Dissertationen zum Thema „Allylamides“

Ces travaux décrivent l’utilisation d’ynamides et de N-allènamides comme intermédiaires clés pour la synthèse d’hétérocycles azotés et fluorés inédits. Une réaction de cyclisation intramoléculaire de mésyl N-allènamides trifluorométhylés promue par le TBAF a permis la synthèse de y-sultames trifluorométhylés alors que, l’utilisation d’un mélange TBAF/acide acétique, a permis l’obtention de y-sultames gem-difluorés. La formation de diènes trifluorométhylés substitués par une fonction amide, obtenue par une réaction de métathèse d’ène-ynamides avec un aldéhyde, a permis la synthèse de plateformes moléculaires hautement fonctionnalisées. Le traitement de ces diènes par des amines primaires, a permis l’obtention stéréosélective de y-lactames trifluorométhylés à l’aide d’un processus domino d’hydroamination/isomérisation/transamidation. Enfin, la réduction régio- et stéréosélective de chaque double liaison des N-allènamides trifluorométhylés a été explorée. Des allylamides ont été obtenus par réduction de la partie énamide des N-allènamides, alors que des énamides ont été formés par isomérisation, en milieu basique des allylamides
This work describes the use of ynamides and N-allenamides as key intermediates for the synthesis of novel nitrogen and fluorine containing heterocycles. A TBAF-promoted intramolecular cyclization reaction of trifluoromethylated mesyl N-allenamides led to the synthesis of trifluoromethylated y-sultams, while the use of a TBAF/acetic acid mixture afforded gem-difluorinated y-sultams. The formation of amide-substituted trifluoromethylated dienes, obtained through metathesis of ene-ynamides with an aldehyde, enabled the synthesis of highly functionalized molecular platforms. Treatment of these dienes with primary amines enabled the stereoselective production of trifluoromethylated y-lactams via a domino hydroamination/isomerization/transamidation process. Finally, the regio- and stereoselective reduction of each double bond of trifluoromethylated N-allenamides was explored. Allylamides were obtained by reduction of the enamide part of N-allenamides, while enamides were formed by isomerization, in basic medium of allylamides

Flavin adenine dinucleotide (FAD) is a coenzyme that participates in the redox process of flavoenzymes. Attempts to characterize the catalytic pathways of these enzymes have relied in part on the use of FAD chemical models. The efforts described in this dissertation focus on the chemical model approach to investigate the mechanism of the monoamine oxidase (MAO) catalyzed oxidation of the cyclic tertiary allylamine 1-methyl-4-(2-methyl-1H-pyrrol-2-yl)-1,2,3,6-tetrahydropyridine (TMMP), which is a close analog of the parkinsonian-inducing designer drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MAO-B catalyzes the conversion of MPTP and its derivatives into active neurotoxins in the brain that subsequently mediate neurogenerative processes that mimic the events leading to idiopathic Parkinson\'s disease. Monoamine oxidase inhibitors are currently used to treat early stages of Parkinson\'s disease. Two FAD chemical models are examined in this project: 5-ethyl-3-methyllumiflavinium perchlorate (5Et3MLF+ClO4-) and 3-methyllumiflavin (3MLF). The flavinium salt 5Et3MLF+ClO4- is an activated form of 3MLF. These FAD chemical models have been used to examine the MAO catalyzed oxidation. MAO-B is expressed in the brain and is known to be involved in the conversion of TMMP into the neurotoxic metabolite 1-methyl-4-phenyl pyridnium (MMP+). MAO-B is responsible for the alpha-carbon oxidation of TMMP to yield 1-methyl-4-(2-methylpyrrol-2-yl)-2,3-dihydropyridinium (DHP+), which then undergoes a second 2-electron oxidation to MMP+. Previous findings demonstrated that 3MLF and 5Et3MLF+ClO4- promoted the oxidation reaction of primary and secondary amines but not tertiary amines. However, the cyclic tertiary allylamine TMMP has not been examined experimentally. Therefore, the alpha-carbon oxidation of TMMP in the presence of the FAD chemical models is reported in this dissertation. The effect of dioxygen and water on the activity of these FAD models is also investigated.
Ph. D.

Tissues constituting mammalian organisms are finitely organised 3-D multicellular structures where cell-cell and cell-matrix interactions are important modulators of functionality. The liver, being the site of metabolism in mammals, is extensively employed in in-vitro studies such as toxicology, drug testing and liver replacement. Most existing liver models have been static, homogenous 2-D models which have shown limited morphological and functional characteristics of in-vivo liver. With the improved understanding of the liver, these models became more sophisticated to comprise various liver and non-liver cell types, various configurations of extracellular matrix and complex scaffold supports, including fluidic systems. Fluidic supports for liver cells in-vitro are reported in this work and two different types were investigated. The first one was a glass based micro fluidic system with hexagonal structure to mimic the liver lobule. The second one was a standard flatplate fluidic chamber made of plastic (Ibidi channel). For the purpose of improving the attachment of the cells and the performance of the bioreactors examined, various substrate coating procedures were evaluated. The main coating techniques employed were collagen in two forms, adsorbed and gel, and plasma polymerised allylamine (ppAAm). The plasma coating procedures utilised in this work changed the surface properties of the substrate used by increasing the levels of nitrogen and improving hydrophilicity as demonstrated by x-ray photoelectron spectroscopy and contact angle measurements. The ppAAm penetrated both etched glass channels of the hexagonal bioreactor and the flat-plate chamber. The ppAAm films on etched glass channels had similar properties to the films produced on the glass surrounding the channels and coverslips. The ppAAm films obtained in the flat-plate chamber were different and they were characterised with a gradient of chemicals and hydrophilicity. This was because the Ibidi chamber is a closed environment and the plasma vapour infiltrated through the inlet and outlet at the ends of the channel. The attachment and functionality of primary rat hepatocytes seeded onto ppAAm films were evaluated using ppAAm coated coverslips and compared to coverslips coated with collagen gel. This demonstrated that both collagen gel and ppAAm improved the attachment, albumin secretion and 7-Ethoxyresorufin-O-deethylase (EROD) activity of the cells compared to uncoated glass. The hexagonal glass bioreactor showed poor attachment of liver cells and this was enhanced with ppAAm coating. The Huh-7 cells incubated into ppAAm coated hexagonal bioreactor died and detached after incubation with media flow. One of the reasons for this was poor cellular attachment. An improved attachment, but not viability, was observed when the cells were seeded using the biotin-avidin technique. The ppAAm coated flat-plate chamber demonstrated low adhesion of primary rat hepatocytes. However, these channels showed good attachment when collagen type I was adsorbed onto the surface. The viability and functionality, when measured using albumin secretion and EROD, of primary rat hepatocytes were maintained for 5 days in closed fluidic circuit in mono-culture and co-culture with 3T3 cells. These promising results could be exploited to further develop these systems for in-vitro culture of liver cells.

