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<p>The adsorption of cationic polyacrylamide (C-PAM) and silicananoparticles onto a model surface of silicon oxide wascompared with the adsorption of C-PAM to fibres and theirinfluence on flocculation of a fibre suspension. An increase inionic strength affects the polyelectrolyte adsorption indifferent ways for these two systems. With the silica surface,an increase in the ionic strength leads to a continuousincrease in the adsorption. However, on a cellulose fibre, theadsorption increases at low ionic strength (1 to 10 mM NaCl)and then decreases at higher ionic strength (10 to 100 mMNaCl). It was shown that the adsorption of nanoparticles ontopolyelectrolyte-covered surfaces has a great effect on both theadsorbed amount and the thickness of the adsorbed layer. Theresults showed that electrostatic interactions were thedominating force for the interaction between both the fibresand the polyelectrolytes, and between the polyelectrolytes andthe silica particles. Furthermore, at higher NaClconcentrations, a significant non-ionic interaction between thesilicon oxide surface/particles and the C-PAM was observed.</p><p>The adsorption rate of C-PAM onto fibres was rapid andquantitative adsorption was detected in the time range between1 and 8 s at polyelectrolyte addition levels below 0.4 mg/g.Conversely, an increase in the amount of added polymer leads toan increased polymer adsorption up to a quasi-static saturationlevel. However, after a few seconds this quasi-staticsaturation level was significantly lower than the level reachedat electrostatic“equilibrium”. The adsorbed amountof charges at full surface coverage after 1 to 8 s contact timecorresponded to only 2 % of the total fibre charge, whereasafter 30 minutes it corresponded to 15 % of the total fibrecharge. This shows that a full surface coverage at shortcontact times is not controlled by surface charge. Based onthese results, it is suggested that a combination of anon-equilibrium charge barrier against adsorption and ageometric restriction can explain the difference between theadsorption during 1 to 8 s and the adsorption after 30 minutes.With increasing time, the cationic groups are neutralised bythe charges on the fibre as the polyelectrolyte reconforms to aflat conformation on the surface.</p><p>The addition of a high concentration of C-PAM to a fibresuspension resulted in dispersion rather than flocculation.This behaviour is most likely due to an electrostericstabilisation of the fibres when the polyelectrolyte isadsorbed. Flocculation of the fibre suspension occurred at lowadditions of C-PAM. A maximum in flocculation was found ataround 50 % surface coverage and dispersion occurred above 100% surface coverage. It was also shown that for a given level ofadsorbed polymer, a difference in adsorption time between 1 and2 seconds influenced the flocculation behaviour. An optimum inflocculation at 50 % surface coverage in combination with theimportance of polymer reconformation time at these shortcontact times showed that the C-PAM induced fibre flocculationagrees with La Mer and Healy’s description of bridgingflocculation.</p><p>A greater degree of flocculation was observed with theaddition of silica nanoparticles to the fibre suspension thanin the single polyelectrolyte system. Flocculation increased asa function of the concentration of added nanoparticles until0.5 mg/g. At higher additions the flocculation decreased againand this behaviour is in agreement with an extended model formicroparticle-induced flocculation. An increase in flocculationwas especially pronounced for the more extended silica-2particles. This effect is attributed to the more extendedpolyelectrolyte layer, since the adsorbed amount wasessentially the same for both silica particles.</p><p>Finally it was found that fines from the wood fibres had asignificant effect on the flocculation. When fines were added,a greater degree of flocculation was detected. Furthermore, itwas also more difficult to redisperse the fibres with polymerin the presence of fines.</p><p><b>Keywords:</b>Adsorption, bridging, cationic polymers,cellulose fibres, electrosteric stabilisation flocculation,ionic strength, nanoparticle, polyelectrolyte, reconformation,retention aids and silica</p>

