La formation de structures nanométriques définies dans l'eau demeure un défi pour les chimistes supramoléculaires. L'interaction entre cyclodextrines beta et adamantane a ici été utilisée pour former des polymères supramoléculaires en solution. L'utilisation d'une structure pontée nous a permis d'éviter la formation de l'espèce auto-incluse et d'augmenter la solubilité de nos dérivés. Les polymères supramoléculaires ont été caractérisés par différentes techniques (ROE, ITC, DOSY, SANS), et forment des espèces linéaires en solution allant jusqu'à 26 unités polymérisées. Celles-ci ont été utilisées pour compacter de l'ADN à des concentrations basses en mettant à profit l'association entre monomères. Un deuxième mécanisme, reposant sur des interactions non spécifiques entre cyclodextrines, a aussi été observé. Enfin, les structures synthétisées ont été utilisées dans la transfection d'ADN plasmidique, mais ne sont pas efficaces. Ce comportement pourrait provenir de leur faible densité de charge. / The synthesis of defined nanometric structures in water remains a challenge for supramolecular chemists. The interaction between adamantane and beta cyclodextrin was thus used to build new supramolecular polymers in solution. The use of a bridged structure enabled us to suppress the self-inclusion phenomenon and to enchance the solubility of our compounds. Supramolecular polymers were characterised using several techniques (ROE, ITC, DOSY, SANS), forming linear species up to 26 polymerised units. These structures were used to condense DNA at low concentrations, taking advantage on their host-guest behavior. Another condensation mechanism was discovered, involving non-specific interactions between cyclodextrins. The same structures were used to transfect plasmidic DNA, but were inefficient. This could be due to their low charge density.

Ce travail s'articule autour de deux grandes parties : i- la synthèse de nouveaux tripodes tris-A,C,E-alpha-cyclodextrine, et l'étude de leurs complexes de coordination avec les métaux. La fonctionnalisation des bis-hétérocycles est une étape importante dans la préparation de ces tripodes moléculaires. En conséquence, la mise au point d'une nouvelle famille de réactifs, les tétrahalogeno-diarylglycoluriles, a permis une halogénation radicalaire sélective des systèmes hétéro-aromatiques pi-déficients non réactifs et impliqués dans la construction des podants cyclodextriniques. La sélectivité et le mécanisme de la réaction ont pu être expliqués en partie par la formation d'un complexe supramoléculaire [réactif /substrat] et l'existence d'interactions halogène-halogène dans le solide; ii- la mise en évidence d'une haute spéciation des tripodes cyclodextrines vis-à-vis d'un certain nombre de métaux et la formation d'hélices métallo-supramoléculaires chirales induite par l'implantation en position 6,6' des unités hétérocycliques. La configuration absolue des hélicates formés est résolue dans quelques cas / This work is structured around two main parts: i- the synthesis of new tris-A,C,E-alpha-cyclodextrin tripods, and studies of their complexes with transition metals. The functionalization of bis-heterocycles is an important step in the preparation of tripods. Therefore, the development of a new family of reagents tetrahalo-diarylglycolurils allowed a selective radical halogenation of heteroaromatic pi-deficient systems, non-reactive and involved in the construction of podants cyclodextrinics. The selectivity and the mechanism of the reaction could be partially explained by the formation of a supramolecular complex [reagent / substrate] and the existence of halogen-halogen interactions in solid state; ii- the highlight of a high speciation tripods cyclodextrins towards a number of metals and formation of supramolecular chiral metallo-helices induced by anchoring in position 6,6' of heterocyclic units. The absolute configuration of helicates formed in some cases is resolved