Fourteen novel comb shaped amphiphilic polymers were successfully synthesised to determine the effect of polymer architecture on the potential of these amphiphilic polymers for hydrophobic drug delivery. Polyallylamine (PAA) was grafted with four different types of hydrophobic pendant groups (Cholesteryl (Ch), (Fmoc), (Dansyl)and (Naphth)). Some amphiphilic polymers were further reacted with methyl orange to form quaternary ammonium moieties. The polymers were characterised by elemental analysis and nuclear magnetic resonance spectroscopy (NMR). In the aqueous environment the amphiphilic polymers formed nano self assemblies with particle size from 99 to 284 nm. The critical aggregation concentration (CAC) of the self assemblies was successfully determined by surface tension measurement. The CAC ranged from the lowest value of 0.093 to the highest 1.5 mgmL-1 (Ch5 and Fmoc5 respectively). The Fmoc and Naphth grafted polymers showed the presence of two CMC values, this phenomenon was due to stacking of the planar hydrophobic ring structures resulting in excimer formation. The theory of excimer formation was confirmed by the observation of peak shifting on the emission spectra of the compounds in water over a large concentration range (0.023 – 3 mgmL-1). The drug loading potential of the polymers was investigated using five model hydrophobic drugs, propofol, prednisolone griseofulvin, etoposide and novel anticancer agent BNIPDaoct. The Ch5 and Dansyl10 showed excellent drug solubilisation capacities. At 6 mgmL-1 the Ch5 achieved propofol solubility 70-fold greater than its aqueous solubility, prednisolone, griseofulvin and etoposide solubility’s were increased 20-fold, 30-fold and 7-fold respectively. Similarly at 6 mgmL-1 the Dansyl10 achieved a 200-fold increase on the aqueous solubility of propofol and increased the solubility of prednisolone, griseofulvin and etoposide by 100-fold and 400-fold and 12-fold respectively. The Ch5 (at 1 mgmL-1) was also used to solubilise the novel anticancer agent Bisnaphthalimidopropyl diaminooctane (BNIPDaoct) which was otherwise insoluble achieving a solubilisation of 0.3 mgmL-1. The sizes of the optimal formulations differed greatly for both modified polymers. This was possibly due to the varying architectures of both the drug and the modified polymers and their ability to expand the hydrophobic core and shield the drugs from the ‘hostile’ aqueous environment. The in vitro drug release profiles, showed controlled release of the hydrophobic drugs from the core of the nano aggregates (Ch5 and Dansyl10), the time span for 100% of the drugs to be released ranged from 48- 96 h. Biological characterisation of the polymers found that most of the polymers showed negligible haemolytic activity over the concentrations tested (0.05 – 1 mgmL-1), the IC50 values for the cytotoxicity assay ranged from 0.01740 - 0.05585 mgmL-1 on Caco-2 cells (Fmoc5 to QCh5 respectively). The quaternized polymers showed a slightly better safety profile than their unquaternized counterparts, despite exhibiting low drug solubilisation capacities. The optimal formulations of Ch5 and Dansyl10 loaded with etoposide and BNIPDaoct were tested for their cytotoxicity in vitro on Caco-2 and HEK293 cells. All formulations were capable of lowering the IC50 values when compared with the free anticancer drugs, thus increasing their therapeutic effect. The Ch5 decreased the solubility of etoposide 2.2-fold and BNIPDaoct 1.3-fold on Caco-2 cells, with Dansyl10 achieving a 14 -fold and 16–fold reduction respectively. In vivo oral administration of Ch5 and Dansyl10, griseofulvin formulations demonstrated significantly enhanced the absorption of griseofulvin absorption in rats compared with griseofulvin in water (8.89-fold and 5.20-fold increase respectively on total concentration of griseofulvin solubilised over 24 h study). A formulation of Ch5, BNIPDaoct was also shown to significantly decrease the tumour growth when treated on tumour bearing nude mice over a 4 week period. This is the first time these novel PAA’s grafted with cholesteryl and dansyl have shown promising potential in hydrophobic drug delivery.

NF-κB activity is critical in the regulation of atherosclerotic vascular smooth muscle cell (vSMC) phenotypes induced following oxidative injury by allylamine. The present studies were designed to detail dysregulation of NF-κB activity in these altered phenotypes, and to assess the importance of NF-κB in the regulation of osteopontin, a cytokine which modulates atherosclerosis. Increased degradation of IκBα was observed in allylamine-induced atherosclerotic vSMC phenotypes (henceforth referred to as allylamine cells). Enhanced phosphorylation of I-κ-kinases was observed by Western immunoblotting. NF-κB DNA binding activity as assessed by electrophoretic mobility shift assay demonstrated changes in the kinetics and magnitude of induction of binding. Enhancement of NF-κB binding activity was evident in allylamine cells compared to controls when seeded on plastic, fibronectin, and laminin, but not collagen I. Posttranscriptional alterations in Rel protein expression and nuclear localization partly account for changes in NF-κB DNA binding activity. Promoter-specific NF-κB binding profiles suggest altered dimer prevalence as a consequence of the changes in Rel protein expression. The expression of NF-κB regulated genes osteopontin and MMP-2 was enhanced in allylamine-treated aortas, while cyclin D1 and MMP-9 were unchanged. As the importance of osteopontin in atherosclerosis has been described in several models, subsequent studies were designed to assess osteopontin promoter activity. Activity of the osteopontin promoter was significantly reduced in allylamine cells compared to controls as assessed using a luciferase reporter. Deletion analysis suggested the presence of inhibitory cis-acting elements in the regulatory region of the gene. Mutation of these elements, including VDRE, AP-1, NF-κB, and USF1, indicated that NF-κB and USF1 mediate suppression of osteopontin promoter activity in allylamine cells. Decreased serine phosphorylation of immunoprecipitated RelA/p65 was observed in allylamine cells, indicating decreased ability of this protein to transactive gene promoters. NF-κB was found to play a role in suppression of osteopontin promoter activity by collagen I-mediated integrin signaling. These findings suggest that enhancements in NF-κB activity suppress osteopontin promoter activity in oxidant-activated vSMC cultures. Dysregulation of NF-κB activity occurs as a result of altered matrix and intracellular signaling upstream of the nucleus and possibly differential dimer assembly leading to cell-specific profiles of NF-κB-dependent gene regulation.

Depuis plusieurs années, notre équipe s’intéresse à l’utilisation d’une résine portant des groupes furyles dans diverses stratégies « traceless ». Cette stratégie implique une étape d’accrochage de diénophiles acétyléniques par réaction de Diels-Alder et de décrochage de la résine par réaction rétro-Diels-Alder thermique après fonctionnalisation de l’adduit de cycloaddition. Différentes fonctionnalisations ont déjà été réalisées. Dans un premier temps, nous avons abordé la préparation de dérivés 7-oxa-bicyclo[2. 2. 1]hepta-2,5-diéniques par réaction de Diels-Alder entre des composés acétyléniques et 2-[4-(benzyloxy)phénylméthyl]furane (analogue en solution de l’attache furanique présentée sur la résine). Dans un second temps, nous avons étudié la réactivité d’ylures d’azométhines avec ces 7-oxabicyclo[2. 2. 1]hepta-2,5-diènes dans diverses conditions. Ceci nous a permis d’obtenir des pyrrolidines par cycloaddition 1,3-dipolaire et des 3-(aminobenzyl)-7 oxabicycloheptènes. Après réaction de rétro-Diels-Alder thermique il a été possible d’accéder à diverses pyrrolines et a des dérivés d’allylamine. Les 3-pyrrolines ainsi obtenues ont pu être oxydé en 2H-pyrroles. Dans une dernière partie, nous avons décrit les premiers essais de réaction des 7-oxabicyclo[2. 2. 1]hepta-2,5-diènes avec des nitrones. Nous avons obtenu, par réaction 1,3 dipolaire, des isoxazolidines qui après réaction de rétro-Diels-Alder thermique ont conduit à des isoxazolines
First, we approached the preparation of 7-oxa-bicyclo[2. 2. 1]hepta-,5-dienic derivatives by Diels-Alde reaction between acetylenic compounds and 2-[4-(benzymoxy)phenylmethyl]furane (smilar in solution of the furan presented on the resin). Then, we studied the reactivity of azomethine ylide with 7-oxabicyclo[2. 2. 1]hepta-2,5-dienes under various conditions. This lead us to obtain pyrrolidines by 1,3-dipolar cycloaddition and 3-(aminobenzyl)-7oxabicycloheptenes. After reaction of thermics retro –Diels-Alder it was possible to reach various pyrrolines and derivatives of allylamine. 2H-pyrroles were obtained by DDQ oxidation of the 3-pyrrolines. In a last part, we described the first tests of reaction of 7-oxabicyclo[2. 2. 1]hepta-2,5-diene with nitrones. We obtained by 1,3-dipolar reaction the isoxazolidines which after reaction of thermics retro-Diels-Alder led to isoxazolines

