Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-02-13T13:18:57Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE colação de grau.pdf: 1971286 bytes, checksum: 680d0e24d2f0efbe0313ab8752f1cc93 (MD5) / Made available in DSpace on 2017-02-13T13:18:57Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE colação de grau.pdf: 1971286 bytes, checksum: 680d0e24d2f0efbe0313ab8752f1cc93 (MD5) Previous issue date: 2013-02-22 / CNPq / A criptococose é uma infecção fúngica predominantemente oportunista cujos principais agentes etiológicos são Cryptococcus neoformans e C. gattii. O tratamento de escolha para a micose é a anfotericina B associada ou não a 5-fluorocitosina seguido de terapia de manutenção com fluconazol. Contudo, falhas no tratamento associadas à toxicidade e ao aparecimento de resistência aos fármacos têm sido relatadas, o que torna essencial a descoberta de novas alternativas terapêuticas, como a ciclopirox olamina (CPO). Neste contexto, o objetivo deste estudo foi caracterizar e avaliar a ação in vitro e in vivo da CPO livre e encapsulada em lipossomas frente a amostras de Cryptococcus neoformans para futura aplicação no tratamento da criptococose sistêmica. Foram obtidas 30 amostras de Cryptococcus neoformans provenientes de pacientes imunocomprometidos. A preparação dos lipossomas convencionais e furtivos de CPO foi realizada pelo método da hidratação do filme lipídico e a caracterização foi realizada avaliando os seguintes parâmetros: tamanho de partícula, Índice de Polidispersão (PDI) e taxa de encapsulação (EE%). Para otimização dos constituintes lipídicos, foi realizado um planejamento fatorial fracionado a 24-1 a partir da melhor formulação obtida nos estudos de pré-formulação. A cinética de liberação in vitro foi conduzida para avaliar e comparar estatisticamente o perfil de liberação dos sistemas convencional e furtivo. Adicionalmente, testes de susceptibilidade antifúngica foram realizados de acordo com Clinical and Laboratotry Standards Institute (CLSI). Para caracterização molecular dos isolados, PCR fingerprinting foi conduzida utilizando os primers M13 e URA5. O estudo in vivo foi conduzido com camundongos imunossuprimidos, infectados com Cryptococcus neoformans (106 cels/mL) e tratados com CPO lipossomal (Lipo-CPO) (0.5 mg/Kg). As concentrações de CPO utilizadas na forma livre e encapsuladas em lipossomas convencionais e furtivos variaram de 0,30 a 625 µg/mL. Os resultados do planejamento fatorial mostraram que o ponto central apresentou características proeminentes com redução do tamanho de partícula em 17,1%; melhora do PDI em 15,34% e da quantidade de fármaco encapsulado (25%). A cinética do lipossoma furtivo apresentou uma velocidade de liberação mais controlada quando comparada ao lipossoma convencional. Com relação ao teste de susceptibilidade, todos os inóculos foram susceptíveis a CPO livre, com atividade fungistática entre 0,30 e 0,61 g/mL e fungicida entre 1,22 e 4,88 g/mL. Não houve diferença relacionada à atividade antifúngica entre as formulações lipossomais convencionais e furtivas. A atividade fungistática dos lipossomas foi observada em concentrações variando de 1,22 e 2,44 g/mL. A faixa das concentrações fungicidas foi de 1,22 a 9,76 g/mL. O padrão de bandas do URA5 revelou que todos os isolados apresentam genótipo VNI, característico de C. neoformans. Lipo-CPO apresentou eficácia antifúngica comparada à anfotericina B após 14 dias de infecção, reduzindo a carga fúngica em aproximadamente 8% no baço, 41% no fígado, 63% no pulmão e 89% no cérebro. O exame histológico evidenciou infiltrado celular no fígado dos grupos tratados com Lipo-CPO e anfotericina B, porém com menor intensidade quando comparado ao grupo controle. O estudo sugere que a CPO encapsulada em lipossomas apresenta significativa ação antimicótica frente às amostras sistêmicas de C. neoformans, reforçando seu potencial na terapêutica da criptococose. / Cryptococcosis is an opportunistic fungal infection whose the main ethiological agents are Cryptococcus neoformans and C. gattii. The treatment of choice for this mycosis is amphotericin B combined or not with 5-fluorocytosine, followed by maintenance therapy with fluconazole. However treatment failures associated with toxicity and drug resistance has been reported, which makes it essential to the discovery of new therapies, such as ciclopirox olamine (CPO). The purpose of this study was to characterize and evaluate the in vitro and in vivo antifungal activity of ciclopirox olamine in its free form and encapsulated in liposomes against thirty Cryptococcus neoformans isolates obtained from immunocompromised patients for future application in systemic cryptococcosis treatment. Preparation of conventional and stealth liposomes was performed to define particle size, polydispersity index (PDI), CPO amount of encapsulated and efficiency of encapsulation (EE%). For optimization of liposomal lipid constituents, a 24-1 fractional factorial design was carried out from the prominent formulation obtained in pre-characterization studies. In vitro release kinetics was conducted to evaluate and compare statistically the release profile of conventional and stealth liposomes. Antifungal susceptibility testing was conducted in accordance with the reference method. Regarding molecular characterization, PCR fingerprinting was carried out by using MT3 and URA5 primers. Concentrations of CPO used in free form and encapsulated into stealth and conventional liposomes ranged from 625 to 0.3 g.mL-1. Despite of the central point of the