	0.39		DMSO	83	45900	53900		1.17
	8	P(GMA 95 -stat-HMM 107 )	70	220/1/0.5	1.10	0.30 e	1.50	DMSO	82	47100	52400		1.18
	9	PGMA 136	40	200/1/0.25		0.40		Toluene	64	18500	19600		1.15
	10	PGMA 136 -b-PHMM 65	70	90/1/0.33	0.55			DMSO	69	41700	42800		1.15
	11	P(GMA 134 -grad-HMM 66 )	70	220/1/0.5	0.52	0.97		DMSO	92	42200	43200		1.11
	12	P(GMA 134 -stat-HMM 66 )	70	220/1/0.5	0.70	0.43 e	2.15	DMSO	88	41200	43200		1.17

Table 2 . 3 .

 23 DLS studies of post-modified copolymers.

				Diameter (nm) a
	Entries	Copolymers	Ethanolamine	(2-aminoethyl)	Taurine
			(EA)	trimethylammonium (AA)	(TR)
	1	PGMA 65 -b-PHMM 133	15	21	25
	2	P(GMA 66 -grad-HMM 130 )	18	13	15
	3	P(GMA 63 -stat-HMM 134 )	20	17	17
	4	PGMA 100 -b-PHMM 107	15	25	n.d b

Chapter 3 Glycopolymers for Anti-adhesive Treatment of E. coli-Induced Inflammatory Diseases 3.1 Introduction

  ..................................................................................................... 3.2 Multivalent FimH antagonists ........................................................................ FimH interactions ..................................................................

	3.3 Glycopolymer/

3.4 Glycopolymer/Bacteria interactions ............................................................. 3.5 Adhesion Tests ............................................................................................... 3.6 Ex-vivo tests.................................................................................................... 3.7 ELLSA tests ..................................................................................................... 3.8 Preliminary results on copolymers anti-adhesive properties ..................... 3.9 Conclusions .................................................................................................... 3.10 References ....................................................................................................

  , N. ; Carvalho, F. A. ; Glasser, A-L.; Darcha, C.; Jantscheff, P.; Allez, M.; Peeters, Bommelaer, G.; Desreumaux, Colombel, J-F.; Darfeuille-Michaud, A. J. Clin. Invest., 2007, 117, 1566-1574. 3. Chen, Y. ; Chen, G. ; Stenzel, M. H. Macromolecules 2010, 43, 8109-8114. 4. Richards, S-J.; Jones, M. W.; Hunaban, M.; Haddleton, D.M, Gibson, M. I. Angew. Chem. Int. Ed., 2012, 51, 7812-7816. 5. Bouckaert, J.; Li, Z.; Xavier, C.; Almant, M.; Caveliers, V.; Lahoutte, T.; Weeks, S. D.; Kovensky, J.; Gouin, S. G. Chem. Eur. J., 2013, 19, 7847-7855.

Table 4 . 1 :

 41 Glycopolymers obtained by RAFT polymerization

	Polymer	M n th (kg.mol -1 ) a	M n NMR (kg.mol -1 ) b	M w (kg.mol -1 ) c	Ð d	b DP n	e (nm) D h
	PHMM1	15.6	17.4	24.0	1.03	47	5.0
	PHMM2	80.1	81.2	102.5	1.02	221	8.9

a) calculated from monomer conversion; b) determined from relative integration of the aromatic chain end group and polymer backbone peaks; c) determined from SEC with RI/light scattering detection in NaNO 3 /NaOH aqueous solution (pH =7); d) Ð from SEC analysis in NaNO 3 /NaOH aqueous solution; e) determined from DLS analysis in pure water.

Table 4 . 2 :

 42 Glycopolymer obtained by RAFT polymerization

	Polymer	M n th	a	M n NMR	b	M w	c	Ð d	D h	e
		(kg.mol -1 )	(kg.mol -1 )	(kg.mol -1 )		(nm)
	P (HMM 206 -co-GMA 17 )	74.0		77.1		42.5	1.10	13

Synthesis of azido-fuctionalized mannoside (4).