Polymers rich in primary amine groups are proposed to be effective adsorbents for the reversible adsorption of CO2 from moderately dilute gas streams (10% CO2) and ultra-dilute gas streams (e.g. ambient air, 400 ppm CO2), with their performance under ultra-dilute conditions being competitive with or exceeding the state-of-the-art adsorbents based on supported poly(ethyleneimine) (PEI). The CO2 adsorption capacity (mmol CO2/g sorbent) and amine efficiency (mmol CO2/mmol amine) of linear poly(allylamine) (PAA), cross-linked poly(allylamine) prepared by post-polymerization crosslinking with epichlorohydrin (PAAEPI), and branched poly(allylamine) prepared by branching of poly(allylamine) with divinylbenzene (PAADVB) are presented here and compared with state-of-the-art adsorbents based on supported PEI, specifically branched and linear, low molecular weight PEI. Silica mesocellular foam, MCF, serves as the support material for impregnation of the amine polymers. In general, branched polymers are found to yield more effective adsorbents materials. Overall, the results of this work show that linear PAA, cross-linked PAAEPI, and branched PAADVB are promising candidates for solid adsorbents with high capacity for CO2.

Embryonic stem (ES) cells have the capacity to form any cell type. However, their propagation and differentiation is limited by current two dimensional (2D) culture techniques which offer little flexibility in terms of surface structure and functionalisation with bioactive molecules. The aim of the current work was to produce a novel scaffold that could manipulate ES cell behaviour using both architectural and biological cues. Electrospinning is a flexible technique that creates nonwoven meshes that mimic the fibrous architecture of the ECM. Initial work focused on investigating the suitability of electrospun poly(lactic-co-glycolic acid) (PLGA) meshes for 2D and three dimensional (3D) culture of mouse ES cells, with the hypothesis that the fibrous architecture would assist in maintaining pluripotency. The study also sought to functionalise the scaffolds with biologically active molecules. Heparan sulphate proteoglycans (HSPGs) reside at the cell surface and within the ECM where they mediate growth factor binding, assist cell attachment and stabilise the ECM. Furthermore, ES cells modulate their own microenvironment by controlling the composition of heparan sulphate (HS), regulating the binding of growth factors such as fibroblast growth factor (FGF) family members. Therefore, we aimed to immobilise HS and heparin (a highly sulphated structural analogue of HS) on the fibre surface in a form that was freely accessible for protein/cell interactions and that retained its biological activity. Electrospinning parameters were optimised to produce microfibre electrospun meshes with an average fibre diameter of 570nm. Cell morphology, proliferation and pluripotency were monitored using an Oct4-GFP reporter cell line and results compared with flat spin coated films. To investigate the potential for 3D culture, spinning parameters were altered to increase fibre diameter to >3micro metre with infiltration assessed using pro-migratory E-cadherin-/- ES cells. Scaffolds were coated with plasma polymerised allylamine (ppAm) to enable non-covalent immobilisation of HS/heparin. Ligand binding assays with the link module of TSG-6 and anti-heparin/HS antibodies were used to probe HS/heparin presentation on the fibre surface. The biological activity of the immobilised HS/heparin was analysed by testing the ability of coated scaffolds to rescue the neural differentiation capacity HS deficient EXT1-/- ES cells. Finally, human ES cells were cultured on the surface of ppAm scaffolds +/- HS in both unconditioned and mouse embryonic fibroblast (MEF) conditioned media for 5 days. Both microfibre meshes and flat spin coated films supported the attachment, growth and pluripotency of mouse ES cells. Cells adopted distinct morphologies, with mouse ES cells aggregating in rounded colonies on microfibre scaffolds and demonstrating increased spreading on spin coated films. Fibres >3micro metre created a thicker mesh with potential for 3D culture supporting the infiltration of E-cadherin-/- ES cells. ppAm enabled non-covalent immobilisation of HS/heparin in a form that was free to participate in protein interactions and which presented essential sulphation motifs within the HS/heparin chains. Bound HS was biologically active and functioned in synchrony with FGF4 to enhance neural differentiation of EXT1-/- ES cells. The constructs also supported the attachment and growth of human ES cells, with HS functionalised scaffolds demonstrating a slight increase in compatibility during culture in unconditioned media. The successful functionalisation of electrospun meshes with HS/heparin creates a highly versatile scaffold for ES cell culture and differentiation. The architecture of the meshes can be manipulated to either serve as a fibrous substrate for maintenance of pluripotency or support the formation of complex cell interactions present in vivo. The immobilisation of HS provides an extra dimension of versatility, as the scaffold can be tailored with specific HS species, potentially enabling the differential regulation of growth factor binding.

Monodispersed nanoparticles of poly-N-isopropylacrylamide-co-allylamine (PNIPAM-co-allylamine) and PNIPAM-co-acrylic acid (AA) have been synthesized and used as building blocks for creating three-dimensional networks. The close-packed PNIPAM-co-allylamine and PNIPAM-co-AA nanoparticles were stabilized by covalently bonding neighboring particles at room temperature and at neutral pH; factors which make these networks amicable for drug loading and release. Controlled release studies have been performed on the networks using dextran markers of various molecular weights as model macromolecular drugs. Drug release was quantified under various physical conditions including a range of temperature and molecular weight. These nanoparticle networks have several advantages over the conventional bulk gels for controlling the release of biomolecules with large molecular weights. Monodispersed nanoparticles of poly-N-isopropylacrylamide-co-allylamine (PNIPAM-co-allylamine) can self-assemble into crystals with a lattice spacing on the order of the wavelength of visible light. By initiating the crystallization process near the colloidal crystal melting temperature, while subsequently bonding the PNIPAM-co-allylamine particles below the glass transition temperature, a nanostructured hydrogel has been created. The crystalline hydrogels exhibit iridescent patterns that are tunable by the change of temperature, pH value or even protein concentration. This kind of soft and wet hydrogel with periodic structures may lead to new sensors, devices, and displays operating in aqueous solutions, where most biological and biomedical systems reside. The volume-transition equilibrium and the interaction potential between neutral PINPAM particles dispersed in pure water were investigated by using static and dynamic light-scattering experiments. From the temperature-dependent size and energy parameters, the Sutherland-like potential provides a reasonable representation of the inter-particle potential for PNIPAM particles in swollen and in collapsed phases. An aqueous dispersion of PNIPAM particles can freeze at both high and low temperatures. At low temperatures, the freezing occurs at a large particle volume fraction, similar to that in a hard-sphere system; while at high temperature, the freezing occurs at low particle concentrations, driven by the strong van der Waals attraction due to the collapsed microgel particles. The calculated phase diagram has been confirmed semi-quantitatively by experiments.

