Hsp90 (heat shock protein 90) est une protéine chaperonne ubiquitaire et conservée impliquée dans le repliement et la réparation de protéines dites « clientes ». Parmi ces protéines, de nombreuses sont impliquées dans des phénomènes oncogéniques, faisant de hsp90 une cible d’intérêt dans le traitement du cancer. Hsp90 est constituée de trois domaines, un domaine N-terminal site de l’hydrolyse de l’ATP, nécessaire à sa fonction ; un domaine intermédiaire où vient se fixer la protéine cliente et un domaine C-terminal impliqué dans la dimérisation, étape indispensable pour le repliement de la protéine cliente. De nombreux inhibiteurs ont été synthétisés en ciblant ces différents domaines. Les inhibiteurs N-terminaux sont efficaces, à l’instar de la Geldanamycine en termes d’activité anti-tumorale mais des effets secondaires ainsi que des résistances au traitement ont limité leur utilisation en pratique clinique. En effet, l’inhibition N-terminale induit une réponse au stress caractérisée par une augmentation de hsp90 et de ses co-chaperonnes, souvent associée à une résistance au traitement et un pronostic défavorable. La novobiocine, un antibiotique coumarinique, est capable d’inhiber le domaine C-terminal de hsp90, sans induire de réponse au stress. Ainsi de nombreux dérivés de cette molécule ont été synthétisés, parmi lesquels on trouve le 6BrCaQ. Cette molécule induit l’apoptose et le blocage dans le cycle cellulaire sur plusieurs lignées cellulaires (dont MCF-7 et MDA-MB-231) et provoque la dégradation de plusieurs protéines clientes impliquées dans le développement tumoral mais sa faible solubilité limite son administration in vivo.Dans cette thèse, une forme liposomale du 6BrCaQ a été développée et étudiée sur des lignées cellulaires de cancer de prostate, de sein et de leucémie aigüe myéloïde in vitro et in vivo sur un modèle orthotopique de cancer du sein (MDA-MB-231 luc-GFP). Le 6BrCaQ liposomal est capable d’ induire de l’apoptose, de bloquer le cycle cellulaire sur différentes lignées cellulaires (PC-3, MDA-MB-231 et MOLM-13) mais également de ralentir la migration cellulaire sur PC-3 (test de comblement de blessure). De plus, le 6BrCaQ liposomal entre en synergie avec la doxorubicine (cellules PC-3) et la daunorubicine (cellules MOLM-13). Au niveau moléculaire, les liposomes de 6BrCaQ modifient l’expression protéique de Hsp90 sans modifier celle d’Hsp70 sur PC-3 alors que les gènes codant pour les Hsp70 semblent être légèrement induits dans MDA-MB-231. Les résultats in vivo ont montré un ralentissement de la croissance tumorale sur un modèle de cancer du sein orthotopique (MDA-MB-231-luc2-GFP) dès 13 jours de traitement pour une dose de 1 mg/kg injectée une fois par semaine. Des analyses histologiques ont révélé une augmentation de la proportion des zones nécrotiques dans le groupe traité par rapport au contrôle et une diminution significative de la prolifération cellulaire (marquage au KI67) intra-tumorale.Par ailleurs, Hsp90 possède également des isoformes et des analogues localisés dans des organites intracellulaires, parmi lesquelles, TRAP-1, localisée au niveau de la mitochondrie, impliquée dans la rgulation du métabolisme mitochondrial et qui pourrait jouer un rôle dans la progression tumorale et les métastases. Le déqualinium (DQ) est capable de cibler la mitochondrie. Dans une seconde partie du travail, des liposomes encapsulant le DQ ont été formulés dans le but de vectoriser le 6BrCaQ vers la mitochondrie. Toutefois, face à la difficulté d’encapsuler le DQ dans des liposomes, une étude de physico-chimie sur l’interaction DQ/liposomes a été mise en place pour comprendre comment le DQ agit sur les bicouches phospholipidiques. Cette étude a révélé que, malgré une capacité de ciblage mitochondrial des liposomes, le DQ était difficile à encapsuler dans les milieux salins et n’était pas inerte sur les bicouches lipidiques ce qui limite son utilisation pour la formulation de liposomes ciblant la mitochondrie. / Hsp90 (Heat shock protein 90) is an ubiquitous and well-conserved chaperone protein involved in the folding and the repair of « client » proteins. Among these proteins, several are involved in oncogenic phenomena making hsp90 an interesting target for cancer therapy. Hsp90 consists of three domains ; a N-terminal domain