The adsorption of cationic polyacrylamide (C-PAM) and silicananoparticles onto a model surface of silicon oxide wascompared with the adsorption of C-PAM to fibres and theirinfluence on flocculation of a fibre suspension. An increase inionic strength affects the polyelectrolyte adsorption indifferent ways for these two systems. With the silica surface,an increase in the ionic strength leads to a continuousincrease in the adsorption. However, on a cellulose fibre, theadsorption increases at low ionic strength (1 to 10 mM NaCl)and then decreases at higher ionic strength (10 to 100 mMNaCl). It was shown that the adsorption of nanoparticles ontopolyelectrolyte-covered surfaces has a great effect on both theadsorbed amount and the thickness of the adsorbed layer. Theresults showed that electrostatic interactions were thedominating force for the interaction between both the fibresand the polyelectrolytes, and between the polyelectrolytes andthe silica particles. Furthermore, at higher NaClconcentrations, a significant non-ionic interaction between thesilicon oxide surface/particles and the C-PAM was observed. The adsorption rate of C-PAM onto fibres was rapid andquantitative adsorption was detected in the time range between1 and 8 s at polyelectrolyte addition levels below 0.4 mg/g.Conversely, an increase in the amount of added polymer leads toan increased polymer adsorption up to a quasi-static saturationlevel. However, after a few seconds this quasi-staticsaturation level was significantly lower than the level reachedat electrostatic“equilibrium”. The adsorbed amountof charges at full surface coverage after 1 to 8 s contact timecorresponded to only 2 % of the total fibre charge, whereasafter 30 minutes it corresponded to 15 % of the total fibrecharge. This shows that a full surface coverage at shortcontact times is not controlled by surface charge. Based onthese results, it is suggested that a combination of anon-equilibrium charge barrier against adsorption and ageometric restriction can explain the difference between theadsorption during 1 to 8 s and the adsorption after 30 minutes.With increasing time, the cationic groups are neutralised bythe charges on the fibre as the polyelectrolyte reconforms to aflat conformation on the surface. The addition of a high concentration of C-PAM to a fibresuspension resulted in dispersion rather than flocculation.This behaviour is most likely due to an electrostericstabilisation of the fibres when the polyelectrolyte isadsorbed. Flocculation of the fibre suspension occurred at lowadditions of C-PAM. A maximum in flocculation was found ataround 50 % surface coverage and dispersion occurred above 100% surface coverage. It was also shown that for a given level ofadsorbed polymer, a difference in adsorption time between 1 and2 seconds influenced the flocculation behaviour. An optimum inflocculation at 50 % surface coverage in combination with theimportance of polymer reconformation time at these shortcontact times showed that the C-PAM induced fibre flocculationagrees with La Mer and Healy’s description of bridgingflocculation. A greater degree of flocculation was observed with theaddition of silica nanoparticles to the fibre suspension thanin the single polyelectrolyte system. Flocculation increased asa function of the concentration of added nanoparticles until0.5 mg/g. At higher additions the flocculation decreased againand this behaviour is in agreement with an extended model formicroparticle-induced flocculation. An increase in flocculationwas especially pronounced for the more extended silica-2particles. This effect is attributed to the more extendedpolyelectrolyte layer, since the adsorbed amount wasessentially the same for both silica particles. Finally it was found that fines from the wood fibres had asignificant effect on the flocculation. When fines were added,a greater degree of flocculation was detected. Furthermore, itwas also more difficult to redisperse the fibres with polymerin the presence of fines. <b>Keywords:</b>Adsorption, bridging, cationic polymers,cellulose fibres, electrosteric stabilisation flocculation,ionic strength, nanoparticle, polyelectrolyte, reconformation,retention aids and silica / NR 20140805

The effect of cationic polymers on the rate of hydrolysis of cornstarch and cellulosic feedstocks was investigated. Poly(diallyldimethylammonium chloride) (p-DADMAC) and cationic polyacrylamides (c-PAMs) were used in the study. Experiments were performed to analyze the effect of both p-DADMAC and c-PAM on cornstarch liquefaction. Measurements were also made on the hydrolysis rates of bleached softwood to determine the mechanism through which cationic polymers accelerate cellulosic hydrolysis. Additional experiments were performed to study the effect of cationic polymers on different lignocellulosic feedstocks such as sludge, wheat straw and brown pulp. Studies on cornstarch hydrolysis showed that p-DADMAC increases the rate of α-amylase-induced cornstarch liquefaction, thereby reducing the enzyme dose necessary for optimal hydrolysis. Studies on bleached softwood showed that cationic polyelectrolytes increase the cellulase-induced hydrolysis rates of bleached wood fiber. It was shown that the polymer associates mainly with the amorphous region of fiber and acts principally on endoglucanase. Both c-PAM and p-DADMAC increased the glucose production of brown pulp at lower kappa numbers. Overall, cationic polymers enhanced the production of glucose from cornstarch and different cellulosic feedstocks. The polymer can reduce the enzyme dosage depending on the conditions and feedstocks used.