>Magister Scientiae - MSc / Efavirenz is a non-nucleoside reverse transcriptase inhibitor used as an anti-retroviral for the treatment of human immunodeficiency virus (HIV) type I. It is classified as a class IΙ drug under the Biopharmaceutical Classification System (BCS) and exhibits a low solubility (aqueous solubility of 9.0 μg/ml) and high permeability (variable oral bioavailability). This study aims to choose a pre-formulation protocol with the best efavirenz derivative in literature between co-crystals and CD inclusion complexes. Upon selection of the efavirenz derivative, the complications of both small scale and large scale laboratory pre-formulation production is highlighted for formulation of a tablet dosage form. Numerous variables were selected for the pre-formulation protocol. Physical, chemical, pharmacological, pharmaceutical and economical variables were investigated. Citric acid monohydrate (CTRC) was chosen as the best co-former for a co-crystal while hydroxypropyl-beta-cyclodextrin (HP-β-CD) was selected as a host for an inclusion complex. Pharmaceutically, the angle of repose, Carr’s index, Hausner’s ratio, moisture content, disintegration time, hardness/resistance to crush, manufacturing process problems and particle size of the CTRC and HP-β-CD were all evaluated. The CTRC was ultimately selected for formulation of a tablet. The preparation of small laboratory scale of EFA/CTRC co-crystal was successfully achieved after several attempts. The large laboratory scale of EFA/CTRC was prepared under various environmental seasons which were indicated as batches 1-6 for purposes of this study. Characterization of the large laboratory scale EFA/CTRC co-crystals was performed by scanning electron microscopy (SEM), hot-stage microscopy (HSM), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and by physical inspection (i.e. season, texture, colour, shape and particle size) of the EFA/CTRC product. Batch 1 and 2 were prepared during the summer season. The SEM analysis showed that the particles were needle-like shaped. The thermal analysis values of batch 1 by HSM, DSC and TGA results were 123 °C, 119 °C and 1.68 % of mass loss, respectively. In batch 2, morphology results by SEM revealed spikes of irregular and agglomerated particles. Batch 2 melted at 123 °C and a small unmelted quantity was observed at 143 °C. The DSC and TGA (mass loss) analysis were 118 °C and 0.75 % respectively. The hardness test of EFA/CTRC tablet prepared in batch 2 was extremely hard hence failed the disintegration test. The EFA/CTRC prepared in batches 3, 4 and 5 was during the winter season which is associated with high humidity and wet weather conditions. The SEM, DSC, TGA results were significantly different from the previous batches. The SEM morphology was highly irregular particles for batch 3, clustered and randomly size particle for batch 4 and irregular, needle-like, spikes and spherical shaped particles for batch 5, respectively. The thermal results HSM, DSC and TGA confirmed the presence of moisture in the prepared EFA/CTRC products. The HSM melting point results of batches 3, 4 and 5 were 123 °C, 115 °C and 121 °C, respectively. The DSC results of 110 °C, 105 °C and 118 °C were observed for batches 3, 4 and 5 respectively. The mass loss i.e. TGA results for batches 3, 4 and 5 were 1.178%, 1.5 % and 2.235 % respectively. In batch 6, EFA/CTRC was prepared using a different commercial batch of EFA and CTRC. The SEM results indicated the formation of needle-like and clustered particles. The values obtained from HSM, DSC and TGA results were 124 °C, 114 °C and 0.54 % in mass loss. The physical appearance of EFA/CTRC prepared from batch 1 and 2 were white in colour while batch 3, 4, 5 and 6 of the prepared EFA/CTRC was pink in colour. The physical appearance of the individual batches differed but the identity of the sample remained intact implying the same pharmacological effects with differing pharmaceutical properties impacting the dosage form preparation.