as the ATP hydrolysis site ; a middle domain where the client proteins binds and a C-terminal domain involved in the dimerization, a necessary step to refold the client protein. Several inhibitors were synthesized to target these different domains. N-terminal inhibitors such as Geldanamycin ; were shown to be very efficient but side effects and resistance to the treatment limited their clinical use. Indeed, N-terminal inhibition induces a stress response characterized by an increase of hsp90 and its co-chaperones which is often associated with resistance to the treatment and poor prognosis. Novobiocin, a coumarin antibiotic, is capable of inhibiting the C-terminal domain of hsp90, without inducing a stress response. Several derivatives of this molecule have been synthesized, including 6BrCaQ. The latter was effective in terms of apoptosis induction and cell cycle blockade on several cell lines (MCF-7, MDA-MB-231) and induced pro-tumoral client protein degradation but its low solubility limits its in vivo administration. In this thesis, a liposomal formulation of 6BrCaQ has been developed and studied on in prostate cancer cell lines and acute myelogenous leukemia in vitro and in vivo in an orthotopic model of breast cancer (MDA-MB-231 luc-GFP). Liposomal 6BrCaQ showed ability to induce apoptosis, to block the cell cycle on several cell lines (PC-3 and MDA-MB-231) and slow down migration of PC-3 cells (wound healing assay). Liposomal 6BrCaQ is able to synergize with doxorubicine or daunorubicine in PC-3 cells and in MOLM-13 cells, respectively. Moreover, protein and RNA expression profiles show that in PC-3 cells liposomal 6BrCaQ downregulates Hsp90 protein and in MDA-MB-231 cells slightly upregulates Hsp70 gene expression. Results obtained during in vivo experiments on the breast orthotopic model revealed a slow downslowdown of tumor growth after 13 days for a dose of 1 mg/kg injected weekly. Histological analysis revealed necrosis in treated groups and aassociated with a decreased cell proliferation (ki67 staining).Hsp90 also has isoforms and analogues localized in intracellular organelles, including, TRAP-1, localized in the mitochondrion and probably implicated in malignant progression through its role in the regulation of the mitochondrial metabolism. Dequalinium (DQ) demonstrated ability to target the mitochondrion. In a second part of the work, liposomes encapsulating DQ have been formulated in order to target 6BrCaQ to mitochondria. However, faced with the difficulty of encapsulating the DQ in liposomes, a physical chemistry study on the interaction DQ / liposomes was established to understand how DQ acts on phospholipid bilayers. Though a mitochondrial targeting capacity, this study revealed DQ was difficult to encapsulate in liposomes in saline medium and not completely inert on lipid bilayers limiting its use to target liposomes to mitochondria.

This diploma thesis focuses on the preparation of the liposomes, containing lipids with gadolinium, which are used for a contrast magnetic resonance imaging. The liposomes were prepared by the lipid film hydration followed by an extrusion and also by a new nanofluid mixing method on the NanoAssemblr Benchtop. The preparation technology has been optimized for parameters such as the composition of lipids, the flow rate ratio and total flow rate. The method of modification of the liposomes surface by gadolinium complexes has been developed. This method is using a conjugation reaction between the lipids containing cyanuric acid and Gd-DOTA. Prepared Gd-liposomes, which contain gadolinium, were complexly defined by the characterization techniques of DLS and NTA. The morphology of liposomes was observed by TEM and cryo-TEM. Methods for the determination of phospholipid content (Stewart test) and residual water in the lyophylisates of liposomes (Karl-Fischer titration) were used. Gadolinium in liposomal preparations was determined by ICP-OES. Using MR, the concept of gadolinium liposomes was verified and designed for MRI imaging of thrombi. The concept describing the mechanism of liposomes formation based on the experimentally proven existence of a phospholipid bilayer fragment has been developed. This concept is based on the experimentally proven existence of a phospholipid bilayer fragment.