  CH 2 ) 5 CH 2 NH), 21.1, 20.8 (OCOCH 3 ), 18.9 (COC(CH 3 )CH 2 ). ESI-MS: m/z calculated for [C 25 H 39 NNaO 11 ] + , 552,2415, found 552.2417.

	removal by evaporation, the crude product (3, pale yellow gum) was dissolved in monomer was finally purified by column chromatography (petroleum ether/ethyl
	anhydrous DMF (200mL), sodium azide (16.6g, 255mmol) and tetra-n-butyl acetate, 3/1, v/v). Yield: 7.4 g (55%) of a colorless gum.
	ammonium iodide (18.9g, 51.2mmol) were added. The mixture was stirred at 70 °C [[α] D = +10 (c= 0.4, Chloroform). 1 H NMR (300 MHz, CDCl 3 ) δ 5.89 (s, 1H, NH), 5.65
	under argon for 24h, cooled to room temperature, filtered and the solid was washed (s, 1H, CCH 2 ), 5.39-5.17 (m, 4H, H-3, CCH 2 , H-4, H-2), 4.78 (d, J =1.5 Hz, 1H, H-1),
	with EtOAc (3×300mL). The filtrate was diluted with EtOAc to reach a total volume of 4.35-4.20 (dd, J = 5.1, 12.3 Hz,1H, H-6), 4.16-4.05 (m, 1H, H-6'), 4.02-3.91 (m, 1H,
	1L. The organic layer was washed with aq. NaHCO 3 (3×500mL), water (3×500mL), H-5), 3.67 (m, 1H, OCH 2 CH 2 ), 3.50 (m, 1H, OCH 2 CH 2 ), 3.28 (q, J =6.3 Hz, 2H,
	brine (500mL) and dried. After concentration, the resulting product (yellow gum) was NHCH 2 CH 2 ), 2.15, 2.09, 2.03, 1.98, (s, 4×3H, 4×CH 3 CO), 1.95 (s, 3H, CCH 3 ),
	purified by column chromatography (petroleum ether/ethyl acetate, 7/1, v/v) to afford 1.67-1.46 (m, 4H, CH 2 (CH 2 ) 3 CH 2 CH 2 NH), 1.34 (s, 6H, CH 2 (CH 2 ) 3 (CH 2 ) 2 NH); 13 C NMR
	the corresponding azido-fuctionalized glycoside (4). Final yield: 12.6 g (50%) obtained (CDCl 3 ) δ 170.8, 170.3, 170.1, 169.9, (OCOCH 3 ), 168.6 (NHCO), 140.4
	as a colorless gum. (COC(CH 3 )CH 2 ), 119.2 (COC(CH 3 )CH 2 ), 97.7 (C-1), 69.8 (C-2), 69.3 (C-3), 68.5 (C-5,
	[α] D = +11 (c= 0.35, Chloroform). 1 H NMR (300 MHz, CDCl 3 ) δ 5.32-5.11 (m, 3H, H-2, OCH 2 (CH 2 ) 5 ), 66.4 (C-4), 62.6 (C-6), 39.8 (CH 2 NHCO), 29.6, 29.2, 29.1, 26.8, 26.1,
	-3, -4), 4.73 (d, J = 1.5 Hz,1H, H-1), 4.20 (dd, J = 5.4, 12.3 Hz, 1H, H-6), 4.03 (dd, J = (OCH 2 (
	2.4, 12.3 Hz, 1H, H-6'), 3.90 (m, 1H, H-5), 3.68-3.54 (m, 1H, H-OCHHCH 2 ), 3.44-3.33
	(m, 1H, OCHHCH 2 ), 3.21 (t, J = 6.9 Hz, 2H, N 3 CH 2 CH 2 ), 2.09, 2.04, 1.98, 1.93 (s,
	4×3H, 4×CH 3 CO), 1.64-1.46 (m, 4H, CH 2 (CH 2 ) 3 CH 2 CH 2 N 3 ), 1.30 (s, 6H,
	CH 2 (CH 2 ) 3 (CH 2 ) 2 N 3 ); 13 C NMR (CDCl 3 ) δ 170.1, 170.1, 169.7, 169.9, (OCOCH 3 ), 97.5
	(C-1), 69.7 (C-2), 69.0 (C-3), 68.4 (C-5, OCH 2 (CH 2 ) 5 ), 66.2 (C-4), 62.5 (C-6), 51.4
	(CH 2 N 3 ), 29.1, 28.9, 28.7, 26.6, 26.0, (OCH 2 (CH 2 ) 5 CH 2 N 3 ), 20.9, 20.7, (OCOCH 3 );
	ESI-MS: m/z calculated for [C 21 H 33 N 3 NaO 10 ] + , 510.2058, found 510.2051.
	SnCl 4 (1M in CH 2 Cl 2 , 154mL, 154mmol) was added dropwise (within 30 min) at room
	temperature to a solution of 7-bromoheptan-ol (1) (15.2g, 77.91mmol), peracetylated Synthesis of HMM (compound 6).
	D-mannose (2) (20g, 51.2mmol) and silver trifluoroacetate (17.0g, 76.8mmol) in 4 (12.6g, 25.8mmol), p-toluenesulfonic acid (5.41g, 28.4mmol) and 10% palladium on
	freshly distilled dichloromethane (250mL). Disappearance of the starting material was activated charcoal (2g) as the catalyst were stirred overnight in ethanol (240mL) at
	observed within 3h and the mixture became pale pink. Saturated aqueous NaHCO 3 room temperature under 1 atm of hydrogen. The solution was filtered over celite and
	(600mL) was added to adjust pH above 8, and the solution was vigorously stirred for evaporated to obtain a colorless gum (5). The resulting product was directly engaged
	20min. A biphasic system formed upon addition of aqueous NaHCO 3 with a white in the next step without further purification. 5 was dissolved in a solution of anhydrous
	suspension in the aqueous layer. The biphasic solution was diluted with 500mL of CH 2 Cl 2 (150mL) containing triethylamine (10.5mL, 75.9mmol). Methacryloyl chloride
	water and the aqueous layer was extracted with CH 2 Cl 2 (3×300mL). The organic (3.8mL, 32.9mmol) was added dropwise into the solution in an ice bath. The solution
	layers were combined, washed successively with saturated aqueous NaHCO 3 was stirred for 1h at room temperature. After reaction, the solution was washed with
	(150mL), water (3×300mL), brine (2×300mL) and dried over MgSO 4 . After solvent water (2×200mL), dried over MgSO 4 and the solvent was removed. The acetylated