Orientador: Carlos Roque Duarte Correira
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química
Made available in DSpace on 2018-08-20T23:15:33Z (GMT). No. of bitstreams: 1 Prediger_Patricia_D.pdf: 29103926 bytes, checksum: bd694717759d7b58fda99bfb77a7fa06 (MD5) Previous issue date: 2012
Resumo: O presente trabalho foi centrado na arilacao de Heck-Matuda de diferentes olefinas com sais de arenodiazonio e a aplicacao dos produtos arilados na sintese de compostos bioativos. O trabalho esta dividido em dois capitulos: 1) Reacoes de acoplamento de Heck-Matsuda entre aminas alilicas e sais de arenodiazonio e suas aplicacoes nas sinteses de compostos bioativos. 2) Estudos da reacao de Heck-Matsuda em liquidos ionicos e suas aplicacoes na sintese de derivados C-azanucleosideos, potenciais compostos antituberculose. Com relacao a primeira parte do trabalho, foi desenvolvida uma metodologia abrangente para a reacao de Heck-Matsuda entre alilaminas e sais de arenodiazonio. Foram empregados diversos derivados de aminas alilicas e diferentes sais de arenodiazonio contendo grupamentos doadores, retiradores de eletrons e grupos volumosos. Para todos os casos foram obtidos rendimentos de bons a excelentes, em alta regio- e estereosseletividades em favor do produto ¿×- arilado de configuracao trans. Adicionalmente, a aplicacao deste protocolo forneceu os compostos bioativos naftifina, abamina, abamina SG, alverina e cloridrato de cinacalcet, tendo como etapa-chave a reacao de Heck altamente seletiva. A segunda parte do trabalho envolveu as reacoes de Heck-Matsuda entre a ftalimida alilica e sais de diazonio liquidos ionicos. Nestas reacoes, foram obtidos rendimentos e seletividades semelhantes aos observados com o solvente convencional benzonitrila. Alem disso, foram estudadas reacoes com geracao in situ de sal de hexafluorfosfato de arenodiazonio em [bmim] [PF6], seguida pela reacao de Heck-Matsuda em procedimento ¡§one pot¡¨. Por fim, foram realizadas as sinteses de dois derivados de C-azanucleosideos, tendo como etapa-chave a reacao de Heck-Matsuda entre um enecarbamato endociclico e sal de arenodiazonio. Estes compostos terao suas atividades antituberculose avaliadas pelo grupo de pesquisa do professor Mourey em Toulouse, na França
Abstract: The present work was focused in the Heck-Matsuda arylation of several olefins with arenediazonium salts and in the application of the arylated products as intermediates in the syntheses of bioactive compounds. The work is divided in two chapters: 1) Heck-Matsuda reactions between allyl amines and arenediazonium salts and their applications in the synthesis of bioactive compounds, 2) Studies of the Heck-Matsuda reaction in ionic liquids and its application to the synthesis of Cazanucleosides, which are potential antituberculosis compounds. Regarding the first part of this work, a comprehensive methodology for the Heck-Matsuda reaction between allylamines and arenediazonium salts was developed. Several allyl amines derivatives and differents arenediazonium salts were sucessfully employed. Both electron donating and withdrawing groups, as well as bulky substituents, were well tolerated in the diazonium salt. In all cases good to excellent yields and high regio- and stereoselectivities in favor of the ¿×-trans arylated products were obtained. Additionally, some of the adducts prepared by this highly selective Heck reaction were employed as intermediates in the total synthesis of the bioactive compounds naftifine, abamine, abamine SG, alverine and cinacalcet hydrochloride. The second part of this work adressed the Heck-Matsuda reaction between allylic phthalimide and arenediazonium salts in ionic liquids. Under the investigated conditions, the yields and selectivities were similar to those previous achieved with the conventional solvent benzonitrile. In addition, we evaluated the in situ generation of the 4-fluorbenzenediazonium hexafluorophosphate salt in [bmim][PF6], followed by the Heck-Matsuda reaction in a one pot procedure. Finally, we carried out the synthesis of two C-azanucleosides using the Heck-Matsuda reaction with an endocyclic enecarbamate as the key step. As a further development of this work, these compounds will be evaluated regarding their potential antituberculosis activity by Professor Mourey's research group in Toulouse, France
Doutorado
Quimica Organica
Doutora em Ciências

Etude et développement de dépôts d'allylamine assistés par plasma basse pression specifiques aux stents coronariens recouverts Les stents coronariens sont des dispositifs médicaux, généralement fabriqués en acier inoxydable 316L, utilisés pour traiter des maladies cardiovasculaires comme l'athérosclérose. Les stents recouverts ou à relargage contrôlé de médicaments sont des solutions prometteuses pour réduire les phénomènes de resténose. Ce travail a pour objectif le développement d'un procédé plasma basse pression capable de déposer une couche de polymère permettant de protéger la surface des stents contre l'agressivité du milieu physiologique. L'allylamine est choisie comme précurseur moléculaire pour assurer un taux élevé de fonctions amines primaires. Ces fonctions pourront être utilisées, successivement, pour l'immobilisation de molécules bioactives afin d'augmenter la biocompatibilité des stents. Les dépôts sont effectués sur des substrats d'acier inoxydable 316L en utilisant un réacteur plasma basse pression (70 kHz). Les différentes techniques d'analyse de surface utilisées (angle de contact, XPS, FTIR-ATR) montrent que les variations de puissance de la décharge et du temps de traitement ne modifient pas significativement la composition chimique de surface des dépôts. Cependant, grâce à une technique de dérivation chimique nous avons mis en évidence une meilleure sélectivité vis-à-vis des fonctions amines primaires pour les couches déposées à faibles valeurs de puissance. En effet, des analyses in-situ de la phase plasmagène (spectrométrie de masse, spectroscopie d'émission optique) révèlent qu'une augmentation de la puissance de la décharge conduit à l'augmentation de son caractère énergétique et, ainsi, à l'augmentation du taux de fragmentation du précurseur. La stabilité des revêtements au lavage dans l'eau de-ionisée a été aussi évaluée. Les dépôts obtenus pour une puissance de la décharge de 2W présentent le meilleur compromis entre rétention des fonctions amines primaires et stabilité. Enfin, nous avons évalué les propriétés d'adhérence des couches après déformation plastique en utilisant le " small punch test ", permettant de reproduire les conditions qu'on retrouve lorsque les stents sont déployés dans les artères. Les dépôts présentent des adéquates propriétés de cohésion et d'adhérence au substrat pour répondre à la déformation sans se fissurer et/ou délaminer. Ces résultats montrent que les couches d'allylamine déposées par procédé plasma basse pression présentent des caractéristiques prometteuses afin d'être utilisées comme revêtement performant pour les stents coronariens. Plasma Polymerized allylamine films deposited on 316L stainless steel

New vigorous regulations have been established for decreasing the allowable formaldehyde emissions from nonstructural wood based composites. Two main sources of formaldehyde emission in non-structural wood based composites are adhesive and wood. Adhesives are quite well known and great efforts have been conducted to decrease their formaldehyde content; however formaldehyde emission from wood has received little attention and it is not completely understood. Wood-borne formaldehyde emission exists in a complex equilibrium in wood matrix. The reaction between formaldehyde and wood hydroxyl groups/water can hinder the complete formaldehyde extraction. In order to have a complete formaldehyde extraction, a stronger nucleophile than hydroxyl and water groups is needed. In this study cross-linked poly (allylamine) (PAA) beads were synthesized and used as a strong nucleophile to extract all the biogenic and synthetic free-formaldehyde within the woody matrix of never-heated and heat-treated Virginia pines; the results were compared to simple water extraction. A new formaldehyde capturing device was also developed using a serum bottle. Results showed that there was no advantage of using PAA beads over simple water extraction for extracting woody matrix free-formaldehyde. This means that simple water extraction can extract all the free-formaldehyde from the woody matrix. It was also found that thermal treatment resulted in generating more wood-borne formaldehyde. The other important finding was the new developed formaldehyde capturing device. The device was very promising for detecting wood-borne formaldehyde from very small pieces of wood (5-70 mg) and can be very useful in future studies.
Master of Science