Précédemment, il a été démontré au laboratoire qu’une vaccination des souris avec des lipopolyplexes (LPR) contenant l’ARNm de l’antigène de mélanome MART1 permet d’induire la formation de lymphocytes T cytotoxiques spécifiques et de retarder le développement de mélanomes B16F10 et de métastases pulmonaires. Les LPR sont des complexes ternaires constitués d’ARNm, d’un polymère cationique histidylé et de liposomes cationiques histidylés. L’objectif de ma thèse était d’améliorer cette vaccination antitumorale en développant de nouveaux liposomes capables de cibler les cellules dendritiques (DC). Le ciblage a été réalisé en incorporant un glycolipide mannosylé aux liposomes afin de favoriser leur reconnaissance par le récepteur mannose. A partir de ces liposomes, des formulations de complexes ternaires à base d’ADN (LPD mannosylés ou Man11-LPD100) ou à base d’ARN (LPR mannosylés ou Man11- LPR100) ont été mis au point. Les résultats montrent que : in vitro les formulations Man11-LPD100 sont mieux internalisés et transfectent plus efficacement les DC que les LPD100 non mannosylés. Les formulations Man11-LPR100 transfectent avec une plus grande efficacité les DC par rapport aux Man11- LPD100. Par ailleurs, une forte réduction de la toxicité des formulations a été obtenue en dialysant les liposomes. Il est également possible de conserver les formulations sous forme déshydratée. Une imagerie par scintigraphie effectuée chez la souris a permis de constater que 9% des LPD sont captés dans la rate après une injection IV. Nous avons mis en évidence après un isolement de DC spléniques que les formulations Man11-LPR100 transfectent 4 fois plus de DC que les LPR non manosylés. Enfin, l’immunisation des souris avec Man11-LPR100 contenant l’ARNm MART1 permet une vaccination plus efficace contre la tumeur B16F10 et une meilleure survie. En conclusion, les LPR Man11-LPD100 sont de bons vecteurs pour cibler et transfecter les DC spléniques avec l’ARNm d’un antigène tumoral et pour induire la réponse immune contre les cellules tumorales. / Previously, it has been demonstrated that mice vaccination with lipopolyplexes (LPR) containing melanoma antigen MART1 mRNA can induce the generation of specific cytotoxic T cells and delay B16F10 melanoma growth and lung metastases. LPR are ternary complexes consisting of mRNA, a histidylated cationic polymer and histidylated cationic liposomes. The objective of my thesis was to enhance this antitumor vaccination through the development of new liposomes that can target specifically dendritic cells (DC). The targeting of DC was achieved by incorporating a mannosylated glycolipid within liposomes to enhance their recognition by the mannose receptor. From these liposomes, formulations based ternary complexes of DNA (mannosylated-LPD or Man11-LPD100) or formulations based on mRNA (mannosylated LPR or Man11 LPR100) were developed. The results show that formulations made with Man11-LPD100 are better internalized and transfect efficiently DC than LPD100. Man11 LPR100 transfect with greater efficiency DC compared to DNA based formulation (Man11-LPD100). Furthermore, a strong reduction of the toxicity of LPD was obtained by liposomes dialysis. It is also possible to preserve their activity by freeze-drying. Mice scintigraphy revealed that 9% of LPD are captured in the spleen following IV injection. We demonstrated after isolation of splenic DC that Man11-LPR100 transfect DC 4 times more than LPR100. Finally, immunization of mice with Man11-LPR100 containing mRNA MART1 allows a more effective vaccination against B16F10 tumor and a better mice survival than non-mannosylated ones. In conclusion, Man11-LPR100 are promising vectors to target and transfect splenic DC with a tumor antigen mRNA aiming to an induction of an immune response against tumor cells.