factorial design have increased the total lipids amount in 35.52%, it showed prominent characteristics when compared with the L4 formulation with improvement of the mean size in 17.1%, PDI in 15.34% and CPO amount in 25%. The minimum concentrations of stearylamine to obtain the stable formulation for one month was 5.88 mM. . Kinetics of stealth liposomes showed a release profile more controlled as compared to conventional liposomes. All inoculations were susceptible to CPO in its free form, presenting fungistatic activity between 0.3 and 0.61 g.mL-1 and fungicidal activity between 1.22 and 4.88 g.mL-1. There was no difference with respect to antimycotic activity between conventional and stealth liposomal formulations. Fungistatic activity of liposomes was observed at concentrations ranging from 1.22 and 2.44 g.mL-1. Fungicidal concentration range was 1.22-9.76 g.mL-1. The URA5 profile analized demonstrated all isolates are VNI genotype (C. neoformans). The treatment with Lipo-CPO showed a reduction of 8% of the C. neoformans population in spleen, 40.8% in liver, 63% in lungs and 89% in brain after 14 days of infection. Histological examination revealed cell infiltrate either Lipo-CPO or Amphotericin B treated groups, but less intense when compared to control group. The results suggest that Ciclopirox olamine loaded-liposomes have significant antimycotic activity against Cryptoccocus spp, reinforcing its potential for in vivo studies and its application in cryptococcosis treatment.

Au cours des dernières années, les autorités sanitaires ont recommandé une réduction de la teneur en sel et en gras dans la consommation alimentaire quotidienne. Cependant, les aliments à teneur réduite en sel et en gras sont souvent peu appréciés par les consommateurs, ce qui a amené au développement des recherches sur les stratégies possibles pour maintenir l’acceptabilité des aliments tout en réduisant les teneurs en ces ingrédients. Dans cette thèse, l’intégration multi-sensorielle et les cinétiques de libération du sel et des arômes mesurées in vitro ont été étudiés pour évaluer leur potentialité à compenser une réduction en sel et matière grasse d’un fromage modèle de composition variable et d’un fromage réel (type trappiste).Dans une première étape, une approche sensorielle a permis d’étudier les interactions multi-sensorielles arômes-saveurs-texture pour les formages modèles et réels. La structure et la perception sensorielle de vingt-quatre modèles fromagers variant en composition (2 niveaux de gras, 2 de sel, 2 de pH à l’emprésurage) et aromatisés avec un arôme de sardine (associé au sel), d'un arôme de beurre (associé au gras) ou non aromatisés (témoin) ont été caractérisés par des mesures rhéologiques (compression uniaxiale) et des mesures sensorielles (profil descriptif). Les résultats ont montré une influence de la composition sur la structure et la texture perçue des produits. Par ailleurs, l’arôme sardine a conduit à une perception plus salé ; l’arôme de beurre a permis de renforcer la perception du caractère gras. Toutefois cette influence des arômes sur les autres dimensions sensorielles est fonction de la texture des produits donc de leur composition et de leur structure. Ces résultats ont été étendus à des fromages réels avec toutefois des spécificités. Si l’arôme sardine renforce la perception du sel, seul l’arôme de beurre associé à l’arôme de sardine permet de renforcer le caractère gras.Dans une deuxième étape, une approche physico-chimique a été développée pour explorer les cinétiques de libération des stimuli odorants et sapides dans des conditions simulant la mastication in vitro à l’aide d’un simulateur de mastication. L’objectif était d’utiliser les données ainsi obtenues pour expliquer l’influence de la structure des produits et de leur déstructuration lors de la mastication sur les effets sensoriels observés dans la première étape. Les modèles fromagers et les fromages réels ont été ainsi étudiés. La libération des composés volatils a été évaluée en connectant le simulateur de mastication à un spectromètre de masse à pression atmosphérique (PTR-MS) ; la libération du sodium a été suivie grâce à une sonde de conductivité. Les résultats ont montré une influence des trois paramètres de composition (teneur en matière grasse, en sel et pH à l’emprésurage) sur les cinétiques de la libération des arômes. Cette variation dépend néanmoins de la nature de l’arôme suivi ; les arômes plus hydrophobes étant moins sensibles aux variations de la teneur en matière grasse et plus sensibles aux variations de pH et donc à la structure du produit. La cinétique de libération du sel lors de la mastication in vitro est aussi largement influencée par la composition et la structure des produits. Outre la teneur en sel qui conditionne les quantités libérées, la teneur en matière grasse et le pH à l’emprésurage module la cinétique de libération du sel. Au final, ces travaux montrent une importante contribution de la cinétique de libération et donc probablement de la temporalité des sensations dans perception globale du sel et de la matière grasse lors de la consommation d’un aliment complexe. / In recent years, health authorities worldwide advise for a reduction of salt and fat in daily food consumption. However, foods with reduced salt and fat content are often not appreciated by consumers, Therefore, the formulation of low-salt-fat foods that maintain acceptability is a major concern in food research. In this thesis, the multi-sensory integration and release kinetics of flavor compounds