In the first part of this study, frictional and normal forces in aqueous solutions were measured between a glass particle and oligopeptide films grafted from a glass plate. Homopeptide molecules consisting of 11 monomers of different amino acids were each "grafted from" an oxidized silicon wafer using microwave-assisted solid phase peptide synthesis. Oligopeptides increased the magnitude of friction compared to a bare hydrophilic silicon wafer. Friction was a strong function of the nature of the monomer unit and was lower for hydrophilic films. There was a strong adhesion and therefore friction between surfaces of opposite charges. Changes in adhesion and friction depended on the hydrophobicity and electrostatic forces: hydrophobic films and oppositely charged films produced high friction, whereas hydrophilic and like-charges produced low friction. Friction was lower in phosphate buffered saline than in pure water due to the screening of the double layer attraction for oppositely charged surfaces and additional lubrication by hydrated salt ions. We also investigated antimicrobial action of poly (allyl amine) (PA) when covalently bonded to glass. Glass surfaces were prepared by a two-step procedure where the glass was first functionalized with epoxide groups using 3-glycidoxypropyltrimethoxy silane (GOPTS) and then exposed to PA to bind via reaction of a fraction of its amine groups. Antibacterial properties of these coatings were evaluated by spraying aqueous suspensions of bacteria on the functionalized glass slides, incubating them under agar, and counting the number of surviving cell colonies. The PA film displayed strong anti-microbial activity against both Gram-positive and Gram-negative bacteria. Films that were prepared by allowing the PA to self assemble onto the solid via electrostatic interactions were ineffective antimicrobials. Such films had an insufficient positive charge and did not extend far from the solid. Thus we found that antimicrobial activity was correlated with a combination of the ability of the polymer chain to extend into solution and a positive surface potential.
Ph. D.

Avec l’avènement des BioMEMS (Bio-MicroElectroMechanical Systems), ce sont toutes les pratiques médicales, biologiques, environnementales et agro-alimentaires qui entament une nouvelle ère. Les enjeux scientifique et industriel se rejoignent dans la miniaturisation des systèmes de détection, l’amélioration de leurs sensibilités et la simplification de leurs procédés de fabrication. Cette thèse hautement interdisciplinaire s’inscrit dans le cadre de ces enjeux. Elle expose la fabrication, la bio-fonctionnalisation et l’application d’un BioMEMS pour l’analyse de réactions enzymatiques en temps réel et à l’échelle micrométrique. Deux choix stratégiques ont été adoptés pour ce travail: le premier concerne l’utilisation de la technologie des plasmas froids ou polymérisation plasma pour la fonctionnalisation de surface à travers le dépôt de films polymères d’allylamine. Ce dépôt a permis ultérieurement l’immobilisation covalente de la trypsine (enzyme modèle protéolytique) au sein du BioMEMS. Cette technologie a été également utilisée pour développer une méthode simple de microfabrication des circuits microfluidiques compatible avec une production à grande échelle. Le second choix concerne l’utilisation de ce bioMEMS autour d’une transduction TeraHertz (THz) mise au point au sein de l’équipe. La spectroscopie THz vise à détecter les événements moléculaires à l’échelle de la picoseconde, sans marqueur et d’une manière non-invasive, en sondant directement les liaisons chimiques de faible énergie. Au cours de ce travail, nous avons donc développé un procédé de fonctionnalisation de surfaces par des amines, optimisé une méthode de greffage des enzymes, et étudié l’activité de la trypsine immobilisée. Nous avons ensuite intégré ces étapes dans le procédé de microfabrication du BioMEMS. Les mesures réalisées dans le domaine sub-THz (0,06-0,11 THz) sur une réaction de biocatalyse confirment la faisabilité d’une telle approche comme méthode analytique en biologie. Les résultats des différentes études montrent également que le mariage des plasmas froids avec les méthodes lithographiques représente une voie efficace, rapide et très compétitive pour le transfert de la technologie des BioMEMS à l’échelle industrielle
The applications of miniaturized devices are no longer limited to electronic industry. Today, a new kind of microsystems called BioMEMS (Bio-MicroElectroMechanical Systems) are spreading in different fields, including biomedical, environmental and food industry applications. Recurring challenges are focusing on enabling processes for smaller, cost-effective, high-functionality devices, with more sensitivity and suitability for industrial scale development. This highly interdisciplinary thesis work attempts to provide new solutions to meet some of the needs mentioned above. It reports the fabrication, functionalization, and applications of a BioMEMS for enzyme reaction monitoring. First, we have developed a PECVD (plasma enhanced chemical vapor deposition) process for the surface functionalization by plasma polymerized allylamine. Films with high amine density and enhanced stability in aqueous environment were obtained. The amine functions were then used for enzymes immobilization. The covalently bonded trypsin molecules were extensively characterized and kinetic parameters determined using several microscopic and spectroscopic methods. Finally, both optimized processes were applied to the biofunctionalization of a TeraHertz (THz)-based BioMEMS. THz spectroscopy is the only non-invasive analytic method able to monitor molecular events at the picosecond timescale by probing low binding energies directly. It is used here for sensing a biocatalysis reaction inside the bioMEMS microchannels. Sub-THz measurements (0.06-0.11 THz) showed that combining microfluidic microsystems technology with THz detection could be a promising alternative for label-free real-time detection of biological interactions at the microscale. Additionally, we have developed a new microchannel fabrication process using direct plasma polymerization of TMDS (TetraMethylDiSiloxane) on micropatterned surfaces. This achievement demonstrates that cold plasma processes could be used not only for functionalization purposes or surface treatment but for the 3D microfabrication as well. This highly reduces processing time and manual handling steps, which is of a great importance for further industrial scale production

Les stents coronariens sont des dispositifs médicaux utilisés pour traiter des maladies cardiovasculaires (ex: athérosclérose). Les stents recouverts sont des solutions prometteuses pour réduire les phénomènes de resténose. Ce travail porte sur le développement d’un procédé plasma basse pression capable de déposer une couche de polymère à haut teneur en fonctions amines permettant de protéger la surface des stents contre l’agressivité du milieu physiologique. Les dépôts sont effectués à l'aide d'un réacteur plasma basse pression (70 kHz). Les résultats obtenus montrent que les variations de puissance de la décharge et du temps de traitement ne modifient pas significativement la composition chimique de surface des dépôts. Cependant, une technique de dérivation chimique a permis de mettre en évidence une meilleure sélectivité vis-à-vis des fonctions amines primaires pour les couches déposées à faibles valeurs de puissance. En effet, les analyses in-situ de la phase plasmagène révèlent qu’une augmentation de la puissance de la décharge conduit à l’augmentation de son caractère énergétique et, ainsi, à l’augmentation du taux de fragmentation du précurseur. Les dépôts obtenus pour une puissance de la décharge de 2W présentent le meilleur compromis entre rétention des fonctions amines primaires et stabilité. Enfin, les dépôts possèdent des adéquates propriétés de cohésion et d’adhérence pour répondre à la déformation sans se fissurer et/ou délaminer. Ces résultats montrent que les couches d’allylamine déposées par procédé plasma basse pression ont des caractéristiques prometteuses pour être utilisées comme revêtement performant pour les stents coronariens.

