Je Souhaite Remercier Grandement

Dr Stéphanie Denis

Dr Simona Mura

Julien Nicolas

Dr Pamela Pasetto

Noury

Je Souhaiterais Aussi Remercier

Anita Loiseau

Emilie Choppé

Aline Lambert

Thi Nguyet Tran

Lionel Lauber

Sylvain, Flavien, Fabien, Kévin, Batiste Justine Thomas

Kévin

δéo Océane…j ' Paul

En

Ludovic, Jérémy (dit Djé Matthieu Maxime

Minidou ) Je

Frère Erwan

Ma Soeur

of (PEGylated) Aminoethyl-based Polyacrylates

Ce travail a été effectué au sein de l'Institut des Molécules et Matériaux du Mans (IMMM) -UMRCNRS-6283 dans l'équipe Méthodologie et Synthèse des Polymères (MSP) à l'Université du εans et δaval. Je voudrais tout d'abord remercier les collectivités locales Sarthoises pour avoir financé ce doctorat. Je souhaite ensuite remercier le Dr Sagrario Pascual, ma directrice de thèse, pour m'avoir accordé sa confiance pour le Master 2 et pour la thèse. Je tiens à présenter mes excuses pour les longues corrections de cette thèse. Mais je dois surtout remercier le Dr Sagrario Pascual pour tous les conseils qu'elle a pu me donner entant que doctorant ou simple étudiant. Je dois dire encore merci pour sa gentillesse, ses encouragements et sa compréhension pour la thèse et aussi dans la vie de tous les jours. Je souhaite remercier tout aussi chaudement le Dr Sandie Piogé, ma co-encadrante de thèse, pour m'avoir accordé aussi sa confiance pour le εaster 2 et la thèse, pour m'avoir poussé pendant le Master 2 pour donner le meilleur de moi-même. Je tiens aussi à remercier le Dr Piogé pour tous ses conseils, sa bonne humeur, son humour et toutes les paroles encourageantes qu'elle a pu me dire. Je souhaite aussi remercier le Pr Laurent Fontaine, mon co-directeur de thèse, pour m'avoir accueilli tout d'abord dans son équipe puis dans son laboratoire. Je tiens aussi à le remercier en tant que co-directeur de thèse pour m'avoir conseillé, aiguillé dans mes choix, pour avoir donné de si bonnes idées et pour m'avoir fait confiance en tant qu'enseignant de chimie organique. Je tiens aussi à le remercier entant qu'enseignant pour avoir fait partie des personnes qui m'ont guidé vers la voie que j'ai choisi. Je tiens aussi à remercier le Pr Rachel O'Reilly et le Pr Richard Hogenboom pour avoir accepté de rapporté ma thèse. Je dois aussi leur dire combien je suis honoré qu'ils aient effectué ce travail.

Remerciements

Je suis très honoré que le Pr. Philippe Guégan ait accepté de présider le comité d'évaluation ma thèse. Je me dois aussi de le remercier pour son évaluation et ses conseils.

Then, I would like to express my sincere gratitude to my supervisor the Dr Sagrario Pascual, to give me her trust for the PhD. I would like to apologize my-self for the numerous corrections of this work. Moreover I would like to thanks the Dr Sagrario Pascual for all the advices during my PhD and when I was a simple student, for her kindness, for her support, her understanding for the PhD and for everyday life.

I would like to thanks warmly my co-supervisor, Dr Sandie Piogé, to give me her trust for my Master 2 internship and the PhD. I would like to thanks the Dr Sandie Piogé to have pushed me during the Master 2 internship to improve myself, for her advices, all her encouraging words, her humor and her good mood.

I would like to give many grateful thanks to my co-supervisor, Pr Laurent Fontaine to welcome me in his laboratory. I would like to thanks him, for his advices, for his ideas and to trust me as a chemistry teacher. I would like to thanks him as the teacher to guide me in my career path.

I would like to express my sincere gratitude to the Pr. Rachel O'Reilly and the Pr Richard Hogenboom to report my PhD work. I am honored that they accepted to do this work.

I am really honored that the Pr. Philippe Guégan accepted to preside the evaluation comitee of my PhD. I would like to thanks him for his advices and his evaluation.

I would like to thanks warmly Dr. Nathalie Casse for her help for the gel retardation assay and her kindness.

I would like to give many grateful thanks to the Pr. Véronique Montembault for her help and for their advices for education. I would like to thanks the Dr. Pamela Passeto and the Dr. Arnaud Noury.

I would like to thanks Patricia Gangnery for HR-MS analyses and Corentin Jacquemmoz for NMR analyses. I would like to thanks warmly Emmanuelle Mebold for the numerous and difficult MALDI-TOF analyses and for the formation on SEC.

I would like to thank Dr Gwenaël Forcher and Alexandre Bénard for the azlactone syntheses. I would like to especially thank Alexandre Bénard for the advices for the chromatography column and for the invitations.

I would like to give grateful thanks to Anita Loiseau, Emilie Choppé, Aline Lamber and Clément Brière for their help and their kindness.

I would like to thanks Maud, my office co-worker, for the song contests and all the "stories" we talk about. I would like to thanks Clémence for all the work she done during her internship and all the discussions. I would like to thanks Joachim for his wisdom and his culture.

I would like all the PhD student I met, Walailuk for all her stories, Cécile for all her journey, Nguyet for sweet potatoes, Lionel (Dr Lionel Lauber) for all politic debates, Julien and Héloïse for the association and Malika (Dr Malika Talantikite) for her humor.

I would like to thanks the interns who worked on this project: Kévin, Stève and Whenao.

General Introduction 1

Gene therapy is a technique that uses genes to treat or prevent diseases due to genetic disorders. Such technique is based on the introduction of healthy or new genes into a patient's cells. Therefore, efficient delivery of exogenous genetic material is crucial in gene therapy.

Different strategies are available to achieve efficient gene delivery. Physical-based ones such as electroporation or sonoporation are not desirable because of complex protocols and the associated high tendency to induce cellular damage leading to necrosis. 1 Viral-based gene delivery systems including retroviruses and adenoviruses 2 have been employed. The first clinical trial was made in 1989 by Rosenberg et al. 3 to treat metastatic melanoma using retroviruses. Despite their high performance, the wide application of adenoviral and retroviralmediated transfection for therapeutic purposes is limited by their toxicity and immunogenicity.

The use of adenoviruses to treat partial ornithine transcarbamylase led to a patient death during a clinical trial in 1999. 4,5 Consequently, the research and the development of synthetic non-viral gene delivery systems is strongly encouraged. Over time, cationic polymers have notably attracted keen attention, due to their ability to interact with cells more effectively and with higher stability compared to other chemically designed non-viral gene delivery systems, such as cationic lipids. [6][7][8][9][10] For instance, Escande and coworkers, 11 have shown that polymers enhanced gene delivery from the cytoplasm to the nucleus in comparison with cationic lipids and that transgene expression in the nucleus was prevented by complexation with cationic lipids but not with cationic polymers. Moreover, the easy control of the physical parameters (e.g., hydrophobicity, charge) of polymer systems represents an additional advantage over lipidic systems. Many reviews have been published on cationic polymeric systems for gene delivery and it appears that each system has its pros and cons to achieve a high transfection efficiency with a low cytotoxicity. [6][7][8][9][10] This is probably due to the fact that an efficient gene delivery necessitates a combination of numerous requirements (e.g., efficient deoxyribonucleic acid (DNA) complexation, polyplex stability under physiological conditions, cell recognition, endosomal escape, cellular uptake, DNA release). For complexation, the positive charges of cationic polymers interact through electrostatic bindings with the anionic charges of DNA due to the phosphate groups of the backbone. The resulting DNA/polymer complex also named polyplex, has to overcome several barriers for an efficient gene delivery.(Scheme 1) 8,[12][13][14][15][16] First, the polyplex has to travel through the blood circulation. Indeed, a naked DNA can be uptaken by different cells as Kupffer hepatic macrophages cells, leading to the phagocytosis of DNA. 17 The cationic polymer allows to "hide" the negative charges of DNA and stop the uptake by macrophages cells. 17,18 Moreover, the polyplex has to be stable in the blood to avoid any accumulation in different tissues. The addition of a hydrophilic part such as poly(ethylene oxyde) (PEO) avoid any binding between the polyplex and specific proteins in the blood. 19 Such interactions lead to the opsonization of the polyplex. The opsonization is a major barrier to the transfection due to the coating of the polyplex by specific plasma proteins, leading to the clearance of the polyplex. Once the polyplex has gone through the blood, it has to go inside the cell.

The next barrier is the cell membrane which has to be penetrated. This step, named cellular uptake, occurs by endocytosis. 20 To go through the membrane, the cationic polymer has to form electrostatic interactions with anionic cell surface proteoglycans. 21 The interaction of the cationic polymer with specific proteins leads to the internalization of the polyplex via endocytosis. Indeed, it has been shown that a poly(L-Lysine) (PLL) cannot transfect Raji cells, as such cells do not possess proteoglycans proteins. 22 This pathway is specific to the cationic polymers which do not get recognition ligands. Such recognition ligands are able to bind the polyplex to specific receptors on the cell surface. This binding allows the polyplex to be internalized by clathrin-dependent endocytosis pathway. In this pathway, the polyplex is trapped in the endocytic vesicles which fuse with the endosome. 23 Then, the polyplex escapes from the endosome to go to the cytoplasm. Once in the cytoplasm, the DNA has to be released from the cationic polymer to enter the nucleus. The polyplex can be dissociated thanks to specific enzymes and partially in the endosome. 13 To improve the DNA release, degradable cationic polymers can be used, such as polymers containing biodegradable bonds or hydrolysable bonds. Furthermore, a better elimination of the cationic polymer by the cell is targeted when using degradable cationic polymers.

Based on previous studies, [6][7][8][9][10] the key structural parameters to overcome all requirements to target a high transfection efficiency and a low cytotoxicity are: (i) cationic amine entities for DNA complexation and endosomal escape, (ii) PEGylation to achieve a high polyplex stability and to hinder the reticuloendothelial recognition allowing longer circulation, (iii) ligands capable to target specific cell membrane receptors for an efficient cell recognition and cellular uptake and (iv) degradable polymers for efficiently release DNA and to reduce cytotoxicity.

Many studies have evaluated some of the previously mentioned key structural parameters from many different polymers. [6][7][8][9][10] However, the adequate combination of all these structural parameters in a unique polymer scaffold has not been reported yet.

In this context, the aim of this research work is to synthesize one single polymer chain containing both cationic amines and degradable entities by using aminoethyl-based polyacrylates, a covalently linked PEO chain and specific chain-ends to anchor recognition ligands. The interest of such polymer is its aptitude to efficiently complex DNA, its selfdegradation capacity and its ability to react with recognition ligands.

The emergence of controlled/living polymerizations including radical deactivation chain transfer polymerization (RDRP) techniques and versatile strategies for polymer chain-ends modification constitutes a scientific breakthrough for polymer chemists. 28,29 The reversible addition-fragmentation transfer (RAFT) polymerization, the thiol-disulfide exchange reaction and the thiol-yne coupling reaction able to be performed under mild conditions have been applied to synthesize our multi-structural single polymer chain. [30][31][32] The structure/complexation and the structure/cells viability relationships have been investigated during this work. Scheme 1. Barriers to overcome and key structural parameters on a polymer for an efficient gene delivery. This Ph. D manuscript is organized in five chapters.

The first chapter is a bibliographic study on cationic polymers used for DNA delivery.

Their synthesis, their efficiency to deliver DNA into cells and their cytotoxicity are discussed.

The second chapter describes the synthesis of macromolecular chain transfer agents based on linear PEO (lPEO) and brushed-like PEO (bPEO). A heterofunctional lPEO with thiol reactive functions at the α-position and a trithiocarbonate chain transfer agent moiety at the ωposition have been synthesized and fully characterized using different strategies. A heterofunctional bPEO has been synthesized by RAFT polymerization using a new alkynebased chain transfer agent.

The third chapter describes the RAFT copolymerization of two aminoethyl acrylates using a lPEO and a bPEO macromolecular chain transfer agents, and an alkyne-based chain transfer agent. Then, the cationization of the repeating units to form the PEGylated cationic copolymers is discussed.

The fourth chapter describes the trithiocarbonate removal reaction using thermal-and photochemical-activation and the thiol-yne and disulfide exchange coupling reactions to functionalize the PEGylated cationic copolymer using thiol-based molecules.

The fifth chapter describes the pDNA complexation and the cytotoxicity of cationic polymers according to the PEO architectures, the trithiocarbonate removal and the molar composition of cationic copolymers.

Introduction

Cationic polymers containing amines such as polyethyleneimine (PEI), 1 PLL, 2 poly(βamino ester)s (PBAEs) 3 and amine-based vinylic polymers are commonly used and well characterized concerning their DNA delivery potential. 4 Modified cationic polymers have been studied in order to satisfy and meet all the following requirements e.g. good DNA complexation, serum stability, cellular uptake, endosomal escape and DNA release with a high transfection efficiency and with minimal cytotoxicity.

The shielding of the cationic polymer by PEO, also named PEGylation, tends to protect it from blood components and to increase its circulation time. PEO possesses many unique physical and biological properties including non-toxicity and excellent biocompatibility. [5][6][7] It has been confirmed that PEGylation increases nuclease resistance of complexed pDNA in serum supplemented physiological saline, providing steric protection against DNAse degradation to entrapped pDNA and reduces non-specific interactions between polyplexes and endogenous biomolecules. [5][6][7] Two main strategies have been developed to target PEGylated cationic polymersμ the "PEO grafting onto" and the "PEO grafting from".

Although PEGylation will reduce cytotoxicity and endow polyplexes with stealth properties, it often leads to decreased transfection efficiency, likely due to interference with the polyplex-cell interactions 8 and thus to a reduced cellular uptake. 9 Therefore, the introduction of several recognition ligands (tripeptide arginine-glycine-aspartate (RGD), folate, anisamide, etc.) has been considered to achieve targeted uptake of polyplexes through receptor-mediated endocytosis. 10,11 The covalent linkage of recognition ligand is mediated through their reactive amine and thiol entities. Amine and thiol-chemistry reactions are performed in soft conditions (room temperature, aqueous solution) to keep the integrity of the recognition ligand.

Other structural parameters such as degradable linkages have been introduced within cationic polymers used for DNA delivery. 12 Depending on their position within the cationic polymer, in the backbone or in the pendant groups, such degradable linkages will either decrease the polymer cytotoxicity or either increases the transfection efficiency. The degradable linkages used for DNA delivery are either hydrolyzable groups or either biodegradable groups.

The hydrolyzable linkages are generally ester function. Peptide linkage and disulfide linkage can be reduced by bioactivity.

In this chapter, the synthesis of PEI, PLL, PBAEs and amine-based vinylic polymers and the strategies, reported in the literature, to target PEGylated cationic polymers, to introduce recognition ligands and degradable moieties on cationic ligands have been described. The influence of the chemical structure of cationic polymer together with the addition of a PEO, of recognition ligands onto cationic polymers and the presence of degradable moieties within the cationic polymers on the polyplexes transfection efficiency and cytotoxicity will be discussed.

I. Cationic polymers for DNA delivery

As PEI, 1 PLL, 2 PBAEs and vinylic polymers widely studied in the literature as synthetic cationic polymers for gene delivery, 1,3,4 their synthesis together with their transfection efficiency and cytotoxicity will be described in this chapter.

A. Poly(ethylene imine) (PEI)

The PEI is called "the gold standard" of synthetic cationic polymers for DNA delivery as it is the most used and widely studied. 1,13 PEIs are classified into three categories depending on their architectures: linear PEI (lPEI), cyclic PEI (cPEI) and branched PEI (bPEI).

lPEIs are synthesized by a two steps reaction. The first step is the cationic Ring-Opening Polymerization (ROP) of 2-ethyl-2-oxazoline mostly initiated by methyl p-toluenesulfonate to form the poly(2-ethyl-2-oxazoline) and the second step is the hydrolysis, in acidic media, of amide functions (Scheme I. 1(A)). 14,15 The polymer backbone is based on protonable secondary amines which pKa values varied from 7.9 to 8.4. 14,16 cPEIs can be formed by an intramolecular Huisgen click reaction of the α-alkynyl-ωazidopoly(2-ethyl-2-oxazoline) followed by the hydrolysis of the resulting cyclic poly(2-ethyl-2-oxazoline) using hydrochloric acid (Scheme I. 1(B)). 15 bPEI is synthesized via an acid-catalyzed ROP of aziridine. 17 Independently of its number-average molar mass, bPEI contains protonable primary (25 %), secondary (50 %) and tertiary amines (25 %) with pKa values equal to 4.5, 6.7 and 11.6, respectively. 18 Scheme I. 1. Synthesis of (A) a lPEI as described by Brissault et al. 14 , (B) a cPEI as described by Cortez et al. 15 and (C) a bPEI described by Zhuk et al. 17 It has been shown that several PEI macromolecular characteristics such as numberaverage molar mass and architecture are important to control the transfection efficiency and cytotoxicity of such polymers.

It is well-known that PEIs with number-average molar masses below 5 kDa are insufficient in polyplex formation and stability. 19 However, for polymers with number-average molar masses above 5 kDa, Remy and co-workers 20 compared three lPEIs with different number-average molar masses (22 kDa, 88 kDa and 220 kDa) and resulting polyplexes with a nitrogen (N from PEI) to phosphorus (P from DNA) ratio (N/P) of 10/1. They showed a better transfection efficiency of pCMV-Luc, a plasmid DNA containing the firefly luciferase gene reporter, with the 22 kDa PEI to transfect NIH-3T3 cells, fibroblasts cells from mouse embryo.

The second important factor for DNA delivery using PEI is its architecture. Even if the comparison of lPEI, cPEI and bPEI with similar number-average molar masses is difficult as amines substitution, charge density or structural flexibility are different, some studies compared them. Cortez et al. 15 have compared lPEI and cPEI. They synthesized a range of lPEI and cPEI with the same number-average polymerization degree ranging from 21 to 84. They tested these polymers with different N/P ratios on transfection efficiency of pCMV-DsREd-express, a pDNA coding for red fluorescent protein on HAE and HFF1 cells, human aortic endothelial and human foreskin fibroblast, respectively. In all cases, cPEI seems to have better transfection efficiency than lPEI with similar number-average polymerization degree. The increase of transfection efficiency is explained by the increase of the charge density due to the more compact conformation of cPEI than the lPEI one. 15 As transfection efficiency, PEI's cytotoxicity depends strongly of its number-average molar mass and architecture. A comparison between two lPEIs with different number-average polymerization degrees, 70 and 84, shows the increase of cytotoxicity with the increase of polymer number-average molar mass on HAE cells. 15 The same result was obtained with two cPEI with two different number-average polymerization degrees, 70 and 84. These results can be explained by the increase of charge density with increasing number-average molar mass of polymers. Indeed, the cationic charges are able to disrupt cell membrane.

However, a study on bPEI showed that the cytotoxicity of polymers can changed with different cell lines. Okon et al. 21 showed that an 800 Da bPEI is less toxic with HeLa cells, cell line derived from human cervical cancer and a 25 kDa bPEI is less toxic with Vero cells, kidney epithelial cells from an African green monkey. Thus, cytotoxicity depends of the physiology of cells too. The comparison of a lPEI and a bPEI with the same number-average molar mass (25 kDa) shows a lower cytotoxicity of lPEI than bPEI to transfect A431 cells, cell line derived from human epidermoid carcinoma, due to the decrease of the charge density. Indeed, the zeta potential analysis of bPEI shows a charge density almost twice greater than lPEI: 28.7 mV for bPEI and 15.7 mV for lPEI. 22

B. Poly(L-lysine) (PLL)

The protonable PLL with different architectures (linear PLL-lPLL, hyperbranched PLL-hbPLL and dendrimeric PLL-dPLL, (Scheme I. 2)) has been employed for DNA delivery thanks to its ability to complex easily DNA with strong electrostatic interactions. 23 PLL is a synthetic polyamide which owns cationic primary amines at neutral pH. lPLLs can be divided in two categoriesμ the α-Pδδ and the -PLL (Scheme I. 2).

lPLLs can be synthesized using an easy reaction: thermal condensation of L-Lysine in bulk or in aqueous solution. However this reaction leads to a mixture of α-and -PLL. 24 To avoid such mixture, Kushwaha et al. 25 proposed to synthesize a linear -PLL using Nα-Butoxylcarbonyl-N -Carbobenzyloxy-L-Lysine (Nα-Boc-N -Cbz-L-Lysine). The polymerization is made step by step, repeat unit by repeat unit. However the process is harsh, using Lindlar palladium, and with several step, leading to a decrease of yield. 25 Another method to form linear -PLL avoiding the use of protecting groups has been reported. To synthesize only the -PLL, Ho et al. 26

used N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDCI).

When the N,N'-dicyclohexylcarbodiimide (DCC) was used, only the α-PLL was formed. 27 hbPLL is synthesized by heating L-Lysine (LL) at high temperature under basic medium and with several metalorganics catalysts as Zr(OnBu)4, Ti(OnBu)4, Sb(OEt)3. These conditions lead to the amidification of the carboxylic acid with the amine of L-lysine. 28 The synthesis of dPLL needs several steps. First step is the protection of amine functions of LL. In the second step, the protected LL reacts by amidification with hexylene diamine using coupling agents. Finally, the amines are deprotected to generate the first generation dPLL. The formation of the next generations follows the same reactions with same conditions. 29 Scheme I. 2. Chemical structures of linear α-PLL and -PLL, hyperbranched PLL and dentritic PLL. [26][27][28][29] As for PEI, the transfection efficiency is correlated to the number-average molar mass and the architecture of PLL. Kadlecova et al. 30 compared the transfection efficiency of PLLs depending on their number-average molar mass and their architecture on CHO DG44 cells, chinese hamster ovary cells, by measuring a green fluorescent protein, eGFP, expression and IgG production, immunoglobulin G. For a same architecture, they proved the increase of transfection efficiency with the number-average molar mass on lPLL, hbPLL or dPLL. For example, with a N/P = 3/1, the green fluorescence is five times higher, representing the internalization of lPLL in CHO DG44 cells when the lPLL number-average molar mass increases from 800 to 19600 Da. These results show a better binding of lPLL on extracellular membrane with a higher number-average molar mass. Similar results have been obtained for hbPLL and dPLL. Moreover, Kadlecova et al. 30 compared the transfection efficiency according to the architecture keeping a comparable number-average molar mass. The hbPLL shows a higher transfection efficiency than dPLL and lPLL. Authors explained this result because hbPLL has the best binding affinity with the cell membrane due to the highest pH buffering effect.

Moreover, PLL number-average molar mass and architecture have an impact also on cytotoxicity. Kadlecova et al 29 compared the effect of number-average molar mass and architecture of PLL cytotoxicity on CHO DG44 cells. For a specific architecture, when the number-average molar mass of PLL increases, the cell viability decreases. When PLL numberaverage molar mass increases, the PLL interactions with cell membranes increase. These interactions lead to disruption of cell membranes by increasing the cationic group of polymer.

As for PEI, PLL architecture plays a key role in cytotoxicity. In the same study, Kadlecova et al. 29 showed the effect of different architectures, lPLL, hbPLL and dPLL. With comparable number-average molar mass, the lPLL seems to be five times less toxic than hbPLL and dPδδ. The comparison of hbPδδ and dPδδ doesn't show a significant difference. They showed the complete degradation of a lPLL with trypsin after 8 hours, a peptidase found in gastric acid, and a partial degradation of hbPLL and dPLL. This degradation can explain the lower cytotoxicity of lPLL. 30 Moreover, there is a difference between the two linear analogues, α-PLL and -PLL. Indeed, De Vries et al. 31 shows that the α-PLL is toxic for mice, guinea pig, rabbits and human kind and causes unacceptable damages to lungs, and membrane cells due to the too high charge of cationic primary amines which react with negatively charged membrane of lungs or kidney. 32 The high solubility of -Pδδ in water, compared to the α-PLL, is a great asset for bio-application indeed the lack of solubility in water can lead to aggregation and block arteries leading to necrosis and heart attack. 27 However, the -PLL polyplex is very stable which avoid destabilization of polyplex leading to the DNA release.

C. Poly(β-amino ester) (PBAE)

PBAE has been studied for DNA delivery as they got the advantage of being protonable due to the amine group and of being biodegradable due to the ester function. 3 The first study of PBAE used for DNA delivery has been made by the team of Langer in 2000. 33 In this work, they prove the efficiency to form several linear PBAEs (lPBAE)s synthesized through Michael addition (Scheme I. 3(A)) able to complex DNA efficiently. In a recent work, a hyper branched poly(β-aminoester) (hbPBAE) was synthesized using a trialkene monomer as a crosslinker in a one-pot Michael addition (Scheme I. 3(B)). 34 The number-average molar mass of PBAE plays a key role on transfection efficiency.

Akinc et al. 35 studied two lPBAEs (Scheme I. 3(C)) with different number-average molar masses (5 and 15 kDa) complexing a pDNA containing luciferase on COS-7 cell line, fibroblast from monkey kidney tissue. The results show the increase of transfection efficiency with the increase of lPBAE number-average molar masses. Like PEI or PLL, this increase is really significant. The values of transfection efficiency for a lPBAE of 5 kDa are nearly 0 ng per well as for a lPBAE with a number-average molar mass twice higher the transfection efficiency is around 50 ng per well, for a polymer/pDNA ratio of 80 (w/w %) (this has been explained by the absence of polyplexes formation with the low number-average molar mass lPBAE as it was not able to retard pDNA on a gel agarose assay).

The effect of PBAE architecture on transfection efficiency has also been studied. As compared to PEI and PLL, PBAE number-average molar mass has an influence on cytotoxicity. The comparison of lPBAE and hbPBAE with similar number-average molar masses shows that lPBAE is less toxic than hbPBAE on HeLa, human cervical cancer cells, NHK, normal human keratinocytes cells, and RDEBK, epidermolysis bullosa keratinocyte cells, because of the poor interaction between the lPBAE/DNA polyplex and the cell membrane and the better hydrophilicity of lPBAE. Unlike PEI and PLL, cytotoxicity tests of hbPBAE of different number-average molar masses showed an increase of cell viability with the numberaverage molar mass on HeLa cells and SHSY-5Y cells, cells of human bone marrow suffering from neuroblastoma. 36 However, Eltoukhy et al. 37 showed no significant difference on cytotoxicity of lPBAE on HeLa cells with different molar masses. The authors explain this result with the increase of number-average molar mass, the size of polyplexes decrease which leads to the decrease of cytotoxicity. Moreover the extremities of PBAEs have also an effect on cytotoxicity. 35 It has been shown that an hydroxyl-terminated PBAE is less toxic than an acrylate-terminated PBAE on COS-7 cells (Scheme I.

D. Cationic vinylic polymers

Cationic polymers based on vinyl monomers such as (meth)acrylates and (meth)acrylamides have been developed as potential transfecting agents. 13,38,[START_REF] Agarwal | PDMAEMA Based Gene Delivery Materials[END_REF] Van de Wetering et al. [START_REF] Van De Wetering | tru ture-A ti it Relatio ships of Water-Soluble Cationic Methacrylate/Methacrylamide Polymers for Nonviral Gene Delivery[END_REF] studied different cationic amine-based polymers (Scheme I. 4) on transfection efficiency on OVCAR-3 using pCMV-LacZ, a pDNA containing a mammalian expression vector. They showed that the transfection efficiency is better with (meth)acrylates than with The first synthesis of linear PDMAEMA (lPDMAEMA) made by RDRP methods was performed using Atom Transfer Radical Polymerization (ATRP) by Matyjaszewski's group in different conditions (Scheme I. 5(A)). [START_REF] Ha G | Mat jasze ski, K. Co trolled/ Li i g Radi al Pol erization of 2-(Dimethylamino)ethyl Methacrylate[END_REF] Furthermore, Synatschke et al. [START_REF] Synatschke | Influence of Polymer Architecture and Molecular Weight of Poly(2-(Dimethylamino)ethyl Methacrylate) Polycations on Transfection Efficiency and Cell Viability in Gene Delivery[END_REF] Among the RDRP methods, RAFT polymerization has been also employed to synthesize PDMAEMA. The RAFT polymerization of 2-(N,N-dimethylamino)ethyl methacrylate (DMAEMA) can occurs in organic solvent, [START_REF] Sahnoun | theti a d Chara terizatio Aspects of Dimethylaminoethyl Methacrylate Reversible Addition Fragmentation Chain Transfer (RAFT) Polymerization[END_REF] in water, [START_REF] Xiong | Synthesis and Characterization of 2-(Dimethylamino)ethyl Methacrylate Homopolymers via Aqueous RAFT Polymerization and Their Application in Miniemulsion Polymerization[END_REF] by heating or UV-exposition. 4,[START_REF] Yuan | Synthesis of Poly(dimethylaminoethyl Methacrylate) with High Cloud Point by RAFT Polymerization under γ-Irradiation[END_REF][START_REF] Wu | Star Polymers: Advances in Biomedical Applications[END_REF] As an example, Sahnoun et al. [START_REF] Sahnoun | theti a d Chara terizatio Aspects of Dimethylaminoethyl Methacrylate Reversible Addition Fragmentation Chain Transfer (RAFT) Polymerization[END_REF] The effect of the architecture of PDMAEMA has been studied on transfection efficiency by Müller and co-workers. [START_REF] Synatschke | Influence of Polymer Architecture and Molecular Weight of Poly(2-(Dimethylamino)ethyl Methacrylate) Polycations on Transfection Efficiency and Cell Viability in Gene Delivery[END_REF] They compared lPDMAEMA with two stars PDMAEMA, the sPDMAEMA-3 and the sPDMEMA-5. They transfected CHO-K1 cells with a pDNA containing a green fluorescent protein. At comparable number-average molar mass and N/P = 2, the lPDMAEMA is more effective than the two stars and the sPDMAEMA-5 is more effective than the sPDMAEMA-3. When the ratio N/P is increases to 20, there are no significant differences between the architectures. The authors explain this result by the difficulty of DNA to reach the nitrogen in the core of stars polymers.