Les plaies chroniques représentent un problème de santé publique dont la prévalence augmente avec l'âge, l'état de santé du patient et la sédentarité. En effet, 1,69% des personnes âgées de plus de 65 ans est atteint d'ulcères de la jambe dans les pays occidentaux. De plus, l'apparition de plaies chroniques augmente chez les patients atteints de diabète avec une probabilité de développer un ulcère du pied de 2,2%/an pour les patients diabétiques atteints de neuropathie (diabète de type I et II confondus). Le risque d'infection des plaies chroniques augmentant avec le temps de guérison de la plaie, il est donc nécessaire d'apporter des soins réguliers afin de prévenir ou lutter contre l'infection de ces plaies jusqu'à leur complète guérison. En cas d'infection, la colonisation critique ou infection localisée peut conduire, en cas d'absence de traitement adapté, à la nécessité d'une intervention chirurgicale afin de prévenir l'infection systémique voire la mort du patient. Cependant, la plaie chronique représente un site de colonisation favorable pour les agents pathogènes qui s'y développent rapidement. La prolifération des bactéries peut alors aboutir à la formation d'un biofilm protecteur diminuant l'efficacité des défenses de l'organisme et des agents antimicrobiens. L'objectif de ce travail de thèse est donc de concevoir un dispositif médical sous forme de pansement capable de libérer de façon prolongée un principe actif antibactérien à large spectre d'action. Le dispositif textile utilisé dans ce projet est un textile non-tissé à base de polyéthylène téréphtalate (PET) fonctionnalisé par le chitosane (CHT) par la méthode "Pad-Dry-Cure". La fonctionnalisation du CHT est réalisée par l'utilisation de deux agents réticulants, la génipine (Gpn) ou l'acide citrique (CTR), qui permet de créer un réseau de chitosane réticulé à la surface des fibres textiles. Le coating apporte ainsi des fonctions ioniques à la surface du PET; positives lorsque l’agent réticulant est la Gpn, positives ou négatives en fonction du ratio de CTR introduit dans la solution quand l’agent réticulant est le CTR.Les charges ioniques du support PET fonctionnalisé par le CHT réticulé par la Gpn (PET-Gpn/CHT) ont été utilisées pour la construction d'un système multicouche (MC) "Layer-by-Layer" auto-assemblé. Les polyélectrolytes (PE) utilisés pour la construction de ce MC par "Dip-Coating" sont le chitosan en tant que PE cationique et un polymère de cyclodextrine (CD) en tant que PE anionique, élaboré et étudié par le laboratoire lors de travaux précédents. La capacité de formation de complexe d'inclusion des CD, apportée par le polymère de CD incorporé au revêtement multicouche, a ensuite été utilisée afin de charger un antiseptique à large spectre d'action, la chlorhexidine (Chx). La construction du système MC a d'abord été optimisée puis une évaluation physico-chimique, biologique et microbiologique a été réalisée sur les textiles PET-Gpn/CHT recouvert d'un système MC chargé par la Chx. Une étude a aussi été menée sur le PET fonctionnalisé uniquement par le CHT réticulé par le CTR (PET-CTR/CHT). Ce système anionique ou cationique en fonction de la quantité d’agent réticulant introduite, a été chargé respectivement par l'argent (Ag+) ou l’'iode (I3-). Une évaluation physico-chimique et microbiologique a été réalisée sur ces supports textiles pour évaluer leur efficacité antibactérienne. Finalement un système multicouche a été construit sur le système PET-CTR anionique chargé d’argent afin de limiter sa libération hors du système MC. Ceci a été ensuite vérifié à travers une évaluation chimique complétée par des tests microbiologiques. / The chronic wounds represent a public health problem health which the prevalence increases with age, patient health and sedentary lifestyle. Indeed, in the occidental countries, 1.69% of the elderly person aged over 65 years is suffering from leg ulcers . Moreover, the chronic wounds appearance increases for the diabetic patients who suffering neuropathy (diabetic type I and II together) with 2.2%/years of probability to developp a leg ulcer. The infection of the chronic wounds increases with wound healing delay, thus, cares need to be repeated to prevent or fight wound infection until their complete healing. In the case of infected wound, the critical colonization or localized infection can result without adapted treatment to surgery operation in order to prevent systemic infection or death. Nevertheless, the chronic wound represents a favourable site for the rapid developpment of the pathogen agents. The bacterial proliferation can lead to the biofilm formation which increases the bacterial protection against the body\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\\'s