Avec l’avènement des BioMEMS (Bio-MicroElectroMechanical Systems), ce sont toutes les pratiques médicales, biologiques, environnementales et agro-alimentaires qui entament une nouvelle ère. Les enjeux scientifique et industriel se rejoignent dans la miniaturisation des systèmes de détection, l’amélioration de leurs sensibilités et la simplification de leurs procédés de fabrication. Cette thèse hautement interdisciplinaire s’inscrit dans le cadre de ces enjeux. Elle expose la fabrication, la bio-fonctionnalisation et l’application d’un BioMEMS pour l’analyse de réactions enzymatiques en temps réel et à l’échelle micrométrique. Deux choix stratégiques ont été adoptés pour ce travail: le premier concerne l’utilisation de la technologie des plasmas froids ou polymérisation plasma pour la fonctionnalisation de surface à travers le dépôt de films polymères d’allylamine. Ce dépôt a permis ultérieurement l’immobilisation covalente de la trypsine (enzyme modèle protéolytique) au sein du BioMEMS. Cette technologie a été également utilisée pour développer une méthode simple de microfabrication des circuits microfluidiques compatible avec une production à grande échelle. Le second choix concerne l’utilisation de ce bioMEMS autour d’une transduction TeraHertz (THz) mise au point au sein de l’équipe. La spectroscopie THz vise à détecter les événements moléculaires à l’échelle de la picoseconde, sans marqueur et d’une manière non-invasive, en sondant directement les liaisons chimiques de faible énergie. Au cours de ce travail, nous avons donc développé un procédé de fonctionnalisation de surfaces par des amines, optimisé une méthode de greffage des enzymes, et étudié l’activité de la trypsine immobilisée. Nous avons ensuite intégré ces étapes dans le procédé de microfabrication du BioMEMS. Les mesures réalisées dans le domaine sub-THz (0,06-0,11 THz) sur une réaction de biocatalyse confirment la faisabilité d’une telle approche comme méthode analytique en biologie. Les résultats des différentes études montrent également que le mariage des plasmas froids avec les méthodes lithographiques représente une voie efficace, rapide et très compétitive pour le transfert de la technologie des BioMEMS à l’échelle industrielle
The applications of miniaturized devices are no longer limited to electronic industry. Today, a new kind of microsystems called BioMEMS (Bio-MicroElectroMechanical Systems) are spreading in different fields, including biomedical, environmental and food industry applications. Recurring challenges are focusing on enabling processes for smaller, cost-effective, high-functionality devices, with more sensitivity and suitability for industrial scale development. This highly interdisciplinary thesis work attempts to provide new solutions to meet some of the needs mentioned above. It reports the fabrication, functionalization, and applications of a BioMEMS for enzyme reaction monitoring. First, we have developed a PECVD (plasma enhanced chemical vapor deposition) process for the surface functionalization by plasma polymerized allylamine. Films with high amine density and enhanced stability in aqueous environment were obtained. The amine functions were then used for enzymes immobilization. The covalently bonded trypsin molecules were extensively characterized and kinetic parameters determined using several microscopic and spectroscopic methods. Finally, both optimized processes were applied to the biofunctionalization of a TeraHertz (THz)-based BioMEMS. THz spectroscopy is the only non-invasive analytic method able to monitor molecular events at the picosecond timescale by probing low binding energies directly. It is used here for sensing a biocatalysis reaction inside the bioMEMS microchannels. Sub-THz measurements (0.06-0.11 THz) showed that combining microfluidic microsystems technology with THz detection could be a promising alternative for label-free real-time detection of biological interactions at the microscale. Additionally, we have developed a new microchannel fabrication process using direct plasma polymerization of TMDS (TetraMethylDiSiloxane) on micropatterned surfaces. This achievement demonstrates that cold plasma processes could be used not only for functionalization purposes or surface treatment but for the 3D microfabrication as well. This highly reduces processing time and manual handling steps, which is of a great importance for further industrial scale production

Les stents coronariens sont des dispositifs médicaux, généralement fabriqués en acier inoxydable 316L, utilisés pour traiter des maladies cardiovasculaires comme l’athérosclérose. Les stents recouverts ou à relargage contrôlé de médicaments sont des solutions prometteuses pour réduire les phénomènes de resténose. Ce travail a pour objectif le développement d’un procédé plasma basse pression capable de déposer une couche de polymère permettant de protéger la surface des stents contre l’agressivité du milieu physiologique. L’allylamine est choisie comme précurseur moléculaire pour assurer un taux élevé de fonctions amines primaires. Ces fonctions pourront être utilisées, successivement, pour l’immobilisation de molécules bioactives afin d’augmenter la biocompatibilité des stents. Les dépôts sont effectués sur des substrats d’acier inoxydable 316L en utilisant un réacteur plasma basse pression (70 kHz). Les différentes techniques d’analyse de surface utilisées (angle de contact, XPS, FTIR-ATR) montrent que les variations de puissance de la décharge et du temps de traitement ne modifient pas significativement la composition chimique de surface des dépôts. Cependant, grâce à une technique de dérivation chimique nous avons mis en évidence une meilleure sélectivité vis-à-vis des fonctions amines primaires pour les couches déposées à faibles valeurs de puissance. En effet, des analyses in-situ de la phase plasmagène (spectrométrie de masse, spectroscopie d’émission optique) révèlent qu’une augmentation de la puissance de la décharge conduit à l’augmentation de son caractère énergétique et, ainsi, à l’augmentation du taux de fragmentation du précurseur. La stabilité des revêtements au lavage dans l’eau de-ionisée a été aussi évaluée. Les dépôts obtenus pour une puissance de la décharge de 2W présentent le meilleur compromis entre rétention des fonctions amines primaires et stabilité. Enfin, nous avons évalué les propriétés d’adhérence des couches après déformation plastique en utilisant le « small punch test », permettant de reproduire les conditions qu’on retrouve lorsque les stents sont déployés dans les artères. Les dépôts présentent des propriétés adéquates de cohésion et d’adhérence au substrat pour répondre à la déformation sans se fissurer et/ou délaminer. Ces résultats montrent que les couches d’allylamine déposées par procédé plasma basse pression présentent des caractéristiques prometteuses afin d’être utilisées comme revêtement performant pour les stents coronariens.
Coronary stents are metallic devices, mainly made of 316L stainless steel (316L SS) used for the treatment of cardiovascular disease such as atherosclerosis. In order to reduce the restenosis rate of bare metal stents, coated stents and drug eluting stents were developed. The aim of this study is to develop a process to isolate metallic surface from the biological environment by depositing a thin plasma polymerized allylamine (PPAA) film on the metallic surface. Allylamine has been chosen as molecular precursor to insure high retention of primary amino groups which can be used, afterwards, to graft biomolecules to improve the biocompatibility of the devices. PPAA films were deposited on flat electropolished 316L SS samples in a low pressure plasma reactor (70 kHz). The different surface analytical methods (water contact angle, XPS, FTIR-ATR) showed that surface chemical composition of the coatings was not significantly influenced by variation of plasma power discharge and treatment time. However, chemical derivatization has shown that high selectivity towards primary amino-groups could be obtained using low discharge power values. In fact, in-situ diagnostic analysis of the plasma discharge, performed by mass spectrometry and optical emission spectroscopy, revealed the increase of the energetic character of the discharge as a function of discharge power that leads to higher fragmentation of the precursor. The coating stability in de-ionised (D.I.) water has been also investigated. We have found an optimum of stability for films deposited at a power of 2 W. For this optimized condition, we have the best trade-off between selectivity and stability upon immersion in D.I. water. In order to mimic stent expansion conditions, a “small punch test” has been used to investigate the adhesive properties of the coating. According to XPS analysis, no significative modification of the chemical composition of the coating was induced by plastic deformation. No cracks, delamination or failures of the coating were observed by FE-SEM indicating that the coating presents sufficient interfacial adhesion and cohesion to resist to plastic deformation. For these reasons, PPAA films presents promising features to be applied as a coating for coronary stents.