were explored as strategies to compensate for salt and fat reduction in cheese products (model cheeses and real cheeses). The objective was to better understand the mechanisms leading to aroma and salt release during mastication and to evaluate how the matrix composition and structure influence salt and aroma release profile.Multisensory integration approach to compensate for salt and fat reduction was studied in a first step. The structure and sensory perception of 24 cheese models varying in composition (2 levels of fat, 2 salt, 2 pH at renneting) and flavored with either a sardine aroma (associated to salt), a butter aroma (associated to fat) or not flavoured (control) were characterised by rheological measurements (uniaxial compression) and sensory evaluation (descriptive analysis). The results demonstrated an influence of the composition on the products structure and perceived texture. Furthermore, a significant saltiness enhancement was induced by sardine aroma while significant fat perception enhancement was induced by butter aroma. However, this influence of the aroma on other sensory dimensions depends on the texture of the products thus on their composition and structure. These results have been extended to real cheeses but with specificities. If the sardine flavor enhanced the perception of salt, only butter-sardine-flavor enhanced the perception of fat.In a second step, a physico-chemical approach was developed to explore the release kinetic of flavor compounds during in vitro breakdown using a chewing simulator. The aim was to use these data to explain the influence of the structure of model cheeses and real cheeses and their breakdown during chewing on sensory effects observed in the first step. Volatile compounds release was monitored by connecting the chewing simulator to a proton transfer reaction-mass spectrometry (PTR-MS), while salt release was monitored using a conductivity probe. Results showed that product composition and structure (fat, salt and pH at renneting) influenced aroma release, which however depends on the nature of the aroma: the more hydrophobic compounds are less sensitive to variations in fat content and more sensitive to variations in pH and therefore to the products structure. The salt release kinetic during in vitro chewing was also influenced by the composition and structure of the products. Indeed, beyond salt content which determined the amount of salt released, fat content and the pH at renneting modulated the release kinetic. In conclusion, this work showed a significant impact of the flavor compounds release kinetic and probably of temporality of sensations on the overall perception of salt and fat when consuming a complex food.

As former studies reveal, the nanoporous confinement could have influence on polymorphic drug crystallization. However, little attention has been paid to the question how crystallization of the commonly polymorphic drugs in nanoporous matrices influences the drug release. As a consequence, sufficient information about the crystallization conditions and their influence on phase behavior, crystal texture, and stability of polymorphs should be retrieved prior to drug delivery experiments. Drug release should be polymorph-selective and even crystal face-specific. Therefore, the topic of this PhD thesis is the systematic investigation of crystallization parameters (e.g., pore morphology, thermal history, presence or absence of a bulk surface reservoir) and their influence on the nucleation and crystal growth of the two selected model compounds in nanoporous matrices: acetaminophen (ACE) and n-tetracosane. Both are confined to two host-systems: AAO containing aligned cylindrical, isolated pores and CPG containing curved, interconnected pores. The guest materials inside the two model matrices have been investigated with X-ray diffraction (WAXS) and differential scanning calorimetry. In the first part it is shown that the nanopore morphology of the host systems determines into which polymorphic form ACE crystallizes. Moreover, the pore morphology influences the kinetics of solid/solid transitions. In AAO uniformly oriented form III crystals are converted into also uniformly oriented form II crystals by a solid/solid transition. Such a phase transition is kinetically suppressed in CPG membranes due to the curved pore morphology. In the second step, polymorph-specific release experiments with ACE from AAO membranes reveal that the drug dissolution is not exclusively diffusion-limited and can be described by the Korsmeyer-Peppas model. Dissolution of crystalline ACE having rough crystal faces exposed to the environment is nearly as fast as release of amorphous ACE. Encapsulating of ACE in AAO nanopores with a PLLA polymer retard the drug dissolution but does not modify the release kinetics. In the third part of this thesis crystallization of n-tetracosane, a saturated hydrocarbon, in nanoporous matrices was studied. n-Tetracosane shows inside AAO membranes the rotator phase sequence: triclinic−RV−RI−RII−liquid. Further, the long axes of the n-tetracosane molecules are oriented normal to the AAO pore axes. In general, n-tetracosane under confinement shows a more complex phase behavior than the polymeric analogue polyethylene. The presented work expands the available strategies for mesoscopic crystal engineering. The methods might be transferred into other areas of interest such as polymorphism screening or preparation of different types of nanowires with customized optoelectronic or ferroelectric properties.