The effect of lPDMAEMA number-average molar masses on transfection efficiency of pCMV-LacZ a mammalian expression vector was investigated in two different cell lines, COS-7 and OVCAR-3, epithelial cells from human ovary suffering from adenocarcinoma. [START_REF] Van De Wetering | Relation between Transfection Efficiency and Cytotoxicity of Poly (2-(Dimethylamino) Ethyl Methacrylate)/Plasmid Complexes[END_REF] In OVCAR-3 cells, the transfection efficiency increases with the lPDMAEMA number-average molar mass from 4 to 817 kDa. In COS-7 cells, the transfection efficiency increases with the number-average molar mass to a limit value (300 kDa) then the transfection efficiency decreases. The increase is due to the free cationic polymers (non-complexed chains) which destabilize the endosomal membrane and help the transfection. Above a number-average molar mass of 300 kDa, the high number of free cationic polymers can limit the transfection by destabilizing directly the cell membrane. Such behavior has a similar impact on cytotoxicity.

When the number-average molar mass increases until 63 kDa for COS-7 cells and 27 kDa for OVCAR-3 cells, the cell viability decreases. Then, for a number-average molar mass around 300 kDa, a plateau is reached and the cell viability at this plateau is around 50 % for both celllines.

The effect of PDMAEMA architecture on cytotoxicity has been studied by Müller and co-workers too. [START_REF] Synatschke | Influence of Polymer Architecture and Molecular Weight of Poly(2-(Dimethylamino)ethyl Methacrylate) Polycations on Transfection Efficiency and Cell Viability in Gene Delivery[END_REF] The sPDMAEMA-3 and sPDMAEMA-5 shows a lower cytotoxicity than a lPDMAEMA on CHO-K1 cells. The authors explained this result because of the charge density.

In the star polymers sPDMAEMA-3 and sPDMAEMA-5, the charge density is hiding in the core of the stars. As for PEI and PLL, 22 the increase of the charge density leads to the decrease of cell viability. aminohexyl methacrylate (AHMA) using tBocAEMA and tBocAHMA, respectively, the P(DMAEMA-co-AEMA) and P(DMAEMA-co-AHMA) contain ammonium groups from primary and tertiary amines (Scheme I. 6(A)). [START_REF] Zhu | Cationic Methacrylate Copolymers Containing Primary and Tertiary Amino Side Groups: Controlled Synthesis via RAFT Polymerization, DNA Condensation, and in Vitro Gene Transfection[END_REF] Such copolymers were employed to transfect COS-7 cells using the pDNA pCMV-Luc as a reporter gene. They compared several molar ratios of primary and tertiary amines for both copolymers, P(DMAEMA-co-AEMA) and P(DMAEMA-co-AHMA). The results show the decrease of the buffer capacity with the increase of primary amine ratios due to the higher pKa. However the increase of primary amine ratio allows improving the DNA condensation as shown on the gel agarose retardation assay.

They showed the better efficiency of P(DMAEMA-co-AHMA) than P(DMAEMA-co-AEMA)

to transfect pCMV-Luc on COS-7 cells. The authors explained this result from: (i) the smallest size of P(DMAEMA-co-AHMA)/pDNA complex than P(DMAEMA-co-AEMA)/pDNA complex, (ii) higher charge density of P(DMAEMA-co-AHMA) and (iii) a better association of P(DMAEA-co-AHMA) with cell membrane due to the hydrophobic interactions of AHMA units. As for the other cationic polymers, transfection efficiency has to be linked with cytotoxicity. The study of the effect of primary amine amount on cell viability didn't show any significant differences with P(DMAEMA-co-AHMA) at different DMAEMA/AHMA molar ratio. However, the P(DMAEMA-co-AEMA) shows a better cell viability with a higher DMAEMA/AEMA molar ratio due to the lower charge density.

Moreover, the acrylate associated monomers have been used because of their efficiency to self-degrade. [START_REF] Truong | Self-Catalyzed Degradable Cationic Polymer for Release of DNA[END_REF] Authors compared the transfection efficiency and the cytotoxicity of PDMAEA with number-average molar mass ranging from 3000 to 8600 g.mol -1 on Hela-cells with fluorescein isothiocyanate (FITC)labeled oligoDNA. Authors showed the increase of transfection with the increase of numberaverage molar mass for PDMAEA (from 10 % to 80 % of transfected cells in 4 hours). The PDMAEA of 8600 g.mol -1 showed interesting results with a high transfection efficiency in just 4 hours. Furthermore, the DNA/PDMAEA polyplexes formed with PDMAEAs of low numberaverage molar mass (from 3000 to 5000 g.mol -1 ) can be dissociated easily to release DNA as shown by gel retarding assay. For PDMAEA with a number-average molar mass ranging from 6900 to 8600 g.mol -1 , a partial dissociation has been observed. The study on cytotoxicity showed the same behavior between PDMAEA with low number-average molar masses and with high number-average molar masses. Indeed, authors showed the non-cytotoxicity of PDMAEA with a number-average molar mass equal or below 5600 g.mol -1 .

Scheme I. 6. (A) Chemical structures of P(DMAEA-co-AEMA) and P(DMAEMA-co-AHMA) copolymers obtained by RAFT polymerization and studied by Zhu et al. [START_REF] Zhu | Cationic Methacrylate Copolymers Containing Primary and Tertiary Amino Side Groups: Controlled Synthesis via RAFT Polymerization, DNA Condensation, and in Vitro Gene Transfection[END_REF] (B) RAFT polymerization of 2-(N,N-dimethylamino)ethyl acrylate (DMAEA) mediated through MCETTC as the chain transfer agent and AIBN as the initiator in 1,4-dioxane at 60°C as described by Monteiro and co-workers. [START_REF] Truong | Self-Catalyzed Degradable Cationic Polymer for Release of DNA[END_REF] Cationic polymers (PEI, PLL, PBAE and vinylic polymers) showed high charge density inducing cytotoxicity. To overcome this issue many reports have been made on the PEGylation of cationic polymers.

II. PEGylation

PEGylation is based on the covalent linkage of PEO on molecules, most tipically peptides, proteins and antibody fragments. PEO possesses many unique properties including noncytotoxicity, non-immunogenesis, non-antigenicity, excellent biocompatibility and miscibility with many solvent. 4,[START_REF] Kolate | PEG -A Versatile Conjugating Ligand for Drugs and Drug Delivery Systems[END_REF] Therefore, the clinical value of PEGylation is well established: increases stability, enhanced protection from proteolytic degradation, extended circulating life,… [START_REF] Kolate | PEG -A Versatile Conjugating Ligand for Drugs and Drug Delivery Systems[END_REF][START_REF] Campa | Anti-VEGF Compounds in the Treatment of Neovascular Age Related Macular Degeneration[END_REF] The addition of PEO on a non-viral DNA delivery system has been reported, on PEI, [START_REF] Pezzoli | RGD-Derivatized PEI-PEG Copolymers: Influence of the Degree of Substitution on the Targeting Behavior[END_REF] on PLL, [START_REF] Thiersch | The Angiogenic Response to PLL-G-PEG-Mediated HIF-1α Plasmid DNA Delivery in Healthy and Diabetic Rats[END_REF][START_REF] Kim | Synthesis and Application of Poly(ethylene Glycol)-Co-Poly(β-Amino Ester) Copolymers for Small Cell Lung Cancer Gene Therapy[END_REF] on PBAE, [START_REF] Kim | Synthesis and Application of Poly(ethylene Glycol)-Co-Poly(β-Amino Ester) Copolymers for Small Cell Lung Cancer Gene Therapy[END_REF][START_REF] Mastorakos | Highly Compacted Biodegradable DNA Nanoparticles Capable of Overcoming the Mucus Barrier for Inhaled Lung Gene Therapy[END_REF] and mostly on cationic vinylic polymers. [START_REF] Rungsardthong | Copolymers of Amine Methacrylate with Poly(ethylene Glycol) as Vectors for Gene Therapy[END_REF][START_REF] Lin | Structural Contributions of Blocked or Grafted poly(2-Dimethylaminoethyl Methacrylate) on PEGylated Polycaprolactone Nanoparticles in siRNA Delivery[END_REF][START_REF] Lai | PEGylated Block Copolymers Containing Tertiary Amine Side-Chains Cleavable via Acid-Labile Ortho Ester Linkages for pH-Triggered Release of DNA[END_REF] A.

How to target PEGylated cationic polymers?

Two main strategies have been developed to covalently attach PEO to a cationic polymer.

The first strategy is based on the linkage of the PEO to a preformed cationic polymer. This strategy will be called "PEO grafted onto". The second strategy is to build the cationic polymer from a PEOν such strategy will be called "PEO grafted from".

The "PEO grafted onto" strategy needs the use of very reactive functions to target an efficient coupling reaction. For example, isocyanate-functionalized PEO has proved to be reactive towards amines and allows to PEGylate a lPEI (Scheme I. 7(A)). [START_REF] Petersen | Synthesis, Characterization, and Biocompatibility of Polyethylenimine-Graft-Poly(ethylene Glycol) Block Copolymers[END_REF] Unfortunately, this strategy has led to a very low functionalization as only 15 % of lPEI has been attached to the PEO. Recently, Pezzoli et al. [START_REF] Pezzoli | RGD-Derivatized PEI-PEG Copolymers: Influence of the Degree of Substitution on the Targeting Behavior[END_REF] used an α-N-hydroxysuccinimide,ω-maleimide PEO (Scheme I.

(B)

) to react with primary amines of a bPEI. From 0.36 % to 5.58 % of primary amines have been grafted with PEO. A PEO was also grafted onto an α-PLL by reacting a Nhydrosuccinimide-PEO onto an α-PLL (Scheme I. 7(C)). [START_REF] Pasche | Relationship between Interfacial Forces Measured by Colloid-Probe Atomic Force Microscopy and Protein Resistance of Poly(ethylene Glycol)-Grafted Poly( L -Lysine) Adlayers on Niobia Surfaces[END_REF] This reaction doesn't need harsh conditions and allows tuning easily the grafting ratio of PEO onto the α-PLL from 4.4 % to 45

%. The versatile "click-chemistry" has also been employed to covalently attach PEO to a cationic polymer. [START_REF] Kolb | Click Chemistry: Diverse Chemical Function from a Few Good Reactions[END_REF][START_REF] Hoyle | Thiol-Click Chemistry: A Multifaceted Toolbox for Small Molecule and Polymer Synthesis[END_REF] Kim et al. [START_REF] Kim | Synthesis and Application of Poly(ethylene Glycol)-Co-Poly(β-Amino Ester) Copolymers for Small Cell Lung Cancer Gene Therapy[END_REF] used the thiol-ene coupling reaction to link a diacrylate-based PEO on a lPBAE (Scheme I. 7(D)). The thiol-ene reaction has been made using soft conditions, at room temperature in basic conditions with a total conversion of the diacrylate-based PEO as determined by 1 H nuclear magnetic resonance (NMR) spectroscopy.

Apart from the thiol-ene coupling reaction such "PEO grafted onto" strategy is generally limited to low attachment efficiency probably due the incompatibility of polymers and also to the accessibility of the chain-end. The PEGylation of cationic vinylic polymers is mostly based on the second strategiesμ the "PEO grafted from".

Scheme I. 7. Covalent linkage of PEO using the grafted-onto strategy on (A) a lPEI thanks to a diisocyanate reagent; 60 (B) a bPEI thanks to a N-hydroxy-succinimide,ω-maleimide-PEO; [START_REF] Pezzoli | RGD-Derivatized PEI-PEG Copolymers: Influence of the Degree of Substitution on the Targeting Behavior[END_REF] (C) a α-Pδδ using a α-N-hydroxysuccinimide-PEO 60 and (D) a PBAE by thiol-ene coupling reaction. [START_REF] Kim | Synthesis and Application of Poly(ethylene Glycol)-Co-Poly(β-Amino Ester) Copolymers for Small Cell Lung Cancer Gene Therapy[END_REF] Such "PEO grafted from" strategy has been possible thanks to RDRPs or living polymerization techniques. Usually the chemical chain-end modification of PEO with a low number-average molar mass molecule is performed in order to introduce initiating group or a chain transfer agent moiety. Such chemical modification is quantitative and leads to target fully PEGylated cationic polymers. ATRP, RAFT polymerization and ROP have been widely

employed to synthesize cationic polymers from functionalized PEO and resulting copolymers have been tested for gene delivery. [START_REF] Choi | Development of Polymeric Gene Delivery Carriers: PEGylated Copolymers of L-Lysine and L-Phenylalanine[END_REF][START_REF] Välimäki | Effect of PEG-PDMAEMA Block Copolymer Architecture on Polyelectrolyte Complex Formation with Heparin[END_REF][START_REF] Venkataraman | The Role of PEG Architecture and Molecular Weight in the Gene Transfection Performance of PEGylated Poly(dimethylaminoethyl Methacrylate) Based Cationic Polymers[END_REF] For instance, to process ATRP, Välimäki et al. [START_REF] Välimäki | Effect of PEG-PDMAEMA Block Copolymer Architecture on Polyelectrolyte Complex Formation with Heparin[END_REF] first quantitavely synthesized an αmethoxy-ω-bromo-PEO macroinitiator by reacting an -hydroxyl-PEO on the 2bromoisobutyrylbromide. Such macroinitiator was then employed to perform the ATRP of DMAEMA using a copper complex (Scheme I. 8(A)). To avoid the use of copper, RAFT polymerization is more and more employed for the "PEO grafted from" strategy. Venkataraman Đ value of 1.18. The PEO "grafted from" strategy has also been used with ROP. Choi et al. [START_REF] Choi | Development of Polymeric Gene Delivery Carriers: PEGylated Copolymers of L-Lysine and L-Phenylalanine[END_REF] have polymerized the carbobenzyloxy-L-Lysine (CbzLL) and L-phenylalanine-L-Lysine (δPheδδ) by ROP using an α-methoxy,ω-amino-PEO of 2000 g.mol -1 as an initiator to form a PEO-b-P(LPheLL-co-CbzLL). They performed the amine deprotection of the poly(carbobenzyloxy-L-Lysine) (PCbzLL) using acidic conditions to target PEO-b-P(LPheLL-co-LL) copolymers with number-average molar masses ranging from 31300 g.mol -1 to 37700 g.mol -1 and ratios of L-Lysine to LPheLL ranging from 10/0 to 6/4.

This method can also be used to link a branched PEO. Venkatamaran et al. [START_REF] Venkataraman | The Role of PEG Architecture and Molecular Weight in the Gene Transfection Performance of PEGylated Poly(dimethylaminoethyl Methacrylate) Based Cationic Polymers[END_REF]

B. Impact of PEGylation on transfection efficiency and cytotoxicity

Zhang et al. [START_REF] Zhang | Poly(ethylene Glycol)-Block-Polyethylenimine Copolymers as Carriers for Gene Delivery: Effects of PEG Molecular Weight and PEGylation Degree[END_REF] studied the PEGylation effect of a bPEI of 25 kDa on transfection efficiency and cytotoxicity on HeLa cells with a plasmid pEGFP-C1, a plasmid containing the enhanced green fluorescent protein. They have shown that with a low grafting ratio (1.89 mole of PEO of 2 kDa for one mole of bPEI or 0.66 mole of PEO of 5 kDa for one mole of bPEI), the transfection efficiency is higher with PEO than without PEO due to the increase of the polyplexes stability. However, a decrease of the transfection efficiency has been observed for a higher grafting ratio (4.17 moles of PEO of 2 kDa for one mole of PEI or 1.67 mole of PEO of 5 kDa for one mole of PEI). This has been explained by the decrease of zeta potential (from 20 mV to 3 mV) with the increase of grafting ratios leading to a decrease of interactions with the cell membrane. [START_REF] Zhang | Poly(ethylene Glycol)-Block-Polyethylenimine Copolymers as Carriers for Gene Delivery: Effects of PEG Molecular Weight and PEGylation Degree[END_REF] Moreover, Zhang et al. studied the effect of the number-average molar mass of the grafted PEO on Hela cells. Two PEOs with number-average molar masses of 2 kDa and 5 kDa have been compared. They have shown that the transfection with a PEO of 5 kDa is less effective than with a PEO of 2 kDa. Indeed the zeta potential of the cationic polymer is lowest with the 5 kDa than with the 2 kDa PEO. This result leads to a lower binding affinity with the membrane cell. Moreover, these two factors, grafting ratio and number-average molar mass, play a role on cytotoxicity. At low concentrations of cationic polymer, ranging from 1 µg.mL -1 and 10 µg.mL -1 , there are no significant differences between PEGylated bPEI and a bPEI. The addition of PEO leads to a decrease of cytotoxicity for concentrations higher than 10 µg.mL -1 . This fall of the cytotoxicity goes on with the number of PEOs unit. Indeed, when the number-average molar mass of the PEO increases the cell viability increases. This phenomenon has been explained by the decrease of charge density with the increase of the PEO numberaverage molar mass. Similar result has been obtained by Fitzsimmons et al. [START_REF] Fitzsi O S | pe ifi Effe ts of PEG latio o Ge e Deli er Effi a of Polyethylenimine: Interplay between PEG Substitution and N/P Ratio[END_REF] on HEK293 cells, Human Embryonic Kidney cells.

Other studies have been reported on the PEGylation effect of PDMAEMA on transfection efficiency. Similar conclusions than previous studies with the bPEI have been obtained. The PEGylation of PDMAEMA decreases both transfection efficiency and cytotoxicity. The comparison between PDMAEMA and PEO-b-PDMAEMA proved the better transfection efficiency of PDMAEMA than of PEO-b-PDMAEMA on A-549 cell line, [START_REF] Rungsardthong | Copolymers of Amine Methacrylate with Poly(ethylene Glycol) as Vectors for Gene Therapy[END_REF] adenocarcinoma human alveolar basal epithelial cells with PRSVlucDNA, containing luciferase reporter gene obtained from cobra. The authors explained these results by a steric hindrance due to the PEO acting like a shield leading to a decrease of interaction of the polyplex with the cell membrane.

The PEO architecture can also influence the transfection efficiency and the cytotoxicity.

Indeed the poly(oligo(ethylene glycol) acrylate) (PPEGA) is a brush-like PEO. Venkataraman et al. [START_REF] Venkataraman | The Role of PEG Architecture and Molecular Weight in the Gene Transfection Performance of PEGylated Poly(dimethylaminoethyl Methacrylate) Based Cationic Polymers[END_REF] than PDMAEMA-b-PPEGA. However, the architecture of the PEO doesn't play a key role on cytotoxicity. Indeed, the authors didn't show any significant differences between the two copolymers.

The positive influence of PEGylation on the cellular viability to the detriment of transfection efficiency has been shown. However, a promising strategy to overcome the decrease of transfection efficiency due to the PEGylation of cationic polymers is the addition of a recognition ligand.

III. Recognition ligands addition

Some diseases, as cancers, lead to a production of specific protein on the cell membrane.

These specific proteins can be targeted with a recognition ligand. [START_REF] Sergeeva | Display Technologies: Application for the Discovery of Drug and Gene Delivery Agents[END_REF] Several targeting ligands are used including antibodies and peptides. [START_REF] Park | A Review of RGD-Functionalized Nonviral Gene Delivery Vectors for Cancer Therapy[END_REF] The recognition ligand has to be added on the cationic polymer by versatile, efficient reactions that can be proceed at room temperature in water to maintain the integrity of ligands.

A. Incorporation of recognition ligand onto cationic polymers

There are different methods to add several kinds of recognition ligands on cationic polymers. For example, Pezzoli et al. [START_REF] Pezzoli | RGD-Derivatized PEI-PEG Copolymers: Influence of the Degree of Substitution on the Targeting Behavior[END_REF] reported the anchoring of a cyclic tripeptide arginineglycine-aspartate (cRGD) and a linear RGD (lRGD) on a PEGylated b-bPEI (Scheme I. 9 (A)).

RGD is able to target several integrins as, α5β1, α8β1, αIIbβ3, αVβ3, α5Vβ5, αVβ6 and αVβ8. [START_REF] Takagi | Structural Basis for Ligand Recognition by RGD (Arg-Gly-Asp)-Dependent Integrins[END_REF][START_REF] Plow | Ligand Binding to Integrins[END_REF] First, the authors grafted a maleimide and a N-hydroxysuccinimide-functionalized PEO onto a bPEI through nucleophilic substitution. Then, they added the amine-based cRGD or lRGD onto maleimide moieties of the PEGylated bPEI through nucleophilic addition. The complete addition of the cRGD and lRGD was checked by the titration of unreacted RGD with Ellman's test after purification by ultra-filtration. The degrees of functionalization was from 0.36 to 5.58 %.

Suh et al. [START_REF] Suh | Anti-JL1 Antibody-Conjugated Poly (L-Lysine) for Targeted Gene Delivery to Leukemia T Cells[END_REF] grafted an antibody on a PLL of 20 kDa containing thiol units through a disulfide coupling exchange between PLL units functionalized by a thiol and an antibody functionalized by a pyridyl disulfide bound (Scheme I. 9(B)). They finally obtained a statistical copolymer containing LL units and LL-grafted-antibody units with a number-average molar ratio of 98.5/1.5, respectively. The degree of functionalization was equal to 68 %.

Moreover, a recognition ligand can be added on the chain-end of linear cationic polymer.

Qian et al. [START_REF] Qian | PEGylated poly(2-(Dimethylamino) Ethyl Methacrylate)/DNA Polyplex Micelles Decorated with Phage-Displayed TGN Peptide for Brain-Targeted Gene Delivery[END_REF] synthesized a PEO-b-PDεAEεA by ATRP with an α-maleimide,ω-bromide PEO used as the macroinitiator (Scheme I. 9(C)). Then they finally added a 12-amino acids peptide, TGNYKALHPHNG (TGN), by nucleophilic addition on the maleimide with a thiolated TGN.

They obtained a complete conversion determined by 1 H NMR spectroscopy.

Scheme I. 9. (A) cRGD functionalization of a PEO-b-bPEI as described by Pezzoli et al. [START_REF] Pezzoli | RGD-Derivatized PEI-PEG Copolymers: Influence of the Degree of Substitution on the Targeting Behavior[END_REF]

B. Impact of recognition ligand on transfection efficiency of polyplexes

The addition of a recognition ligand onto cationic polymers has an impact on their transfection efficiency through the recognition of some proteins on cell membrane.

Oba et al. [START_REF] Oba | Cyclic RGD Peptide-Conjugated Polyplex Micelles as a Targetable Gene Delivery System Directed to Cells Possessing α v β 3 and α v β 5 Integrins[END_REF] PEGylation, the addition of a recognition ligand has no effect on cytotoxicity.

IV. Cationic polymers degradability and its effect on transfection efficiency and cytotoxicity

Degradable linkages have been introduced within cationic polymers used for DNA delivery. Depending on their position within the cationic polymer, in the backbone or in the pendant groups, such degradable linkages either decrease the polymer cytotoxicity or either increases the transfection efficiency. The degradable linkages used for DNA delivery are either hydrolyzable groups or either biodegradable groups. The hydrolyzable linkages are generally ester function. Peptide linkage and disulfide linkage can be reduced by bioactivity.

A. Hydrolyzable functions

Hydrolyzable ester functions are present within the pendant groups of amine-based poly(meth)acrylate, they can be introduced on a non-degradable polymer as PEI and in the

PBAEs backbone

The PDMAEMA with pendant esters functions can be hydrolyzed depending on the pH.

Van de Wetering et al. [START_REF] Van De Wetering | A Mechanistic Study of the Hydrolytic Stability of Poly(2-(Dimethylamino)ethyl Methacrylate)[END_REF] have studied the hydrolytic stability of polyplexes based-on pCMV-LacZ etiher PDMAEMA of a number-average molar mass of 17000 g.mol -1 or with PDMAEA of a number-average of 9000 g.mol -1 , at different pH. After 6 months of incubation at 37°C, authors didn't observe traces of the degradation products by high performance liquid chromatography (HPLC). To increase the hydrolysis rate, they increased the temperature. They showed that the hydrolysis rate of PDMAEA is 50 % at pH 7 and 80 % at pH 1 in 12 days. The hydrolysis rate of PDMAEMA is lower than the hydrolysis rate of PDMAEA as 45 % at pH 1 and less than 10 % at pH 7 in 12 days. This is probably due to a better solubility of PDMAEA in water than PDMAEMA. Therefore, Monteiro and co-workers studied more deeply the hydrolysis of PDMAEA leading to the formation of poly(acrylic acid) (PAA) and 2-N,Ndimethylaminoethanol (Scheme I. 10 (A)). [START_REF] Truong | Self-Catalyzed Degradation of Linear Cationic Poly(2-Dimethylaminoethyl Acrylate) in Water[END_REF] PAA is a non-toxic polymer already used in oral chemotherapy. [START_REF] Bromberg | Polymeric Micelles in Oral Chemotherapy[END_REF] Several PDMAEAs have been synthesized with number-average molar mass ranging from 3300 to 8600 g.mol -1 . In 200 hours, all PDMAEAs reached 80 % of degradation whatever the pH (pH = 5.1 and 7.4).

Hydrolyzable ester functions have been added on non-degradable 800 Da bPEI-NH2 (800 Da) by Forrest et al. [START_REF] Forrest | A Degradable Polyethylenimine Derivative with Low Toxicity for Highly Efficient Gene Delivery[END_REF] . The nucleophilic addition of a bPEI-NH2 onto a diacrylate has been performed (Scheme I. 10(B)) and has led to bPEI with higher number-average molar masses, 14 kDa (named P1) and 30 kDa (named P2), which are more effective to transfect pDNA containing luciferase gene on MDA-MB-231, human breast carcinoma cell line and C2C12, murine myoblast cell line than a bPEI with lower number-average molar mass. 20 Forrest et al. [START_REF] Forrest | A Degradable Polyethylenimine Derivative with Low Toxicity for Highly Efficient Gene Delivery[END_REF] showed the degradation of 40 % of ester linkages at pH 5.1 and 80 % at pH 7.4 for P1 and P2.

Such P1 and P2 have been compared with a non-degradable bPEI of 25 kDa in terms of transfection efficiency and of cytotoxicity. [START_REF] Forrest | A Degradable Polyethylenimine Derivative with Low Toxicity for Highly Efficient Gene Delivery[END_REF] They tested the transfection efficiency of such polymers using pGL3, a pDNA containing a luciferase gene onto two cell lines, MDA-MB-231 and C2C12. P2 is more efficient to transfect MDA-MB-231 cells lines at low N/P ratios, 4/1, but at higher N/P, from 8/1 to 12/1 ratios, P1 is more efficient. With C2C12 cells, both polymers don't show any significant difference. εoreover, P1 and P2 showed higher transfection efficiency than bPEI (from 2 fold to 6 fold for MDA-MB-231 and 9 fold to 16 fold for C2C12). [START_REF] Forrest | A Degradable Polyethylenimine Derivative with Low Toxicity for Highly Efficient Gene Delivery[END_REF] The authors explained these results by a better pDNA release with degradable P1 and P2.

Moreover, all bPEIs (degradable and non-degradable) don't show any significant cytotoxicity on MDA-MB-231 cells. But with C2C12 cells, P1 and P2 showed a lower cytotoxicity than non-degradable bPEI.

Hydrolyzable ester functions which are present within the PBAEs backbone can be easily degraded at 37°C in less than 40 hours at pH 5.1 and in less than 70 hours at pH 7.4. 33 The degradation has been followed by size exclusion chromatography (SEC) and the resulting byproducts were identified by 1

B. Biodegradable linkages

The disulfide bounds can be reduced by glutathione, an enzyme found in the cytoplasm. [START_REF] Lin | The Role of the Disulfide Group in Disulfide-Based Polymeric Gene Carriers[END_REF] You et al. [START_REF] You | Reducible poly(2-Dimethylaminoethyl Methacrylate): Synthesis, Cytotoxicity, and Gene Delivery Activity[END_REF] synthesized a reducible PDMAEMA (named rPDMAEMA) and compared it to a usual PDMAEMA. An α,ω-dithiobenzoate PDMAEMA synthesized by RAFT polymerization (Scheme I. 11). The rPDεAEεA was synthesized thanks to the coupling between two α,ω-dithiolPDMAEMAs (HS-PDMAEMA-SH) resulting from the aminolysis of α,ωdithiobenzoate PDMAEMA. Then the transfection efficiency of rPDMAEMAs with a numberaverage molar mass of 16700 g.mol -1 (rPDMAEMA1) and with a number-average molar mass of 53000 g.mol -1 (rPDMAEMA2) was compared with PDMAEMAs with a number-average molar mass of 13000 g.mol -1 (PDMAEMA1) and with a number-average molar mass of 26000 g.mol -1 (PDMAEMA2), respectively. The transfection efficiency was measured, on B16F10 cells, cells from mouse muscle with gWiz TM , with high-expression luciferase pDNA. The authors showed the best transfection efficiency of rPDAEMA1 compared to PDMAEMA1. The cytotoxicity of PDMAEMAs and rPDMAEMAs was also compared at different concentrations (from 10 to 50 µg.mL -1 ). At a concentration of 10 µg.mL -1 , the different polymers don't show any significant differences. For a concentration of 20 µg.mL -1 , the two rPDMAEMAs did not show any cytotoxic contrary to PDMAEMAs showed a cytoxicity towards human endothelial EA.hy926 cells and human pancreatic adenocarcinoma MiaPaCa cells. The authors explained such results by the degradation of rPDMAEMA leading to PDMAEMA with low numberaverage molar masses.