The present research involves two projects: a surface science study of the room-temperature adsorption and thermal evolution of allylamine and ethanolamine on Si(100)2×1, studied by using temperature-dependent X-ray photoelectron spectroscopy (XPS) and thermal desorption spectrometry (TDS), as well as Density Functional Theory (DFT) calculations; and a materials science study on the shape control of copper nanoparticles (Cu NPs) deposited on H-terminated Si(100) substrate with an extended size regime of 5-400 nm, by using a simple, one-step electrochemical method. The Cu NPs of three primary shapes were characterized with scanning-electron microscopy (SEM), glancing-incidence X-ray diffraction (GIXRD) and XPS. In the first surface science study, the presence of broad N 1s XPS features at 398.9-399.1 eV, corresponding to N–Si bonds, indicates N–H dissociative adsorption for both allylamine and ethanolamine on Si(100)2×1. For allylamine, the presence of C 1s features at 284.6 eV and 286.2 eV, corresponding to C=C and C−N, respectively, and the absence of the Si−C feature expected at 283.5 eV show that the reactions involving the ethenyl group such as the [2+2] C=C cycloaddition or those producing the [N, C, C] tridentate adstructures do not occur at room temperature. For ethanolamine, the O 1s feature at 533.1 eV indicates the formation of Si−O bond and O−H dissociation, which confirms an [O, N] bidentate adstructure and excludes the N−H and O−H dissociation unidentate structures. These XPS data are consistent with the N−H unidentate, and N−H and O−H double dissociation [O, N] bidentate adstructures for allylamine and ethanolamine, respectively, as predicted by the DFT calculations. TDS and temperature-dependent XPS data further show the desorption of propene and ethylene at 580 K and of acetylene at 700 K for allylamine and the desorption of ethylene at 615 K for ethanolamine, while the lack of N- or O-containing desorbates suggests that the dissociated N and O species are likely bonded to multiple surface Si atoms or diffused into the bulk at elevated temperatures (as confirmed by the corresponding temperature-dependent XPS spectra). Unlike the multidentate allyl alcohol and allylamine adstructures that have been found to be not favored kinetically, the present [O, N] bidentate ethanolamine adstructure appears to be kinetically favored on Si(100)2×1. In the second materials science study, Cu NPs of three primary shapes have been deposited on H-terminated Si(100) by a simple, one-step electrochemical method. By precisely manipulating the electrolyte concentration [CuSO4.5H2O] below their respective critical values, cubic, cuboctahedral, and octahedral Cu NPs of ranges of average sizes and number densities can be easily obtained by varying the deposition time. Combined GIXRD and depth-profiling XPS studies show that these Cu NPs have a crystalline core-shell structure, with a face-centered cubic metallic Cu core and a simple cubic Cu2O shell with a CuO outerlayer. The shape control of Cu NPs can be understood in terms of a progressive growth model under different kinetic conditions as dictated by different [CuSO4.5H2O] concentration regimes. The two studies in the present work lay the foundation for future investigation of surface biofunctionalization of these fascinating Cu NPs with different shapes and therefore different surface chemistries as controlled by the relative amounts of the (100) and (111) facets, and their boundaries.