Une nouvelle génération d’assemblages nano-organisés, constitués de couches lipidiques enveloppant des cœurs particulaires sphériques, a été développée ces dernières années. Un des nombreux intérêts de cette génération d’assemblages, à structure bio-inspirée, est de constituer in fine des vecteurs ou réservoirs pour des applications biomédicales de délivrance contrôlée de principes actifs. Ce type d’architecture originale est obtenu par réorganisation de membranes lipidiques à la surface de nanoparticules de polymère. Au cours de ce travail, la sélection du support colloïdal s’est portée sur des nanoparticules de chitosane, un biopolymère aux propriétés physico-chimiques et biologiques remarquables. Ces nanoparticules ont été élaborées par une méthode dite de « gélification ionique » à l’aide de tripolyphosphate de sodium. L’optimisation de ce procédé de synthèse a permis l’obtention de nanoparticules cationiques et sphériques, de taille reproductible d’une centaine de nanomètres, et de distribution en taille étroite. Aucune modification de ces caractéristiques de taille n'a été montrée durant au moins 4 mois à 23°C dans l'eau. Quant aux membranes lipidiques, il a été choisi d’élaborer des vésicules ou des nano-disques modèles et anioniques de façon à présenter une charge électrostatique opposée à celle des surfaces particulaires. La caractérisation de ces entités en termes de taille, distribution en taille, charge de surface et morphologie a été effectuée par diffusion quasi-élastique de la lumière, microscopie électronique à transmission, diffusion de neutrons aux petits angles et zêtamétrie. L’influence de différents paramètres physico-chimiques impliqués dans la formation des assemblages (i.e., force ionique et pH de la phase continue, rapport lipides/nanoparticules) a été examinée, permettant d’une part, une meilleure compréhension des interactions s’établissant entre les chaînes de chitosane et les membranes lipidiques, et d’autre part, l’obtention d’assemblages de taille et de distribution en taille satisfaisantes. Une étude de l’incorporation de différentes molécules thérapeutiques (isoniazide, ibuprofène et rose bengale) dans le support particulaire a ensuite été réalisée afin d’évaluer le potentiel des nanoparticules de chitosane, puis des assemblages lipidiques en tant que vecteurs de principes actifs. Des études préliminaires ont ainsi pu mettre en évidence que ce recouvrement surfacique lipidique apportait une modification satisfaisante du profil de relargage du principe actif. / A novel generation of nano-organized assemblies, composed of lipid layers surrounding spherical nanoparticle cores, has been recently developed for drug delivery applications. This original architecture results of the lipid membrane reorganization onto colloidal polymer surfaces. In the present study, the colloidal supports are constituted of chitosan nano-hydrogels, a biopolymer with exceptional physicochemical and biological properties. These colloidal supports have been elaborated via an ionic gelation process using sodium tripolyphosphate as gelation agent. The optimization of all the parameters involved in this process has led to cationic spherical nanoparticles, with reproducible sizes in nanometer range, and narrow size distributions. No alteration of these characteristics has been observed for at least 4 months in water at 23 °C. Regarding the lipid membranes, two different morphologies (i.e., vesicle and nanodisc) have been prepared. Lipid formulation has been designed to obtain objects with a negative surface charge, opposed to chitosan nanoparticle one. These entities were characterized in terms of size, size distribution, surface charge and morphology using dynamic light scattering, transmission electron microscopy as well as small angle neutron scattering. The assembly of colloidal supports and lipid membranes has been investigated by studying the influence of the different physicochemical parameters involved in the synthesis. During this study, phenomena involved in the adsorption process were pointed out, and experimental conditions were optimized in order to control the assembly elaboration process. Finally, a study on drug incorporation (isoniazid, ibuprofen and rose bengale) into chitosan nanoparticles has been carried out. Preliminary experiments on drug release from nanoparticles and assemblies revealed a promising effect of the surface modification of assemblies on their drug release profile.

Small molecule synthesis has become a valuable tool in the study of biological systems. Biologically active compounds can be designed based on well-characterized endogenous systems or they can be found through the screening of large libraries of small molecules. This work involves the development of a small library of cyclic thiourea-based small molecules by use of an unreported synthetic pathway. Briefly, parent thioureas were cyclized by reaction with bromoacetyl bromide, and one or two isomeric heterocycles were found to form. Further studies indicated that the reaction could be easily manipulated by temperature or solvent to effectively control the product distribution. These iminothiozolidinones were characterized by single crystal x-ray analysis. The new reaction was explored in an