natural defences and antimicrobial agents. So, the aim of this work is elaborate a medical device as wound dressing for extended broad spectrum antibacterial drug release. The textile device used in this project is a non-woven textile based on polyethylene terephtalate (PET) functionalized with Pad-Dry-Cure method with chitosan (CHT). In the present work, two crosslinking agents are evaluated, genipin (Gpn) and citric acid (CTR), which allows obtaining a CHT network coated at the surface of the textile fibers. Thus, there are ionic functions at the surface of the PET; positive charges when the crosslink agent is Gpn, positive or negative charges in function of the amount of the CTR used in the solution.The ionic charges from the PET functionalized by CHT crosslinked by Gpn (PET-Gpn/CHT), are used to build a multilayer system layer-by layer self-assembly. The polyelectrolytes (PE) used to build this multilayer system by dip-coating are: the CHT, as cationic PE, and cyclodextrin (CD) polymer, as anionic PE (it was developed and studied by the team in previously work). The ability of the CD to form inclusion complex, from the CD polymer included in the multilayer coating, is used in the case of PET-Gpn/CHT to load one antiseptic with broad spectrum, the chlorhexidine (Chx). This Chx loaded multilayer system is optimized and evaluated physic-chemically, biologically and microbiologically. The study is also realized on the functionalized PET-CTR/CHT. This system, anionic or cationic, in function of the quantity of the crosslinking agent used, is loaded respectively by silver (Ag+) or iodide (I3-). A physico-chemical and microbiological evaluation are realized to evaluate their antibacterial efficiency. Finally, the multilayer system is built-up on the anionic PET-CTR/CHT loaded by silver to reduce its release out of the multilayer system. Chemical completed by microbiology tests are realized to verify the influence on the antibacterial activity and the kinetic of the release of the multilayer system

Ce travail vise à obtenir un matériau possédant des propriétés de complexation grâce aux cyclodextrines (CyD) et à démontrer la faisabilité de la synthèse en milieu CO2 supercritique (scCO2). L'adaptation en scCO2 de la polyaddition directe entre une α-CyD et un diisocyanate n’a montré qu’une faible conversion après 7 heures de réaction (presque totale en 4 heure en milieu liquide), en raison de la complexation possible du diisocyanate par la CyD, de l’insolubilité de la CyD dans le scCO2 ou d’une diminution de la vitesse de réaction nécessitant un temps supérieure pour atteindre une conversion plus élevée. Des études supplémentaires sont nécessaires pour confirmer ses hypothèses. La conception d’un monomère original pouvant être scCO2-soluble, par acétylation des hydroxyles de la CyD, fonctionnalisé par 3 isocyanates a montré, par polyaddition avec une alkyldiamine, en milieu solvant, une forte dépendance de la masse molaire avec la concentration (jusqu’à 16,89 kg/mol à 50 %pds en monomères). En milieu scCO2, la conversion du monomère original a été totale, avec des masses molaires atteintes supérieures à celles en milieu liquide à concentration identique, démontrant l’intérêt d’un tel milieu en remplacement de solvants organiques dans le cas d’une conception bien adaptée. L’extension de la gamme de concentrations et des conditions opératoires (température, pression) reste à réaliser pour conclure à l’intérêt du scCO2 par rapport à ce monomère original à base de CyD / This work aims at obtaining a polymeric material possessing complexation abilities thanks to cyclodextrins (CyD) within its framework and demonstrating the feasibility of the synthesis in supercritical CO2 medium (scCO2). Contrary to liquid medium, the polymerization of α-CyD and a diisocyanate in scCO2 shows only low conversion after 7 hours due to the possible complexation of the diisocyanate by the CyD, the insolubility of the CyD in scCO2 medium or insufficient time to achieve high conversion due to diminution of the reaction rate. New studies have to be done in order to confirm these hypothesis. The design of an original monomer which could be soluble in scCO2 thanks to acetyl groups fixed on the CyD and functionalized by 3 isocyanates, leads to a strong dependency of the molar mass with the concentration of monomers (up to 16.89 kg/mol at 50 %w of monomers) when reacted with a diaminoalkyle chain extender in solvent medium. In scCO2 medium, full conversion of this monomer is achieved, with higher molar masses obtained than in liquid medium at the same concentration. This demonstrates the potential of such a synthesis medium instead of organic solvents when design is well adapted. However, more experiments are needed to study the influence of concentration, temperature and pressure on larger scales to conclude in the interest of scCO2