Peptide linkages are very stable and can't be hydrolyzed at room temperature but there are sensitive to enzymatic degradation. [START_REF] Islam | Major Degradable Polycations as Carriers for DNA and siRNA[END_REF] Kito et al.

Conclusions

Cationic polymers have been studied for gene delivery in order to replace the adenovirus systems which are known to be toxics. The PEI, the PLL, the PBAE and cationic vinylic polymers are the most synthetic cationic polymers studied till now to transfect DNA. Each of them has its pros and cons. The PEI and the PLL showed high transfection but higher cytotoxicity. The PBAE did not show a high transfection efficiency but the degradability leads to a very low cytotoxicity. The cationic vinylic polymers are easily designed and tunable.

Among the cationic vinylic polymers, the cationic acrylate-based copolymers present hydrolysable functions which allow high transfection efficiency and low cytotoxicity.

The PEGylation of cationic polymers is one step further to decrease cytotoxicity by decreasing the charge density and increasing the hydrophilicity. Moreover, the linkage of recognition-ligands onto cationic polymers increases transfection efficiency by improving the cellular uptake. The degradation of cationic polymers, by addition of biodegradable or hydrolyzable linkages, increases the transfection efficiency by helping the DNA release.

In our work, following the literature to obtain a high transfection efficiency and a low cytotoxicity of polyplex we developed a new cationic copolymer. A cationic acrylate-based polymer containing primary amine and tertiary amines has been chosen. The coupling of tertiary and primary amines allows to tune the charge density to improve the cell binding affinity, the endosomal escape and cytotoxicity. [START_REF] Zhu | Cationic Methacrylate Copolymers Containing Primary and Tertiary Amino Side Groups: Controlled Synthesis via RAFT Polymerization, DNA Condensation, and in Vitro Gene Transfection[END_REF] Moreover, the acrylate functions are hydrolysable leading to a decrease of cytotoxicity and an increase of the transfection efficiency. [START_REF] Truong | Self-Catalyzed Degradable Cationic Polymer for Release of DNA[END_REF][START_REF] Truong | Self-Catalyzed Degradation of Linear Cationic Poly(2-Dimethylaminoethyl Acrylate) in Water[END_REF] The PEGylation of cationic polymer has been realized by RAFT using the "PEO grafting from" process. The addition of a PEO allows to decrease the cytotoxicity and increase the polyplex stability. [START_REF] Zhang | Poly(ethylene Glycol)-Block-Polyethylenimine Copolymers as Carriers for Gene Delivery: Effects of PEG Molecular Weight and PEGylation Degree[END_REF] This PEO was functionalized with reactive functions to add a thiol-based RGD recognition ligand.

The addition of a recognition ligand allows to increase the transfection efficiency by increasing the cellular uptake and to target specific cells. 68

Introduction

PEO is a commercial water soluble, non-toxic and biocompatible polymer widely used in several applications such as cosmetic or biology and it has been approved by the food and drugs administration (FDA). [1][2][3][4] PEO is used in biopharmaceuticals processes to increase the circulating half-life of drugs. 2 The addition of a PEO on a molecule or a polymer is named PEGylation.

In gene therapy, the PEGylation of cationic polymers was studied on PEI, 5 PLL, 6,7 PBAE, 8,9 or cationic vinylic polymers. [10][11][12] It has been shown that the PEGylation decreases cytotoxicity of cationic polymers by decreasing the charge density. 13,14 Moreover, this decrease of the charge density leads to a lower binding affinity with cell membrane leading to a decrease of transfection efficiency. 13 However, with a low grafting ratio of PEO on polyplex, the transfection efficiency increases due to the better stability of the polyplex. The PEO acts like a "shield" to protect the polyplex from destabilization, opsonization and rapid clearance due to the salts, proteins of macrophages in the blood. 3,4,15,16 As reported in the literature chapter (Chapter I- §A), there are two strategies to covalently attach a linear PEO onto a cationic polymer to target block copolymersμ either the "PEO grafted onto" or either the "PEO grafted from". The "PEO grafted onto" strategy based on the covalent linkage between the PEO block and the second cationic polymer block, leads too low yields and partial functionalization due the incompatibility of both polymer blocks. In this work, the "PEO grafting from" strategy was employed. It consists in the formation of the second polymer block by the direct polymerization of appropriate monomer(s) from a first PEO block used as a macromolecular initiator or a macromolecular chain transfer agent. It is well-known that such "PEO grafting from" strategy allows a high PEO functionalization with good yields. As the versatile RAFT polymerization has been chosen in our study to synthesize cationic amine-based polyacrylates, a linear PEO (lPEO) and a bPEO macromolecular chain transfer agents are aimed in this work. The influence of the POE architecture will be studied on the polyplex formation and the cationic polymer cytotoxicity. Moreover, in order to introduce a recognition ligand, heterofunctionalized PEOs are targeted.

In this chapter, the first part is devoted to the heterofunctionalization of lPEO. The chosen recognition ligand possesses a thiol group. A pyridyldithio group and an alkyne group able to react with thiol functionalizd recognition ligand under mild conditions, have been added on the α-position of the lPEO. The thiol function of the recognition ligand can be added to the lPEO by disulfide echange reaction or by thiol-yne coupling reaction with the pyridyldithio group and the alkyne group, respectively. [17][18][19][20] On the ω-position of the lPEO has been a trithiocarbonate group, by nucleophilic substitution using different methods. Trithiocarbonate allows to control the RAFT polymerization of aminoethyl acrylate 21 . The trithiocarbonate has been chose because of its better stability and lower toxicity than dithioesters. 22,23 In a second part, the heterofunctionalization of the bPEO has been studied by RAFT polymerization of PEGA using an alkyne-based chain transfer agent.

A wide range of groups can be introduced and monitored at the a-position of the RAFT synthesized polymer by carefully choosing the R-group of the initial chain transfer agent. The direct RAFT polymerization using a clickable functionalized RAFT agent allows the thiol-yne reaction to functionalize the α-position of a polymer by alkyne-click chemistry. 11,[24][25][26] This chain-transfer agent contains an alkyne R-group and a trithiocarbonate Z-group. The alkyne group is linked via an amide function. The stability of the alkyne bond could avoid side reactions during the polymerization, and the amide group is a robust functionality that can resist hydrolysis in view of further (biological) applications in aqueous media. The alkyne group can react with a thiol by thiol-yne coupling reaction to functionalize the α-position of the PPEGA.

I. Synthesis of an heterofuntional linear poly(ethylene oxide) (lPEO) for

PEGylation of cationic copolymers

On the α chain-end of the lPEO, reactive groups including pyridyldisulfide or alkyne will allow the incorporation of thiol-based recognition ligand through disulfide exchange reaction and thiol-yne coupling reaction, respectively. [17][18][19][20] Such reactions have been chosen because they could be performed under mild conditions in order to preserve the recognition ligand.

Then, on the ω chain-end, a trithiocarbonate group will permit to control the RAFT copolymerization of aminoethyl acrylate.

A. Synthesis of an α-pyridyldithio,ω-hydroxylPEO

The α-pyridyldithio,ω-hydroxylPEO was synthesized through the nucleophilic substitution of the succinimidyl 3- All FT-IR and 1 H NMR spectroscopies, MALDI-TOF mass spectrometry and SEC characterizations confirmed the successful synthesis and purification of the α-pyridyldithio,ω-hydroxylPEO.

B. Synthesis of an α-alkynyl,ω-hydroxylPEO

Two different strategies have been explored to introduce an alkyne group on the α position of a PEO: the first one is based on the nucleophilic substitution of the propargyl bromide using the α,ω-dihydroxylPEO under basic condition (Williamson reaction) and the second one is based on the nucleophilic addition of the α-amino,ω-hydroxyPEO on L-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl)ethylhex-5-ynoate, an azlactone-based linker containing the alkyne group (named alkyne-AZL).

Using the propargyl bromide

An α,ω-dihydroxylPEO with a number-average molar mass determined by MALDI-TOF mass spectrometry (DP ̅̅̅̅ n,MALDI-TOF = 44) of 1938 g.mol -1 was employed as starting PEO. The reaction was performed using 1 equivalent of α,ω-dihydroxylPEO, 1.9 equivalent of potassium hydroxide and 1.3 equivalent of propargyl bromide in toluene during 7 days at 70°C (Scheme II. 2). The final product was purified by precipitation in cold diethyl ether and dried under vacuum. The resulting polymer was obtained with a yield of 87 % and was analyzed by 1 H NMR spectroscopy and MALDI-TOF mass spectrometry.

Scheme II. 2. Synthesis of an α-alkynyl,ω-hydroxylPEO using the propargyl bromide. (C) α,ω-dihydroxylPEO which were clearly identified. This result shows that this reaction is not selective and the difficulty comes probably from the homofunctional α,ω-dihydroxylPEO.

Therefore, the heterofunctional α-amino,ω-hydroxylPEO will be employed for our follow-up work.

Using the L-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl) ethyl hex-5-ynoate (named alkyne-AZL)

The azlactone (also named oxazolidin-5-one) is an electrophilic heterocycle which can react easily with amines through a ring-opening reaction. This reaction leads to complete conversion without the formation of by-products and can be performed in organic solvent or water. 27,28 In this work, the alkyne-AZδ has been chosen as a linker to modify the α-amino,ω-hydroxylPEO to target the α-alkynyl,ω-hydroxylPEO. The alkyne-AZL was synthesized according to the reported procedure from our laboratory. 28 One equivalent of α-amino,ω-hydroxylPEO was added to 1.1 equivalent of alkyne-AZL previously solubilized in dichloromethane (CH2Cl2). The reaction was performed at room temperature for 24 hours (Scheme II. 3). The final polymer was separated and purified by one precipitation in cold diethyl ether. After purification, a white powder was obtained with a yield of 99 % and was analyzed by MALDI-TOF mass spectrometry and 1 H and 13 C NMR spectroscopies.

Scheme II. 3. Synthesis of an α-alkynyl,ω-hydroxylPEO using the alkyne-AZL.

The purified polymer was first analyzed by MALDI-TOF mass spectrometry (Figure II. All NMR spectroscopies and MALDI-TOF mass spectrometry characterizations confirmed the successful synthesis and purification of the α-alkynyl,ω-hydroxylPEO using the alkyne-AZL linker.

The effective synthesis of the α-pyrydyldithio,ω-hydroxylPEO and the α-alkynyl,ω-hydroxylPEO will allow the incorporation of thiol-based recognition ligand through disulfide exchange reaction and thiol-yne coupling reaction under mild conditions, respectively. Then, a trithiocarbonate will be attached to the ω-position of both PEOs in order to be able to use them as macromolecular chain transfer agents to mediate the RAFT copolymerization of aminoethyl acrylates.

C. Synthesis of a linear PEO macromolecular chain transfer agent (named PEO-CTA)

The synthesis of the PEO-CTA is based on the esterification of the α-pyridyldithio, -hydroxylPEO and of the α-alkynyl, -hydroxylPEO previously synthesized and the 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDP).

Several activation methods have been used to process the esterification. The first one is based on the esterification of α-pyridyldithio, -hydroxylPEO mediated through the acyl chloride derived from CDP.

1.

Esterification of α-pyridyldithio,ω-hydroxylPEO a) Esterification mediated through acyl chloride

To perform the esterification through the acyl chloride from CDP, one equivalent of CDP is previously modified with 5 equivalents of oxalyl chloride in anhydrous CH2Cl2 at 50°C under argon (Scheme II. 4(A)). After three hours of reaction the unreacted oxalyl chloride was evaporated under vacuum. Then a solution of one equivalent of α-pyridyldithio,ω-hydroxylPEO in CH2Cl2 was added slowly under argon at room temperature (Scheme II. 4(B)). The reaction was launched for 24 hours. The product was purified by precipitation in cold diethyl ether and analyzed by 1 H NMR spectroscopy and SEC in DMF (LiBr 1 g.L -l ).

Scheme II. 4. Synthesis of (A) the acyl chloride derived from CDP and (B) the αpyridyldithio,ω-dodecyltrithiocarbonate PEO by esterification using the acyl chloride based CDP in CH2Cl2 at room temperature during 24 hours. The SEC traces of the final purified compound using RI detection show two populations (Figure 11). These two populations also absorb at 309 nm which is the characteristic wavelength of trithiocarbonate group. The signal at higher elution time show the functionalization of αpyridyldithio,ω-hydroxylPEO with the trithiocarbonate. The signal at lower elution time can be explained by the coupling of two PEOs due to the cleavage of the disulfide bond. The 1 H NMR spectrum and the SEC trace obtained with the UV-vis detector (fixed at 309 nm) prove the addition of the dodecyltrithiocarbonate group on the α-pyridyldithio,ω-hydroxylPEO. However the degree of functionalization calculated by 1 H NMR is still low, 69 %, and the SEC trace, obtained with the RI detector, shows disulfide exchange reaction.

c) Esterification mediated through DCC/DMAP

DCC is a coupling agent used with 4-(dimethylamino)pyridine (DMAP) to improve esterification. 30 Even if the degree of functionalization is not quantitative (53 %), the esterification of the α-pyridyldithio,ω-hydroxylPEO with CDP mediated through DCC/DMAP is the best choice as there is no evidence of coupling reactions between two PEOs.

Esterification of α-alkynyl,ω-hydroxylPEO a) Esterification mediated through acyl chloride

To perform the esterification through the acyl chloride from CDP, the same conditions were used than for the synthesis of the α-pyridyldithio,ω-dodecyltrithiocarbonate PEO (Chapter II- §C-1-a, Scheme II. 8). The polymer was characterized by 1 H NMR spectroscopy and SEC in DMF (LiBr 1 g.L -1 ).

Scheme II. 8. Synthesis of (A) the acyl chloride derived from CDP and (B) the α-alkynyl,ωdodecyltrithiocarbonate PEO by esterification using the acyl chloride based CDP in CH2Cl2 at room temperature during 24 hours. 1 H NεR spectroscopy confirmed the low degree of functionalization (13 %) of the αalkynyl,ω-hydroxylPEO using acyl chloride derived from CDP as it was shown during the esterification of the α-pyridyldithio,ω-hydroxylPEO. Therefore, the esterification of the αalkynyl,ω-hydroxylPEO was performed using the best conditions highlighted for the esterification of the α-pyridyldithio,ω-hydroxylPEO with CDP mediated through DCC/DMAP.

b) Esterification mediated through DCC/DMAP

To perform the esterification, a solution of 2 equivalents of DCC in anhydrous CH2Cl2

was added slowly to a cold solution containing 1 equivalent of α-alkynyl,ω-hydroxylPEO, 0.2 equivalent of DMAP and 2 equivalents of CDP. After 4 days at room temperature, the polymer has been purified by several washes and by chromatography column. The product was analyzed by SEC in DMF, 1 H and 13 C spectroscopies and MALDI-TOF mass spectrometry.

Scheme II. 9. Synthesis of an α-alkynyl,ω-dodecyltrithiocarbonate PEO by esterification between the α-alkynyl,ω-hydroxylPEO and CDP using DCC and DMAP as a coupling agents in CH2Cl2 at room temperature during 4 days.

The SEC trace using UV-vis detection fixed at 309 nm of the polymer before purification The SEC in DMF, the 1 H and 13 C NMRs spectroscopies and MALDI-TOF mass spectrometry analyses showed the efficiency of the esterification and purification.

The combination between the addition of an alkyne with an azlactone group and the esterification using DCC and DMAP, allows to obtained a well-defined α-alkynyl,ωdodecyltrithiocarbonate PEO (aPEO-CTA). This polymer can be used as a macromolecular chain transfer agent to perform RAFT polymerizations.

II. Synthesis of an heterofuntional brush-like PEO (bPEO) for PEGylation of cationic copolymers

The role of PEO architecture has been already studied on PDMAEMA. 31

A. Synthesis of 2-cyano-5-oxo-5-(prop-2-yn-1-ylamino)pentan-2yldodecyltrithiocarbonate (COPYDC)

The synthesises of various alkyne-terminated chain transfer agents have been previously reported. 25,26,32 In contrast with previous studies, our contribution relies on the presence of an amide group, known to be more stable than the ester group to hydrolysis and less sensitive to side reactions during thiol-yne reaction. 33 The alkyne-based chain transfer agent was The analysis of FT-IR, 1 H and 13 C NMR spectroscopies and of HR-MS spectrometry showed the efficient synthesis and purification of the COPYDC which could be used as an alkyne-based chain transfer agent to mediate the RAFT polymerization of PEGA.

B. RAFT polymerization of PEGA using COPYDC

Several conditions have been already used to perform the RAFT polymerization of PEGA. Boyer et al. 34 Such inhibition period is well-known to be dependent on the structure and concentration chain transfer agent and is related to the establishment of the addition-fragmentation equilibrium. 24 Moreover, the slope of the ln([M]0/[M]t) versus time is higher using the [COPYDC]0/[ACVA]0 ratio of 1/0.5 in comparison with the [COPYDC]0/[ACVA]0 ratio of 1/0.1. Such behavior is relevant with an apparent propagation rate constant (kp app = kp[P • ]) higher using the

[COPYDC]0/[ACVA]0 ration of 1/0.5. Same behavior has been observed by Wood et al. 37 when they studied the influence of initiator concentration on RAFT polymerization of methacrylate. This result is well-known and is called "the initialization" effect. 38 Moreover, the apparent propagation rate constant decreases with the decrease of the [PEGA]0/[COPYDC]0 ratios. This phenomenon is known as the "retardation"effet. 38 Among all these RAFT polymerization conditions, the [PEGA]0/[COPYDC]0 ratio of 30/1 together with the [COPYDC]0/[ACVA]0 ratio of 1/0.1 gave the best results in terms of kinetics, molar masses and dispersity values (Ð < 1.2). Indeed, using such conditions, a good control of the RAFT polymerization of PEGA allowed to target well-defined heterofunctional α-alkynyl,ω-dodecyltrithiocarbonatePPEGA.

Synthesis of a high amount of α-alkynyl,ω-dodecyltrithiocarbonate PPEGA

To target a high amount of well-defined heterofunctional α-alkynyl,ωdodecyltrithiocarbonate PPEGA, the optimal experimental conditions previously defined for the RAFT polymerization of PEGA were used on a larger initial amount of PEGA The SEC and the 1 H NMR spectrum and the SEC showed the efficiency of RAFT polymerization to target well-defined α-alkynyl,ω-dodecyltrithiocarbonatePPEGAs able to be used as macromolecular chain transfer agents for the copolymerization of aminoethyl acrylate.

Conclusion

Different pathways have been used to target well-defined heterofunctional lPEO and bPEO containing reactive function which can react with a thiol group at the α-position and a chain transfer agent moiety able to control a RAFT polymerization at the ω-position.

Two different thiol-reactive groups have been added on the α-position of lPEO: a pyridyldithio group able to react with a thiol group by disulfide exchange and an alkyne group able to react with a thiol group by thiol-yne coupling reaction. The addition of the pyridyldithio group moiety was efficient with a high degree of functionalization (95 %) using SPDP. The addition of the alkyne on the lPEO has been studied using two different strategies. The nucleophilic substitution of the propargyl bromide using α,ω-dihydroxylPEO led to low degree of functionalization (78 %) meanwhile the nucleophilic addition of α-amino, ω-hydroxylPEO onto the alkyne-AZL led to complete functionalization under mild conditions.

Experimental part

A. Reagents

All HPLC grade, Fisher Chemical) and tetrahydrofuran (THF, 99.9 %) were dried over dry solvent stations GT S100. Oligo(ethylene glycol) acrylate (PEGA, pure, sigma Aldrich) was passed through a column of aluminum oxide prior to polymerization. The L-(4,4-dimethyl-5-oxo-4,5dihydrooxazol-2-yl) ethyl hex-5-ynoate (alkyne-AZL) have been synthesized following a and a delay of 300 ns, 500 single shot aquisitions were summed to give the spectra and the data were analyzed using Bruker FlexAnalysis and Polytools softwares. Samples were prepared by dissolving the matrix (DCTB or DIT) in the solvent (CH2Cl2, 30 mg.mL -1 ) and mixing with the polymer (10 mg.mL -1 ) in the ratio 1:10 (v/v). 1 µL was spotted directly onto a thin layer of NaTFA in acetone (concentration 19 mg.mL -1 ) that had been deposited to act as a cationizing agent.

C. Synthesis of an α-pyridyldithio,ω-hydroxylPEO

In a round bottom flask a magnetic stir bar was carged together with α-amino,ω-hydroxylPEO (50 mg, 0.016 mmol) and SPDP (6 mg, 0.019 mmol) and acetonitrile (4 mL).

The reaction was launched at room temperature during 5 hours. The solution was concentrated and the solid obtained was dissolved in 10 mL of ultra-pure water. The solution was filtered onto a nylon filter with a porosity of 0.22 µm. The solution was dialyzed with a 10 kDa membrane during 24 hours. The solution was lyophilized and the solid obtained was dried under vacuum with P2O5. The solid was obtained with a yield of 89 % (m = 44.5 mg).

FT-IR ( in cm

D.

Synthesis of an α-alkynyl,ω-hydroxylPEO

1.

Using the propargyl bromide α,ω-dihydroxylPEO (10.0504 g, 5.186 mmol) was previously purified by azeotropic distillation in toluene. After complete water removal, the temperature was decreased to 70°C and a dried powder of KOH (0.5500 g, 9.980 mmol) was added to the solution. The mixture was stirred during 1 hour at 70°C and propargyl bromide (0.8248 g, 6.933 mmol) was injected through a syringe previously degassed. The solution was stirred at 70°C during 7 days. Toluene is removed by evaporation and the residual mixture was dissolved in CH2Cl2. The organic phase is washed three times with a solution of HCl (3N) and three times with a saturated solution of NaCl. The organic phase was concentrated and precipitated three times in cold diethyl ether.

The mass obtained was 8.8339 g with a yield of 87 %.

2.

Using the L-(4,4-dimethyl-5-oxo-4,5-dihydrooxazol-2-yl) ethyl hex-

5-ynoate (named alkyne-AZL)

In a round bottom flask were charged α-alkynyl,ω-hydroxylPEO (2,301 g, 0.764 mmol), alkyne-AZL (222.9 mg, 0.888 mmol) and CH2Cl2 (50 mL). The reaction was launched during 24 hours at room temperature. The solution was concentrated and precipitated once in cold diethyl ether. The solid obtained was dried under vacuum in the presence of P2O5. The final product was obtained with a yield of 99 %.

FT-IR ( in cm

b) Esterification mediated through DMT-MM

A solution of CDP (8.2 mg, 0.02 mmol), α-pyridyldithio,ω-hydroxylPEO (67.0 mg, 0.02 mmol) and NMM (6.1 mg, 0.06 mmol) in 0.5 mL of anhydrous THF was stirred at room temperature for 10 minutes. The DMT-MM (16.6 mg, 0.06 mmol) was added and the mixture was stirred at room temperature for 24 hours. Solvent was evaporated and the residual mixture was dissoled in CH2Cl2. The solution was filtered and washed 3 times by a NaCl saturated solution. The organic phase was dried on MgSO4 and filtered. The solution was concentrated and the residual mixture was precipitated in cold diethyl ether. The solid was dried under vacuum in the presence of P2O5.

c) Esterification mediated through DCC/DMAP

In a round bottom flask a stirred bar was charged together with CDP (17.1 mg, 0.043 mmol) and DCC (8.9 mg, 0.043 mmol). Under argon, were added 0.4 mL of a solution of DMAP (6.0 mg, 0.049 mmol) in 4 mL of anhydrous CH2Cl2. At 0°C and under argon was added a solution of -pyridyldithio, -hydroxylPEO (72.6 mg, 0.022 mmol) in 0.6 mL of anhydrous CH2Cl2. After complete addition, the solution was stirred at 0°C during 6 hours and at room temperature during 4 days. The solution was filtered and concentrated. The product was dialyzed into ethanol during 24 hours with a membrane of 1kDa, dried, solubilized in a minimum of CH2Cl2 and precipitated three times in cold diether. The solid obtained was dried under vacuum in the presence of P2O5. The solid was obtained with a yield of 47 % (30 mg). SEC in DMF (LiBr 1 g.L -1 ): M n,SEC ̅̅̅̅̅̅̅̅ = 8500 g.mol -1 and Ð = 1.10.

Esterification of -alkynyl, -hydroxylPEO a) Esterification mediated through acyl chloride

A solution of CDP (68.3 mg, 0.170 mmol) in 2.1 mL of anhydrous CH2Cl2 was degassed under argon for 30 minutes and oxalyl chloride (108.0 mg, 0.850 mmol) was added to the solution. The mixture was heated to reflux during 3 hours at 50°C. CH2Cl2 and oxalyl chloride were removed under vacuum and under argon was added α-alkynyl,ω-hydroxylPEO (500.0 mg, 0.154 mmol) in 6.4 mL of anhydrous CH2Cl2. The solution was agitated during 24 hours at room temperature. The solution was evaporated and the solid was precipitated in cold diethyl ether and dried under vacuum. The solution was degassed during 10 minutes and the reaction was launched at room temperature for 4 days. Organic phase was washed twice with a solution of HCl 3 M and twice with a saturated solution of NaCl. The organic phase was evaporated and the residual mixture was kept in ice. Aliquots of 500 mg of the crude product were purified using a silica column (15 mL of silica) with CH2Cl2/MeOH mixture (from 100/0 to 90/10 in volume). The solvent was evaporated and the product was dried under vacuum at 40°C. HCl solution. The organic phase was dried with MgSO4 and evaporated. The crude product was purified using a silica gel column with cyclohexane/ethyl acetate mixture (2/1 by volume) as the eluent. A brown oil was obtained in a yield of 70 %.

RAFT polymerization of PEGA using COPYDC

General procedure of RAFT polymerization of PEGA using COPYDC:

In a typical successful RAFT polymerization procedure, a magnetic stir bar was charged to a schlenk flask together with ACVA, COPYDC and PEGA in DMF. The solution was degassed with argon for 20 minutes and immersed in an oil bath thermostated at 70°C. Aliquots were withdrawn every 30 minutes and are analyzed by 1 H NMR spectroscopy to determine PEGA conversion and SEC in DMF to determine the M n,SEC ̅̅̅̅̅̅̅̅ and the Ð. The reaction was stopped after 300 minutes by opening the reaction to the air and cooling the schlenk flask in liquid nitrogen.

Introduction

Interest in cationic polymers results from their potential to mediate transfection via the condensation of nucleic acids, to provide protection from enzymatic degradation and to facilitate cellular uptake and endosomal escape. [1][2][3][4] Such feature is provided by the charge density of the cationic polymer. [1][2][3][4] The charge density is driven by the pKa of cationic amine groups which can be protonated to form polyplexes with negatively charged nucleic acids through electrostatic interactions. Amine groups with a high pKa allow efficient DNA complexation and protection whereas amine groups with a low pKa facilitate endosomal escape. Several works showed the addition of primary amine allows to increase the charge density and the transfection efficiency. 5,6 The incorporation of protonatable tertiary amine within the copolymer reduces its cellular toxicity. 7 It has been shown that the combination of both primary and tertiary amines with a wide buffer range rise to modulate transfection efficiency and cytotoxicity. 8,9 Zhu et al. 6 synthesized polymethacrylates containing tertiary amines DMAEMA units, and primary amine groups, AEMA units. Authors studied the effect of DMAEMA/AEMA molar ratios on transfection efficiency and cytotoxicity. They showed that the decrease of DMAEMA/AEMA molar ratio from 91/9 to 70/30 led to the increase of the complexation of pDNA and a better transfection efficiency together with a decrease of cell viability.

In our work, polyacrylates containing primary and tertiary amines group are targeted. The interest of polyacrylates in comparison with polymethacrylates for gene delivery is their high water solubility and self-catalyzed hydrolyzation leading to a lower cytotoxicity. 10,11 Therefore, N-aminoethyl acrylate (AEA) and 2-(N,N-dimethylamino)ethyl acrylate (DMAEA) were chosen as repeating units. Recent studies dealing with block copolymers versus statistical copolymers with similar composition, showed that statistical copolymers lead to greater cell viabilities and gene expression, in the presence and absence of serum, as compared to diblock copolymers, even if diblock copolymers form polyplexes with a higher colloidal stability. [12][13][14] Therefore, statistical copolymers based on DMAEA and AEA units are studied on this work as they are supposed to be better candidates for transfection than block copolymers. The RAFT polymerization has been chosen as it paves the way for the synthesis of polymers not only with defined molar mass, narrow molar mass distribution but also with homogeneous chain composition and monomer sequences. 15 The RAFT polymerization of aminoethyl acrylates is well-known and have been previously described. 10,11,16 Furthermore, the synthesis and the RAFT polymerization of N-(tert-butoxycarbonyl)aminoethyl acrylate (tBocAEA) has been described for the first time by our group. 17 In such study, 17 to avoid any aminolysis of chain transfer agent moiety in the presence of primary amine-derived AEA, the protected tBocAEA was used as a precursor for the RAFT polymerization.