effort to uncover the factors influencing the control of the isomer formation. Furthermore, these iminothiozolidinones underwent a novel decyclization reaction that resulted in the loss of the parent thiourea connectivity and incorporation of an external nucleophile to yield an aminooxoethylcarbamothionate. The reaction proceeds through a termolecular mechanism. These reactions can be combined to a one-pot reaction series. These compounds share similarities with a class of compounds reported to be known HIV-1 reverse transcriptase inhibitors94. In addition to these new synthetic reactions, work was conducted with a previously developed cyclen thiourea receptor for the anionic dye HPTS and its derivatives50-52. This system was used to develop a cell labeling assay that led to the amplification of fluorescent labeling of target cells through the use of liposomes. Briefly, a dye-ligand conjugate for the glycine receptor was synthesized. Liposomes functionalized with the cyclen receptor were prepared encapsulating Rhodamine B. Confocal microscopy studies demonstrated the binding of the HPTS-ligand to the cell membranes. Addition of the liposomes resulted in quenching of the green fluorescence, indicating binding of the cyclen to HPTS. Subsequent excitation of Rhodamine B showed red fluorescence associated with the cells. The intensity of the red signal was demonstrably higher than for the signal resulting from the binding of the ligand-dye to the receptor. Together, these projects increase the synthetic usefulness of thiourea based small molecules and demonstrate the potential biological applications of related compounds.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Nous avons optimisé des liposomes thermosensibles, encapsulant un principe actif anticancéreux, le 5-Fluorouracile (5-FU), afin de déclencher sa libération par une hyperthermie locale modérée (39-42°C) induite par des ultrasons focalisés. L'hyperthermie sera appliquée au niveau de la tumeur, afin d'améliorer l’efficacité thérapeutique et de réduire la toxicité systémique. Les liposomes ont été formulés par hydratation du film lipidique en mélangeant la 1,2-dipalmitoyl-sn-glycéro-3-phosphocholine (DPPC) pour sa thermosensibilité à 41,5 ± 0,5°C, le cholestérol (CHOL) pour favoriser la stabilité des liposomes vis-à-vis des composants du sang, et le 1,2-distéaroyl-sn-glycéro-3-phosphoéthanolamine-N-[méthoxy(polyéthylène glycol)-2000] (DSPE-PEG) pour assurer la furtivité de la formulation. Les expériences ont confirmé que les liposomes formulés à base de DPPC/CHOL/DSPE-PEG dans un ratio molaire 90 : 5 : 5 mol% sont thermosensibles. Des liposomes composés du même mélange lipidique dans un rapport 65 : 30 : 5 mol% ont été considérés comme contrôle négatif non thermosensible. L’optimisation de l’encapsulation passive du 5-FU a permis d’obtenir une efficacité d’encapsulation (5-FU encapsulé/5-FU total) de 13%, mais le 5-FU est très faiblement retenu (12%) dans la cavité aqueuse des liposomes du fait du gradient osmotique à la dilution. La rétention du 5-FU a été optimisée (93%) par la technique d’encapsulation active basée sur la complexation intraliposomale du 5-FU avec le complexe cuivre-polyéthylèneimine préalablement encapsulé dans les liposomes. Cette technique a également permis d’améliorer l’efficacité d’encapsulation d’un facteur trois environ (37%), avec un taux de charge (ratio final 5-FU/lipides, mole/mole) de 50% environ. Nous avons alors obtenu des liposomes thermosensibles d'un diamètre hydrodynamique de 65 nm et de charge de surface de -10 mV. Les liposomes non thermosensibles, ont été caractérisés par un diamètre hydrodynamique de 105 nm et une charge de surface de -4,9 mV. La libération du 5-FU déclenchée par une hyperthermie induite par des ultrasons focalisés a été mesurée in vitro. En réponse à une hyperthermie de 42°C, les liposomes thermosensibles libèrent 68% de leur contenu, au bout de 10 min, alors que les liposomes non thermosensibles en libèrent moins de 20%. En outre, la cytotoxicité des liposomes encapsulant le complexe 5-FU-cuivre-polyéthylèneimine a été évaluée vis-à-vis de la lignée cellulaire HT-29 du carcinome colorectal humain. Les résultats ont révélé que les lipides à une concentration de 800 µM ne sont pas cytotoxiques (80% de viabilité). De plus, la complexation du 5-FU n’influence pas sa cytotoxicité ce qui prouve que la toxicité provient du 5-FU et non des excipients. En revanche, l’encapsulation du complexe 5-FU-cuivre-polyéthylèneimine dans les liposomes induit une diminution de la concentration inhibitrice médiane de 115 (solution du complexe) à 49 µM environ, corrélée à leur internalisation cellulaire. La pharmacocinétique chez des souris porteuses d’un modèle de tumeur colorectale HT-29 xénogreffée a montré que les liposomes permettent de prolonger d’un facteur 1,4 la demi-vie plasmatique de distribution du 5-FU. De plus, les aires sous la courbe des concentrations plasmatiques sur 24 h sont 1,9 et 2,9 fois plus élevées lorsque le 5-FU est administré sous forme de liposomes thermosensibles et non thermosensibles, respectivement, par rapport à la solution de 5-FU. Enfin, les liposomes non thermosensibles augmentent significativement d'un facteur 2 l'accumulation du 5-FU dans la tumeur