Beim Tissue Engineering ist die Vaskularisierung von größeren Zell-Matrix-Konstrukten nach Implantation bis heute ein großes Problem. Durch das initiale Fehlen eines mikrovaskulären Netzwerkes kommt es zu einem raschen Zellsterben im Scaffold. Aufgrund dessen war das Ziel dieser Arbeit, im Sinne des in situ-Tissue Engineering ein Scaffold auf Basis von mineralisiertem Kollagen zu entwickeln, welches mit dem angiogenen Wachstumsfaktor VEGF funktionalisiert wird, um den Prozess der Vaskularisierung – die Einsprossung von Blutgefäßen – zu fördern und gleichzeitig durch Chemoattraktion in vivo Zellen aus dem umliegenden Knochengewebe in das Innere des Scaffolds migrieren zu lassen, so dass eine beschleunigte Defektheilung erzielt wird. Poröse Scaffolds aus mineralisiertem Kollagen wurden durch zwei unterschiedliche Strategien funktionalisiert und durch in vitro-Testungen charakterisiert. Die erste Strategie umfasste die Heparin-Modifizierung der gesamten Scaffolds, während die zweite Strategie die Injizierung eines zentralen VEGF-haltiges Depots in das Scaffoldinnere darstellte. Neben der Charakterisierung der Scaffolds wurde die Freisetzungskinetik des Modellwachstumsfaktors VEGF aus den modifizierten Scaffolds untersucht und die biologische Aktivität des freigesetzten Faktors auf Endothelzellen getestet. Zusätzlich wurde bei der 2. Strategie, der Injizierung eines Wirkstoffdepots, die Ausbildung eines Wirkstoffgradienten und die zielgerichtete Migration von Endothelzellen in Richtung des Wirkstoffdepots analysiert.

Beim Tissue Engineering ist die Vaskularisierung von größeren Zell-Matrix-Konstrukten nach Implantation bis heute ein großes Problem. Durch das initiale Fehlen eines mikrovaskulären Netzwerkes kommt es zu einem raschen Zellsterben im Scaffold. Aufgrund dessen war das Ziel dieser Arbeit, im Sinne des in situ-Tissue Engineering ein Scaffold auf Basis von mineralisiertem Kollagen zu entwickeln, welches mit dem angiogenen Wachstumsfaktor VEGF funktionalisiert wird, um den Prozess der Vaskularisierung – die Einsprossung von Blutgefäßen – zu fördern und gleichzeitig durch Chemoattraktion in vivo Zellen aus dem umliegenden Knochengewebe in das Innere des Scaffolds migrieren zu lassen, so dass eine beschleunigte Defektheilung erzielt wird. Poröse Scaffolds aus mineralisiertem Kollagen wurden durch zwei unterschiedliche Strategien funktionalisiert und durch in vitro-Testungen charakterisiert. Die erste Strategie umfasste die Heparin-Modifizierung der gesamten Scaffolds, während die zweite Strategie die Injizierung eines zentralen VEGF-haltiges Depots in das Scaffoldinnere darstellte. Neben der Charakterisierung der Scaffolds wurde die Freisetzungskinetik des Modellwachstumsfaktors VEGF aus den modifizierten Scaffolds untersucht und die biologische Aktivität des freigesetzten Faktors auf Endothelzellen getestet. Zusätzlich wurde bei der 2. Strategie, der Injizierung eines Wirkstoffdepots, die Ausbildung eines Wirkstoffgradienten und die zielgerichtete Migration von Endothelzellen in Richtung des Wirkstoffdepots analysiert.