The RAFT copolymerizations of DMAEA and tBocAEA mediated through an αalkynyl,ω-dodecyltrithiocarbonate PEO (aPEO-CTA), an α-alkynyl,ω-dodecyltrithiocarbonate PPEGA (PPEGA-CTA) and the COPYDC was studied to target: aPEO-b-P(DMAEA-co-tBocAEA), PPEGA-b-P(DMAEA-co-tBocAEA) and P(DMAEA-co-tBocAEA) copolymers, respectively. These copolymers with lPEO and bPEO blocks have been synthesized with different DMAEA/tBocAEA molar ratios. The associated cationic copolymers was obtained by cationization and deprotection of tBocAEA repeating unit in acidic media. In our study, the influence of the PEO architecture and the DMAEA/AEA molar ratio onto pDNA complexation and onto resulting polyplexes cytotoxicity will be highlighted.

I.

Copolymers based on DMAEA and tBocAEA units.

A. Determination of DMAEA and tBocAEA reactivity ratios ǂ

In conventional free radical copolymerization, if reactivities of two monomers are different, chains formed at different times in the process have different composition and the final polymer is a mixture of chains with different microstructures. In a RAFT copolymerization, since all chains grow at the same time, the composition drift takes place at the same extent in each single chain. The copolymer composition is influenced by the reactivity ratios. Several methods are currently used to determine reactivity ratios among which the Fineman-Ross method, 18 the Mayo-Lewis terminal model, 19 and the Kelen-Tüdos method. 20 Usually, in conventional free radical copolymerization, monomers reactivity ratios are measured by fitting the copolymer composition at low conversion as a function of initial monomer feed composition. However, in RDRPs, the copolymer chain composition at low conversion could be affected by the initiation step considering that different reactivities are usually detected. 21 Therefore, to obtain reliable results, it is necessary to measure the cumulative copolymer composition from 10% to high conversion. The extended Kelen-Tüdos method is the right choice to calculate both monomer reactivity ratios (rDMAEA and rtBocAEA) because higher conversions could be considered. 22 The extended Kelen-Tüdos equation is expressed by the following equations:

η = r DεAEA + r tBocAEA α -
x 0 = f DεAEA 1-f DεAEA Equation (III. 12)
where, fDMAEA is the molar ratio of DMAEA in the comonomer feed, FDMAEA is the molar ratio in the copolymer, MDMAEA is the molar mass of DMAEA (143.2 g.mol -1 ), MtBocAEA is the molar mass of tBocAEA (215.2 g.mol -1 ), ω represents the total conversion of both DMAEA and tBocAEA monomers and α = 1.04323.

To determine such parameters, several RAFT copolymerizations of DMAEA and tBocAEA were carried out (Table III. 1) The extended Kelen-Tüdos plot reveals that the dependence of η versus is linear as indicated by the R 2 parameter (R 2 0.991) and the linear fit allows the calculation of both reactivity ratios: rDMAEA 0.81 and rtBocAEA 0.99 (Figure III. 4). These closed r values confirm the random composition of these copolymers. The copolymerization is characteristic of an "ideal" copolymerization. Same behavior has been observed starting from DMAEA and methyl acrylate (MA) comonomers.10 When DMAEA is combined with an acrylamide monomer such as N,N-dimethylacrylamide, 24 2-acrylamido-2-methyl-1-propanesulfonic acid, 25 and 3aminopropylmethacrylamide, 26 the acrylamide possesses a much higher reactivity ratio and a higher tendency of homopropagation. Therefore, chain growth reaction proceeds predominantly by the addition of DMAEA monomer to polyacrylamide macroradicals yielding statistical copolymers. The FT-IR, 1 H NMR spectroscopies and SEC in DMF (LiBr 1 g.L -1 ) showed that the RAFT copolymerization of DMAEA and tBocAEA mediated through the α-alkynyl,ω-dodecyltrithiocarbonate PEO used as the macromolecular chain transfer agent and ACVA used as the initiator allows to target a well-defined aPEO-b-P(DMAEA-b-tBocAEA).

Therefore, similar conditions have been used to synthesize copolymers with different DMAEA/tBocAEA molar ratios (Table III. 3). III. 4). III.

4).

The SEC was performed in DMF (LiBr 1 g.L III. 4).

The 1 H NMR spectroscopy and SEC in DMF (LiBr 1 g.L -1 ) showed that the RAFT copolymerization of DMAEA and tBocAEA mediated through the α-alkynyl,ωdodecyltrithiocarbonate PPEGA as the macromolecular chain transfer agent and ACVA as the initiator allows to target a PPEGA-b-P(DMAEA-b-tBocAEA) copolymers.

Therefore, similar conditions have been used to synthesize copolymers by RAFT polymerization using different DMAEA/tBocAEA molar ratios (Table III. 4). Determined by SEC in DMF (LiBr 1 g.L -1 ) using PS standards.

In order to be able to study the influence of PEGylation of cationic P(DMAEA-co-tBocAEA) copolymers using lPEO and bPEO on pDNA complexation and onto resulting III. 5).

The SEC in DMF (LiBr 1 g.L -1 ) using the RI detection shows a unimodal trace with a M n,SEC ̅̅̅̅̅̅̅̅ = 5200 g.mol -1 (PS equivalents) and Ð = 1.50 (Figure III. 14). The trace obtained with a UV-vis detector fixed at 309 nm (characteristic wavelength of the trithiocarbonate group)

shows that the polymer absorbs at this wavelength. The shape of both SEC traces obtained with RI detection and UV-vis detection is not a Gaussian. Such behavior is probably due to the interactions of amine groups of the copolymer with SEC columns. This phenomenon, well- III. 5).

The 1 H NMR spectroscopy and the SEC in DMF (LiBr 1 g.L -1 ) showed the efficiency of the RAFT copolymerization of DMAEA and tBocAEA to target a well-defined P(DMAEA-co-tBocAEA) copolymers. The experimental conditions previously used for RAFT copolymerizations have been employed to synthesize several copolymers with different DMAEA/tBocAEA molar ratios (Table III. 5). RAFT copolymerizations of DMAEA and tBocAEA using ACVA as the initiator and aPEO-CTA, PPEGA-CTA and COPYDC as the (macromolecular) chain transfer agents has led to well-defined aPEO-b-P(DMAEA-co-tBocAEA), PPEGA-b-P(DMAEA-co-tBocAEA) and P(DMAEA-co-tBocAEA), respectively. To target corresponding cationic copolymers able to complex pDNA, the amine deprotection of the tBocAEA repeating units is performed.

II. Amine deprotection of tBocAEA repeating units of aPEO-b-P(DMAEA-co-tBocAEA), PPEGA-b-P(DMAEA-co-tBocAEA) and P(DMAEA-co-tBocAEA)

tBocAEA units have been deprotected under acidic conditions to form aminoethyl acrylate (AEA) units. The deprotection of primary amines using trifluoroacetic acid (TFA) and

CH2Cl2 has been widely used in peptide chemistry and organic chemistry. 29 Maynard and coworkers showed the efficiency of TFA in CH2Cl2 to deprotect a primary amine on the α-position of a PEO-b-Poly(oligo(ethylene glycol)methacrylate) (PPEGMA) and PEO-b-Poly((hydroxyethyl)methacrylate) (PHEMA) synthesized by ATRP and of a poly(Nisopropylacrylamide) PNIPAM synthesized by RAFT polymerization. 30,31 Moreover, our group showed the efficiency of TFA to deprotect tBocAEA. 17

C. Characterization of a cationic P(DMAEA-co-AEA)

A P(DMAEA-co-tBocAEA) copolymer DP ̅̅̅̅ n,NMR PDMAEA and PtBocAEA equal to 9 and 13 respectively (named P(DMAEA9-co-tBocAEA13), Run 3, Table III. 5) has been analyzed

by 1 H NMR spectroscopy after tBoc deprotection and cationization in acidic medium (Figure

III. 19).

The overlaid 1 H NMR spectra of P(DMAEA9-co-tBocAEA13) copolymers before and after deprotection shows the disappearance of the signal at 1. 44 The 1 H NMR spectrum showed that the deprotection and the cationization of P(DMAEAco-tBocAEA) is successful using TFA. Several cationic P(DMAEA-co-AEA) resulting from the cationization and deprotection reaction have been employed to complex pDNA.

The 1 H NMR spectra show the deprotection and cationization mediated through acidic media allows to obtain well-defined cationic polymer. Same primary amine deprotection has been performed on P(DMAEA-co-tBocAEA) copolymers with different DMAEA/AEA molar ratios.

Conclusion

Cationic copolymers based on protonated DMAEA and AEA units with different PEO architectures or without PEO have been successfully synthesized by sequential reactions.

In a first part, the RAFT copolymerization of DMAEA and tBocAEA has been studied using three different alkyne-base (macromolecular) chain transfer agents: the aPEO-CTA, the PPEGA-CTA and the COPYDC. An additional study on the determination of DMAEA and tBocAEA reactivity ratios exhibits rDMAEA = 0.81 and rtBocAEA = 0.99 suggesting random monomer unit distribution resulting in "ideal" copolymers. εoreover it has been highlighted that the molar composition of random copolymers is equal to the molar composition of the feed comonomers whatever the conversion. Well-defined aPEO-b-(DMAEA-co-tBocAEA) copolymers (6100 < M n,NMR ̅̅̅̅̅̅̅̅̅ < 11500 and 1.21 < Ð < 1.52), PPEGA-b-P(DMAEA-co-tBocAEA) copolymers (10100 < M n,NMR ̅̅̅̅̅̅̅̅̅ < 10700 and 1.57 < Ð < 1.62) and P(DMAEA-co-tBocAEA) (6400 < M n,NMR ̅̅̅̅̅̅̅̅̅ < 9400 and 1.20 < Ð < 1.50) copolymers with different DMAEA/AEA molar ratios have been successfully obtained through RAFT copolymerization using aPEO-CTA, PPEGA-CTA and COPYDC respectively as the (macromolecular) chain transfer agent using ACVA as the initiator at 70°C in 1,4-dioxane or/and DMF.

In a second part, the tBoc amine group of the copolymers previously synthesized have been deprotected using TFA in CH2Cl2 to form: cationic aPEO-b-(DMAEA-co-AEA) copolymers, cationic PPEGA-b-P(DMAEA-co-AEA) copolymers and cationic P(DMAEA-co- The resulting polymers will be used to study the impact of hydrophilic block architecture and DMAEA/AEA ratio on pDNA complexation and on cell viability. rate of 1.0 mL.min -1 at 60°C and was calibrated with narrow linear polystyrene (PS) standards ranging in molar mass from 580 g.mol -1 to 483 000 g.mol -1 . Molar masses and dispersities were calculated using Waters EMPOWER software.

FT-IR spectra of copolymers were recorded using a Nicolet iS5 FT-IR spectrometer operating with an attenuated total reflection (iD5 ATR Diamond) gate. Spectra were analyzed with the OMNIC software.

C.

Copolymers based on DMAEA and tBocAEA units.

1.

Determination of DMAEA and tBocAEA reactivity ratios.

In a successfull RAFT copolymerization procedure (Run 3, Table III. 1), a magnetic stir bar was charged to a round bottom flask together with tBocAEA (0.350 g, 1.63x10 -3 mol), DMAEA (0.548 g, 3.83x10 -3 mol), mPEO-CTA (0.130 g, 5.42x10 -5 mol), ACVA ( SEC in DMF (LiBr 1 g.L -1 ): M n,SEC ̅̅̅̅̅̅̅̅ = 9600 g.mol -1 ; Đ = 1.17.

FT-IR ( , cm

RAFT copolymerization of DMAEA and tBocAEA using aPEO-CTA

In a successfull RAFT copolymerization (Run 9, Table III. 3) procedure, a magnetic stir bar was charged to a round bottom flask together with tBocAEA (0.4166 g, 1.93x10 -3 mol), DMAEA (0.2758 g, 1.93x10 -3 mol), aPEO-CTA (0.1350 g, 3.86x10 -5 mol), ACVA (2.2 mg, 7.71x10 -6 mol), 1,4-dioxane (4.1 mL) and DMF (0.07 mL) used as the internal reference. The mixture was deoxygenated by bubbling argon for 30 min. The solution was then immersed in an oil bath thermostatted at 70°C to allow the polymerization to occur. The reaction was stopped after 5h30 by opening the reaction mixture to oxygen. Total of conversion of DMAEA and tBocAEA were determined to be 24% by 1

RAFT copolymerization of DMAEA and tBocAEA using PPEGA-CTA

In a successfull RAFT copolymerization (Run 1, Table III. 4) procedure, a magnetic stir bar was charged to a round bottom flask together with tBocAEA (0.3958 g, 1.84x10 -3 mol), DMAEA (0.2712 g, 1.89x10 -3 mol), PPEGA-CTA (0.3340 g, 3.68x10 -5 mol), ACVA (2.0 mg, 7.32x10 -6 mol), 1,4-dioxane (10 mL) and DMF (0.3 mL) used as the internal reference. The mixture was deoxygenated by bubbling argon for 30 min. The solution was then immersed in an oil bath thermostatted at 70°C to allow the polymerization to occur. The reaction was stopped after 27 h by opening the reaction mixture to oxygen. Total conversion of DMAEA and

Characterization of a cationic PPEGA-b-P(DMAEA-co-AEA) copolymer

In a typical procedure, 220.2 mg of PPEGA-b-P(DMAEA-co-tbocAEA) (2.19x10 -5 mol) block copolymer was dissolved in dichloromethane (CH2Cl2, 4.5 mL). TFA (500 µL, 6.51x10 - 3 moL) was added dropwise at 0°C. After complete addition, the solution was stirred 5 h at room temperature. The CH2Cl2 was then removed using a rotary evaporator and the polymer was dissolved in acetone, precipitated into diethyl ether/hexane (50/50 v/v), centrifuged (7000 rpm, 0°C, 5 min) and dried in a vacuum. The product obtained was a yellow solid and analyzed by 1 H NMR spectroscopy.

, HC≡CCH2, OCH2CHO(CH2CH2O), OCH2CH2N + H(CH3)2, OCH2CH2N + H3).

Characterization of a cationic P(DMAEA-co-AEA)

In a typical procedure, 500 mg of P(DMAEA-co-tbocAEA) (9.7x10 -5 mol) copolymer was dissolved in dichloromethane (CH2Cl2, 10 mL). TFA (1.5 mL, 1.93x10 -2 moL) was added dropwise at 0°C. After complete addition, the solution was stirred 5 h at room temperature. The CH2Cl2 was then removed using a rotary evaporator and the polymer was dissolved in acetone, precipitated into diethyl ether/hexane (50/50 v/v), centrifuged (7000 rpm, 0°C, 5 min) and dried in a vacuum. The product obtained was a yellow solid and analyzed by 1 H NMR spectroscopy.

Introduction

The incorporation of new functionality or removal unwanted chain-end reactivity is a major theme and essential tool for polymer researchers. Several reviews and recent highlight have been published on established and emerging strategies for polymer-chain end modification. 1,2 More often, such strategies are focused on the most popular chain-growth processes included RDRP and post-polymerization modifications. When polymers for gene delivery are concerned for post-polymerization modification, soft reaction conditions (room temperature, aqueous solution) are necessary to maintain the integrity of the cationic polymer or/and the bio(macro)molecules structures. In this project the post-polymerization modification of cationic aPEO-b-P(DMAEA-co-tBocAEA) copolymers, PPEGA-b-P(DMAEA-co-tBocAEA) copolymers and P(DMAEA-co-tBocAEA) copolymers has been studied to both decrease cytotoxicity and increase transfection efficiency.

Among organic synthetic methods able to modify alkyne-based RAFT copolymers chainend under mild conditions the photochemical-mediated trithiocarbonate removal, the thiol-yne coupling reaction and the thiol-disulfide exchange have been chosen. The first one has been studied to decrease polymer cytotoxicity and the two following ones has been employed to insert a recognition ligand to increase transfection efficiency.

Even though, it has been shown the cytotoxicity of trithiocarbonyl group is lower compared to dithioester group, 3 it is necessary to remove the thiocarbonylthio group of copolymers to use them in non-viral gene delivery. Indeed, the thiocarbonylthio moieties are sulfur analogs of esters designed to readily undergo radical homolysis and may also be prone to hydrolysis and polymer-polymer coupling. 3,4 Moreover, the removal of the thiocarbonylthio group is preferable to avoid side reactions between such group and thiol groups included in biological molecule. 5 Several reviews reported chain transfer agent removal procedures. 6,7 Among click chemistry reactions, the thiol-yne coupling reaction does not need any metallic catalyst and is a robust tool for polymer modification. [8][9][10][11] Such reaction can proceed through nucleophilic addition or radical addition. To use the nucleophilic addition, the unsaturated bond has to be activated and sensible to nucleophilic reagents. When the unsaturated bond is not activated, the thiol-yne reaction has to be made through radical addition.

One or two thiol-based molecules could be added by thiol-yne reaction depending on experimental conditions. [8][9][10][11] The thiol-disulfide exchange reaction can be reduced by enzymes as gluthatione. 12,13 In non-viral gene delivery, the disulfide bond has been employed as a reducible cross-linker or moiety to decrease cytotoxicity by increasing the degradation into the cytoplasm. [14][15][16] Furthermore, the disulfide bond remains stable in extra-cellular media.

In this work, the radical-induced trithiocarbonate group removal was modeled using a PNIPAM under thermo-activation. Then, the reaction was performed using photochemicalactivation on the cationic aminoethyl polyacrylates copolymers, in order to maintain the integrity of copolymers under softer conditions. After the trithiocarbonate removal, the addition of a thiol compound was modeled using the disulfide coupling exchange on α-pyridyldithio,ω-hydroxylPEO and the thiol-yne coupling reaction on a PNIPAM. The challenging "one-pot" cascade thiol-yne click reaction at the α-chain end and the trithiocarbonate removal at the ωchain-end through radical mechanisms will be explored.

I. Trithiocarbonate removal

Several fast and facile trithiocarbonate group removal procedures have been reported that do not require expensive reagents or harsh conditions. 6,7 Perrier et al. suggested in 2005 an easy way to achieve the complete removal of trithiocarbonate end-group thanks to a radical initiator such as AIBN. 17 Furthermore, the trithiocarbonate moiety can be changed to a different group by using a functional radical initiator. The authors showed that using ACVA as radical initiator, it is possible to functionalize the ω-position of the polymer chain-end by a carboxylic acid group. 17 The thermal-mediated trithiocarbonate removal reaction conditions used by Perrier et al. 17 are studied in our work on a PNIPAM used as a polymer model easy to characterize by 1 H NMR spectroscopy and MALDI-TOF mass spectrometry. 18

A. PNIPAM used as a model polymer to trithiocarbonate removal ǂ

A PNIPAM has been first synthesized by RAFT polymerization using COPYDC as the chain transfer agent. Then, the trithiocarbonate has been removed using AIBN as the initiator at 80°C.

RAFT polymerization of NIPAM with COPYDC

A RAFT polymerization of NIPAM was launched with COPYDC as chain transfer agent a Monomer conversion monitored by 1 H NMR spectroscopy by comparing the integration values of the formamide proton of DMF at 8.02 ppm, and of the alkene protons of NIPAM between 6.0 and 6.3 ppm. b M n,th ̅̅̅̅̅̅ = Conv.

[NIPAε] 0

[COPYDC] 0 ×ε NIPAε +ε EXT where MNIPAM is the molar mass of NIPAM (113.16 g.mol -1 ) and MEXT is the molar mass of the chain-ends (434.20 g.mol - 1 ). c Determined by SEC in DMF (LiBr 1 g.L -1 ) using PS standards. The 1 H NMR spectroscopy, the SEC in DMF (LiBr 1g.L -1 ) and MALDI-TOF spectrometry showed that the alkyne-based chain transfer agent COPYDC efficiently mediates the RAFT polymerization to prepare well-defined heterofunctional PNIPAM. This PNIPAM is now used to model the reaction of trithiocarbonate removal using Perrier et al. 17 conditions.

Trithiocarbonate removal mediated through AIBN at 80°C

Methods for thiocarbonylthio group removal from polymers have been previously discussed in different reviews. 6,7 Among all the methods used to remove a trithiocarbonate, 6,7 the addition of an excess of radical initiator such as AIBN is a facile and efficient technique. 17 Hu et al. have shown that while this method is effective for removing chain-ends of polymers with a terminal methacrylate unit, it provides incomplete end group removal when applied to Chapter 4: Chain-ends modification of (PEGylated) aminoethyl-based polyacrylates.

polymers with a terminal acrylate or styrene unit. 20 When a polyacrylamide such as PNIPAM is considered, Zhang et al. 21 have shown that the dodecylthiocarbonylthio end group can be successfully removed by increasing the polymer concentration compare to Perrier's conditions.

In this work, the reaction was performed using a PNIPAM initial concentration of 0.012 mol.L -1 with 20 equivalents of AIBN in 1,4-dioxane at 80°C (Scheme IV. 2). Samples were withdrawn to follow the reaction by SEC using RI detection and UV-vis detection at 309 nm, the characteristic absorption wavelength of the trithiocarbonate group. and At,UV are peak areas at a given time (t) using respectively a RI detector and a UV-vis detector at 309 nm and At0,RI and At0,UV are peak areas before the reaction using respectively RI detector and UV-vis detector at 309 nm, respectively.

After 2 weeks of reaction, the reaction has been stopped and the resulting copolymer has been purified by several precipitation/decantation in cold diethyl ether/hexane mixture (50/50 v/v). The pure product was analyzed by 1 H NMR spectroscopy and SEC in DMF (LiBr 1 g.L - 1 ).

The

II. Addition of a thiol based compound

To target specific cells, recognition ligands have been already added on polyplexes to increase their transfection efficiency. [23][24][25] In our work, the RGD has been chosen as recognition ligand due to its ability to target tumorous specific cells. 26,27 The RGD recognition ligand, as all peptides, is thermo-degradable. 28 Therefore, reactions able to be proceed in soft conditions (room temperature, aqueous solution, no by-products) were chosen to add the recognition ligand. As thiolated-RGD can be linked directly onto pyridyldithio and onto alkyne moieties, two strategies were studied: (i) by disulfide bond exchange using previously synthesized polymers containing the pyridyldithio-group at the -position (Chapter 2 § I-A), and (ii) by thiol-yne coupling reaction using previously synthesized polymers containing the alkyne group at the α-position (Chapter 3 § I-B, C and D).

The thiol-disulfide coupling exchange has been modeled on an α-pyridyldithio,ω-hydroxylPEO synthesized in Chapter 2 I §A and the thiol-yne coupling reaction has been modeled on an α-alkynylPNIPAM synthesized in this Chapter ( §I-A-2).

A. Thiol-disulfide coupling reaction

The reaction has been performed on the α-pyridyldithio,ω-hydroxylPEO (M n,SEC ̅̅̅̅̅̅̅̅ = 8800 g.mol -1 and Ð = 1.07) using 1.5 equivalent of N-acetylcysteine in phosphate buffering solution x 100 after purification with At,UV and At,RI peaks areas at a given moment using an UV-vis detector at 266.2 nm and a RI detector respectively and At0,UV and At0,RI are peaks areas a t = 0 using an UV-vis detector at 266.2 nm and a RI detector, respectively.

The PNIPAM resulting from optimal experimental conditions (Entry 3 in Table IV. As previously shown, two steps are needed to be able to add thiol-based molecules onto RAFT polymers through a radical mechanism. The challenging one-pot radical mediated process via tandem thiol-yne/thiocarbonylthio chain-end removal reactions was studied.

This one-pot two-steps has been modeled using a α-alkynyl,ω-dodecyltrithiocarbonate-PNIPAM previously synthesized in this Chapter ( §I-A-1) (Scheme IV. 6). Thus, such heterofunctional PNIPAM was first reacted with an excess of AIBN in 1,4-dioxane at 80°C for 24 hours; then 15 equivalents of benzyl mercaptan and 15 equivalents of DMPP were added and the reaction was continued for 24 hours (Scheme IV. 6). Samples were withdrawn to assess the trithiocarbonate removal efficiency using SEC with UV-Vis detection at 309 nm and the final sample was purified in order to determine the functionalization degree with the benzyl mercaptan by 1 H NMR spectroscopy analysis (Scheme IV. 6). Results are presented in Table IV )x100 where At,UV nm and At,RI are peak areas at given time (t) using an UV-vis detector operating at 309 nm and a RI detector, respectively and At0,UV and At0,RI are peak areas before the reaction using an UV-vis detector operating at 309 nm and a RI detector, respectively. b τμ thiol-yne coupling degree of functionalitation determined by 1 H NMR spectroscopy (in CD2Cl2) analysis of purified PNIPAM using the following equation: τ (%) = Results showed that the trithiocarbonate moiety was completely removed after 24 hours of reaction (Entry 1 in Table IV 5). Such result is in good agreement with the first thiol-yne result (Entry 3, Table IV. 5). In this one-pot reaction, while the trithiocarbonate removal conversion was high, the thiol-yne degree of functionalization remained limited. However, it should be noted that such a conversion allows for sufficient functionalization when applied to the bioconjugation of biologically active molecules or probes at polymer chain-end. 34,35 As shown in I §B of this chapter, the trithiocarbonate removal reaction on aPEO-b-P(DMAEA-co-tBocAEA) copolymers, and P(DMAEA-co-tBocAEA) copolymers was used successfully through photochemical-activation. Therefore, the one-pot radical mediated process via tandem trithiocarbonate removal/thiol-yne reactions will be employed using UV photochemical-activation in future work. This combination of reaction was never published and can be a good platform to introduce thiolated recognition ligand on RAFT copolymers through a radical process.

Conclusion

The incorporation of thiolated recognition ligand and the removal of unwanted trithiocarbonate group have been studied. Moreover, the combination of trithiocarbonate removal and thio-yne coupling reactions has been target via radical "one-pot" process.

The trithiocarbonate removal on aminoethyl acrylate based monomers was first modeled under thermo-activation using a PNIPAM. This PNIPAM was synthesized by RAFT polymerization using COPYDC as the chain transfer agent and AIBN as the initiator equivalents of AIBN as the radical source in 1,4-dioxane at 80°C. The reaction was followed by SEC using UV-vis detection at 309 nm. The conversion was equal to 97 % after 24 hours of reaction. This conversion was confirmed by 1 H NMR spectroscopy and SEC after purification.

From this result and to maintain the integrity of the cationic polymer the trithiocarbonate removal of aminoethyl acrylate-based copolymers (aPEO-b-P(DMAEA-co-AEA) and P(DMAEA-co-AEA)) have been mediated through photochemical-activation using AIBN as the radical initiator. The reactions were followed by SEC and the conversions of trithiocarbonate removal were superior to 81 %. The 1 H NMR spectroscopy showed maintain of the integrity of the aminoethyl acrylate copolymers.

Chapter 4: Chain-ends modification of (PEGylated) aminoethyl-based polyacrylates.

186

Two different pathways have been studied to covalently link a thiolated-recognition ligand. First, the thiol-disulfide exchange has been performed using an α-alkynyl,ω-hydroxylPEO and N-acetylcysteine as model (macro)molecules. The degree of functionalization was determined by UV-vis spectrophotometry and was equal to 0.56 thiol group per polymer chain. Then, the thiol-yne coupling reaction was studied using an αalkynyl,ω-cyanoisopropylPNIPAM and benzyl mercaptan as the models (macro)molecules with AIBN as the radical initiator. The best conditions was obtained with 15 equivalents of benzyl mercaptan and 15 equivalents of DMPP leading to 0.76 thiol group per polymer chain, determined by 1 H NMR spectroscopy.

Then the "one-pot" cascade thiol-yne click reaction and radical-induced trithiocarbonate removal through radical mechanism was explored. The reaction was performed on an αalkynyl,ω-dodecyltrithiocarbonate PNIPAM using AIBN to remove the trithiocarbonate on the ω-positions and AIBN, benzyl mercaptan and DεPP to functionalize the α-position by thiolyne "click" chemistry. The SEC after 24 hours of reaction showed a complete removal of trithiocarbonate group and the 1 H NMR spectroscopy showed a degree of functionalization of 0.80 thiol molecule onto the alkyne bond, as shown ny 1 H NMR spectroscopy.

The aminoethyl acrylate-based copolymer after trithiocarbonate removal reaction will be cationized, deprotected and used to study the impact of the dodecyltrithiocarbonate on cytotoxicity and DNA binding affinity.

Chromatography (SEC) using DMF-LiBr (1 g.L -1 ) as an eluent and carried out using a system equipped with a guard column (Polymer Laboratories, PL Gel 5 µm) followed by two columns (Polymer Laboratories, 2 PL gel 5 µm MIXED-D columns) and with a Waters 410 differential refractometer (RI) and a Waters 481 UV-Visible detector. The instrument operated at a flow rate of 1.0 mL.min -1 at 60°C and was calibrated with narrow linear polystyrene (PS) standards ranging in molar mass from 580 g.mol -1 to 483000 g.mol -1 . Molar masses and dispersities were calculated using Waters EMPOWER software.

MALDI-TOF Mass Spectrometry analyses were performed on a Bruker UltraFlex II instrument equipped with a nitrogen laser operating at 337 nm, pulsed ion extraction source and reflectron. Spectra were recorded in the reflectron mode with an acceleration voltage of 25 kV and a delay of 300 ns, 500 single shot aquisitions were summed to give the spectra and the data were analyzed using Bruker FlexAnalysis and Polytools softwares. Samples were prepared by

dissolving the matrix (trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-
propenylidene]malononitrile, DCTB) in the solvent (CH2Cl2, 30 mg.mL -1 ) and mixing with the polymer (10 mg.mL -1 ) in the ratio 1/10 (v/v). 1 µL was spotted directly onto a thin layer of sodium trifluoroacetate (NaTFA) in acetone (concentration 19 mg.mL -1 ) that had been deposited to act as a cationizing agent. UV-vis spectra of polymers were recorded using a VWR UV-3090PC. Spectra were analyzed with UV-vis Analyst software. where At,UV and At,RI represent peak areas at a given reaction time (t) using a UV-vis detector operating at 309 nm and a RI detector, respectively and where At0,UV and At0,RI represent peak areas at t = 0 using a UV-vis detector operating at 309 nm and a RI detector, respectively.