Un nouveau dendron phénol trialcyne a été développé par analogie avec le dendron phénol triallyle déjà connu. En nous appuyant sur les cœurs dendritiques polyiodés du laboratoire, nous avons obtenu une famille de dendrimères polyalcynes comportant 27, 81 et 243 branches. Des ferrocènes ont ensuite été greffés par chimie « click » à la périphérie de ces dendrimères. La série obtenue permet la reconnaissance électrochimique d’oxo-anions d’intérêt biologique, comme l’ATP, et de cations métalliques. Grâce aux propriétés d’adsorption des grand dendrimères, nous avons obtenu des électrodes de platine modifiées, robustes et recyclables, permettant de réaliser cette reconnaissance. Une série de glycodendrimères comportant 27, 81 et 243 xylopyranosides terminaux a également été synthétisée. Les méthodes de caractérisation de nanoobjets ont été investiguées, et en particulier, les techniques permettant d’obtenir la taille des molécules en solution comme la RMN DOSY et la diffusion dynamique de la lumière (DLS). Enfin, une série de dendrimères robustes comportant 4 à 6 branches alcynes très longues a été développée. Le greffage périphérique par chimie « click » de beta-cyclodextrines méthylées aléatoirement est également rapporté. / A new easily accessible trialkyne phenol dendron has been developed mimicking the already known triallyl phenol dendron. A family of polyalkynyl containing dendrimers with 27, 81 and 243 terminal branches was obtained starting from the classical polyiodo dendritic cores of the laboratory. Ferrocenes were then grafted using “click” chemistry. The dendrimers obtained allowed electrochemical sensing of both biologically interesting oxo-anions like ATP and metallic cations. Robust and recyclable modified platinum electrodes were obtained thank to the adsorption properties of large dendrimers. These electrodes are able to recognize the same ions as the dendrimer in solution. A novel series of glycodendrimers with 27, 81 and 243 modified xylopyranosides termini was synthesized too. The characterization methods for such nanoobjects were investigated, and particularly technics that allow to obtain the size of the molecules like dynamic light scattering (DLS) and DOSY NMR. Finally, a family of robust polyalkynyl containing dendrimers with 4 to 6 enlarged branches was developed. The functionalization with randomly methylated beta-cyclodextrins using “click” chemistry is also reported.

Niemann-Pick type C1 (NPC1) disease is a lysosomal storage disorder caused by defective intracellular trafficking of exogenous cholesterol. Purkinje cell (PC) degeneration is the main sign of cerebellar dysfunction in both NPC1 patients and animal models. It has been recently shown that a significant decrease in Sonic hedgehog (Shh) expression reduces the proliferative potential of granule neuron precursors in the developing cerebellum of Npc1(-/-) mice. Pursuing the hypothesis that this developmental defect translates into functional impairments, we have assayed Npc1-deficient pups belonging to the milder mutant mouse strain Npc1(nmf164) for sensorimotor development from postnatal day (PN) 3 to PN21. Npc1(nmf164)/Npc1(nmf164) pups displayed a 2.5-day delay in the acquisition of complex motor abilities compared to wild-type (wt) littermates, in agreement with the significant disorganization of cerebellar cortex cytoarchitecture observed between PN11 and PN15. Compared to wt, Npc1(nmf164) homozygous mice exhibited a poorer morphological differentiation of Bergmann glia (BG), as indicated by thicker radial shafts and less elaborate reticular pattern of lateral processes. Also BG functional development was defective, as indicated by the significant reduction in GLAST and Glutamine synthetase expression. A reduced VGluT2 and GAD65 expression also indicated an overall derangement of the glutamatergic/GABAergic stimulation that PCs receive by climbing/parallel fibers and basket/stellate cells, respectively. Lastly, Npc1-deficiency also affected oligodendrocyte differentiation as indicated by the strong reduction of myelin basic protein. Two sequential 2-hydroxypropyl-beta-cyclodextrin administrations at PN4 and PN7 counteract these defects, partially preventing functional impairment of BG and fully restoring the normal patterns of glutamatergic/GABAergic stimulation to PCs. These findings indicate that in Npc1(nmf164) homozygous mice the derangement of synaptic connectivity and dysmyelination during cerebellar morphogenesis largely anticipate motor deficits that are typically observed during adulthood.