par rapport à la solution de 5-FU. En conclusion, les liposomes thermosensibles développés présentent un fort intérêt pour une application thérapeutique en combinaison avec des ultrasons focalisés. / We optimized thermosensitive liposomes encapsulating an anticancer drug, 5-Fluorouracil (5-FU), in order to trigger the release upon focused ultrasound-mediated mild hyperthermia at the tumor. This approach would improve drug efficacy and would lower side effects. Liposomes were prepared by the lipid hydration method by mixing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) for its temperature sensitivity at 41.5 ± 0.5°C, cholesterol (CHOL) to promote liposome stability towards blood components, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG) to confer stealthiness to the formulation. The experiments confirmed that the liposomes formulated with DPPC/CHOL/DSPE-PEG in a molar ratio 90:5:5 mol% are thermosensitive, while liposomes composed of the same lipid mixture in a ratio 65:30:5 mol% were considered non thermosensitive negative control. The optimization of passive encapsulation of 5-FU yielded an encapsulation efficacy (encapsulated 5-FU/total 5-FU) of 13%. 5-FU was, however, very weakly retained (12%) in the aqueous core of liposomes following dilution due to the generation of an osmotic gradient. The retention of 5-FU has been optimized (93%) by the active encapsulation technique based on the intraliposomal complexation of 5FU with copper-polyethylenimine complex encapsulated beforehand into liposomes. This technique also improved 5-FU encapsulation efficacy by 3-fold (37%), yielding a loading efficiency (final drug/lipid ratio, mol/mol) of approximately 50%. The resulting thermosensitive liposomes and non thermosensitive liposomes have a hydrodynamic diameter and a surface charge around 65 nm and -10 mV, and 105 nm and -4.9 mV, respectively. Heat-triggered drug delivery was evaluated using focused ultrasound, and showed a release of 68% of the encapsulated 5-FU from thermosensitive liposomes, within 10 min, whereas release remained below 20% for the non thermosensitive formulation. Furthermore, the cytotoxicity of 5-FU-copper-polyethylenimine complex-loaded liposomes towards HT-29 human colorectal carcinoma cell line was evaluated. Results revealed that lipids at a concentration of 800 µM are not cytotoxic (80% viability). Moreover, 5-FU complexation has no impact on its cytotoxic activity, disclosing that liposomes toxicity arose from 5-FU and not from the excipients. Nevertheless, 5-FU-copper-polyethylenimine complex-loaded liposomes exhibited a lower half maximal inhibitory concentration of 49 µM compared to 115 µM for complex solution. This enhancement of cytotoxicity was attributed to the cellular internalization of liposomes. Pharmacokinetics in mice bearing HT-29 xenograft tumor showed that liposomes can extend the plasma distribution half-life of 5-FU by a factor 1.4. Furthermore, areas under the concentration-time curve over 24 h were higher by 1.9- and 2.9-fold when the drug was encapsulated into thermosensitive and non thermosensitive liposomes, respectively, compared to free 5-Fluorouracil. Finally, non thermosensitive liposomes significantly increased 5-FU accumulation in tumor by 2-fold, compared to 5-FU solution. In conclusion, these 5-FU-loaded thermosensitive liposomes represent valuable carriers to investigate the therapeutic efficacy following focused ultrasound-mediated heat application.

As doenças alérgicas representam um problema de saúde pública mundial, sendo a rinite e a asma as doenças mais comuns. Essas doenças exigem o uso contínuo de medicamentos cujos efeitos colaterais comprometem a qualidade de vida dos alérgicos e justifica a busca por novos fármacos, ou formulações, relevantes no tratamento das alergias. Neste contexto, destaca-se a planta medicinal Mangifera indica L. cujo extrato aquoso, o Vimang®, tem sido utilizado para o tratamento de diversas patologias. O estudo fitoquímico deste extrato levou ao isolamento de vários compostos fenólicos, dentre os quais a mangiferina (Mgf) é a majoritária (~46 %) e responsável por propriedades farmacológicas importantes tal como a antialérgica. Esta molécula é metabolizada no intestino e transformada na sua aglicona, o noratiriol (Nrt), que é encontrada no plasma em maior concentração que a Mgf. Como a relação estrutura-atividade destas substâncias ainda é pouco explorada, este estudo propôs a síntese de novas moléculas, a partir do esqueleto da Mgf, com modificações estruturais em seus substituintes para avaliação, in vitro, do potencial antialérgico. Foram sintetizadas moléculas análogas da Mgf (compostos 4, 5 (Nrt) e 6), através de modificações pontuais de hidroxilas do anel xantônico, por meio de uma estratégia sintética que proporcionou maior rendimento reacional. O Nrt apresentou maior potencial antialérgico que a Mgf sugerindo que o açúcar, de fato, interfere na biodisponibilidade. A presença de hidroxilas nas posições 6 e 7 do anel xantônico (grupo catecol) é crucial para a bioatividade, pois estes substituintes influenciam consideravelmente a solubilidade da molécula. Os grupos hidroxila nas posições 1 e 3 do anel xantônico também são importantes, uma vez que a substituição da hidroxila, apenas