C. Trithiocarbonate Removal

The reaction was stopped after 14 days of reaction and the crude mixture was evaporated under vacuum. The crude oiled was dissolved in a minimum of acetone and precipitated in a cold mixture of hexane and diethyl ether (50/50 v/v), then the mixture was centrifuged (7000 rpm, 5 min, 0°C). The operation was repeated three times.

The solid obtained was dried under vacuum and analyzed by 1 H NMR spectroscopy and SEC in DMF (LiBr 1 g.L -1 ).

D. Addition of a thiol based compound 1. Thiol-disulfide coupling reaction

A buffering solution of ethylenediaminetetraacetic acid (EDTA) (14.9 mg, 0.051 mmol), Na2HPO4 (433.1 mg, 3.05 mmol) and NaH2PO4 (168.2 mg, 1.17 mmol) at pH = 7.5 was prepared in 50 mL of ultra-pure water. In a round bottom flask, the α-pyridyldithio,ω-hydroxylPEO (25.1 mg, 0.0079 mmol) and the N-acetylcysteine (2.0 mg, 0.012 mmol) were dissolved in 8 mL of the PBS solution previously prepared. The reaction was launched at room temperature. A t= 18 h, 1 mL of media was withdrawn of an initial molar concentration equal to 0.001 mmol.L -1 , diluted by ten in PBS and analyzed by UV-vis spectrophotometry. The conversion of 56 % was calculated by UV-vis spectrophotometry using the following equations:

τ = 1- C PEOt18h C PEOt0 Equation (IV. 1) C PEOt18h = C PEOt0 -C εPt18h Equation (IV. 2) τ = C εPt18h C PEOt0 Equation (IV. 3) A 343nm = ×l×C εPt18h 10 Equation (IV .4) τ = A 343nm ×10 ×l×C PEOt0 Equation IV. 5)
Where, τ is the degree of functionalization, CPEO is the molar concentration of αpyridyldithio,ω-hydroxylPEO after 18 h of reaction, CPEOt0 is the intial molar concentration of α-pyridyldithio,ω-hydroxylPEO (CPEOt0 = 0.001 mmol.L -1 ), CMPt18h is the molar concentration of 2-mercaptopyridine after 18 h of reaction, A343nm is the absorbance at 343 nm (A343nm = 0.4518), is the molar attenuation coefficient of 2-mercaptopyridine ( = 8080 L.mol -1 .cm - 1 ) 29,30 and l is the length of the UV-vis cell (l = 1 cm).

Thiol-yne coupling reaction.

To a round bottom flask were added α-alkynyl,ω-dodecyltrithiocarbonate PNIPAM (50 mg, 1.3 × 10 -2 mmol, M n,SEC ̅̅̅̅̅̅̅̅ = 11600 g.mol -1 ), AIBN (0.1 equivalent toward benzyl mercaptan), benzyl mercaptan (from 10 to 20 equivalents toward PNIPAM), DMPP (from 0 to 10 equivalents toward benzyl mercaptan) and 1,4-dioxane (0,9 mL). The solution was degassed with argon during 15 minutes and then heated at 80°C fot 24 hours. The solvent was evaporated, the polymer was dissolved with a minimum of CH2Cl2 and precipitated in cold diethyl ether three times. The solid obtained was dried under vacuum at 40°C. The degree of functionalization of the thiol-yne coupling reaction was determined by SEC comparing peaks areas using a RI detector and peaks areas using a UV-vis detector (fixed at 266.2 nm) using the following equation:

τ % = A t,UV A t, RI ⁄ - A t0,UV A t0,RI ⁄ A t0,UV A t0, RI ⁄ ×100 Equation (IV. 7)
where τ is the degree of functionalization of thiol-yne coupling, At,UV represents the peak area after the reaction using a UV-vis detector operating at 266.2 nm, At,RI represents the peak area after the reaction using a RI detector, At0,UV represents the peak area at t = 0 the reaction using a UV-vis detector operating at 266.2 nm and At0,RI represents the peak area at t = 0 the reaction using a RI detector.

The degree of functionalization was also determined by 1 H NMR spectroscopy using the following set of equations:

I 7.52 = 4 × x × y Equation (IV. 8)
Chapter 4: Chain-ends modification of (PEGylated) aminoethyl-based polyacrylates.

194

where I7.52 is the intensity of the signal at 7.52 ppm, τ is the degree of functionalization, 4 is the number of aromatic protons at 7.52 ppm for a 100 % conversion and y is the integration coefficient. E. Tandem trithiocarbonate removal/thiol-yne reactions in a one-pot process.

In a round bottom flask a stir bar was charged together with PNIPAM (200.1 mg, 0.040 mmol), AIBN (132.2 mg, 0.806 mmol) and 1,4-dioxane (3.45 mL). This first solution was degassed with argon during 15 minutes and the reaction was launched at 80 °C for 24 hours.

An aliquot was withdrawn to determine the trithiocarbonate removal reaction using SEC. A second solution previously degassed containing benzyl mercaptan (73.2 mg, 0.589 mmol), AIBN (9.7 mg, 0.059 mmol) and DMPP (85.0 mg, 0.615 mmol) was added to the first solution under argon for further 24 h. The solution was precipitated in cold diethyl ether three times.

The solid obtained was dried under vacuum at 40°C and analyzed by 1 H NMR spectroscopy in CD2Cl2 and by SEC in DMF (LiBr 1 g.L -1 ). A white powder was obtained with a yield of 50 %. The conversion of the trithiocarbonate removal reaction was determined by SEC comparing peaks areas using a RI detector and a UV-vis detector operating at 309 nm. The degree of functionalization of the thiol-yne coupling reaction was determined by 1 H NMR spectroscopy using the following set of equations:

I 7.25 =10 × x × y Equation (IV. 11)
where I7.25 is the intensity of the signal at 7.25 ppm, 10 is the number of aromatic protons at 7.25 ppm for a 100 % conversion, x is the degree of functionalization and y is the integration coefficient. Chapter 5μ Cationic (PEGylated) aminoethyl-based polyacrylatesμ pDNA complexation and cytotoxicity.

Introduction

DNA-delivery mediated via cationic polymers is driven by several important parameters influencing transfection efficiency and/or cytotoxicity.

The effect of PEGylation on transfection efficiency and cytotoxicity has been widely studied on different non-viral gene delivery system including PEI or PLL (Chapter 1 §II). [1][2][3] The different studies showed a decrease of cytotoxicity and a decrease of transfection efficiency with the PEGylated polymers which has been explained by the "hiding" of the cationic charges leading to a limitation of polyplex instability under physiological conditions (salt, proteins) and by the aid to the decrease of cell binding affinity, respectively. Moreover, the influence of the PEO architecture was not signification as reported by Venkataraman et al. 4 Authors just noticed a slight better transfection efficiency of the PEO-b-PDMAEMA copolymers based polyplexes than the PPEGA-b-PDMAEAMA copolymers based polyplexes at high N/P ratios, 15 and 20.

Introducing protonable primary and tertiary entities and tuning their molar ratio improved transfection efficiency by modifying the pKa of cationic polymer. Zhu et al. 5 synthesized P(DMAEMA-co-AEMA) copolymers with different DMAEMA/AEMA molar ratios and they showed that a decrease of DMAEMA/AEMA molar ratio allowed to reduce of the N/P ratios to target full DNA complexation. However, such DMAEMA/AEMA molar ratio decreases led to a reduction of COS-7 cells viability.

As it mentioned in Chapter 4 §I-A the trithiocarbonate can be hydrolyzed leading to polymer-polymer coupling, even if, is more stable than dithioester groups. Moreover, it has been shown that the polymer chain-end can influence cytotoxicity. Akinc et al. 6 showed by replacing alcohol chain-end extremities by acrylate functions on PBAEA the drastic decreases of cell viability.

I. Size and zeta potential of a pDNA/aPEO-b-P(DMAEA-co-AEA) polyplex

The polyplex size plays a key role for cytotoxicity and transfection efficiency. 7 It has been shown that polyplexes with a sizes below 150 nm lead to non-cytotoxic sytem. 8 Moreover, Xu et al. 9 showed the pDNA/bPEI polyplex size impact the transfection efficiency of A549 cell line, Hela cell line, BGC-823, a human gastric adenocarcinoma cell line and Bel7402, human hepatocellular carcinoma, cell line. Authors, synthesized several polyplex with sizes ranging from, 38 nm to 167 nm. They showed the best that transfection efficiency was targeted with a size of polyplex ranging from below 87 nm.

The determination of the polyplex size and zeta potential has been done by dynamic light scattering (DLS) and zetametry, respectively. The analyses have been made on an aPEO64-b-P(DMAEA10-co-AEA12) (Run 9, Table III. 3, Chapter 3) with a trithiocarbonate group. First, the optimal N/P ratio to obtain a fully complexed pDNA has been determined by gel retardation Chapter 5: Cationic (PEGylated) aminoethyl-based polyacrylates: pDNA complexation and cytotoxicity 200 assay; then the size and the zeta potential of the polyplex have been characterized using the polyplex obtained with this optimal N/P ratio.

The optimum N/P ratio has been determined using a pDNA containing 4631 base pairs.

The electrophopherogram shows a migration of pDNA for N/P ratios of 0, 1 and 2 (Figure V.

1). At N/P ratios above 3 no migration was observed due to the fully complexation of pDNA. Further by DLS and zeta potential analyses were performed on the polyplex formed with a N/P = 3 to measure the hydrodynamic diameter (Dh), the size disperisity (PD) and zeta potential of the polyplex.

The formed polyplex was analyzed by DLS a Dh equal to 38 nm with a PD of 0.39 were observed. Moreover, it has been that shown the polyplex size below 87 nm leads to low cytotoxicity. 9 The zeta potential of a aPEO64-b-P(DMAEA11-co-AEA9) cationic copolymer and associated polyplex has been compared.

The zeta potential of the cationic aPEO64-b-P(DMAEA11-co-AEA9) copolymer is positive and equal to + 12 mV and the zeta potential of associated polyplex is around -1 mV. This result shows the neutralization of cationic charge during the complexation, the fully complexation of pDNA and the formation of the polyplex by electrostatic binding. To study the influence of the PEGylation of cationic DMAEA/AEA based copolymers on polyplex formation, the behavior of polyplexes based on (i) pDNA/aPEO-b-P(DMAEA-co-AEA) copolymers (6100 g.mol -1 < M n,SEC ̅̅̅̅̅̅̅̅ < 11500 g.mol -1 and 1.21 < Ð < 1.52), (ii) pDNA/PPEGA-b-P(DMAEA-co-AEA) copolymers (10100 g.mol -1 < M n,SEC ̅̅̅̅̅̅̅̅ < 10700 g.mol -1 and 1.57 < Ð < 1.62) and pDNA/P(DMAEA-co-AEA) copolymers (6400 g.mol -1 < M n,SEC ̅̅̅̅̅̅̅̅ < 9400 g.mol -1 and 1.20 < Ð < 1.50) were compared using gel retardation assays. The cell viability of these polymers were studied by cell viability tests.

A. Gel retardation assay

To study the influence of PEGylation a PPEGA12-b-P(DMAEA7-co-AEA14) copolymer (Run 2, Table III. 4., Chapter 3) has been compared with a P(DMAEA9-co-AEA13) copolymer (Run 3, Table III. 5., Chapter 3). N/P ratio equal to 5 for bPEO based cationic copolymer to fully complex DNA. The decrease of the ability to complex DNA can be explained by the increase of the molar mass of the M n ̅̅̅̅ of the PEO block, from 3000 g.mol -1 for the lPEO to 4800 g.mol -1 for the bPEO leading to a lower pDNA complexation ability. The agarose gel electropherograms show that the addition of a PEO block leads to the decrease of pDNA binding affinity. Moreover, this decrease is more important for a bPEO compared to a lPEO.

B. Cell viability test

The Cell viability decreased to 22 % when J774.A1 cells were incubated with the aPEO64-b-P(DMAEA11-co-AEA9). This lower cell viability could be explained taking into account the composition of copolymers, which will be further discussed in the following paragraphs. The MTT assays show a decrease of cell viability using PEGylated copolymer compare to non-PEGylated copolymer. The bPEO has been employed for PEGylation of P(PDMAEAco-AEA), the copolymer is toxic just on HUVEC cells.

Chapter 5: Cationic (PEGylated) aminoethyl-based polyacrylates: pDNA complexation and cytotoxicity

206

In conclusion of this part, the gel retardation assay shows a decrease of the complexation of pDNA with the PEGylation of the cationic P(DMAEA-co-AEA) copolymer. However, the PEGylation tends to decrease the cytotoxicity depending on the PEO architecture. lPEO is a better candidate than bPEO to decrease the cytotoxicity of P(DMAEA-co-AEA) copolymers.

III. Influence of the DMAEA/AEA molar ratios on pDNA binding and cell viability

The RAFT copolymerization of tBocAEA and DMAEA followed by the cationization and deprotection allow to tune efficiently the DMAEA/AEA molar ratios. The influence of the DMAEA/AEA ratio on pDNA binding and cell viability has been by gel retardation assay and MTT assay on NIH-3T3 cells, HUVEC cells and J774.A1 cells.

A. Gel retardation assays

The Figure V. 5 shows the comparison of gel retardation assay of pDNA polyplexes using an aPEO64-P(DMAEA11-b-AEA4) (Run 4, Table III. 3, Chapter 3) copolymer and an aPEO64-P(DMAEA5-co-AEA10) copolymer (Run 5, Table III. 3, Chapter 3). The gel retardation assays using an aPEO64-P(DMAEA11-b-AEA4) copolymer and an aPEO64-P(DMAEA5-co-AEA10) copolymer show an optimum N/P ratios above 8 and of 5, respectively to fully complex DNA.

This results show the better pDNA binding affinity using copolymers with a higher amount of protonated primary amine AEA as shown with P(DMAEMA-co-AEMA) by Zhu et al. The gel retardation assays of pDNA/polyplexes using aPEO-b-P(DMAEA-co-AEA) copolymers and PPEGA-b-P(DMAEA-co-AEA) copolymers show that the increase of DMAEA/AEA molar ratio leads to the increase of the N/P ratio to fully complex pDNA due to a decrease of the protonated primary amine entities.

B. Cell viability test

The influence of cationic copolymer DMAEA/AEA molar ratio has been studied by comparing (i) an aPEO64-b-P(DMAEA5-co-AEA10) copolymer (Run 5, Table III The gel retardation assay showed an increase of cell binding affinity with the decrease of DMAEA/AEA molar ratio. The cell viability showed opposites behaviors of the influence of DMAEA/AEA molar ratio between bPEO and lPEO depending of the cell lines. Further investigations will be made to evaluate more precisely the impact of DMAEA/AEA ratio on cytotoxicity.

A. Gel retardation assays

The influence of the dodecyltrithiocarbonate group on pDNA binding has been compared by gel retardation assay using the aPEO64-P(DMAEA10-co-AEA12)-CTA copolymer with a dodecyltrithiocarbonate (-CTA) group and the aPEO64-P(DMAEA11-co-AEA9) (Run 9, Table III. 3, Chapter 3) copolymer without the dodecyltrithiocarbonate group (Figure V. 8). The comparison doesn't show any significant difference between the aPEO64-P(DMAEA10-co-AEA12)-CTA and the aPEO64-P(DMAEA10-co-AEA12). Indeed, the optimum N/P ratio to fully complex pDNA for both copolymers is equal to 3. This result shows the presence of the trithiocarbonate doesn't lead to a better complexation of the pDNA. Even if the presence of the trithiocarbonate on copolymers did not show any effect on the pDNA complexation may plays a role in copolymers cytotoxicity.

B. Cell viability tests

The influence of the presence of the trithiocarbonate on the cytotoxicity have been measured on two cell lines: NIH-3T3 and HUVEC cell lines (Figure V. 9). On NIH-3T3 cells, the comparison between an aPEO64-b-P(DMAEA11-co-AEA9)-CTA and an aPEO64-b-P(DMAEA11-co-AEA9) shows a decrease of the cell viability from 89 % to 30 % when the trithiocarbonate has been removed (Figure V. 9(A)). Analogous results have been observed on NIH-3T3 and HUVEC cells when exposed to a P(DMAEA9-co-AEA13)-CTA and a P(DMAEA9-co-AEA13). At 2 g.L -1 , the P(DMAEA9-co-AEA13), cell viabilities was equal to 76 % and 66 % for NIH-3T3 and HUVEC cells, respectively. On the contrary a drop to ~50% in both cell lines was measured after incubation with the copolymer P(DMAEA9-co-AEA13)-CTA. These results highlight the cytotoxocity due to the presence of dodecyltrithiocarbonate.

Conclusion

The pDNA polyplexes obtained from aPEO-b-P(DMAEA-co-AEA) copolymers, PPEGA-co-P(DMAEA-co-AEA) copolymers and P(DMAEA-co-AEA) copolymers with different DMAEA/AEA ratio with our without trithiocarbonate group have been characterized by gel retardation assay and MTT assays on three different cell lines.

In a preliminary study an aPEO64-co-P(DMAEA10-co-AEA12) with a thrithiocarbonate function has been characterized by gel retardation assay. The electropherograms showed the between the aPEO64-co-P(DMAEA11-co-AEA9) and its associated polyplex showed the neutralization of cationic charge after complexation, + 12 mV and -1 mV, respectively.

The influence of the PEGylation has been studied by gel retardation assay. The gel retardation assays showed optimal N/P ratio of 4 for a pDNA/PPEGA12-b-P(DMAEA7-co-AEA14) polyplex and N/P ratio of 1 for a pDNA/P(DMAEA9-co-AEA13) polyplex due to the addition of the bPEO block. The comparison between a bPEO based polyplex and a lPEO based polyplex with the same DMAEA/AEA molar ratio shows a fully complexation for a N/P of 5 and 3, respectively. This result is explained by the higher M n ̅̅̅̅ of bPEO compare to the lPEO.

Moreover, the MTT assays on three different cell lines showed a decrease of cytotoxicity on the three different cell lines after PEGylation using a lPEO. The cytotoxicity of bPEO based cationic copolymers is more important that lPEO based cationic copolymers but results are cell dependents.

The influence of DMAEA/AEA molar ratio has been studied. Electropherograms of pDNA/aPEO64-b-P(DMAEA11-co-AEA4) polyplex and pDNA/aPEO64-b-P(DMAEA5-co-AEA10) polyplex showed a decrease of optimal N/P with DMAEA/AEA molar ratio, from 8 to 5, due to the increase of primary amine ratio. Opposite behavior has been noticed using bPEO based polyplex.

The influence of the trithiocarbonate chain-end on the binding DNA affinity has been analyzed by gel retardation assay. The presence of this group did not show any significant differences on binding affinity. However, the MTT assay showed the high cytotoxicity of the trithiocarbonate, indeed using a lPEO based polyplex the cell viability of NIH-3T3 increases from 30 to 89 %, with and without dodecyltrithiocarbonate chain-end, respectively.

D. Cells culture

Embryonic murine fibroblast (NIH-3T3) and human endothelial umbilical vein cells (HUVEC) were cultured in DMEM, supplemented with 50 U.mL -1 penicillin, 50 U.mL -1 streptomycin and 10% FBS. The J774.A1 murine macrophage-monocyte cell line was cultured in RPMI 1640 medium supplemented with 50 U.mL -1 penicillin, 50 U.mL -1 streptomycin, and 10% heat inactivated FBS. All cell lines were obtained from ATCC and maintained at 37 °C in a humidified 5% CO2 atmosphere.

E. MTT assay

The in vitro cytotoxic activity of the copolymers was evaluated on the three cell lines, using the MTT test. Cells were seeded in 100 L of growth medium (HUVEC and J774.A1: 8

The aim of this work was to synthesize a unique multi-structural copolymer with low cytotoxicity that can be used for efficient DNA delivery. To attain this goal, several heterofunctional PEGylated cationic polymers with different DMAEA and tBocAEA molar ratios with different PEO architectures (with or without a trithiocarbonate end-group) have been synthesized by RAFT polymerization. By using alkyne-based (macro)molecular chain transfer agents, well-defined P(DMAEA-co-tBocAEA), PEO-b-P(DMAEA-co-tBocAEA), PPEGA-b-P(DMAEA-co-tBocAEA), and P(DMAEA-co-tBocAEA) were successfully prepared. In parallel, a study was conducted to anchor a thiolated recognition ligand by thiol-yne coupling reaction and thiol-disulfide coupling reaction on model polymers (lPEO and PNIPAM). The associated cationic copolymers have been synthesized and characterized and their ability to complex DNA was studied by light scattering, zeta-potential and gel retardation assay.

Cytotoxicity was evaluated using MTT assay on NIH-3T3, HUVEC and A1.J774 cell lines.

The influence of (i) PEGylation, (ii) molar composition of cationic copolymer, and (iii) chainend modification were investigated.

The synthesis of heterofunctional lPEO was studied first. Two different thiol-reactive groups, namely pyridyldithio and alkynyl groups, were covalently linked at the α-chain-end of lPEO. α-Pyridyldithio,ω-hydroxylPEO was obtain quantitatively by reacting commercial αamino,ω-hydroxylPEO with an azlactone-based alkynyl linker under mild conditions. Then, a Moreover, after complexation the hydrodynamic diameter of the polyplex was measured and equal to 38 nm. As shown in literature, this size led to non-toxic polyplex. 3 The influence of the PEGylation, of the presence of the dodecyltrithiocarbonate group and the DMAEA/AEA ratio on pDNA binding affinity and cytotoxicity of cationic copolymers were studied. The gel retardation assay on PEGylated copolymers showed a complete pDNA complexation with a higher N/P ratio that non-PEGylated copolymer. Moreover, the MTT assay on three different cell lines showed an increase of cell viability using PEGylated copolymer.

The comparison between the lPEO and the bPEO didn't show any significant differences on

General Conclusion

224

cell viability. Gel retardation assays of different PEO-b-P(DMAEA-co-AEA) showed the decrease of optimal N/P ratios with the decrease of the DMAEA/AEA molar ratio due to the increase of primary amine ratio. The result has been confirmed with two bPEO-based aminoethyl polyacrylates. Furthermore, the decrease of the DMAEA/AEA ratio led to a decrease of cell viability using a lPEO as shown by MTT assay. Opposite behavior have been noticed using PPEGA based cationic polymer. The presence of the dodecyltrithiocarbonate group didn't play a role on pDNA binding affinity as shown by gel retardation assay. However, copolymer with dodecyltrithiocarbonate chain end exhibited higher cytotoxicity than without this group as shown by MTT assay. The best results were obtained using a PEGylated copolymer, to decrease cytotoxicity, with a low DMAEA/AEA ratio, to increase pDNA binding affinity and without trithiocarbonate chain-end.

To summarize, this work established efficient pathways to synthesize a small library of innovative PEGylated cationic copolymers containing primary and tertiary amine-based repeating units by combining efficient and versatile organic reactions with RAFT polymerization processes. The combination of a primary and tertiary amines entities allows to tune both the binding of pDNA affinity and cytotoxicity. Furthermore, PEGylation increases cell viability, leading to non-toxic cationic copolymers.

With these non-toxic poly(aminoethyl acrylates) copolymers in hand, investigation of the influence of PEGylation and DMAEA/AEA ratio on polymer degradation, zeta potential, polyplex size, and stability and transfection efficiency will be continued in the next future.

Furthermore, it is expected that the addition of a recognition ligand will increase the transfection efficiency of these copolymers by increasing cellular uptake. Thanks to the methodologies implemented during this work and results using model reactions, the recognition ligand can be efficiently introduced into the non-toxic PEGylated cationic copolymer chains using photochemically-activated thiol-yne coupling.

( 39 Scheme I. 4 .

 394 Scheme I. 4. Cationic amine-based polymers studied on transfection efficiency by Van deWetering et al.[START_REF] Van De Wetering | tru ture-A ti it Relatio ships of Water-Soluble Cationic Methacrylate/Methacrylamide Polymers for Nonviral Gene Delivery[END_REF] , PDMAEMA, poly(3-(N,N-dimethylamino)propyl methacrylate)

  Scheme I. 5. (A) Synthesis of a lPDMAEMA by ATRP using different solvents, ligands and

  studied the RAFT polymerization of DMAEMA in 1,4-dioxane using 2,2'-azobis(isobutyronitrile) (AIBN) and 4-cyano-2-propyl dithiobenzoate (CPDB) using different ratios of [CPDB] 0 [AIBN] 0 ⁄ and of [DεAEεA] 0 [CPDB] 0 ⁄ . For all conditions, they reached well-controlled polymerizations with relatively low dispersity (values) (Đ = 1.09-1.4) and they reached high DMAEMA conversions (93 %).

et al. fixed a 4 -

 4 cyanopentanoic acid dithiobenzoate (CPADB) onto a hydroxyl-PEO by esterification using a diisopropyl carbodiimide (DIC) as a coupling agent in dichloromethane (Scheme I. 8(B)). They used the resulting PEO as a macromolecular RAFT agent to perform the RAFT polymerization of DMAEMA in 1,4-dioxane using 4,4-azobis-4-cyanolaveric acid (ACVA) as the initiator. They obtained a PEO-b-PDMAEMA copolymer of 10100 g.mol -1 with

  studied the effect of the architecture of PEO on transfection efficiency of pCMV-Luc, a plasmid containing the firefly luciferase gene, on Hep-G2 (human liver cell lines) and HEK-293 cells lines and on cytotoxicity by comparing a PDMAEMA-b-PEO and a PDMAEMA-b-PPEGA. They synthesized these two copolymers by ATRP. The results of transfection efficiency don't show any significant differences in the different cell lines on HEK 293 cell lines but, authors shown the better transfection efficiency of PDMAEMA-b-PEO on Hep-G2 cell lines than PDMAEMA-b-PPEGA due to the better cellular uptake of PDMAEMA-b-PEO

  studied the impact of the addition of cRGD on PEGylated-b-PLL on transfection efficiency of pCAcc+Luc, a pDNA containing the luciferase firefly gene on HeLa and HEK293 cells. cRGD is able to target αVβ3 and αVβ5 integrins. The authors showed, by flow cytometric analysis, that HEK293 cells don't expressed the αVβ3 and expressed a little amount of αVβ5 while the HeLa cells expressed both of integrins. The comparison of transfection efficiency between the cRGD-PEO-b-PLL and PEO-b-PLL on HeLa cells showed the better transfection efficiency of polymer functionalized by cRGD. Moreover, the comparison on 293T cells didn't show any significant differences between cRGD-PEO-b-PLL and PEO-b-Pδδ. These results showed the ability of cRGD to target αVβ3 and αVβ5 integrins and the better transfection efficiency of polymers with a recognition ligand. The work of Qian et al. 73 on TGN-PEO-b-PDMAEMA showed the effect of the recognition ligand on transfection efficiency. This ligand is able to target brain cells. The authors showed the best transfection efficiency of TGN-PEO-b-PDMAEMA of pGL2 pDNA on brain capillary endothelial cells, BCECs compared to a PEO-b-PDMAEMA. Moreover, the authors showed not significant differences of cell viability on BCECs. Therefore, as

( 2 - 1 (

 21 Scheme II. 1. Synthesis of an α-pyridyldithio,ω-hydroxylPEO.

Figure II. 1 .

 1 Figure II. 1. Comparison of FT-IR spectra of α-amino,ω-hydroxylPEO (top) and α-

Figure II. 2 .

 2 Figure II. 2. MALDI-TOF mass spectrum of an α-pyridyldithio,ω-hydroxylPEO. Matrix:

Figure II. 3 . 1 H

 31 Figure II. 3. 1 H NMR spectrum (400 MHz, CDCl3) of the α-pyridyldithio,ω-hydroxylPEO.

Figure II. 4 .

 4 Figure II. 4. Overlaid SEC traces of purified α-pyridyldithio,ω-hydroxylPEO (solid line: RI

The 1 H

 1 NMR spectroscopy (Figure II. 5) shows the characteristic signals of CH2CH2O of EO repeating unit at 3.65 ppm (labeled b in Figure II. 5) and two signals characteristics of the propargyl moiety at 4.20 ppm for the methylene protons of HC≡CCH2O (labeled c in Figure II. 5) and at 2.40 ppm for the methine proton of HC≡CCH2O (labeled d in Figure II. 5). The degree of functionalization was calculated by comparing the integration areas of the CH2CH2O signals of EO unit at 3.65 ppm (176 protons, corresponding to a DP n ̅̅̅̅̅ = 44 determined by MALDI-TOF mass spectrometry, labeled b in Figure II. 5) and of the HC≡CCH2O signal of the methine proton at 2.40 ppm (0.78 proton, labeled d in Figure II. 5). The degree of functionalization is equal to 78 %.

Figure II. 5 . 1 H

 51 Figure II. 5. 1 H NMR spectrum (400 MHz, CDCl3) of the α-alkynyl,ω-hydroxylPEO

Figure II. 6 .

 6 Figure II. 6. MALDI-TOF spectrum between m/z of 1740 and 1900 Da of purified polymer obtained after reaction of α,ω-dihydroxylPEO with propargyl bromide and KOH in toluene at

  7). The spectrum shows a single series of peaks separated by 44.023 Da, corresponding to the molar mass of the EO repeating unit (calculated value: 44.026 Da). The peak at 3108.780 Da corresponds to a polymer chain with 64 EO unit, an alkynyl moiety at one chain-end, a hydroxyl group at the other chain-end, and a sodium entity as the atom responsible of the ionization (calculated value: 3108.809 Da). A DP n ̅̅̅̅̅ of 64 has been determined by MALDI-TOF mass spectrometry.