Synthesis of CPADB-based trifunctional RAFT agent.

  CCN), 38.23 (NCH 2 CH 2 NH), 34.36 (COCH 2 CH 2 ), 31.88 (CH 2 CH 2 C), 24.23 (CH 3 ) (Spectra given in Fig S6). ESI-MS: m/z calculated for C 45 H 52 N 7 O 3 S 6 [M+H] + 930.2450, found 930.2409.

	The activated RAFT agent (1.24g, 3.3mmoL) was dissolved in THF (100mL) and a
	solution of tris(2-aminoethyl)amine (146 mg, 1mmol) in methanol (10mL) was added
	dropwise at room temperature. The solution was stirred for 1h. After reaction, the
	solvent was evaporated and the crude product was purified by column
	chromatography (CH 2 Cl 2 /Methanol, 30/1, v/v). 520mg (41%) as pink powder. 1 H NMR
	(250 MHz, CDCl 3 ) δ 7.86 (m, 6H, phenyl), 7.53 (m, 3H, phenyl), 7.35 (m, 6H, phenyl),
	6.93 (s, 3H, NH), 3.29 (s, 6H, NHCH 2 CH 2 N), 2.88-2.21 (m, 18H, CH 2 CH 2 CO and
	NHCH 2 CH 2 N ), 1.90 (m, 9H, CH 3 ). 13 C NMR (CDCl 3 ) δ 222.89 (SCS), 171.43 (CONH),
	144.61, 133.16 , 128.70, 126.85 (phenyl), 118.94 (CN), 54.80 (NCH 2 CH 2 ), 46.34
	(CH 3 Synthesis of CPADB-based octafunctional RAFT agent. To a solution of Octa
	Ammonium POSS purchased from Hybrid Plastics (200 mg, 0.17mmol) in methanol
	(10mL) was added 0.33mL of DIPEA (2 mmol)). After 2h under stirring, the solution
	was introduced drop by drop in a solution of activated RAFT agent (650mg, 1.7mmoL
	in 100mL) of THF. After 1h of reaction, the solvent was evaporated and the RAFT
	agent was finally purified by column chromatography (CH 2 Cl 2 /Methanol, 30/1, v/v),
	Typically, in an argon conditioned Schlenk tube, 4-cyano-4-(phenylcarbonothioylthio) obtained pink powder 151mg (30%). 1 H NMR (250 MHz, CDCl3) δ 7.88 (d, J = 7.5 Hz,
	pentanoic acid (CPADB, 297mg, 1mmol) was dissolved in chloroform (20 mL). After 16H, phenyl), 7.54 (t, J = 7.5 Hz, 8H, phenyl), 7.37 (t, J = 7.5 Hz, 16H, phenyl), 6.56 (s,
	cooling 8H, NH), 3.45-3.22 (m, 16H, CH 2 CH 2 NH), 2.75-2.24 (m, 32H, CH 2 CH 2 CO), 1.89 (s, to 0°C, N-hydroxysuccinimide (115mg, 1mmol) and
	N,N′-dicyclohexylcarbodiimide (206mg, 1mmol) were successively added. After one 24H, CH 3 ), 1.78-1.45 (m, 16H, SiCH 2 CH 2 ), 0.78-0.52 (m, 16H, SiCH 2 CH 2 ). 13 C NMR