na posição 3, inibiu totalmente a bioatividade. Como a Mgf e o Nrt (os mais bioativos) são pouco solúveis em água e susceptíveis à oxidação química, ou degradação enzimática, foi planejada a incorporação em nanocarreadores lipossomais, a fim de melhorar a biodisponibilidade. O estudo da melhor formulação foi desenvolvido para a Mgf pela maior quantidade disponível e por seu merecido destaque nas mais diversas aplicações biológicas. Os lipossomos de PC:PE:COL (52:28:20), nas concentrações lipídicas de 2,9; 14,5 e 29 mmol/L, foram preparados pelo método de hidratação do filme lipídico em diferentes razões Mgf/lipídeo (0,01; 0,02; 0,04 e 0,10), nos pHs 6,5; 7,0 e 7,4. Todas as formulações foram estáveis no período de 29 dias, com tamanhos ao redor de 100 nm e índices de polidispersividade ao redor de 0,3, apropriados para aplicação intravenosa. A formulação com maior capacidade de carga (3,4 %) foi a de 2,9 mmo/L de lipídeos totais e razão Mgf/lipídeo de 0,1 a qual foi mantida nos ensaios biológicos. A formulação lipossomal manteve o perfil de inibição da desgranulação observado para os bioativos livres. Este resultado é relevante para a aplicação biológica, uma vez que o bioativo está protegido de degradação química e ainda exerce suas propriedades farmacológicas. Como os lipossomos são sistemas que mimetizam a membrana biológica, a localização da Mgf e os efeitos na membrana foram estudados por anisotropia de fluorescência, DSC e RPE, no pH 7,4. Os resultados mostraram que a Mgf interage com a membrana lipídica, aumentado sua fluidez e diminuindo a cooperatividade lipídica, reforçando a bioatividade dessa molécula vinculada à sua ação em proteínas de membrana, o que é comum para moléculas polifenólicas. / Allergic diseases represent a worldwide public health issue, being asthma and rhinitis the most common ones. These diseases demand the continuous use of medications whose side effects impinge on the affected people´s quality of life, justifying the search for new drugs, or formulations, relevant to the treatment of allergies. In this context, we highlight the medicinal plant Mangifera indica L., whose aqueous extract, Vimang®, has been utilized for the treatment of several pathologies. The phytochemical study of this extract led to the isolation of various phenolic compounds, among which Mangiferin (Mgf) is predominant (~46 %) and responsible for important pharmacological properties, such as the antiallergic one. This molecule is metabolized in the intestine and transformed into its aglycone, norathyriol (Nrt), which is found in the plasma in a higher concentration than Mgf. Seeing as the structure-activity relationship of these substances is still little explored, this study proposed the synthesis of new molecules from the core structure of Mgf, with structural modifications in its substituent groups for in vitro evaluation of the antiallergic potential. Analogous molecules to Mgf were synthesized (compounds 4, 5 (Nrt) and 6), through specific modifications of hydroxyl groups of the xanthonic ring, by means of a synthesis strategy that allowed for a greater reactional yield. Nrt showed a higher antiallergic potential than Mgf, suggesting that sugar does in fact interfere in bioavailability. The presence of hydroxyl groups in the 6 and 7 positions of the xanthonic ring (catechol group) is crucial for bioavailability, because these substituents considerably influence the molecule´s water solubility. The hydroxyl groups in the 1 and 3 positions of the xanthonic ring are also important, seeing as the substitution of the hydroxyl group only in position 3 completely inhibited bioactivity. Seeing as Nrt and Mgf (the most bioactive) are slightly soluble in water and susceptible to chemical oxidation or enzymatic degradation, the incorporation in liposomal nanocarriers was planned to improve bioavailability. The study for the best formulation was developed for Mgf due to the greater quantity available and its well-earned spotlight in various biological applications. The liposomes of PC:PE:COL (52:28:20), in the lipid concentrations of 2.9, 14.5 and 29 mmol/L, were prepared by the lipid film hydration method in different Mgf/lipid ratios (0.01, 0.02, 0.04 and 0,10), in pHs 6.5, 7.0 and 7.4. All the formulations were stable in the period of 29 days, with sizes around 100 nm and polydispersity indexes around 0.3, appropriate for intravenous application. The formulation with the highest loading capacity (3.4 %) was the one with 2.9 mmol/L of total lipids, and a Mgf/lipid ratio of 0.1, which was used in the biological essays. The liposomal formulation maintained the antiallergic potential profiles observed for free drugs. This result is relevant for biological application since the drugs are protected from chemical degradation and still exerts its pharmacological properties. Seeing as liposomes are systems that mimic the biological membrane, the location of Mgf and its effects on the membrane were studied by fluorescence anisotropy, DSC and EPR, in pH 7.4. The results showed that Mgf interacts with the lipid membrane, increasing its fluidity and reducing the lipid cooperation, reinforcing the bioactivity of Mgf linked to its action on membrane proteins, which is common for polyphenolic molecules.