Figure II. 7 .

 7 Figure II. 7. MALDI-TOF mass spectrometry of α-alkynyl,ω-hydroxylPEO obtained by ringopening reaction of the alkyne-AZL by α-amino,ω-hydroxylPEO in CH2Cl2 at room

Figure II. 8 . 1 H

 81 Figure II. 8. 1 H NMR spectrum (400 MHz, CDCl3) of α-alkynyl,ω-hydroxylPEO obtained by ring-opening reaction of the alkyne-AZL with α-amino,ω-hydroxylPEO in CH2Cl2 at room

Figure II. 9 .

 9 Figure II. 9. 13 C NMR spectrum (100 MHz, CDCl3) of α-alkynyl,ω-hydroxylPEO obtained by ring-opening reaction of the alkyne-AZL with α-amino,ω-hydroxylPEO in CH2Cl2 at room

The 1 H

 1 Figure II. 10) and a triplet at 4.25 ppm characteristic of the CH2CH2OC(=O) at the αposition

Figure II. 10 . 1 H

 101 Figure II. 10. 1 H NMR spectrum (400 MHz, CDCl3) of α-pyridyldithio,ω-dodecyl trithiocarbonate PEO obtained by esterification of α-pyridyldithio,ω-hydroxylPEO with the

Figure II. 11 .Scheme II. 5 .

 115 Figure II. 11. Overlaid SEC traces of purified α-pyridyldithio,ω-dodecyltrithiocarbonate PEO

Figure II. 12 .Scheme II. 6 .

 126 Figure II. 12. Overlaid of SEC traces using RI detection of α-pyridyldithio,ω-

Figure II. 13 . 1 H

 131 Figure II. 13. 1 H NMR spectrum (400 MHz, CDCl3) of α-pyridyldithio,ωdodecyltrithiocarbonate PEO obtained by esterification of α-pyridyldithio,ω-hydroxylPEO and

Figure II. 14 .

 14 Figure II. 14. Overlaid SEC traces of purified α-pyridyldithio,ω-dodecyltrithiocarbonate PEO

Scheme II. 7 .

 7 Scheme II. 7. Synthesis of α-pyridyldithio,ω-dodecyltrithiocarbonate PEO by esterification of α-pyridyldithio,ω-hydroxylPEO with CDP mediated through DCC and DMAP at room

Figure II. 15 . 1 H

 151 Figure II. 15. 1 H NMR spectrum (400 MHz, CDCl3) of the α-pyridyldithio,ωdodecyltrithiocarbonate PEO obtained by esterification of the α-pyridyldithio,ω-hydroxylPEO

Figure II. 16 .

 16 Figure II. 16. Overlaid SEC traces of purified α-pyridyldithio,ω-dodecyl trithiocarbonate PEO

The 1 H

 1 NMR spectrum shows the characteristic signals of the CH2CH2O of EO unit at 3.65 ppm (labeled j in Figure II. 17), the quadruplet of the CH3CHC(=O)NH at 5.10 ppm (labeled e in Figure II. 17) and the triplet at 3.12 ppm characteristic to the SCH2(CH2)10CH3 (labeled o in Figure II. 17). The integration values ratio was calculated by comparing the intensity areas of methylene protons CH2CH2O of the repeating unit (labeled j in Figure II. 17) and of the methylene proton CH2CH2OC(=O) (labeled k in Figure II. 17). The integration values ratio is equal to 254/0.26 corresponding to a degree of functionalization of 13 %.

Figure II. 17

 17 Figure II. 17. 1 H NMR spectrum (400 MHz, CDCl3) of the α-alkynyl,ω-

Figure II. 18 .

 18 Figure II. 18. Overlaid SEC traces of purified α-alkynyl,ω-dodecyltrithiocarbonate PEO using

  shows a signal at higher elution time due to the CDP(Figure II. 19). After purification the SEC trace using UV-vis detector shows the complete disapearance of the CDP signal. The signal at lower elution time is narrow and unimodal (M n,SEC ̅̅̅̅̅̅̅̅ = 8200 g.mol -1 (PS equivalent) and Ð = 1.13).

Figure II. 19 .

 19 Figure II. 19. Overlaid of SEC traces using a UV-vis detection fixed at 309 nm of α-alkynyl,ω-

Figure II. 20

 20 Figure II. 20. 1 H NMR spectrum (400 MHz, CDCl3) of the α-alkynyl,ωdodecyltrithiocarbonate PEO obtained by esterification of the α-alkynyl,ω-hydroxylPEO with

Figure II. 21 .

 21 Figure II. 21. 13 C NMR spectrum (100 MHz, CDCl3) of the α-alkynyl,ωdodecyltrithiocarbonate PEO obtained by esterification of the α-alkynyl,ω-hydroxylPEO with

Figure II. 22 .

 22 Figure II. 22. MALDI-TOF mass spectrometry of the α-alkynyl,ω-dodecyltrithiocarbonate PEO obtained by esterification of the α-alkynyl,ω-hydroxylPEO with CDP mediated through

Chapter 2 :

 2 Synthesis and Characterisization of PEO Macromolecular Chain Transfer Agent 78 ǂ Le Bohec, M.; Pascual, S.; Piogé, S.; Fontaine, L.: Heterofunctional RAFT-derived PNIPAM via cascade trithiocarbonate removal and thiol-yne click coupling reaction. Journal of Polymer Science Part A: Polymer Chemistry 2017, accepted manuscript

  In our work, two main architectures are studied: the lPEO and the bPEO. bPEO is synthesized through the polymerization of the PEGA for instance. In this work, the synthesis of PPEGA by RAFT polymerization has been studied using a new alkyne-based chain transfer agent: the 2-cyano-5oxo-5-(prop-2-yn-1-ylamino)pentan-2-yldodecyltrithiocarbonate (COPYDC). Such chain transfer agent will lead to bPEO containing an alkyne group at α-chain-end position and a trithiocarbonate at the -chain-end position.

  Scheme II. 10. Synthesis of COPYDC by amidification of CDP with propargyl amine in

Figure II. 23 .

 23 Figure II. 23. FT-IR spectrum of 2-cyano-5-oxo-5-(prop2-yn-1-ylamino)pentan-2-

Figure II. 24 .

 24 Figure II. 24. Superposition of 1 H NMR spectra (400 MHz, CDCl3) of COPYDC (top) and

Chapter 2 :

 2 Synthesis and Characterisization of PEO Macromolecular Chain Transfer Agent the trithiocarbonate entity is confirmed by the signal of the thiocarbonyl S(C=S)S at 217 ppm (labeled g, Figure II. 25).

Figure II. 25 .

 25 Figure II. 25. 13 C NMR spectrum (100 MHz, CDCl3) of COPYDC.

  ̅̅̅̅̅̅̅̅ and Ð values by SEC in DMF (LiBr 1 g.L -1 ). The influence of the [COPYDC]0/[ACVA]0 and of the [PEGA]0/[COPYDC]0 initial molar ratios were studied on polymerization kinetics and on the evolution of M n ̅̅̅̅ and Đ with conversion. 1. Influence of the [COPYDC]0/[ACVA]0 initial molar ratio RAFT polymerizations of PEGA were performed using a constant [PEGA]0/[COPYDC]0 ratio ([PEGA]0/[COPYDC]0 = 150) and changing the [COPYDC]0/[ACVA]0 ratio from 1/0.5 to 1/0.1. The Figure II. 26 shows the evolution of ln([M]0/[M]t) versus time for both[COPYDC]0/[ACVA]0 ratios. In both cases, a short inhibition period (30 minutes) is observed.

  At PEGA conversions above 70 % (for [COPYDC]0/[ACVA]0 = 150/1/0.5) and 75% (for [COPYDC]0/[ACVA]0 = 150/1/0.1), a decrease of the apparent propagation rate constant is observed due to a decrease of the propagating radicals concentration due to the presence of irreversible termination reactions.

Figure II. 26 .

 26 Figure II. 26. Evolution of ln([M]0/[M]t) and conversion versus time of RAFT polymerization

  1 H NMR spectroscopy (400 MHz, CDCl3) by comparing the integration values of the formamide proton of DMF at 8.02 ppm and of the alkene protons of PEGA between 5.7 and 6.5 ppm. b Determined by SEC in DMF (LiBr 1 g.L -1 ) using PS equivalents. c M n,NMR ̅̅̅̅̅̅̅̅̅ = [PEGA] 0 [ACVA] 0 ×conv×M n,PEGA ̅̅̅̅̅̅̅̅̅̅ +M COPYDC where conv is the conversion, M n,PEGA ̅̅̅̅̅̅̅̅̅̅ is the number-average molar mass of PEGA (M n,PEGA ̅̅̅̅̅̅̅̅̅̅ = 480 g.mol -1 ) and MCOPYDC is the molar mass of COPYDC (MCOPYDC = 440 g.mol -1 ). To get a better control, the influence [PEGA]0/[COPYDC]0 initial molar ratio on the RAFT polymerization will be studied. The chosen [COPYDC]0/[ACVA]0 initial molar ratio is equal to 1/0.1. The literature shows the increase of the control of RAFT polymerization by decreasing the chain transfer agent initiator initial molar ratio. 24,37 2. Influence of the [PEGA]0/[COPYDC]0 initial molar ratio RAFT polymerizations of PEGA were performed in DMF at 70°C using a constant initial concentration of PEGA, a constant [COPYDC]0/[ACVA]0 initial molar ratio of 1/0.1 and changing the [PEGA]0/[COPYDC]0 ratios (150/1, 50/1, and 30/1). The Figure II. 27 shows the comparison of the evolution of ln([M]0/[M]t) versus time of three RAFT polymerizations of PEGA. The inhibition period increases with the decrease of the [PEGA]0/[COPYDC]0 initial molar ratio: from 30 minutes for 150/1 to 90 minutes for 30/1.

  As shown by the decrease of the slope of ln([M]0/[M]t) versus time at high conversions, a loss of control is observed for a PEGA conversion of 70 % for [PEGA]0/[COPYDC]0 = 150/1, of 80 % for [PEGA]0/[COPYDC]0 = 50/1 and of 85 % for [PEGA]0/[COPYDC]0 = 30/1.

Figure II. 27 .

 27 Figure II. 27. Evolution of ln([M]0/[M]t) versus time during the RAFT polymerizations of

Figure II. 28 .

 28 Figure II. 28. Evolution of M n,SEC ̅̅̅̅̅̅̅̅ , M n,NMR ̅̅̅̅̅̅̅̅̅ (lines) and Ð versus conversion of RAFT

(

  [PEGA]0/[COPYDC]0/[AVCA]0 = 30/1/0.1 in DMF at 70°C). The reaction was launched and several aliquots were taken to follow the reaction by SEC in DMF (LiBr 1g.L -1 , Figure II. 29).The SEC traces show the decrease of the intensity of the PEGA signal at higher elution time corresponding to the remaining PEGA concentration and the shift of the PPEGA signal to the higher elution time due to the increase of the number-average molar mass with time. The final PEGA conversion, determined by 1 H NMR spectroscopy was equal to 52 %. The polymer was purified by several dialysis in pure water and lyophilized. The pure product was analyzed by SEC in DMF (LiBr 1 g.L -1 ) and 1 H NMR spectroscopy.

Figure II. 29 .

 29 Figure II. 29. Overlaid of SEC traces using RI detection of the crude mixture of the RAFT

Figure II. 30 .

 30 Figure II. 30. Overlaid SEC traces of purified α-alkynyl,ω-dodecyltrithiocarbonate PPEGA

Figure II. 31 . 1 H

 311 Figure II. 31. 1 H NMR spectrum (400 MHz, CDCl3) of PPEGA synthesized by RAFT

  Several strategies have been studied to functionalize the ω-position of the lPEO with a trithiocarbonate. The esterification of α-pyridyldithio,ω-hydroxylPEO with CDP has been mediated through acyl chloride based CDP, DMT-MM and DCC. The esterification using the acyl chloride based CDP and DMT-MM lead to low degree of functionalization (42 and 69 %, respectively) and polymer-polymer coupling. The esterification of α-pyridyldithio,ω-hydroxylPEO with CDP mediated through DCC/DMAP led to a low degree of functionalization (53 %) under mild conditions but doesn't lead to polymer-polymer coupling. The esterification of α-alkynyl,ω-hydroxylPEO with CDP has been mediated through acyl chloride based CDP and DCC. The addition of the trithiocarbonate group using the acyl chloride based CDP led to low conversion (13 %) meanwhile the esterification of α-alkynyl,ω-hydroxylPEO with CDP using DCC led to the synthesis of a well-defined α-alkynyl,ω-dodecyltrithiocarbonate PEO fully functionalized as shown by FT-IR, 1 H and 13 C NMRs spectroscopies, SEC and MALDI-TOF mass spectrometry. The bPEO, PPEGA, has been synthesized by RAFT polymerization using an alkynebased chain transfer agent the COPYDC. The influence of the [COPYDC]0/[ACVA]0 and [PEGA]0/[COPYDC]0 initial molar ratios have been studied. The best conditions was [PEGA]0/[COPYDC]0/[ACVA]0μ 30/1/0.1 to form an α-alkynyl,ω-dodecyltrithiocarbonate PPEGA (M n,SEC ̅̅̅̅̅̅̅̅ = 14700 g.mol -1 , Ð = 1.26).

- 1 )

 1 : 2875 (C-H), 1662 (C-O amide), 1569 (C=C aromatic and C-N,amide), 1100 (C-O ether).

13 C

 13 NMR (100 MHz, CDCl3), (ppm)μ 17 (CH≡CCH2), 23 (CH2CH2C(=O)O), 25 ((CH3)2CC(=O)NH), 33 (CH2CH2C(=O)O), 40 (CH3CHC(=O)NH), 57 ((CH3)2CC(=O)NH), 62 (NHCH2CH2O(CH2CH2O)63), 69 (CH≡CCH2), 70 (NHCH2CH2(OCH2CH2)63), 72 (CH3CHC(=O)NH), 83 (CH≡CCH2), 170 ((CH3)2CC(=O)NH), 172 (CH2CH2C(=O)O, 175 (CH3CHC(=O)NH) SEC (DMF): M n,SEC ̅̅̅̅̅̅̅̅ = 8900 g.mol -1 (PS equivalent), Đ = 1.13 Esterification mediated through acyl chloride A solution of CDP (8.3 mg, 0.02 mmol) in 0.25 mL of anhydrous CH2Cl2was degassed under argon for 15 minutes. Oxalyl chloride (13 mg, 0.1 mmol) was added to the solution. The mixture was heated to reflux at 50°C during 3 hours. CH2Cl2 and oxalyl chloride were removed under vacuum. Then after, a solution of αpyridyldithio,ω-hydroxylPEO (68 mg, 0.021 mmol) in 0.5 mL of anhydrous CH2Cl2 was added under argon. The solution was kept during 24 hours at room temperature. The solution was evaporated and the solid obtained was dissolved in a minimum of CH2Cl2 and was precipitated in cold diethyl ether. The solid obtained was dried under vacuum in the presence of P2O5. The mass obtained was 38 mg.

101 13 C 33 (

 1333 NMR (100 MHz, CDCl3), (ppm)μ 14 (SCH2(CH2)10CH3), 18 (HC≡CCH2), 23 (CH3CHC(=O)NH), 24 (CH2CH2C(=O)O), 25 ((CH3)2CC(=O)NH), 28 (SCH2CH2(CH2)9CH3), 29 (CH2CH2C(CH3)(CN)S, SCH2CH2(CH2)9CH3), 32 (CH2CH2C(CH3)(CN)), =O)C(CH3)2), 46 (CH2CH2C(CH3)(CN)), 57 (C(CH3)2NH), 64 (CH2OC(=O)CH2), 69 (HC≡CCH2), 70 (CH3CHC(=O)NH), 71 ((CH2CH2O)64), 83 (HC≡CCH2), 120 (CH2CH2C(CH3)(CN)), 170 (C(=O)NHC(CH3)2), 172 (C(=O)OCH(CH3) and C(=O)CH2CH2C(CN)(CH3)), 175 (C(=O)NH(CH2CH2O)64), 217 (SC(=S)S), SEC in DMF: M n,SEC ̅̅̅̅̅̅̅̅ = 8200 g.mol -1 (PS equivalent), Đ = 1.13 F. Synthesis of an heterofuntional brush-like PEO (7479 g, 4.88 mmol) and EDCI (0.7740 g, 4.99 mmol) were charged with a stir bar in a 100 mL round bottom flask. Under argon at 0°C were added DMF (2.8 mL) and anhydrous CH2Cl2 (5 mL). In another round bottom flask, CDP (2.0004 g, 4.96 mmol) was weighted and 15 mL of anhydrous CH2Cl2 were added. This second solution was degassed under argon during 10 minutes and was transferred in the first round bottom flask with a doubletipped needle under argon. Propargyl amine (0.2764 g, 5.02 mmol) was added with a degassed syringe under argon. The final mixture was degassed under argon during 10 minutes. The ice bath was removed and the reaction was launched for 24 hours at room temperature. The reaction was stopped by opening it to air. The solution was dissolved in 200 mL of CH2Cl2 and the organic phase was washed with 600 mL of a saturated solution of Na2CO3 and 600 mL of 1M

13 C

 13 NMR (400 MHz, CDCl3), (ppm)μ 15 (SCH2(CH2)10CH3), 23 (S(CH2)10CH2CH3), 25 (CH2CH2C(CH3)(CN)S), 27 (CH2CH2C(CH3)(CN)), 29 (HC≡CCH2NH, SCH2CH2(CH2)9CH3), 32 (SCH2(CH2)8CH2CH2CH3), 34 (CH2C(CH3(CN)), 37 (SCH2CH2), 47 (C(CN)(CH3)S), 72 (HC≡CCH2), 80 (HC≡CCH2), 119 (N≡C), 170 (C=O), 217 (SC(=S)S) FT-IR ( in cm -1 ): 3292 (N-H), 2919 (C-H, CH2). 2232 (C≡N), 1651 (C=O, amide), 1532 (N-H), 1453 (C-H, CH2), 1375 (C-H, CH3), 1275-1230 (C-N), 1064 (C=S). HRMS: [M+Na] + exp = 463.1869 Da, [M+Na] + theo = 463.1888 Da.

Chapter 3 :

 3 Synthesis and Characterization of (PEGylated) aminoethyl-based polyacrylates 109 ǂ Ho, H.T.; Bohec, M. L.; Frémaux, J.; Piogé, S.; Casse, N. ; Fontaine L.; Pascual, S. Tuning the molar composition of «charge-shifting» cationic copolymers based on 2-(N,Ndimethylamino) ethyl acrylate. Macromolecular Rapid Communications 2017, 38 (5), 1600641-600646

Chapter 3 :Scheme III. 1 .Table III. 2 .Chapter 3 :

 3123 Scheme III. 1. RAFT copolymerization of tBocAEA and DMAEA mediated through ACVA used as the initiator and α-methoxy,ω-dodecyltrithiocarbonate PEO (mPEO-CTA) used as the

Figure III. 4 .

 4 Figure III. 4. Kelen-Tüdos plot for the RAFT copolymerizations of DMAEA and tBocAEA at

Figure III. 5 . 3 ) 3 :

 533 Figure III. 5. Overlaid of 1 H NMR spectra (400 MHz, CDCl3) between 4.6 and 8.5 ppm of the

Figure III. 6 .

 6 Figure III. 6. FT-IR spectrum of aPEO-b-P(DMAEA-co-tBocAEA) synthesized by RAFT

Figure III. 7 . 1 H

 71 Figure III. 7. 1 H NMR spectrum (400 MHz, CDCl3) of purified aPEO-b-P(DMAEA-co-

Figure III. 8 .

 8 Figure III. 8. Overlaid SEC traces using RI detection (top) and UV-vis detection (fixed at 309

C

  Scheme III. 3. RAFT copolymerization of tBocAEA and DMAEA mediated through ACVA used as the initiator and α-alkynyl,ω-dodecyltrithiocarbonate PPEGA (PPEGA-CTA) used as

Figure III. 9 .

 9 Figure III. 9. Overlaid 1 H NMR spectra (400 MHz, CDCl3) between 5.4 and 8.6 ppm of the

The 1 H

 1 NMR spectrum (Figure III. 10) shows the characteristic signals of the methylene protons CH2CH2O (PEGA units) at 3.65 ppm (labeled g in Figure III. 10), of the methyl protons of the OCH2CH2N(CH3)2 (DMAEA units) at 2.27 ppm (labeled k in Figure III. 10) and of the methyl protons NHC(=O)OC(CH3)3 (tBocAEA units) at 1.44 ppm (labeled n in Figure III. 10). The DP n ̅̅̅̅̅ of PtBocAEA has been calculated by comparing the integration area values of the of the methylene protons CH2CH2O of PEGA repeating unit at 3.65 ppm (432.00 protons, labeled g in Figure III. 10) and of the methyl protons (CH2CH2O)9CH3 of PEGA units and methylene protons of OCH2CH2NH of tBocAEA units and of SCH2(CH2)10CH3 at 3.38 ppm (59.19 protons, labeled h, m and o in Figure III. 10). The DP n ̅̅̅̅̅ of PtBocAEA is equal to 10. The DP n ̅̅̅̅̅ of PDMAEA has been calculated by comparing the integration area values of the methylene Chapter 3: Synthesis and Characterization of (PEGylated) aminoethyl-based polyacrylates 128 protons CH2CH2O of EO repeating unit at 3.65 ppm (432.00 protons, labeled g in Figure III. 10) and of the methylene protons of the HC≡CCH2NH of the alkyne moiety, of the OCH2CH2O(CH2CH2O)8 of PEGA units, of the OCH2CH2N(CH3)2 of DMAEA units, of the OCH2CH2NHC(=O) of tBocAEA units at 4.16 ppm (65.25 protons, labeled b, f, i and l in Figure III. 10) The DP n ̅̅̅̅̅ of PDMAEA is equal to 9.

Figure III. 10 . 1 H

 101 Figure III. 10. 1 H NMR spectrum (400 MHz, CDCl3) of PPEGA-b-P(DMAEA-co-tBocAEA)

- 1 )

 1 (Figure III. 11). The overlaid SEC traces of PPEGA-CTA and PPEGA-b-P(DMAEA-co-tBocAEA) copolymer using RI detection shows an insignificant shift of the SEC trace of PPEGA-CTA from higher retention times to lower Chapter 3: Synthesis and Characterization of (PEGylated) aminoethyl-based polyacrylates 129 retention times. This result could be explained by a relative broad molar masses distribution of PPEGA-CTA (Ð = 1.26). The SEC trace of the purified copolymer allowed to determine M n,SEC ̅̅̅̅̅̅̅̅ = 11200 g.mol -1 (equivalent PS) and Ð = 1.57. The SEC traces using the UV-vis detection at 309 nm, characteristic wavelength of the trithiocarbonate group, show the covalent linkage of the trithiocarbonate moiety.

Figure III. 11 .

 11 Figure III. 11. Overlaid of SEC traces using RI detection (top) and UV-vis detection (fixed at

Chapter 3 :Scheme III. 4 .

 34 Scheme III. 4. RAFT copolymerization of DMAEA and tBocAEA mediated through COPYDC

Figure III. 12 .

 12 Figure III. 12. Overlaid of 1 H NMR spectra (400 MHz, CDCl3) between 5.4 and 8.6 ppm of

Figure III. 13 . 1 H

 131 Figure III. 13. 1 H NMR spectrum (400 MHz, CDCl3) of purified P(DMAEA-co-tBocAEA)

Chapter 3 : 28 Figure III. 14 .

 32814 Figure III. 14. Overlaid SEC traces of purified P(DMAEA-co-tBocAEA) (solid line: RI

  Same procedure is used to deprotect the amine of tBocAEA repeating units of aPEO-b-P(DMAEA-co-tBocAEA) copolymers, several PPEGA-b-P(DMAEA-co-tBocAEA) copolymers and several P(DMAEA-co-tBocAEA) copolymers in order to target corresponding cationic aPEO-b-P(DMAEA-co-AEA) copolymers, PPEGA-b-P(DMAEA-co-AEA) copolymers and P(DMAEA-co-AEA) copolymers, respectively. Reactions are performed in CH2Cl2 at a concentration of copolymer equal to 50 g.L -1 with 200 equivalents of TFA at room temperature during 5 hours (Scheme III.5). Copolymers were purified by several decantation and precipitation in cold diethyl ether/hexane mixture (50/50, v/v) and dried under vacuum. The purified copolymers were analyzed by FT-IR and 1 H NMR spectroscopies. Fully characterizations of aPEO-b-P(DMAEA-co-AEA), PPEGA-b-P(DMAEA-co-AEA) and P(DMAEA-co-AEA) are reported on following paragraphs A, B and C, respectively.

Chapter 3 :Scheme III. 5 .

 35 Scheme III. 5. Amine deprotection of tBocAEA repeating units mediated through TFA in

Figure III. 15 .

 15 Figure III. 15. Overlaid of FT-IR spectra of aPEO64-b-P(DMAEA11-co-tBocAEA4) (top) and

Figure III. 16 .

 16 Figure III. 16. Overlaid of 1 H NMR spectra (400 MHz, DMSO d6) of aPEO64-b-P(DMAEA11-

1 H

 1 NMR spectra for aPEO64-b-P(DMAEA11-co-tBocAEA4) and for an aPEO-b-P(DMAEA-co-AEA) have been both done in CDCl3 and D2O (Figure III.17). The 1 H NMR spectra show the same result than with deuterated DMSO. The following 1 H NMR studies have been made by dissolving the copolymer before amine deprotection in CDCl3 and after deprotection in D2O.

Figure III. 17 .Chapter 3 :

 173 Figure III. 17. Overlaid of 1 H NMR spectra (400 MHz) of aPEO64-b-P(DMAEA11-co-

Figure III. 18 .

 18 Figure III. 18. Overlaid 1 H NMR spectra (400 MHz) of PPEGA-b-P(DMAEA7-co-

Figure III. 19 .

 19 Figure III. 19. Overlaid 1 H NMR spectra (400 MHz) of P(DMAEA9-co-tBocAEA13) (top,

Chapter 3 :

 3 Synthesis and Characterization of (PEGylated) aminoethyl-based polyacrylates 145 AEA) copolymers. The efficiency of the deprotection and cationization reaction has been highlighted by FT-IR and 1 H NMR spectroscopies.

  .0 mL) and DMF (0.10 mL) used as the internal reference. The mixture was deoxygenated by bubbling argon for 30 min. The solution was then immersed in an oil bath thermostatted at 70°C to allow the polymerization to occur. The reaction was stopped after 12 h by opening the reaction mixture to oxygen. Total conversion of DMAEA and tBocAEA were determined to be 24% by1 H NMR spectroscopy by comparing the integration areas of vinylic proton of DMAEA and tBocAEA at 5.81-6.46 ppm and with the integral area value of the CH of DMF at 8.02 ppm. 1,4-Dioxane and DMF were removed under vacuum. The resulting product was then dissolve in acetone and precipitated several times in cold hexane/diethyl ether (90/10 v/v), filtered and dried under vacuum. The final product obtained as a yellow solid was characterized by 1 H NMR spectroscopy, FT-IR spectroscopy and SEC analysis. DP ̅̅̅̅ n,PDMAEA (= 18) and DP ̅̅̅̅ n,PtBocAEA (= 9) were determined by comparing the integration area values of the signal of CH2CH2O units at 3.65 ppm, of the signal of CH3O(CH2CH2O), of OCH2CH2NHC(=O)O, of SCH2(CH2)10CH3 at 3.38-3.40 ppm, of the signal of CH2CH2OC(=O), of OCH2CH2NH of tBocAEA and of OCH2CH2N(CH3)2 of the DMAEA at 4.15 ppm on 1 H NMR spectrum. M n,NMR ̅̅̅̅̅̅̅̅̅ = 6910 g.mol -1 .

  H NMR spectroscopy by comparing the integration areas of vinylic proton of DMAEA and tBocAEA at 5.81-6.46 ppm and with the integral area value of the CH of DMF at 8.02 ppm. 1,4-Dioxane and DMF were removed under vacuum. The resulting product was then dissolve in CH2Cl2 and precipitated in cold hexane/diethyl ether (90/10 v/v), filtered and dried under vacuum. The final product obtained as a yellow solid was characterized by 1 H NMR and FT-IR spectroscopies and SEC analysis. DP ̅̅̅̅ n,PDMAEA (= 10) and DP ̅̅̅̅ n,PtBocAEA (= 12) were determined by comparing the integration area values of the signal of CH3CHC(=O)NH at 5.10 ppm, of the signal of OCH2CH2NH and of SCH2(CH2)10CH3 at 3.35 ppm and of the signal of CH2CH2OC(=O) , of OCH2CH2N(CH3)2 and of OCH2CH2NH at 4.16 ppm on 1 H NMR spectrum. M n,NMR ̅̅̅̅̅̅̅̅̅ = 7500 g.mol -1 SEC in DMF (LiBr 1 g.L -1 ): M n,SEC ̅̅̅̅̅̅̅̅ = 11700 g.mol -1 ; Đ = 1.31FT-IR (υ, cm -1 ): 3367 (N-H carbamate), 2883 (C-H), 1731 (C=O ester), 1518 (C=O carbamate), 1454 (C-H), 1108 (C-O). 1 H NMR (400 MHz, CDCl3), (ppm)μ 0.88 ppm (t, SCH2(CH2)10CH3, 3.07 H), 1.21 (m, SCH2CH2(CH2)9CH3, 19.96 H), 1.35-2.41 (m, HC≡CCH2CH2CH2-, CH3CHO(C=O)NH, NHC(CH3)2C(=O)NH, CH2CH2C(CN)(CH3), (CH2CH)n, OCH2CH2N(CH3)2, C(=O)OC(CH3)3, SCH2CH2, 293.06 H), 2.56 (m, OCH2CH2N(CH3)2, 32.72 H), 3.35 (m, OCH2CH2NHC, SCH2(CH2)10CH3, 26.79 H), 3.65 (m, CH2CH2O, 292.29 H), 4.16 (m, CH2CH2OC(=O), OCH2CH2N(CH3)2, OCH2CH2NH, 48.50 H), 5.10 (q, CH3CHC(=O)NH, 1.00 H).