	hour at 0°C, the solution was stirred overnight at room temperature. The solution was (CDCl3) δ 222.89 (SCS), 170.94 (CONH), 144.64, 133.21, 128.74, 126.84 (phenyl),
	filtrated and the solvent was evaporated to afford the activated RAFT agent as a pink 118.95 (CN), 46.33 (CH 3 CCN), 42.19 (NHCH 2 CH 2 ), 34.35 (COCH 2 CH 2 ), 31.98
	powder (95%, 357 mg). 1 H NMR (250 MHz, CDCl 3 ) δ 7.91 (d, J = 7.5 Hz, 2H, phenyl), (CH 2 CH 2 C), 29.83 (CH 2 CH 2 CH 2 ), 24.28 (CH 3 ), 23.02 (CH 2 CH 2 CH 2 ). (Spectra given in
	7.58 (t, J = 7.5 Hz, 1H, phenyl), 7.41 (t, J = 7.5 Hz, 2H, phenyl), 3.11-2.45 (m, 8H, Fig S7)
	CH 2 CH 2 CO and CCH 2 CH 2 C), 1.96 (s, 3H, CH 3 ). 13 C NMR (CDCl 3 ) δ 220.01, 168.93,
	167.22, 144.52, 133.31, 128.76, 126.87, 118.28, 45.59, 32.88, 27.05, 25.71, 24.38 ESI-MS: m/z calculated for C 128 H 154 N 16 O 20 S 16 Si 8 [M+2H] 2+ 1485.2600, found
	(Spectra given in Fig S5). 1485.2639.

  1. Brument, S.; Sivignon, A.; Dumych, T. I.;Moreau, N.; Roos, G.; Guerardel, Y.; Chalopin, T.;Deniaud, D.;Bilyy, R. O.; Darfeuille-Michaud, A.;Bouckaert, J.; Gouin, S.G. J. Med. Chem., 2013, 56, 5395-5406. 2. Tardy, A.; Delplace, V.; Siri, D.; Lefay, C.; Harrisson, S.; de Fatima Albergaria Pereira, B.; Charles, L.; Gigmes, D.; Nicolas, J.; Guillaneuf, Y. Polym. Chem. 2013, 4, 4776-4787.

Cette thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2015ISAL0011/these.pdf © [X. Yan], [2015], INSA de Lyon, tous droits réservés

Cette thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2015ISAL0011/these.pdf © [X. Yan], [2015], INSA de Lyon, tous droits réservés

Cette thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2015ISAL0011/these.pdf © [X. Yan], [2015], INSA de Lyon, tous droits réservés

Cette thèse est accessible à l'adresse : http://theses.insa-lyon.fr/publication/2015ISAL0011/these.pdf © [X. Yan], [2015], INSA de Lyon, tous droits réservés

Acknowledgement

Great thanks to Chinese Scholarship Council for my Ph. D grant. Initial C(T51)=2.25μM C(FimH)=83μM C (T51)=0.28μM C (FimH)=73μM C (T51)=0.68μM C (FimH)=58μM C (T51)=0.93μM C (FimH)=48μM C (T51)=1.70μM C Initial C(T74)=1.55μM C(FimH)=83μM C (T74)=0.19μM C (FimH)=73μM C (T74)=0.47μM C (FimH)=58μM C (T74)=0.64μM C (FimH)=48μM C (T51)=1.25μM C (FimH)Hepes Buffer, 20℃ C (O74)=0.516μM C (FimH)=55μM C