A utilização de bioativos naturais como ingredientes está em constante expansão na indústria alimentícia, devido ao aumento das exigências pelos consumidores por alimentos mais saudáveis. Por isso, há uma busca constante de tecnologias que possibilitem a incorporação de tais substâncias em alimentos. O β-caroteno é uma substância hidrofóbica, cujos benefícios estão relacionados principalmente à sua ação antioxidante. Devido à sua característica hidrofóbica, a utilização deste pigmento implica em desafios tecnológicos para ser incorporado em formulações alimentícias de base aquosa. Por este motivo, a encapsulação em lipossomas pode ser uma ótima alternativa, devido à capacidade de englobar tais substâncias em sua bicamada lipídica. Além da proteção, essas matrizes encapsulantes podem proporcionar a liberação controlada dos ingredientes encapsulados, bem como aumento de sua biodisponibilidade. O objetivo deste trabalho foi produzir e caracterizar dispersões de lipossomas encapsulando β-caroteno estabilizadas com a adição de hidrocolóides(goma xantana ou mistura de goma xantana e goma guar). O diâmetro médio hidrodinâmico, a distribuição de tamanho das partículas e a morfologia foram avaliadas. Os lipossomas produzidos foram vesículas multilamelares (MLV), as distribuições de tamanho dos lipossomas apresentaram-se heterogêneas e as micrografias revelaram a forma esférica dos lipossomas dispersos no meio aquoso, assim como a integridade da sua bicamada lipídica. Foram realizadas análises de quantificação do β-caroteno e colorimetria instrumental, sendo que todas as dispersões mostraram-se eficientes na preservação do β-caroteno ao longo do período de armazenamento. Os hidrocolóides adicionados foram eficazes no aumento da viscosidade da fase contínua, evitando a agregação das vesículas ao longo do tempo, exceto para dispersão estabilizada com a mistura de goma xantana e goma guar, com 0,15% de goma total. Em relação à adição das dispersões de lipossomas em iogurte, as formulações mostraram-se homogêneas, com ausência de grumos ou qualquer tipo de separação de fases, e também foram aprovados por uma parcela de painelistas na análise sensorial. / The use of natural bioactives as ingredients is in constant expansion in the food industry, due to increasing consumer demands for healthier foods. Therefore, there is a constant search for technologies capable of incorporating such substances in food. β-carotene is a hydrophobic substance, whose benefits are mainly related to its antioxidant action. Because of its hydrophobic characteristics, the use of this pigment implies technical challenges to be incorporated into aqueous-based food formulations. For this reason, encapsulation in liposomes may be a good alternative, because of their ability to incorporate such substances in their lipid bilayer. Besides the protection, these encapsulants matrix can provide controlled release of the encapsulated ingredients, as well as increasing its bioavailability. The objective of this study was to produce and characterize dispersions of liposomes encapsulating β-carotene, which were stabilized with the addition of hydrocolloids: xanthan gum or a mixture of xanthan gum and guar gum. The mean hydrodynamic diameter, distribution of particle size and its morphology were studied. The obtained dispersions were multilamellar vesicles (MLV), the liposomes size distributions were heterogeneous and the micrographs revealed the liposomes spherical shape dispersed in aqueous medium, as well as the integrity of their lipid bilayer. The quantification of β-carotene and instrumental colorimetry analyses indicated the liposomes were efficient in the preservation of β-carotene during the storage period. The hydrocolloids added in the dispersions were highly efficient to increase the viscosity of the continuous phase. Therefore, the hydrocolloids were responsible for the prevention of aggregation of the vesicles during the storage period, except for stabilized dispersion with the mixture of xanthan gum and guar gum, with 0.15% gum total. Regarding the dispersions of liposomes added in yoghurt, the formulations were homogeneous, with absence of lumps or any phase separation, and also have been approved by a significant number of the panelists.

Bone is the most commonly replaced organ, with nearly 1 million grafting procedures performed annually in the United States. Inherent limitations associated with bone grafts, such as graft availability and donor site morbidity, leave room for alternative grafting solutions. Current mineralized tissue engineering approaches include the use of synthetic hydroxyapatite as cement or as nano- or micro-particles pre-incorporated into a tissue engineering scaffold prior to cell seeding or implantation. While promising results have been reported with such methods, these constructs are not biomimetic as they fail to replicate neither the size, distribution, nor density of mineral inherent in the native bone, leading to inferior mechanical properties and supra-physiologic levels of calcium phosphate that can disrupt healing, alter cell response and inhibit normal tissue homeostasis. To address these issues, inspiration is taken from the native biomineralization process which is often facilitated by matrix vesicles, a lipid-based nanocarrier within which calcium and phosphate ions are combined to form calcium phosphate mineral in hard tissues such as bone. Synthetic matrix vesicles (SMV) formulated from self-assembling liposomes have emerged as a promising model both for studying the biomineralization process as it relates to matrix vesicles and for use in regenerative medicine. The ideal SMV system is defined as follows: the mineral formed should match the native calcium phosphate in both structure and chemistry, the mineral must be stable in the physiological environment and can continue to grow in size when necessary and the matrix vesicles should also be able to work in conjunction with a scaffold tailored for bone tissue engineering. It is hypothesized that the formation of native bone-like calcium phosphate can be achieved