This thesis explores the capabilities of cationic polymers modified with lipids of different carbon chain length to deliver DNA molecules to primary cells and transformed cell lines. Our studies focus on two different polymers: polyethylenimine (PEI) and poly(L-lysine) (PLL). Firstly, PEI and PLL were conjugated to palmitic acid (C16). The delivery of plasmid DNA to rat bone marrow stromal cells (rat-BMSC) was evaluated by using a Green Fluorescent Protein gene expressing plasmid (pEGFP-N2) as a reporter system. The rationale for lipid substitution is to give the polymer an amphiphilic character so as to improve the transfection efficiency of native polymers by improving the DNA/polymer translocation through the phospholipid-rich cell membranes. In the case of PLL-C16, transfection efficiency was significantly increased (5 fold) as compared to native PLL, and it was significantly higher than commercially available cationic lipids (LipofectamineTM 2000 and FugeneTM). We further explore the use of other lipids with variable chain lengths (carbon chain length ranging from 8 to 18 saturated and unsaturated) in order to identify other candidates to enhance the gene delivery properties of the PLL. Lipid-modified PLL of high molecular weight (25 vs. 4 kDa) was found to be more effective in delivering plasmid DNA in rat-BMSC. We noted that C14-, C16- and C18-substituted PLL gave the most effective DNA delivery. Moreover, a correlation between the extent of lipid substitution and the plasmid DNA delivery efficiency was found Additionally, transgene expression by BMSC significantly increased when amphiphilic PLLs were used as compared to native PLL. The modified polymers were able to transfect the cells up to 7 days, after which the expression decreased. Encouraged by the successful transgene expression agents obtained by modifying low molecular weight PEI with the same series of lipids described above, we explored the possibility of modifying low molecular weight PEI (2 kDa) with longer lipids; saturated fatty acid (C22), trans fat (C18:1T) and essential fatty acids (C22:1, C22:6 and C18:3). Transfection efficiency proved to be cell dependent. Only the transformed 293T cells were able to express GFP compared to human-derived BMSC. The highest transfection efficiency was found with highly unsaturated lipid-substituted PEI (C18:3 and C22:6) and were able to increase transgene expression overtime (6 days). Furthermore, internalization studies indicated that effective transfection of these carries do not follow any known endocytosis pathway instead the DNA/carrier penetrates the plasma membrane directly. / Pharmaceutical Sciences

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Le développement de la thérapie génique dépend des systèmes utilisés pour le transport de gènes vers les cellules eucaryotes. Les systèmes à base de virus sont les plus efficaces. Cependant, il est urgent de trouver une alternative à de tels systèmes viraux pathogènes et oncogènes. Les polymères cationiques sont des vecteurs synthétiques prometteurs ; toutefois, une question cruciale reste en suspens : quelle structure de polymère cationique visée pour une efficacité de transfection élevée et une faible cytotoxicité ? Face à ce questionnement scientifique, de nouveaux polymères cationiques offrant une grande flexibilité en termes de structure et de fonctionnalité sont développés dans cette thèse. Les différents paramètres structuraux pertinents étudiés sont : (i) des entités amines primaire et tertiaire pH-sensibles pour la complexation de l'ADN et pour la libération des polyplexes ADN/polymère, (ii) un groupe alcyne destiné à l’ancragepar chimie click de ligands capables de viser des récepteurs spécifiques de membrane cellulaire pour une reconnaissance efficace des cellules, (iii) des entités polyacrylates à « charge modulable » pour libérer l'ADN et diminuer la cytotoxicité du polymère et (iv) un poly (oxyde d'éthylène) (PEGylation) pour une meilleure stabilité en milieu physiologique et une meilleure biocompatibilté. / The clinical success of gene therapy is really dependent on the development of new efficient gene transfer systems. Viral-based gene transfer systems are remarkably efficient in transfecting body cells. However, viral-based systems raised some concerns in terms of immunogenicity, pathogenicity, and oncogenicity. Cationic polymers are promising candidates as they show low host immunogenicity, are cheaper and easier to produce in a large scale than viral ones. However, a crucial question is still pending: which cationic polymer structures and functionalities give the highest transfection efficiency and the lowest cytotoxicity? In dealing with this scientific issue, new cationic polymers with key structural parameters and functionalities were developped during this PhD thesis. The key structural features studied are : (i) pH sensitive primary and tertiary amine entities for DNA complexation and to ensure the endosomal escape, (ii) an alkyne group to attach ligands capable to target specific cell membrane receptors for an efficient cell recognition and receptor-mediated cellularuptake, (iii) “charge-shifting” amino-based polyacrylates for DNA release and to decrease cytotoxicity and (iv) PEG chains (PEGylation) to achieve high stability, longer circulation in physiological conditions and a better biocompatibility. The synthesis of such multi-structural cationic polymers has been achieved through the combination of RAFT polymerization and thiol-yne click coupling reaction. The structure/complexation and the structure/cells viability relationships have been investigated during this work.

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