1 H

 1 NMR (D2O, 400 εHz), (ppm)μ 0.60-2.81 (m, HC≡CCH2NH, CH2CH2C(CN)(CH3), (CH2CH)n, SCH2(CH2)10CH3), 2.88 (m, OCH2CH2N + H(CH3)2), 3.32 (m, (CH2CH2O)8CH3, OCH2CH2N + H3, SCH2(CH2)10CH3), 3.43 (m, OCH2CH2N + H(CH3)2),

Chapter 4 :

 4 Chain-ends modification of (PEGylated) aminoethyl-based polyacrylates. 158 ǂ Le Bohec M., Pascual S., Piogé S., Fontaine L., Heterofunctional RAFT-derived PNIPAM via cascade trithiocarbonate removal and thiol-yne click coupling reaction. Journal of polymer science Part A: Polymer chemistry 2017

  and AIBN as initiator in DMF solvent at 70°C with [NIPAM]0/[COPYDC]0/[AIBN]0 = 100/1/0.1 (Scheme IV. 1).

Figure IV. 1 .

 1 Figure IV. 1. Overlaid SEC traces of polymers obtained from the RAFT polymerization of

Figure IV. 2 .

 2 Figure IV. 2. Ln([M]0/[M]t vs. time plot for the RAFT polymerization of NIPAM using

Figure IV. 3 .

 3 Figure IV. 3. M n,SEC ̅̅̅̅̅̅̅̅ (filled marks, solid line: experimental values and dashed line: theoretical

Figure IV. 4 .

 4 Figure IV. 4. 1 H NMR (400 MHz) spectrum in CDCl3 of purified PNIPAM obtained from the

Figure IV. 5 .

 5 Figure IV. 5. Overlaid SEC traces of purified PNIPAM (solid line: RI response and dashed

Figure IV. 6 .

 6 Figure IV. 6. MALDI-TOF mass spectrum of a PNIPAM synthesized by RAFT polymerization

Scheme IV. 2 .Figure IV. 7 .

 27 Scheme IV. 2. Removal of trithiocarbonate chain-end of α-alkynyl,ω-dodecyltrithiocarbonate

  detector and UV-vis detector at 309 nm, respectively. Efficient trithiocarbonate removal was also supported by discoloration of the polymer at the end of the reaction (Figure IV. 8).

Figure IV. 8 .

 8 Figure IV. 8. Picture of α-alkynyl,ω-dodecyltrithiocarbonate PNIPAM before (left) and after

Figure IV. 9 . 1 H 22 .B.

 9122 Figure IV. 9. 1 H NMR spectra (400 MHz, CDCl3) of α-alkynyl,ω-dodecyltrithiocarbonate

  overlaid 1 H NMR spectra of an aPEO64-b-P(DMAEA11-co-tBocAEA4) before and after trithiocarbonate removal shows the intensity decrease of the triplet at 0.88 ppm characteristic of the methyl protons SCH2(CH2)10CH3 of the dodecyl trithiocarbonate moiety (labelled v in Figure IV. 11). Moreover, the integration areas values of the methylene protons signal CH2CH2OC(=O), OCH2CH2N(CH3)2 and OCH2CH2NH at 4.09 ppm (labelled i, m and p in Figure IV. 11) remain constant before and after the trithiocarbonate removal. The results show the trithiocarbonate removal together with the non-degradation of acrylate groups during reaction.

Figure IV. 11 . 1 H

 111 Figure IV. 11. 1 H NMR spectra (400 MHz, CDCl3) of aPEO64-b-P(DMAEA11-co-tBocAEA4)

Figure IV. 12 .Chapter 4 :Table IV. 4 .a

 1244 Figure IV. 12. Overlaid SEC traces of aPEO64-b-P(DMAEA11-co-tBocAEA4) before (in grey)

(pH = 7 . 5 )Scheme IV. 4 .

 754 Scheme IV. 4. Thiol-disulfide coupling exchange reaction of α-pyridyldithio,ω-hydroxylPEO

Figure IV. 13 .

 13 Figure IV. 13. UV-vis spectra of the crude mixture at tf = 18 h of the disulfide coupling

4 ) 33 Figure IV. 14 . 1 H

 433141 Figure IV. 14). The thiol-yne reaction efficiency was determined by 1 H NMR spectroscopy

I 7 . 5 ×

 75 (DP n +2) 10×I 3.90 -3×I 7. 5 ×100 where I7.25 and I3.90 are intensities of signals at 7.25 and 3.90 ppm, respectively.

. 6 )

 6 . The 1 H NMR spectrum (Figure IV. 15) of the purified PNIPAM shows a signal at 7.25 ppm corresponding to aromatic protons of the benzyl entity (labelled g in Figure IV. 15) and a signal at 3.54 ppm corresponding to methylene protons SCH2C6H5 (labelled f in Figure IV. 15). The thiol-yne efficiency was determined by 1 H NMR spectroscopy using the signal at 3.90 ppm relative to methine protons of the PNIPAM units Chapter 4: Chain-ends modification of (PEGylated) aminoethyl-based polyacrylates. 184 (labelled b in Figure IV. 15), methylene and methine protons of the resulting -chain-end (labelled c, d and e in Figure IV. 15), and the signal at 7.25 ppm relative to aromatic protons (labelled g in Figure IV. 15). The degree of functionalization calculated was equal to 40 % (Entry 2 in Table

Figure IV. 15 . 1 H

 151 Figure IV. 15. 1 H NMR spectrum (400 MHz, CD2Cl2) of PNIPAM resulting from the one pot thrithiocarbonate removal/thiol-yne coupling reaction of the α-alkynyl,ω-

(

  [NIPAM]0/[COPYDC]0/[AIBN]0 = 100/1/0.1). A conversion of 40 %, determined by 1 H NMR spectroscopy, was targeted to obtain a well-defined α-alkynyl,ω-dodecyltrithiocarbonate PNIPAM (M n,SEC ̅̅̅̅̅̅̅̅ = 11600 g.mol -1 , Ð = 1.17 and DP ̅̅̅̅ n = 40). The structure was confirmed by MALDI-TOF mass spectrometry. Then the trithiocarbonate has been removed using 20

I 4 .

 4 01 = DP n +2 (1-x) + 5 x × y Equation (IV. 9) where I4.01 is the intensity of the signal at 4.01 ppm, DPn is the number-average degree of polymerization of NIPAM, 2(1-x) is the number of methylene protons in α position of the alkyne bond according to the conversion and 5x is the number of protons for a 100 % conversion at 4.01 ppm. x = I 7.52 ×(DP n +2) (4×I 4.01 -3×I 7.52 ) Equation (IV. 10) The product was characterized by 1 H NMR spectroscopy in CDCl3 and by SEC in DMF (LiBr 1 g.L -1 ). 1 H NMR (400 MHz, CDCl3) (ppm) = 0.90-2.50 (m, CH2CH2C(CH3)(CN), CH2CH, HC≡CCH2, NHCH(CH3)2, CHC(CH3)2(CN)), 3.64 (d, SCH2C6H5, 1.5 H); 4.01 (m, SCH2CH(S)CH2NH, HC≡CCH2NH and NHCH(CH3)2, 42.0 H), 6.33 (m, NHCH(CH3)2, NHCH2CH), 7.30 (m, (CHCH=)2CCH2S), 7.52 (m, (CH=)2CCHS, 1.5 H), 7.90 (m, CH(CHCH=)2CCH2S, 0.6 H).

I 3 .

 3 90 = DP n + 2 (1-x) + 5 x × y Equation (IV. 12) where I3.90 is the intensity of the signal at 3.90 ppm, DPn is the number-average degree of polymerization of NIPAM, 2(1-x) is the number of methylene protons in α-position of the alkyne bond according to the conversion and 5x is the number of protons at 3.90 ppm for a 100 % conversion. x = I 7.25 ×(DP n +2) (10×I 3.90 -3×I 7.25 ) Equation (IV .13) 1 H NMR (400 MHz, CD2Cl2) (ppm) = 0.90-2.50 (m, CH2CH2C(CH3)(CN), CH2CH, HC≡CCH2, NHCH(CH3)2, CHC(CH3)2(CN)), 3.54 (d, SCH2C6H5); 3.90 (m, SCH2CH(S)CH2NH, HC≡CCH2NH, NHCH(CH3)2, 48 H), 6.28 (NHCH(CH3)2, NHCH2CH), 7.25 (C6H5, 4.06 H).

Chapter 5 :

 5 Cationic (PEGylated) aminoethyl-based polyacrylates: pDNA complexation and cytotoxicity 199In this chapter, the influence of (i) the PEGylation, (ii) the DMAEA/AEA molar ratio and (iii) the chain-end nature within DMAEA and AEA based copolymers were studied on polyplex formation and cytotoxicity. A previous study of polyplex size and pDNA complexation has been previously done on a PEO-b-P(DMAEA-co-AEA). Gel retardation assays allow to evaluate the DNA/polymer binding affinity and to target optimal N/P ratios. In vitro cytotoxicity of cationic polymers has been evaluated using the 3-[4,5-dimethylthiazol-2-yl]-3,5-diphenyltetrazolium bromide (MTT) assay on three different cell lines (i) NIH-3T3, the most commonly used fibroblast cell lines, with a rapid response to external stimuli, (ii) endothelial umbilical vein cells (HUVEC) responds fast to external stimuli and is sensitive and (iii) murine macrophage-monocyte cells (J774.A1) which may highlight the cytotoxicity during the endocytosis process because of its key role in phagocytosis.

Figure V. 1 .

 1 Figure V. 1. Agarose gel electropherogram of a pDNA/aPEO64-b-P(DMAEA10-co-AEA12)

Chapter 5 :

 5 Cationic (PEGylated) aminoethyl-based polyacrylates: pDNA complexation and cytotoxicity 201 The aminoethyl acrylate-based cationic copolymers have been designed to study the influence of the PEGylation, DMAEA/AEA molar ratio and the chain-end nature. II. Influence of the PEGylation of cationic DMAEA/AEA-based copolymers on pDNA binding and cell viability.

Figure V. 2

 2 Figure V. 2 shows electropherograms of a based on pDNA/P(DMAEA9-co-AEA13)

Chapter 5 :Figure V. 2 .

 52 Figure V. 2. Agarose gel electropherogams of pDNA/P(DMAEA9-co-AEA13) polyplexes (top)

Chapter 5 :Figure V. 3 .

 53 Figure V. 3. Agarose gel electropherograms of pDNA/aPEO64-b-P(DMAEA10-co-AEA12)

1 Chapter 5 :

 15 influence of PEGylation on cell viability has been tested by comparing (i) a aPEO64b-P(DMAEA11-co-AEA9) copolymer and one aPEO64-b-P(DMAEA5-co-AEA10) copolymer (Runs 1 and 5, respectively TableIII. 3, Chapter3) with a P(DMAEA9-co-AEA13) copolymer (Run 3, TableIII. 5, Chapter 3) and (ii) a P(DMAEA9-co-AEA13) copolymer with a PPEGA12b-P(DMAEA7-co-AEA14) (Run 2, TableIII. 4, Chapter 3) copolymer on different cell lines, NIH-3T3, HUVEC and J774.A1 (Figure V. 4). The MTT assays on NIH-3T3 cell lines (Figure V. 4(A)), contrary to HUVEC (Figure V. 4(B)) and J774.A1 cell lines (Figure V. 4 (C)), shows low or no cytotoxicity at 0.5 and 1 g.L -Cationic (PEGylated) aminoethyl-based polyacrylates: pDNA complexation and cytotoxicity 204 (over 80 % of cell viability) for all copolymers. Moreover, at 2 g.L -1 only the P(DMAEA9-co-AEA13) shows cell viability of NIH-3T3 below 80 % At a higher concentration (2 g.L -1 ), the non-PEGylated P(DMAEA9-co-AEA13) polymer induced a decrease of viability in all cell lines, revealing a greater sensibility of J774.A1 (22 % of cell viability, standard deviation of 2 %) compared to HUVEC (66 % of cell viability, standard deviation of 2 %) and NIH-3T3 cells (76 % of cell viability, standard deviation of 4 %). The influence of PEO architecture has been studied on HUVEC cell line (Figure V. 4 (A)). The MTT assay using the aPEO64-b-P(DMAEA5-co-AEA10) shows a cell viability around 80 % for all tested concentrations, meanwhile the MTT assay of the PPEGA12-b-P(DMAEA7co-AEA14) shows a decrease of cell viability from 1 g.L -1 to 2 g.L -1 (from 72 % to 58 %).

Chapter 5 :Figure V. 4 .

 54 Figure V. 4. Cell viability (MTT assay) after incubation of (A) NIH-3T3 cells, (B) HUVEC

5

 5

Chapter 5 :Figure V. 5 .

 55 Figure V. 5. Agarose gel electropherograms of pDNA/aPEO64-b-P(DMAEA11-b-AEA4)

Chapter 5 :Figure V. 6 .

 56 Figure V. 6. Agarose gel electropherograms of pDNA/PPEGA12-b-P(DMAEA7-co-AEA14)

. 3 ,Figure V. 7 .

 37 Figure V. 7. Cell viability (MTT assay) after incubation of (A) NIH-3T3 cells (B), HUVEC

Chapter 5 :

 5 photochemical-activation. The influence of the dodecyltrithiocarbonate removal has been

Figure V. 8 .

 8 Figure V. 8. Agarose gel electopherograms of pDNA/aPEO64-b-P(DMAEA10-co-AEA12)-CTA

Chapter 5 :Figure V. 9 .

 59 Figure V. 9. Cell viability (MTT assay) after incubation of (A) NIH-3T3 cells (B) and HUVEC

Chapter 5 :

 5 Cationic (PEGylated) aminoethyl-based polyacrylates: pDNA complexation and cytotoxicity 214 fully pDNA complexation with a N/P ratio of 3. The polyplex hydrodynamic diameter was equal to 38 nm with a size dispersity of 0.39, determined by DLS. The zetametry comparison

  trithiocarbonate function was introduced by esterification at the ω-position of the so-obtained lPEO. The resulting PEO-based macro chain transfer agent was used to mediate the RAFT polymerization of aminoethyl acrylates.Three different pathways were studied to anchor a chain-transfer agent on the lPEO: the esterification of lPEO with CDP was mediated through (i) an acyl chloride based on CDP, (ii) DMT-MM and (iii) DCC. The esterification of lPEO through the acyl choride based on CDP and DMT-εε led to low degree of functionalization. εoreover, the esterification of the αpyridyldithio,ω-hydroxylPEO using acyl chloride based on CDP and DMT-MM led to polymerpolymer disulfide coupling reaction. The esterification of lPEO with CDP with the αpyridyditio,ω-hydroxylPEO mediated through DCC led to a low functionalization (53 %) but no polymer-polymer reactions occurred. The same pathway was used with the α-alkynyl,ω-hydroxylPEO allowing to obtain a fully functionalized and well-defined α-alkynyl,ωdodecyltrithiocarbonate PEO (with a M n,SEC ̅̅̅̅̅̅̅̅ = 8200 g.mol -1 and a Ð = 1.13). The esterification mediated through DCC led to the best result with the α-pyridyditio,ω-hydroxylPEO and the αalkynyl,ω-hydroxylPEO. Furthermore, this reaction led to a well defined α-alkynyl,ωdodecyltrithiocarbonate PEO, this polymer was used to perform the RAFT polymerization of DMAEA and tBocAEA.In order to study the influence of the PEO architecture on pDNA complexation and cytotoxicity, bPEO was synthesized by RAFT polymerization of PEGA using a new alkynebased chain transfer agent (COPYDC). The optimal conditions were studied and was [PEGA]0/[COPYDC]0/[ACVA]0 = 30/1/0.1. An α-alkynyl,ω-dodecyltrithiocarbonate PPEGA with a DP ̅̅̅̅ n of 12 as calculated by 1 H NMR spectroscopy and a M n,SEC ̅̅̅̅̅̅̅̅ = 14700 g.mol -1 and a Ð = 1.26 was obtained for further study and used to copolymerize DMAEA and tBocAEA. α-alkynyl,ω-dodecyltrithiocarbonate PEO (aPEO-CTA) and α-alkynyl,ωdodecyltrithiocarbonate PPEGA (PPEGA-CTA) were then used for RAFT copolymerization of DMAEA and tBocAEA. Reactivity ratios determination led to rDMAEA = 0.81 and rtBocAEA = 0.99, showing characteristics of an "ideal" copolymerization system.εoreover, the DP ̅̅̅̅ n of DMAEA and tBocAEA units calculated by 1 H NMR spectroscopy shows that final DMAEA/AEA molar ratio in the copolymers are in agreement with the comonomer feeds. From these results the RAFT copolymerization of DMAEA and tBocAEA with different General Conclusion 222 DMAEA/AEA molar ratios has been performed mediated through aPEO-CTA, PPEGA-CTA and COPYDC used as (macromolecular) chain transfer agents to obtain several aPEO-b-(DMAEA-co-tBocAEA), PPEGA-b-P(DMAEA-co-tBocAEA) and P(DMAEA-co-tBocAEA) copolymers, respectively. Associated aPEO-b-P(DMAEA-co-AEA) cationic copolymers, PPEGA-b-P(DMAEA-co-AEA) cationic copolymers and P(DMAEA-co-AEA) cationic copolymers with different DMAEA/AEA ratios were obtained through the primary amine deprotection of tBocAEA units under acidic medium. The cationic (PEGylated) cation aminoethylacrylated were used to complex pDNA to form the polyplexes. To increase the transfection efficiency and decrease the cytotoxicity, the incorporation of a thiolated recognition ligand and the trithiocarbonate removal have been studied. To avoid too much synthesis steps needed to target copolymers based on EO, DMAEA, and (tBoc)AEA units; the trithiocarbonate removal reaction has been first modeled. The PNIPAM has been chosen as a model polymer. The RAFT polymerization of NIPAM has been mediated using COPYDC as chain transfer agent and AIBN as the initiator leading to a well-defined αalkynyl,ω-dodecyltrithiocarbonate PNIPAM with a DP ̅̅̅̅ n of PNIPAM equal to 40. The trithiocarbonate removal reaction has been realized by thermal-activation (80°C) using 20 equivalents of AIBN, after 24 h the trithiocarbonate group was fully removed. The trithiocarbonate removal reaction has been adjust for the PEO-b-P(DMAEA-co-tBocAEA) and P(DMAEA-co-tBocAEA) copolymers using a photochemical-activation and the trithiocarbonate was fully removed in 2 weeks without aminoethyl polyacrylates modifications. The modification of chain end to anchor thiolated recognition ligand has been studied on the lPEO and the PNIPAM without trithiocarbonate. First, the thiol-disulfide exchange coupling reaction has been performed using an α-pyridyldithio,ω-hydroxylPEO and N-acetylcysteine as the thiol model molecule and the conversion was equal to 56 % in 18 hours. Then, the thiol-yne coupling reaction has been performed on a free trithiocarbonate PNIPAM using AIBN as the radical initiator, benzyl mercaptan as the thiol model molecule and DMPP to reduce disulfide bonds during 24 hours. The highest functionalization degree has been obtained using a [PNIPAM]0/[AIBN]0/[Benzyl mercaptan]0 initial molar ratio of 1/15/15, 0.78 aromatic group for 1 alkyne group. As the trithiocarbonate removal reaction and the thiol-yne coupling reaction have been performed in the same conditions the two reactions have been performed in one-pot two steps process. This combination led to the complete removal of the trithiocarbonate and the addition of 0.80 thiol group for 1 alkyne group. This reaction allow sufficient functionalization for bio-applications. 1,2 Moreover, the thiol-yne coupling reaction showed a better degree of functionalization than the thiol-disulfide coupling reaction. The cationic aPEO-b-P(DMAEA-co-AEA) copolymers, PPEGA-b-P(DMAEA-co-AEA) copolymers and P(DMAEA-co-AEA) copolymers with and without trithiocarbonate group (CTA) have been characterized. First a PEO-b-P(DMAEA-co-AEA)-CTA and a DP ̅̅̅̅ n,PDMAEA = 10 and a DP ̅̅̅̅ n,PtBocAEA = 12 was used to complex DNA. Gel retardation assay showed an optimum ratio of N/P of 3. Then, zeta potentials of cationic PEO64-b-P(DMAEA10-co-AEA12) copolymer and the pDNA/PEO64-b-P(DMAEA10-co-AEA12) polyplex were compared. The zeta-potential showed a complete neutralization of cationic charge after complexation.

  Cutlaret al.34 compared the transfection efficiency of a hbPBAE and a lPBAE with comparable number-average molar masses (hPBAE and lPBAE4 Scheme I. 3(B)). They found that hbPBAE has a better transfection efficiency than lPBAE with HeLa cells and RDEBK cells, keratinocyte cell lines derivated from epidermolysis bullosa. The authors explained this result by the strong interactions of hbPBAE/DNA polyplexes with cells as shown by gluciferase activity in different cell lines. Moreover, the hbPBAE protects more DNA against DNAse, an enzyme able to degrade DNA, than lPBAE.

  The best conditions were obtained using ACVA and DMF. Moreover, ACVA and DMF are water-soluble and water-miscible respectively and they can be eliminated together easily by dialysis with the remaining PEGA. A study has been performed to find the

	best conditions to target well-defined PPEGA by RAFT polymerization and different
	[PPEGA]0/[COPYDC]0/[ACVA]0 initial molar ratios were studied for the RAFT
	polymerizations of PEGA at 70°C (Scheme II. 11).
	synthesized a PPEGA by RAFT polymerization using AIBN in
	acetonitrile. Song et al. 35 synthesized a PPEGA by RAFT polymerization using AIBN but in
	1,4-dioxane. Qiao et al. 36 recently synthesized a PPEGA in acetate buffer (pH = 5.2) using

ACVA a water soluble initiator. In our hands, several studied have been performed on the RAFT polymerization of PEGA using, ACVA or AIBN as the initiator and 1,4-dioxane, toluene and DMF as the solvent.

Scheme II. 11. RAFT polymerization of PEGA using COPYDC as the chain transfer agent, ACVA as the initiator in DMF at 70°C. During RAFT polymerizations, aliquots have been taken to determine the PEGA conversion by 1 H NMR spectroscopy, M n,SEC

Table II

 II These results show the initial molar ratio [COPYDC]0/[ACVA]0 = 1/0.1 or 1/0.5 doesn't play a key role in the control of RAFT polymerization. Indeed, in both cases the RAFT polymerization show a poor control of the RAFT polymerization.

	Table II. 1. Monomer conversion and macromolecular characteristics of polymers obtained by
	RAFT polymerization of PEGA with COPYDC as chain transfer agent and ACVA as the
	initiator in DMF at 70°C. [PEGA]0/[COPYDC]0/[ACVA]0 = 150/1/0.1
	Time (min)	Conv. (%) a	M n,SEC ̅̅̅̅̅̅̅̅ (g.mol -1 ) b	Ð b	M n,NMR ̅̅̅̅̅̅̅̅̅ (g.mol -1 ) c
	0		0	1300	1.12	440
	30		3.8	13300	1.13	3200
	72		16.2	17300	1.28	12000
	90		40.1	24700	1.46	29300
	120		62.0	33500	1.48	45100
	241		83.0	35200	1.83	60200
	300		86.7	36600	1.87	63000
	356		90.2	40900	1.74	65400
	M n,NMR ̅̅̅̅̅̅̅̅̅ =	[PEGA] 0 [ACVA] 0	×conv×M n,PEGA ̅̅̅̅̅̅̅̅̅̅ +M COPYDC where conv is the conversion, M n,PEGA ̅̅̅̅̅̅̅̅̅̅ is the
	. 1 and table II. 2 show the monomer conversion and macromolecular number-average molar mass of PEGA (M n,PEGA ̅̅̅̅̅̅̅̅̅̅ = 480 g.mol -1 ) and MCOPYDC is the molar mass
	characteristic of RAFT polymerization of PEGA using [PEGA]0/[COPYDC]0/[ACVA]0 = of COPYDC (MCOPYDC = 440 g.mol -1 ).
	150/1/0.1 and [PEGA]0/[COPYDC]0/[ACVA]0 = 150/1/0.5, respectively. The two tables show

the increase of the number-average molar masses with time and conversion. However, the dispersities are high at low conversion (Ð = 1.46 at 40.1 % of conversion, Table

II

.1).

a Determined by

1

H NMR spectroscopy (400 MHz, CDCl3) by comparing the integration values of the formamide proton of DMF at 8.02 ppm and of the alkene protons of PEGA between 5.7 and 6.5 ppm. b Determined by SEC in DMF (LiBr 1 g.L -1 ) using PS equivalents. c

Table II . 2 .

 II2 Monomer conversion and macromolecular characteristics of polymers obtained byRAFT polymerization of PEGA with COPYDC as chain transfer agent and ACVA as the

	initiator in DMF at 70°C. [PEGA]0/[COPYDC]0/[ACVA]0 = 150/1/0.5
	Time (min)	Conv. (%) a	M n,SEC ̅̅̅̅̅̅̅̅ (g.mol -1 ) b	Ð b	M n,NMR ̅̅̅̅̅̅̅̅̅ (g.mol -1 ) c
	0	0	1320	1.12	440
	15	1.7	1330	1.13	1700
	30	9.5	12900	1.27	7300
	61	70.1	40300	1.47	51000
	92	83.0	42200	1.73	60200
	120	87.0	42500	1.79	63100

a Determined by

  reagents were purchased from Sigma-Aldrich unless otherwise noted. Sodium

	trifluoroacetate	(NaTFA,	≥	98	%),	trans-2-[3-(4-tert-butylphenyl)-2-methyl-
	2propenylidene]malononitrile (DCTB, ≥ 98 %), dithranol (DIT, 97 %) DL-dithiothreitol
	(DITH, ≥ 98 %), dicyclohexylcarbodiimide (DCC, 99%), 4-(dimethylamino)pyridine (DMAP,
	99 %) 4-cyano-4-[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoic acid (CDP, 97 %), N-(3-
	hydroxybenzotriazole (HOBT, 98 %, Acros-Organics), potassium hydroxide (KOH, Acros-
	Organics, 85 %), sodium chloride (NaCl, Acros-Organics, 99.5 %) and silica gel for
	chromatography (SiO2, 0.035 -0.070 mm, 60 Å, Acros Organics). Dichloromethane (CH2Cl2,

dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDCI, ≤ 97 %), oxalyl chloride (99 %); 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM, 96 %), 4-methylmorpholine (NMM, 99 %), propargyl bromide (80 % in toluene), 4,4-azobis(4cyanolaveric acid) (ACVA, 98 %), sodium carbonate (Na2CO3, 99,5 %), hydrochloric acid (HCl, 37 %), N,N-dimethylformamide (DMF), toluene (99.5 %), acetonitrile (99.8 %), succinimidyl 3-(2-Pyridyldithio)propionate (SPDP, ≥ 95 %), phosphorus pentoxide (P2O5, ≥ 99.8 %), aluminum oxide (basic activated), α-amino,ω-hydroxylpoly(ethylene oxide) (DP ̅̅̅̅ n,MALDI = 64 pure, Rapp polymers), α,ω-dihydroxylpoly(ethylene oxide) (DP ̅̅̅̅ n,MALDI = 44, pure, Rapp polymers), magnesium sulfate (MgSO4, laboratory reagent grade, Fischer), cyclohexane (99.8 %, Quaron), diethyl ether (99.8 %, Carlo Erba), methanol (99.8 %, Carlo Erba), ethyl acetate (pure, Carlo Erba), propargyl amine (≥ 99 %, Acros Organics), 1-

  In a round bottom flask was charged a stir bar together with α-alkynyl,ω-hydroxylPEO (2190 mg, 0.674 mmol), DMAP (17.6 mg, 0.144 mmol), and CDP (543.1 mg, 1.34 mmol). 10 mL of anhydrous CH2Cl2 were added under argon. In another round bottom flask, DCC (302.4 mg, 1.47 mmol) was solubilized in 7 mL of anhydrous CH2Cl2 under argon. The solution of DCC was injected dropwise into the first solution via a syringe. The solution became trouble.