W ramach rozprawy doktorskiej zbadano oddziaływania polielektrolitów, pochodnych poli(chlorowodorku alliloaminy) (PAH), z błonami lipidowymi oraz komórkowymi. Oddziaływania pomiędzy polikationami, a błonami komórkowymi odgrywają istotną rolę w wielu zastosowaniach biofizycznych. Do najbardziej istotnych należy zaliczyć: dostarczanie materiału genetycznego do komórek w celowany i bezpieczny sposób, użycie polikationów jako czynników biobójczych, czy uzyskiwanie układów liposomowych stabilizowanych wielowarstwami polimerowymi. W pierwszym etapie badań przeprowadzono modyfikacje chemiczne PAH celem uzyskania pochodnych o pożądanych właściwościach fizykochemicznych i biologicznych. W tym celu wykonano syntezy pochodnych polikationowych, które różniły się: gęstością ładunku, pozycją naładowanych grup amoniowych w stosunku do głównego łańcucha polimerowego, brakiem lub obecnością ugrupowań hydrofobowych oraz długością łańcucha alkilowego obecnego w podstawniku hydrofobowm. Ponadto zsyntetyzowano pochodną polianionową - N-sulfonowany PAH (PASulf). Otrzymane pochodne, jako mocne polielektrolity charakteryzowały się stabilnymi właściwościami w roztworach o różnym pH oraz sile jonowej. Dowiedziono, że biologiczna aktywność badanych polimerów (bakteriostatyczność, bakteriobójczość, cytotoksyczność) jest silnie zależna od struktury przestrzennej łańcucha polimerowego, głównie od lokalizacji naładowanych dodatnio grup amoniowych w stosunku do łańcucha głównego, a także od obecności ugrupowań hydrofobowych. Gęstość ładunku w strukturze polimeru jest jednak mniej istotnym czynnikiem. Różnice w toksyczności pochodnych PAH względem bakterii (E. coli i S. aureus) i fibroblastów skóry ludzkiej (HSF) są silnie związane z naturą ich oddziaływań ze ścianą bakterii i błoną komórek. Następnie przeprowadzono charakterystykę uzyskanych pochodnych PAH w postaci sporządzonych z nich filmów wielowarstwowych. Na podstawie otrzymanych wyników stwierdzono, że grubość filmów polimerowych otrzymywanych metodą warstwa po warstwie silnie zależy od gęstości ładunków polimerowych oraz od ich lokalizacji w stosunku do głównego łańcucha polimerowego. Wielowarstwy o parzystych liczbach warstw, czyli te zakończone PASulf charakteryzowały się większą hydrofilowością, w porównaniu z układami gdy warstwę terminalną tworzył polikation. Zbadano proliferację i ruchliwość komórek HSF na powierzchniach filmów. Z badań wynika, że gęstość ładunku w łańcuchu polimerowym jest mniej istotna z punktu widzenia ruchliwości komórek. Ma ona jednak duży wpływ na proliferację komórek na warstwach polimerowych, gdzie korzystniejsze jest zmniejszenie zawartości 4o grup amoniowych. Na podstawie parametrów fizykochemicznych można stwierdzić, iż najistotniejszy wpływ na zdolność adhezji komórek miał potencjał zeta warstwy terminalnej filmów, a także struktura chemiczna użytego polikationu, mająca zasadniczy wpływ na organizację kolejnych warstw. Komórki chętnie adherowały i proliferowały na warstwach o ujemnym ładunku powierzchniowym filmu tworzącego najbardziej zewnętrzną, hydrofilową powierzchnię. Celem zbadania oddziaływań polielektrolitów z dwuwarstwą liposomową (obojętną i ujemnie naładowaną), przeprowadzono szereg eksperymentów pokrywania błony lipidowej przez badane polimery. Przeprowadzono badania optymalizacji liczby warstw polielektrolitów optymalnych do pokrycia liposomów. Ponadto, zbadano stabilności oraz przepuszczalność błon poddanych działaniu polimerów i surowicy krwi. Stwierdzono, iż bardziej stabilne układy otrzymuje się po pokryciu tylko jedną warstwą polimeru. Wykazano, że polimery amfifilowe mają zdolność szybkiego i łatwego wbudowywania się w błony lipidowe przy zachowaniu ich morfologii. Badania te znajdują swoje potwierdzenie w wykonanych symulacjach dynamiki molekularnej. Badania wykazały, że pojedyncza warstwa polikationów osadzona na anionowych liposomach powoduje w pewnym stopniu stabilizację pęcherzyków przed niszczącym działaniem surowicy. Jednocześnie, wyniki pokazały, że struktura chemiczna polimeru ma kluczowy wpływ na zmniejszenie oddziaływań między anionowymi liposomami, a składnikami surowicy. Istotnym zdaje się być optymalne rozmieszczenie ładunku dodatniego w stosunku do szkieletu polimeru. Ostatnim krokiem było zbadanie użyteczność pochodnych PAH (ogólnie określonych PAG) jako nośników materiału genetycznego. Przy pomocy niezbyt skomplikowanych modyfikacji chemicznych otrzymano nowe pochodne PAH o różnym stopniu zmetylowania i podstawienia chlorkim glicydylotrimetyloamoniowym (GTMAC). Polimery PAG wykazały różną pojemność buforową, ściśle zależną od obecności różnorzędowych grup aminowych. Ponadto, PAG efektywnie kompleksowały DNA, co prowadziło do otrzymania stabilnych polipleksów. Polimery te wykazały bardzo dobrą efektywność transfekcji komórek przy całkowitym braku toksyczności. Dodatkowo stwierdzono, że otrzymane polipleksy wnikają do komórek poprzez endosomy. Otrzymane wyniki mogą posłużyć do rozwijania nowych nośników dla wydajnej i bezpiecznej transfekcji.
The main objective of the dissertation was to examine the interactions of new derivatives of poly(allylamine hydrochloride) (PAH) polymer with lipid membranes of model systems such as liposomes and with the native plasma membrane of cells. The interaction between polycations and cell membrane is a fundamental issue in cell biophysics and plays an important role in different biological applications in particular: delivery of genetic material into cells in a targeted and safe manner, the use of polycations as biocidal agents, or obtaining stabilized vesicles by covering liposome surface with multilayer films. In the first stage of the study, chemical modifications of PAH were carried out to obtain derivatives with desired physicochemical and biological properties. The performed synthesis of new polycations, resulted in products, which differed in: charge density, position of the ammonium groups in relation to the polymer chain, presence or absence in the polymer structure hydrophobic groups and length of the alkil groups of the hydrophobic substituents. In addition, one polyanionic derivative was synthesized - N-sulfonated PAH (PASulf). All of the obtained derivatives, as strong polyelectrolytes, were characterized by stable properties in solutions at different pH and ionic strength. Furthermore, biological activity of the tested polymers i.e. antibacterial, bactericidal and cytotoxicity strongly depended on the spatial structure of the polymer, especially on the location of the positively charged ammonium groups in relation to the polymer backbone, as well as the presence of hydrophobic moieties. On the other hand, charge density in the polymer structure turned out to be a less important factor. The obtained differences in toxicity of PAH derivatives against two pathogenic bacteria (E. coli and S. aureus) and human skin fibroblasts (HSF) were strongly related to the nature of their interactions with the bacteria wall and cells membrane. In the next stage of the work characteristics of multilayer films composed from all of the polymer derivatives were carried out. It was found that the thickness of polymer films strongly depended on the charge density of the polymer and its location in relation to the main polymer chain. Multilayers with even number of layers, namely these with PASulf as a terminal layer, were characterized by higher hydrophilicity, when compared to systems in which the terminal was formed by polycations. Subsequently, cell proliferation and motility were examined on the film surfaces. The obtained results demonstrate that the charge density in the polymer chain did not have a significant impact on cell motility. On the other hand, it had a major impact on cell. Based on physico-chemical characteristic of the polymers it was concluded that the zeta potential of the terminal film layer played a key role in the cells adhesive abilities. Moreover, chemical structure of the polycations had a significant impact on the organization of the subsequent layers. The cultured cells were more adherent and revealed higher levels of proliferation on hydrophilic polymeric films with negative charge of the terminal layer. To explore the interactions of polyelectrolytes with the liposome bilayer (neutral and negatively charged), a number of experiments by the lipid membrane coating with polymers were carried out. Optimization studies were performed to cover the liposomes with appropriate amount of polyelectrolytes. Furthermore, the stability and permeability of the polymer-coated membranes in the presence of blood serum were examined. It was found that only one polymer layer is required to obtain the most stable systems. It has been shown that the amphiphilic polymers were capable of quick and easy incorporation into lipid membranes, while maintaining their morphology. These studies were confirmed by molecular dynamics simulations. Results showed that polycation single layer deposited on the anionic liposomes causing a certain degree of stabilization of the vesicles against the destructive effects of the serum. Simultaneously, the results indicated that the chemical structure of the polymer had a significant impact on the reduction of interactions between the anionic liposomes and serum components. It seems that the most important was the optimal location in relation to the main polymer chain. The final step was to examine transfection abilities of the new PAH derivatives (generally named as PAGs) as carriers of genetic material. Using relatively simple chemical modifications, novel PAH derivatives, obtained with varying degrees of methylation, and substitution by glicydyltrimethyloammonium chloride (GTMAC). PAG polymers showed different buffering capacity, strictly dependent on the balanced of the order of amino groups. All the derivatives showed the ability to complex pDNA into stable polyplexes. These polymers had very good transfection efficiency, while showing no toxicity against the examined cells. The polyplexes were routed via early and late endosomes. The obtained results can be used to develop new carriers for efficient and safe transfection.

The main objective of this Thesis was to encapsulate single viable cells within polyelectrolyte films using the Layer-by-Layer (LbL) technique. Most of the experiments used human mesenchymal stem cells (MSCs) whose characteristics (capacity of selfrenewal and potential to differentiate into several types of cells) make them particularly interesting to be used in biomedical applications. Also, most of the experiments used alginate (ALG) as the anionic polyelectrolyte and chitosan (CHI) or poly(allylamine hydrochloride) (PAH) as the cationic polyelectrolyte. Hyaluronic acid (HA) was also tested as an anionic polyelectrolyte. At the beginning of the work, the experimental conditions necessary to obtain the encapsulation of individual cells were studied and established. Through fluorescence microscopy visualization by staining the cell nucleus and using polyelectrolytes conjugated to fluorescent dyes, it was possible to prove the obtainment of capsules containing one single cell inside. Capsules aggregation was an observed problem which, despite the efforts to design an experimental process to avoid this situation (namely, by playing with cell concentration and different means of re-suspending and stirring the cells), was not completely overcome. In a second part of the project, single cells were encapsulated within polyelectrolyte layers made of CHI/ALG, PAH/ALG and PAH/HA and their viability was evaluated through the resazurin reduction assay and the Live/Dead assay. In these experiments, during the LbL process, polyelectrolyte solutions were used at a concentration of 1mg/mL based on literature. In general, the viability of the encapsulated cells was shown to be very low/absent. Then, as a consequence of the lack of viability of cells encapsulated within polyelectrolyte layers, the LbL technique was applied in cells growing adherent to the surface of cell culture plates. The cells were cultured like in a sandwich, between the surface of the cell culture dish and the polyelectrolyte layers. Also here, the polyelectrolyte solutions were used at a concentration of 1mg/mL during the LbL process. Surprisingly, cell viability was also absent in these systems. A systematic study (dose-effect study) was performed to evaluate the effect of the concentration of the individual polyelectrolytes (ALG, CHI and PAH were studied) in cell viability. Experiments were performed using cells growing adherent to the surface of cell culture plates. The results pointed out that a very high (cytotoxic) concentration of polyelectrolytes had been in use. Also, in general, PAH was much more cytotoxic than CHI, whereas ALG was the less cytotoxic polyelectrolyte. Finally, using alginate and chitosan solutions with adequate concentrations (low concentrations: 50ng/mL and 1μg/mL), the encapsulation of single viable cells was again attempted. Once again, the encapsulated cells were not shown to be viable. In conclusion, the viability of the encapsulated cells is not only dependent on the cytotoxic characteristics (or combined cytotoxic characteristics) of the polyelectrolytes but it seems that, when detached from the culture plates, the cells become too fragile and lose their viability very easily.
Universidade da Madeira