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-07-28T13:28:28Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Thiers Tese versão digital.pdf: 2738186 bytes, checksum: 9403cdd3615d510302f8e562a4b2df09 (MD5) / Made available in DSpace on 2017-07-28T13:28:28Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Thiers Tese versão digital.pdf: 2738186 bytes, checksum: 9403cdd3615d510302f8e562a4b2df09 (MD5) Previous issue date: 2016-02-17 / CNPQ / O 17-N-alil-amino-17-demetoxigeldanamicina (17-AAG) é um inibidor da proteína chaperona Hsp90 e tem sido estudado extensivamente como um agente anticancerígeno. O seu uso terapêutico ainda é limitado devido a sua baixa hidrofilicidade e toxicidade. Diante disso, o estudo tem como objetivo principal desenvolver um nanossistema contendo 17-AAG e verificar a sua ação antiproliferativa com efeitos tóxicos reduzidos. Primeiro, foram desenvolvidos lipossomas catiônicos contendo 17-AAG avaliando sua ação através de análises in vitro (viabilidade celular para linhagem de células Hela, MCF-7, J774 e Sarcoma 180) e in vivo (atividade antintumoral utilizando o tumor de Ehrlich). No segundo momento, foram preparados complexos de inclusão (17-AAG:HPβCD) obtidos por liofilização e caracterizados físicoquimicamente. Por último, os lipossomas contendo complexo de inclusão do 17-AAG foram preparados pelo método de hidratação do filme lipídico e submetidos a análise de citotoxicidade frente as células Hela e MCF-7. Os lipossomas catiônicos desenvolvidos apresentaram monodispersos e com tamanho inferior a 200 nm. O 17-AAG na bicamada dos lipossomas influenciou apenas na diminuição do potencial zeta passando de +21,13 para +11, 43 mV. O tratamento nas três células de câncer com estes lipossomas contendo o 17AAG e nas diferentes concentrações estudadas, demonstraram percentuais maiores de inibição em relação ao composto livre. Já os lipossomas branco não foram tóxicos frente as células sadias como macrófagos (J774). Nos estudos in vivo foi verificado que não houve diferença estatística quanto ao volume do tumoral, peso dos animais, peso do tumor, peso dos órgãos e índice mitótico. No entanto, alterações foram visíveis na histomorfologia do baço, fígado e rins. Os complexos de inclusão obtidos demonstraram a integridade do 17-AAG identificado através do teor (99 ± 0.79%).O diagrama de solubilidade de fases de 17-AAG:HPβCD apresentou uma curva do tipo AL, com K1:1 = 5,3 CAMPOS, T.A., (2016) Desenvolvimento de um nanosistema contendo 17-AAG ... 11 M-1. Na presença de 1000 mM de HPβCD, a solubilidade do 17-AAG aumentou 39 vezes. O complexo de inclusão 17-AAG:HPβCD apresentou modificações no IV, difração de raios-X, microscopia e DSC e TG, sugerindo a formação do complexo de inclusão. A citotoxicidade frente a células sadias (J774) evidenciaram que o complexo de inclusão apresentou efeitos inibitórios similares ao composto livre em concentrações <10 µM. Após o processo de preparação, os lipossomas contendo complexo de inclusão apresentaram diâmetro médio variando iando entre 142 e 157 nm, e o índice de polidispersão não superior a 0,37. As formulações mantiveram-se estáveis após 30 dias quando armazenadas a 4ºC. Os resultados apresentaram, que não há diferença significativa nas formas de encapsular o 17-AAG nos lipossomas, revelando uma EE% de 99% para ambos tipos. Os lipossomas desenvolvidos neste estudo, obtiveram uma carga positiva e sua morfologia microscópica confirma o tamanho e o tipo de lipossomas preparados. Nos estudos de citotoxicidade a ação inibitória do LP-17-AAG e o LP-17-AAG:HPβCD demonstraram-se mais eficientes comparado ao fármaco livre. Estes resultados demonstram que a complexação do 17-AAG a ciclodextrina, e sua incorporação em lipossomas, proporcionou uma melhoria na estabilidade e hidrossolubilidade do composto, viabilizando, portanto, seu uso terapêutico. / The 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) is an inhibitor of Hsp90 chaperone protein and has been extensively studied as an anticancer agent. The therapeutic use is still limited due to its low hydrophilicity and hepatotoxicity. Thus, the study aims to develop a nanossistema containing 17AAG and check their antiproliferative action with reduced toxicity. First, cationic liposomes containing 17-AAG were developed evaluating their action through in vitro tests (cell viability for line of Hela cells, MCF-7, J774 and Sarcoma 180) and in vivo (antintumoral activity using the Ehrlich tumor). In the second phase, they were prepared inclusion complexes (17-AAG: HPβCD) obtained by lyophilization and physico-chemically characterized. Finally, liposomes containing the inclusion complex of 17-AAG were prepared by lipid film hydration method and subjected forward cytotoxicity assay Hela and MCF-7 cells. Developed cationic liposomes showed monodisperse and smaller than 200 nm. The 17-AAG in the bilayer of the liposomes only influenced the decrease in zeta potential of passing +21.13 +11 to 43 mV. The treatment in all three cancer cells with these liposomes containing 17-AAG and different concentrations studied showed greater percentage inhibition compared to the free compound. Already the white liposomes were no toxicity in healthy cells such as macrophages (J774). In vivo studies it was found that there was no statistical difference in the volume of tumor, animal weight, tumor weight, organ weight and mitotic index. However, changes were visible on histomorphology of spleen, liver and kidneys. The solubility phase diagram of 17-AAG: HPβCD showed a curved-type LA, K1: 1 = 5.3 m-1. In the presence of HPβCD 1000 mm, the solubility of 17-AAG increased 39 times. The inclusion complex of 17-AAG: HPβCD presented modifications IR, X-ray diffraction, microscopy and DSC and TG, suggesting the formation of the inclusion complex. After the manufacturing process, liposome proved to be homogeneous with a mean diameter ranging between 142 and 157 nm and a polydispersity not exceeding 0.37.The front cytotoxic to healthy cells (J774) showed that the inclusion complex showed similar inhibitory effects to the free compound in concentrations of <10 µM. After the process of preparation of the inclusion complex containing liposomes had an average diameter between 142 and 157 nm and the polydispersion index not greater than 0.37. The formulations were stable after 30 days when stored at 4 ° C. The results showed that there is no significant difference in the ways of encapsulating 17-AAG in liposomes, revealing an EE% 99% for both types. The Liposomes developed in this study obtained a positive charge and their microscopic morphology confirms the size and type of liposomes. In cytotoxicity studies the inhibitory action of the LP-17-AAG and the LP-17-AAG: HPβCD proved to be more efficient compared to the free drug.These results demonstrate that the complexation of cyclodextrin 17-AAG, and their incorporation into liposome gave an improvement in stability and water solubility of the compound, allowing therefore their therapeutic use.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21819号 / 農博第2332号 / 新制||農||1067(附属図書館) / 学位論文||H31||N5191(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 三上 文三, 教授 植田 充美, 教授 宮川 恒 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Recent pharmaceutical developments have investigated using supramolecular nanoparticles in order to increase the bioavailability and solubility of drugs delivered in various methods. Modification of the carbohydrate cyclodextrin increases the ability to encapsulate hydrophobic pharmaceutical molecules by forming a carrier with a hydrophobic core and hydrophilic exterior. Guest molecules are commonly added to these inclusion complexes in order to add stability and further increase targeting abilities of the carriers. One such guest molecule is adamantine combined with a poly(ethylene glycol) chain. Vesicles are formed by hydrating a thin film of amphiphilic cyclodextrin and guest molecules in buffer solution that mimics physiological conditions. The solution is subject to freeze-thaw cycles and extrusion, and the complexes are separated out via size exclusion chromatography. Dynamic Light Scattering instrumentation is used to observe the particle size distribution. Cargo release can be observed in fluorescent dye-loaded vesicles by addition of a membrane-cleaving agent under a fluorimeter instrument. Future work involving this drug delivery technology includes synthesizing a chemically sensitive guest that will cleave in the presence of an intra-cellular anti-oxidant, and finally observing the uptake of these vesicles into live cells and testing the delivery of cargo in vitro under physiological conditions.