	HC≡CCH2, 0.93 H), 2.29 (td, HC≡CCH2, 2.20 H), 2.57 (t, CH2CH2C(=O)OCH, 1.59 H), 2.65
	(m, CH2CH2C(CN)(CH3)), 3.12 (t, -SCH2(CH2)10CH3, 0.23 H), 3.65 (m, (CH2CH2O)64, 254 H),
	4.27 (t, CH2CH2OC(=O), 0.26 H), 5.10 (q, CH3CHC(=O)NH, 0.73 H), 6.57 (m, NHCH2CH2O,
	0.61 H), 7.12 (m, NHC(CH3)2, 0.53 H).
	SEC in DMF (Libr 1 g.L -1 ): M n,SEC ̅̅̅̅̅̅̅̅ = 9300 g.mol -1 , Ð = 1.13
	b)	Esterification mediated through DCC/DMAP
	1 H NMR (400 MHz, CDCl3),	(ppm)μ 0.88 (t, S(CH2)11CH3, 1.56 H), 1.26 (m,

SCH2CH2(CH2)9CH3), 1.45 (d, CH3CHC(=O)NH), 1.60 (d, (CH3)2CC(=O)NH, 4.77 H), 1.70 (m, SCH2CH2(CH2)9CH3), 1.88 (m, HC≡CCH2CH2, C(CH3)(CN)S, 1.94 H), 2.00 (t,

Table III . 1 .

 III1 Data for the RAFT (co)polymerizations of DMAEA and tBocAEA at different loadings using mPEO-CTA as macromolecular chain transfer agent and ACVA as the initiator

	in 1,4-dioxane using ([DMAEA]0/[tBocAEA]0)/[mPEO-CTA]0/[ACVA]0 = 100/1/0.2
	Run	[DεAEA]0/ [tBocAEA]0	Time (h)	Conv (%) a	DP ̅̅̅̅ n, PDMAEA b	DP ̅̅̅̅ n, PtBocAEA b	M n,NMR ̅̅̅̅̅̅̅̅̅ (g.mol -1 ) c	M n,SEC ̅̅̅̅̅̅̅̅ (g.mol -1 ) d	Ð d
	1	100/0 e	8	44	23	0	5690	8900	1.07
	2	50/50 e	18	22	10	10	5980	9400	1.16
	3	70/30 e	12	24	18	9	6910	9600	1.17
	4	0/100 f	4	14	0	14	5410	9400	1.15
	90 °C
		RAFT (co)polymerizations were performed at 70°C in organic solvents such as 1,4-
	dioxane in which DMAEA and tBocAEA are stable. 10,11,17,23 RAFT agents bearing a 4-
	cyanopentanoic acid as a R leaving group have previously been used with success for RAFT
	polymerizations involving DMAEA and tBocAEA. 17

a Total monomers conversion determined by

1

H NMR spectroscopy (400 MHz, CDCl3) comparing the peak area of the vinylic protons of DMAEA and tBocAEA at 5.81-6.46 ppm with the peak area of the CH of DMF (used at internal standard) at 8.02 ppm. b Determined by 1 H NMR spectroscopy (400 MHz in CDCl3) c M n,NMR ̅̅̅̅̅̅̅̅̅ = M n,mPEO-CTA ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ + DP ̅̅̅̅ n,PDMAEA ×M DMAEA +DP ̅̅̅̅ n,PtBocAEA ×M tBocAEA with M n,mPEO-CTA ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ = 2400 g.mol -1 , MDMAEA = 143.2 g.mol -1 and MtBocAEA = 215.2 g.mol -1 . d Determined by SEC in DMF (LiBr g.L -1 , PS equivalents). e Solvent: 1,4-dioxane and T = 70°C f Solvent: N,N-dimethylformamide and T =

Table III

 III CTA ̅̅̅̅̅̅̅̅̅̅̅̅̅̅ = 3500 g.mol -1 , MDMAEA = 143.2 g.mol -1 and MtBocAEA = 215.2 g.mol-1 . d Determined by SEC in DMF (LiBr 1 g.L -1 ) using PS standards.

	. 3. Macromolecular characteristics of aPEO-b-P(DMAEA-co-tBocAEA) synthesized
	by RAFT copolymerization of DMAEA and tBocAEA mediated through the aPEO-CTA as the
	macromolecular chain transfer agent and ACVA as the initiator under argon in 1,4-dioxane and
	DMF at 70°C using the following ratio: ([DMAEA]0+[tBocAEA]0)/[aPEO-CTA]0/[ACVA]0 =
	100/1/0.2.
	Run	[DεAEA]0/ [tBocAEA]0	Time (h)	Conv (%) a	DP n, ̅̅̅̅̅̅ PDMAEA	b	DP n, ̅̅̅̅̅̅ PtBocAEA	b	M n,NMR ̅̅̅̅̅̅̅̅̅ (g.mol -1 ) c	M n,SEC ̅̅̅̅̅̅̅̅ (g.mol -1 ) d	Ð d
	1	50/50	18	23	11		9		7200	9900	1.48
		70/30	18	38	25		10		9400	11700	1.34
	3	30/70	18	16	5		9		6400	11900	1.21
	4	70/30	12.5	20	11		4		6100	11300	1.22
	5	30/70	19	19	5		10		6600	11500	1.30
	6	30/70	14	20	10		20		8700	13700	1.27
	7	50/50	18	40	18		15		9500	12000	1.43
	8	50/50	17	42	21		23		11500	11700	1.52
	9	50/50	5.5	24	10		12		7500	11700	1.31
	a Total monomers conversion monitored by 1 H NMR spectroscopy of the crude product (400
	MHz, CDCl3). b DP n ̅̅̅̅̅ determined by 1 NMR spectroscopy of the purified polymer (400 MHz,
	CDCl3).

c M n,NMR ̅̅̅̅̅̅̅̅̅ determined by

1

H NMR spectroscopy (400 MHz, CDCl3) using the DP n ̅̅̅̅̅ previously calculated, M n,NMR ̅̅̅̅̅̅̅̅̅ = DP ̅̅̅̅ n,PDMAEA ×M DMAEA + DP ̅̅̅̅ n,tBocAEA ×M tBocAEA +M n,aPEO-CTA ̅̅̅̅̅̅̅̅̅̅̅̅̅̅ with M n,aPEO-

Table III .

 III DP ̅̅̅̅ n,PDMAEA ×M DMAEA + DP ̅̅̅̅ n,PtBocAEA ×M tBocAEA +M n,PPEGA ̅̅̅̅̅̅̅̅̅̅̅ .with M n,PPEGA-CTA ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ = 6700 g.mol -1 , MDMAEA = 143.2 g.mol -1 and MtBocAEA = 215.2 g.mol -1 . d

		4. Macromolecular characteristics of PPEGA-b-P(DMAEA-co-tBocAEA)
	copolymers synthesized by RAFT copolymerization of DMAEA and tBocAEA using α-
	alkynyl,ω-dodecyltrithiocarbonate PPEGA (PPEGA-CTA) as the macromolecular chain
	transfer agent and ACVA as the initiator in 1,4-dioxane and DMF at 70°C.
	Run	[DεAEA]0/ [tBocAEA]0	Time (h)	Conv (%) a	DP n ̅̅̅̅̅ PDMAEA b	DP n, ̅̅̅̅̅̅ PtBocAEA b	M n,NMR ̅̅̅̅̅̅̅̅̅ (g.mol -1 ) c	M n,SEC ̅̅̅̅̅̅̅̅ (g.mol -1 ) d	Ð d
	1	50/50	27	20	9	10	10100	11200	1.57
	2	18/42	16	46	7	14	10700	9700	1.62
	3	70/30	2	27	13	8	10300	10900	1.58
	a Total monomers conversion monitored by 1 H NMR spectroscopy of the crude product (400
	MHz, CDCl3). b DP n ̅̅̅̅̅ determined by 1 NMR spectroscopy of the purified polymer (400 MHz,
	CDCl3). c M n,NMR ̅̅̅̅̅̅̅̅̅ determined by 1 H NMR spectroscopy (400 MHz, CDCl3) using the DP n ̅̅̅̅̅
	previously calculated, M n,NMR ̅̅̅̅̅̅̅̅̅ =

Table III . 5 .

 III5 Macromolecular characteristics of P(DMAEA-co-tBocAEA) synthesized by RAFT copolymerization of DMAEA and tBocAEA mediated through the COPYDC used as the chain transfer agent and ACVA used as the initiator in DMF at 70°C.

	Run [DεAEA]0/ [tBocAEA]0	Time (h)	Conv (%) a	DP n, ̅̅̅̅̅̅ PDMAEA b	DP n, ̅̅̅̅̅̅ PtBocAEA b	M n,NMR ̅̅̅̅̅̅̅̅̅ (g.mol -1 ) c	M n,SEC ̅̅̅̅̅̅̅̅ (g.mol -1 ) d	Ð d
	1	70/30	4	26	10	4	7200	4300	1.20
	2	18/42	38	19	4	14	9400	4200	1.23
	3	50/50	21	19	9	13	6400	5200	1.50

a Total monomers conversion monitored by

1

H NMR spectroscopy of the crude product (400 MHz, CDCl3). b DP n ̅̅̅̅̅ determined by 1 NMR spectroscopy of the purified polymer (400 MHz, CDCl3). c M n,NMR ̅̅̅̅̅̅̅̅̅ determined by 1 H NMR spectroscopy (400 MHz, CDCl3) using the DP n ̅̅̅̅̅ previously calculated M n,NMR ̅̅̅̅̅̅̅̅̅ = DP ̅̅̅̅ n,PDMAEA ×M DMAEA + DP ̅̅̅̅ n,PtBocAEA ×M tBocAEA +M COPYDC with MCOPYDC = 440 g.mol -1 , MDMAEA = 143.2 g.mol -1 and MtBocAEA = 215.2 g.mol -1 . d Determined by SEC in DMF (LiBr 1 g.L -1 ) using PS standards.

  ppm of the methyl protons NHC(=O)OC(CH3)3 of the tBoc group (labeled k in Figure III. 19). Moreover, the shift of the signal of methylene protons OCH2CH2N + H(CH3)2 of DMAEA units, from 2.56 to 3.52 ppm (labeled g in Figure III. 19) and the shift of the signal of the methyl protons OCH2CH2N + H(CH3)2 of DMAEA units, from 2.27 to 2.99 ppm (labeled h in Figure III. 19) demonstrate the cationization of DMAEA units.

Table IV . 1 .

 IV1 Monomer conversion and macromolecular characteristics of polymers obtained by RAFT polymerization of NIPAM with COPYDC as chain transfer agent and AIBN as initiator in DMF at 70°C. [NIPAM]0/[COPYDC]0/[AIBN]0 = 100/1/0.1.

	Entry	Time (min)	Conv. (%) a	(g.mol -1 ) b M n,th ̅̅̅̅̅̅	(g.mol -1 ) c ̅̅̅̅̅̅̅̅ M n,SEC	Đ c
	1	115	29.8	3810	7460	1.18
	2	180	51.9	6309	11000	1.25
	3	240	60.4	7270	13600	1.19
	4	290	64.5	7730	14600	1.19
	5	349	68.7	8200	15200	1.20
	6	406	70.3	8380	14700	1.26

Table IV . 2 .

 IV2 Kinetic of the trithiocarbonate removal reaction of the α-alkynyl,ω-

	a Determined by SEC in DMF with LiBr (1 g.L -1 ) using PS standards. b Conversion
	calculated according to the following equation: conv. (%) = 100 -	[At,UV/At,RI] [At0,UV/At0,RI] x 100 where At,RI
	and At,UV are peak areas at a given time (t) using respectively a RI detector and an UV-vis
	detector at 309 nm and At0,RI and At0,UV are peak areas before the reaction using respectively RI
	dodecyltrithiocarbonate PNIPAM using AIBN in 1,4-dioxane at 80°C.
	Entry	Time (h)	M n,SEC ̅̅̅̅̅̅̅̅ (g.mol -1 ) a	Đ a	Conv. (%) b
	1	0	12300	1.14	0.0
	2	1	12300	1.14	4.5
	3	2	11900	1.14	12.8
	4	3	11900	1.14	15.5
	5	5	12400	1.14	23.4
	6	7	12200	1.18	41.7
	7	24	12800	1.17	97.0

Table IV . 3 .

 IV3 Kinetic of the trithiocarbonate removal reaction of aPEO64-b-P(DMAEA11-co-

	tBocAEA4) using AIBN in ethanol under photochemical activation. Entry Time (days) M n,SEC ̅̅̅̅̅̅̅̅ (g.mol -1 ) a t0 0 13500 t1 3 11700 t2 6 11600 t3 7 11900 t4 9 12600 t5 12 12200 t6 14 12300 1.31 a Determined by SEC in DMF with LiBr (1 g.L -1 ) using PS standards. b Conversion Ð a Conv. (%) b 1.19 0 1.32 37 1.31 56 1.31 68 1.25 78 1.36 87 calculated according to the following equation: conv. (%) = 100 -[At,UV/At,RI] [At0,UV/At0,RI] x 100 where At,RI

Table IV . 5 .

 IV5 Experimental conditions for the thiol-yne coupling reaction on α-alkynyl,ω-cyanoisopropylPNIPAM using benzyl mercaptan, DMPP as the reducing agent, and AIBN as the radical source in 1,4-dioxane at 80°C for 24 h.

	Entry	Benzyl mercaptan (molar eq.)	DMPP (molar	τ (%) a
				eq.)
	1	10		0	6.2
	2	20		0	25
	3	15		15	39
	a τμ degree of functionalization determined by SEC using an UV-vis detector operating at
	266.2 nm using the following equation: τ (%) =	[At,UV/At,RI]-[At0,UV/At0,RI] [At0,UV/At0,RI]

Table IV . 6 .

 IV6 . 6. Conversions of trithiocarbonate removal/thiol-yne coupling during the one-pot reaction on the α-alkynyl,ω-dodecyltrithiocarbonate PNIPAM using AIBN, benzyl mercaptan and DMPP in 1,4-dioxane at 80°C.

	Chapter 4: Chain-ends modification of (PEGylated) aminoethyl-based polyacrylates.
				Trithiocarbonate removal
	Entry	Time (h)		τ (%) b
				conv. (%) a
	1	24	99	0
	2	48	97	40
	a Conversion determined by SEC using UV-vis detection at 309 nm using the following
	equation: conv. (%) = (1-	[At,UV/At,RI] [At0,UV/At0,RI]
	Scheme IV. 6. One-pot trithiocarbonate removal and thiol-yne coupling reactions of the α-
	alkynyl,ω-dodecyltrithiocarbonate PNIPAM using AIBN, benzyl mercaptan, and DMPP in 1,4-
	dioxane at 80°C.
					183

  LiBr 1 g.L -1 ). The conversion of 89 % was calculated by SEC by comparing peaks areas using a RI detector and a UV-vis detector (fixed at 309 nm) using the following equation:

	Trithiocarbonate removal conversion (%) = 100-	A t,UV A t, RI ⁄ A t0,UV A t0, RI ⁄	×100 Equation (IV. 6)
	1.	PNIPAM used as a model polymer to trithiocarbonate removal
		a)	RAFT polymerization of NIPAM with COPYDC
	A magnetic stir bar was charged to a round bottom flask together COPYDC (200.2 g,

0.455 mmol), AIBN (7.5 mg, 0.045 mmol), NIPAM (5.1354 g, 45.45 mmol) and DMF (8.63 mL). The solution was degassed during 30 minutes with argon then heated at 70°C The solution DMF (

Table V . 1 .

 V1 Preparation of polyplexes at different N/P

	N/P	VpDNA (µδ)	VDNA loading (µδ)	VpH buffering	Vcopolymer solution (µδ)
	0	2	2	7	0
	1	2	2	5	2
	2	2	2	3	4
	3	2	2	1	6
	4	2	2	0	8
	5	2	2	0	10
	6	2	2	0	12
	7	2	2	0	14
	8	2	2	0	16

H NMR (400 MHz, CDCl3), (ppm): 0.88 (t, SCH2(CH2)10CH3, 1.02 H) , 1.25 (m, -SCH2CH2(CH2)10CH3, 6.11 H), 1.55-1.95 (m, CH2CH2C(CN)(CH3), SCH2CH2(CH2)10CH3),

Further investigations on the influence of (i) DMAEA/AEA molar ratio (ii) the transfection efficiency, (iii) polyplex sizes and (iv) zeta potential will be made.

Acknowledgements

This work has been performed in the Institut des Molécules et Matériaux du Mans (IMMM)-UMRCNRS-6283 Laboratory in the Méthodologie et Synthèse des Polymères (MSP) team in Le Mans université. First, I would like to thanks the Collectivité Locales Sarthoises for the financial support. Acknowledgements I would like to express my sincere gratitude to the Dr Simona Mura, Stéphanie Denis and the Dr Julien Nicolas from the Galien institute for their warm welcome and their help for cell viability assays. I would like to thanks Pr Elias Fattal to allow me to perform cell viability assay within his team. I thank all the PhD student and interns for their welcome and their kindness. Acknowledgements I would like to thanks all the people I met during this PhD: Thomas, Sylvain, Flavien, Fabien, Kévin, Batiste, Justine, Kévin, Paul, Léo, Océane, Antoine… I forget many of them and I would like to thanks them too. I would like to thanks all my good friends who support me and especially: Maxime, Ludovic, Jérémy (aka Djé), Matthieu, Vincent (aka Mouth) and Jéremy (aka Minidou). I would like to thanks my family, my parents, André et Marie-Christine, my brother Erwan, my sister and her husband, Anne et Benoit, my step-father, Fabien and my nieces, Ninon and Maïlys, who help me to be in good spirit.

I would like to finish by thanking, the one who has support me the most during these three years, who has listen me, the one who give me her energy and courage. Thank you for all Laura.

Remerciements

Chapter 1μ Bibliographic Study

reported procedures. 28 Ultra-pure water was obtained from a Millipore Direct Q system and had a conductivity of 18.2 εΩcm at 25°C. All the deuterated solvents are purchased from Eurisotop

B. General Characterization

Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker AC-400 spectrometer for 1 H NMR (400 MHz) and 13 C NMR (100 MHz). Chemical shifts are reported in ppm relative to the deuterated solvent resonances.

The average molar masses (number-average molar mass ε n,SEC ̅̅̅̅̅̅̅̅ , weight-average molar mass ε w,SEC ̅̅̅̅̅̅̅̅̅ and dispersity (Đ = ε w,SEC ̅̅̅̅̅̅̅̅̅ /ε n,SEC ̅̅̅̅̅̅̅̅ ) values were measured by Size Exclusion Chromatography (SEC) using DMF-LiBr (1 g.L -1 ) as an eluent and carried out using a system equipped with a guard column (Polymer Laboratories, PL Gel 5 µm) followed by two columns (Polymer Laboratories, 2 PL gel 5 µm MIXED-D columns) and with a Waters 410 differential refractometer (RI) and a Waters 481 UV-Visible detector. The instrument operated at a flow rate of 1.0 mL.min -1 at 60°C and was calibrated with narrow linear polystyrene (PS) standards ranging in molar mass from 580 g.mol -1 to 483 000 g.mol -1 . Molar masses and dispersities were calculated using Waters EMPOWER software.

FT-IR spectra of polymers were recorded using a Nicolet iS5 FT-IR spectrometer operating with an attenuated total reflection (iD5 ATR Diamond) gate. Spectra were analyzed with OMNIC software.

High-resolution Mass Spectra (HR-MS) were recorded on a Bruker MicroTOF-QIII (ESI + ).

MALDI-TOF Mass Spectrometry analyses were performed on a Bruker UltraFlex II instrument equipped with a nitrogen laser operating a 337 nm, pulsed ion extraction source and reflectron. Spectra were recorded in the reflectron mode with an acceleration voltage of 25 kV

Conversion of PEGA was determined by 1 H NMR spectroscopy by comparing the integration area values of the vinylic protons signals of PEGA between 5.7 and 6.5 ppm and of the formamide proton signal of DMF used at internal standard at 8.02. The DMF was removed under vacuum and the product was dialyzed in water during 3 days. The solution was lyophilized and the solid obtained was dried under vacuum in the presence of P2O5. The final product was analyzed by

For the large amount of PPEGA:

A magnetic stir bar was charged, to a schlenk flask together with ACVA (8.5 mg, 0.030 mmol), COPYDC (134.2 mg, 0.305 mmol) and PEGA (4.3275 g, 9.02 mmol) in 4 mL of DMF.

The solution was homogeneized, and degassed with argon during 30 minutes. The reaction was launched at 70°C during 1h30. The reaction was stopped by freezing the schlenk in liquid nitrogen and opening it to the air. Conversion of PEGA was determined to be 52 % by 1 H NMR spectroscopy by comparing the integration area values of the formamide proton of DMF at 8.00 ppm and the vinylic protons of PEGA between 5.7 and 6.5 pppm. The crude polymer was purified with several dialysis in pure water, lyophilized and dried under vacuum in the presence of P2O5. The pure product was analyzed by 1 H NMR spectroscopy and SEC in DMF (LiBr g.L - 1 ).

DP ̅̅̅̅ n,PEGA = 12 was calculated by comparing the integration area values of the signal at 0.88 ppm corresponding to the methyl protons S(CH2)11CH3 and of the signal at 4.17 ppm corresponding to the methylene protons CH≡CCH2NHC(=O) and CH2CH2O(CH2CH2O)8CH3. . III. 1).

The number-average polymerization degrees of DMAEA (DP ̅̅̅̅ n,,PDMAEA ) and tBocAEA (DP ̅̅̅̅ n,PtBocAEA ) were determined by 1 H NMR spectroscopy analysis of purified copolymers. III. 2).

B. RAFT copolymerization of DMAEA and tBocAEA using aPEO-CTA

The synthesis of aPEO-b-P(DMAEA-co-tBocAEA) copolymers with different DMAEA/tBocAEA molar ratios has been performed by RAFT copolymerization of DMAEA and tBocAEA using similar conditions than the one previously reported (Chapter 3 §I-A).

Polymerizations were performed using ACVA as the initiator in 1,4-dioxane and DMF at 70°C under argon (Scheme III.

Experimental part

A. Reagents

All reagents were purchased from Sigma-Aldrich unless otherwise noted. Trifluoroacetic acid (TFA, ≥ 98 %), 4,4-azobis(4-cyanolaveric acid) (ACVA, 98 %), hexane (≥ 95 %), 1,4dioxane (≥ 99.8 %), N,N-dimethylformamide (DεF, ≥ 99.9 %), lithium bromide (δiBr, ≥99.9 %), aluminum oxide (basic activated), diethyl ether (99.8 %, Carlo Erba), Acetone (pure, Carlo Erba) and dichloromethane (CH2Cl2, 99.8 %, Carlo Erba) were used without purification. 2-(N,N-dimethylaminoethyl) acrylate (DMAEA, 98 %) was passed through a column of aluminum oxide prior to polymerization. tBocAEA has been synthesized following a reported procedure. 23 mPEO-CTA has been synthesized following a reported procedure (M n,NMR ̅̅̅̅̅̅̅̅̅ = 2400 g.mol -1 , Ð = 1.08). 32 COPYDC (MCOPYDC = 440 g.mol -1 ), aPEO-CTA (M n,NMR,aPEO-CTA ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ = 3500 g.mol -1 ) and PPEGA-CTA (M n,NMR,PPEGA-CTA ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅ = 6700 g.mol -1 ) have been synthesized following procedures reported in Chapter 2. All deuterated solvents were purchased from Euriso-top.

B. General characterization

Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker DPX-200 and a Bruker AC-400 spectrometer for 1 H NMR (400 MHz) and 13

RAFT copolymerization of DMAEA and tBocAEA using COPYDC

In a successfull RAFT copolymerization (Run 3, Table III. 5) procedure, a magnetic stir bar was charged to a round bottom flask together with tBocAEA (3.6124 g, 1.68x10 -2 mol), DMAEA (2.1602 g, 1.51x10 -2 mol), COPYDC (0.1348 g, 3.07x10 -4 mol), ACVA (8.4 mg, 3.00x10 -5 mol), DMF (6.3 mL). The mixture was deoxygenated by bubbling argon for 30 min.

The solution was then immersed in an oil bath thermostatted at 70°C to allow the SEC in DMF (LiBr 1 g.L -1 ): M n,SEC ̅̅̅̅̅̅̅̅ = 5200 g.mol -1 (PS equivalents); Đ = 1.50 The Figure IV. 10 shows the decrease of SEC trace intensity of the copolymer with time using UV-vis detection at 309 nm. As for the model reaction using PNIPAM, the trithiocarbonate removal conversion was calculated by comparing the SEC trace areas of the copolymer at t = 0 and at t using UV-vis detection. Unlike the thermally activated reaction with PNIPAM, the reaction was launched during 2 weeks to finally reach a conversion of 89 % (Table IV. 3). Where, τ is the degree of functionalization, CPEOt18 is the molar concentration of αpyridyldithio,ω-hydroxylPEO after 18 h of reaction, CPEOt0 is the intial molar concentration of α-pyridyldithio,ω-hydroxylPEO (CPEOt0 = 0.001 mmol.L -1 ), CMPt18h is the molar concentration of 2-mercaptopyridine after 18 h of reaction, A343nm is the absorbance at 343 nm (A343 nm = 0.4518), is the molar attenuation coefficient of 2-mercaptopyridine ( = 8080 δ.mol -1 .cm - 1 ) 29,30 and l is the length of the UV-vis cell (l = 1 cm).

The degree of functionalization is equal to 56 %. The thiol-yne coupling reaction on αalkynyl,ω-cyanoisopropylPNIPAM was then carried out in order to compare the functionalization degree obtained by the those two strategies.

B. Thiol-yne coupling reaction ǂ

The thiol-yne coupling reaction is performed using α-alkynyl,ω-cyanoisopropylPNIPAM and benzyl mercaptan as models using AIBN to generate radicals at 80°C in 1,4-dioxane (Scheme IV. 5). Benzyl mercaptan was used as a model molecule because it is easy to characterize by 1 H NMR spectroscopy thanks to the aromatic moiety leading to the characteristics signals between 7 and 8 ppm, and by SEC using UV-vis detection due to the characteristic absorption at 266.2 nm.

Chapter 4: Chain-ends modification of (PEGylated) aminoethyl-based polyacrylates.

Scheme IV. 5. Thiol-yne coupling reaction of α-alkynyl-ω-cyanoisopropylPNIPAM with benzyl mercaptan using AIBN as radical initiator and dimethylphenyl phosphine (DMPP) as reducing agent in 1,4-dioxane at 80°C.

Different experimental conditions were tested in order to target a high thiol-yne coupling efficiency (Table IV. 5). The degree of functionalization was determined by SEC using UV-vis detection at 266.2 nm and confirmed by 1 H NMR spectroscopy. Results showed that the increase of the equivalent number of benzyl mercaptan leads to a better conversion: 6.2 % with 10 molar equivalents (Entry 1 in Table IV. 5) and 25 % with 20 equivalents (Entry 2 in Table IV. 5). Then, to increase the thiol-yne efficiency through the decrease of undesirable disulfide coupling reactions, DMPP was added as reducing agent. 31 Results showed that the addition of DMPP increased the conversion from 25 % to 39 % (Entries 2 and 3 in Table IV. 5).

Chapter 4: Chain-ends modification of (PEGylated) aminoethyl-based polyacrylates.

Experimental part A. Reagents

All reagents were purchased from Aldrich unless otherwise noted. Sodium (CH2Cl2, HPLC grade, Fisher Chemical) were dried over dry solvent stations GT S100. Ultrapure water was obtained from a Millipore Direct Q system and had a conductivity of 18.2 εΩcm at 25°C. All deuterated solvent were purchased from Euriso-top.

B. General characterization

Nuclear Magnetic Resonance (NMR) spectra were recorded on a Bruker AC-400 spectrometer for 1 H NMR (400 MHz). Chemical shifts are reported in ppm relative to the deuterated solvent resonances. SEC in DMF (LiBr 1 g.L -1 ): M n,SEC ̅̅̅̅̅̅̅̅ = 11600 g.mol -1 , Đ = 1.17.

b) Trithiocarbonate removal mediated through AIBN at 80°C.

A round bottom flask was charged with a stir bar together with PNIPAM (400. where At,UV and At,RI represent peak areas at a given reaction time (t) using a UV-vis detector operating at 309 nm and a RI detector, respectively and where At0,UV and At0,RI represent peak areas at t = 0 using a UV-vis detector operating at 309 nm and a RI detector, respectively.

The solvent was evaporated from the final sample, the polymer was dissolved in a minimum of CH2Cl2 and precipitated dropwise three times in cold diethyl ether. The white solid was dried under vacuum at 40°C. The yield of 80 % was determined by gravimetry.

The trithiocarbonate removal conversion was also confirmed by 1 H NMR spectroscopy (400 MHz, CDCl3) verifying the disappearance of the methyl signal at 0.88 ppm of the CH3 of the dodecyl chain before and after reaction and purification. The product was characterized by 1 H NMR spectroscopy in CDCl3 and by SEC in DMF (LiBr 1 g.L -1 ). SEC in DMF (LiBr 1 g.L -1 ): M n,SEC ̅̅̅̅̅̅̅̅ = 12800 g.mol -1 , Đ = 1.17.

Trithiocarbonate removal on PEO

B. General characterization

Zeta potential (ξ) and size measurements. ξ were performed using a dynamic light scattering instrument (Zetasizer Nano ZS, Malvern Instrument) equipped with a He-Ne laser beam at 658 nm. The average value was calculated from four replicate measurements. Dh of polyplex were calculated using multi narrow mode algorithm at a polyplex concentration of 0.5 µg.mL -1 .

C. Gel retardation assay

Cationic copolymers/pDNA polyplexes have been prepared by mixing pDNA (0.5 µg.L - 1 ) with a solution of copolymers in tris-HCl solution at pH = 7.4 (nitrogen concentration = 0.5 µg.µL -1 ), DNA loading and completed with the pH buffering solution (Table V. 1). The solution was homogenized with a Vortex-2 Genie scientific industries. A gel agarose was prepared to characterize the polyplex (0.8 % of agarose in TAE 1 X buffer) with ethidium bromide. The gel retardation assay was carried out at 50 mV with running TAE 1 X buffer.