with the controlled optimization of matrix vesicles in terms of fabrication parameters, ion transport, cell response and interactions with a gelatinous matrix. To this end, a liposome-based, biomimetic matrix vesicle system was designed to facilitate vesicle-mediated biomineralization for regeneration of calcified tissues. Synthetic matrix vesicles were fabricated from two different phospholipids, DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DMPC (1,2-bis(myristoyl)-sn-glycero-3-phosphocholine) and optimized in terms of membrane composition, alkaline phosphatase bioconjugation, and ion encapsulation. Calcium (Ca2+) and phosphate (Pi) ions were successfully encapsulated within the liposomes. Ion permeability across the bi-layer membrane, which is necessary for Ca2+ and Pi to combine within the SMV for mineralization, was found to increase with increasing DMPC composition, validated through ion release studies and diffusion modeling through Fick's 2nd Law. In addition, alkaline phosphatase (ALP), an enzyme which cleaves Pi from organic phosphate molecules for mineral formation with Ca2+, was successfully conjugated to the SMV membrane through the use of biotin-functionalized phospholipids and streptavidin-ALP. Human osteoblast-like cells were dosed with the optimized SMV and the effects of SMV type and dosage on mineralization response was evaluated. Mineralization potential of human osteoblast-like cells was found to decrease through exposure to Pi-encapsulated SMV similar to the response found for human osteoblast-like cells supplemented with beta-glycerophosphate (beta-GP), an organic phosphate source typically used in mineralization in vitro studies. Human osteoblast-like cells were also dosed with two different configurations of ALP SMV liposomes with ALP bound within (ALP-inside SMV) and liposomes with ALP bound to the membrane on the outside (ALP-outside SMV). ALP-outside SMV were ultimately selected for further study since the location of the ALP in the outside configuration more closely mimics the structure of native matrix vesicles. While mineral-like structures were observed in several types of SMV under cryo-electron microscopy, no bulk mineralization was observed by human osteoblast-like cells from SMV supplementation alone. This motivated a dosage study conducted with the Pi SMV which optimized the cell-to-liposome ratio and the concentration of Pi encapsulated. The optimized ALP-outside SMV and Pi SMV were individually combined with an electrospun gelatin nanofiber scaffold to further promote cell mineral deposition by acting as a biomimetic substrate for calcium phosphate nucleation. It was demonstrated that in the absence of growth factor stiumulation, culture of human osteoblast-like cells with SMV+beta-GP and Pi SMV resulted in mineral deposition on the gelatin nanofiber scaffold. Human mesenchymal stem cells (hMSC), a more clinically relevant cell type, were also cultured on the SMV-gelatin scaffold system. Mineralization potential was found to increase for hMSC cultured with ALP SMV, and the osteogenic marker osteocalcin was upregulated for cultures with Pi SMV. Dosage of hMSC with SMV+beta-GP and Pi SMV alone resulted in the formation of a mineralized matrix. In summary, this thesis focuses on the design of a biomimetic, liposome-based synthetic matrix vesicle system and elucidates the compositional and dosage parameters for the formation of calcified tissue by human osteoblast-like cells and MSCs. The synthetic matrix vesicle system developed in this thesis can be utilized for further investigation into the mechanisms of biomineralization, in addition to its potential for use in promoting cell-mediated regeneration of a variety of calcified tissues, including bone, teeth and mineralized soft-to-hard tissue interfaces.

In considering the concept of surface-immobilised liposomes as a drug release system, two factors need to addressed, the interfacial surface density of the liposomes for maximum drug loading and the stability of these liposomes to allow for controlled drug release. This thesis investigates a multilayer system for the affinity immobilisation of liposomes and their stability to various applied stresses. In the work presented here an allylamine monomer was used to create plasma coatings that were stable, thin and amine-rich. The aging studies using AFM showed these films to rapidly oxidise on exposure to water. The freshly deposited films were used for further surface modifications, by the covalent grafting of PEG layers of different interfacial densities under the conditions of varying polymer solvation. The AFM was used to measure the interaction forces between the grafted PEG layers and modified silica interfaces. It was found that the polydispersity of the PEG species resulted in bridging interactions of ???brush???-like PEG layers with the silica surface. These interactions were screened minimised by increasing the ionic strength of the solution. Although the densely grafted PEG layers were found to be highly protein-resistant by the XPS and QCM-D some minor protein-polymer adhesions were observed by the AFM. The densely anchored biotinylated PEG chains served as an optimum affinity platform for affinity-docking of NeutrAvidinTM molecules, which assembled in a rigid, 2-D layer as confirmed by the QCM-D. The submonolayer surface density of NeutrAvidin, as determined by Europium-labelling, was attributed to steric hindrance of the immobilised molecules. The final protein layer enabled specific binding of biotin-PEG-liposomes as a highly dissipative, dense and stable layer verified by tapping mode AFM and QCM-D. We found that these liposomes were also stable under a range of stresses induced by the shearing effects of water, silica probe and HSA layer at increased loads and velocities. The frictional response of the liposome layer also demonstrated the viscoelasticity and stability of these surface immobilised liposomes. Finally, the minimal adhesive interaction forces, as measured by the AFM, demonstrated the repellency of these liposomes to commonly found proteins, such as HSA.