Cannabinoids (CBs) and particularly CB2 agonists have been shown to reduce pain andinflammation without eliciting any apparent psychotropic effect conversely to CB1agonist compounds. CBs candidates are usually lipophilic non drug-like compoundswith poor bioavailability. To serve the purpose of evaluating new synthetic CB2 agonistsdeveloped by our group, on in vivo neuropathic pain models, an enabling formulationstrategy has been set up and four Drug Delivery Systems (DDS) developed. Forparenteral administration, cyclodextrin (CD)-based inclusion complexes, liposomes andsurfactants/co-solvents micellar solution have been investigated whereas Self-Emulsifying DDS (SEDDS) was selected for oral administration. A pharmacologicalstudy conducted with lead compound MDA7, formulated in CD-based DDS resulted inthe higher antinociceptive activity. A comprehensive study of the inclusion mechanismof MDA7 in the CD supramolecular complexes prepared was carried out. MDA7pharmacokinetic profile was also generated formulated in micellar solution and SEDDS.Besides, cationic polymeric nanocapsules (NCs) have been designed to serve as aprotective DDS for oral administration of a dietary phytocannabinoid CB2 agonist.Studies were undertaken to characterize and evaluate the influence of differentparameters on NCs formation prepared by nanoprecipitation. The cationic NCsdeveloped have been explored for their property to yield proportion of counterioniccondensation in the presence of macrocycles bearing anionic groups such assulfobutylether-beta-cyclodextrin or to form electrostatic interactions/host-guestcomplexion with cucurbit[n]uril.

Le cancer est un problème de santé publique dans le monde entier et d'importantes ressources en soins de santé sont dépensées pour le diagnostic. Plus précoce sera le dépistage des tumeurs, meilleures sont les chances de rémission sans rechute. Les techniques d'imagerie permettent de suivre l’évolution du traitement et de réorienter la stratégie en cas d’échec. En combinaison avec des agents de contraste ciblés, les modalités d'imagerie permettent même de sonder les structures à l’échelle moléculaires ce qui pourrait laisser envisager un traitement personnalisé du cancer [1, 2]. L’imagerie par résonance magnétique (IRM) et l’échographie sont deux techniques complémentaires et non invasives qui permettent la détection de plusieurs cancers (sein, colon, cerveau ...). L'échographie est rentable, portable et fournit, en temps reel, des informations anatomiques. L'IRM profite d’une pénétration profonde dans les tissus mous, d’un contraste élevé et d’une meilleure sensibilité que l’échographie [3]. Néanmoins, l'utilisation de ces techniques en combinaison avec des agents de contraste est difficile, surtout parce que la concentration locale atteint dans la tumeur est souvent inférieure à la plage de sensibilité de détection [4]. Au cours des 20 dernières années, les agents de contraste multifonctionnels ont été construits sur mesure pour atteindre une accumulation préférentielle dans les tissus malades [5]. Dans cette étude, des stratégies de ciblage passif et actif de la tumeur ont été envisagées pour renforcer la concentration locale de nanocapsules polymère, contenant un noyau liquide de bromure de perfluorooctyle (PFOB). L’approche de ciblage passif est basée sur l’effet de pénétration et la rétention accrue (EPR). Les nanocapsules doivent avoir un diamètre inférieur à 400nm une demi-vie plasmatique prolongée. L’approche de ciblage actif est basée sur la reconnaissance spécifique d’un ligand pour une cible biologique surexprimée par la tumeur ou la néovascularisation. Pour le ciblage passif, les nanocapsules ont été préparées avec PLGA-b-PEG par un procédé d'émulsion-évaporation. La morphologie cœur-couronne a été confirmée par RMN du Fluor et cryo microscopie électronique. La surface des nanocapsules est densément couverte par des chaînes de PEG qui adoptent une conformation en brosse, telle qu'évaluée par XPS et diffusion des neutrons aux petits angles. La furtivité des nanocapsules a été démontrée in vitro par des mesures d'activation du complément et in vivo par une étude cinétique de la capture hépatique, réalisée après l'administration intraveineuse de nanocapsules chez la souris nude. L'imagerie des tumeurs, par IRM du Fluor, a révélé que seulement 1% de la dose injectée a été accumulée dans le tissu malade. Par échographie aucun réhaussement du contraste n’a été observé. Ainsi, une autre approche de ciblage a été nécessaire afin d’augmenter l’accumulation des nanocapsules au sein de la tumeur. Les nanocapsules ont été fonctionnalisées avec un peptide RGD (Arginine-Glycine-Acide aspartique afin de cibler les intégrines avß3, qui sont des protéines transmembranaires surexprimées par les néovaisseaux. Deux stratégies, appelées bottom-up et top-down, ont été élaborées pour mener à une décoration satisfaisante du peptide à la surface des nanocapsules. L'efficacité du couplage a été mesurée par RMN du proton. La morphologie des nanocapsules a été étudiée par CryoTEM. / Cancer is a worldwide public health concern and significant health care resources are spent on diagnosis. The sooner the tumor detection, the better the chance of remission without relapse. Furthermore, imaging modalities facilitate the treatment monitoring and feedback, and support decision making to change the strategy when the treatment fails. When used in combination with targeted contrast agents, imaging modalities even enable to probe molecular structures on specific cells opening the doors to personalized cancer therapy [1, 2]. Ultrasonography and Magnetic Resonance Imaging (MRI) are two complementary and non invasive imaging modalities, which allow the detection of a broad range of cancers (breast, colon, brain…). Ultrasonography is cost-effective, portable and provides real-time anatomical information. MRI imparts deep penetration into soft tissues with high contrast and better sensitivity [3]. Nevertheless the use of these techniques in combination with contrast agents is challenging, mostly because the local concentration reached in the tumor is often below the sensitivity detection range [4]. In the last 20 years, multifunctional contrast agents were custom-built to achieve preferential accumulation in the diseased tissue [5]. In this study, passive and active tumor targeting strategies were considered to enhance the local concentration of polymeric nanocapsules, containing a liquid core of perfluorooctyl bromide (PFOB). The passive tumor targeting approach is based on the enhanced permeation and retention (EPR) effect. The related nanocapsules require to be small enough (< 400nm) and have extended plasmatic half life. The active tumor targeting approach is based on the specific receptor-ligand recognition.For passive tumor targeting, the nanocapsules were prepared with PLGA-b-PEG by an emulsion-evaporation process. The core shell morphology was confirmed by cryoTEM and 19F NMR. The surface of nanocapsules was densely covered by PEG chains with brush conformation, as assessed by XPS and Small Angle Neutrons Scattering. The related stealthiness of nanocapsules was evidenced in vitro by complement activation measurements and in vivo by a kinetic study of the mice liver uptake, performed after intravenous administration of nanocapsules. The tumor imaging, by 19F MRI, revealed that only 1% of the injected dose was accumulated in the diseased tissue whereas, by ultrasonography no contrast enhancement was observed. Thus, another targeting approach was required to increase nanocapsule distribution within the tumor. Nanocapsules were functionalized with an Arginine-Glycine-Aspartic acid (RGD) peptide to target the αvβ3 integrins, which are overexpressed proteins on neovessels. Two strategies, called bottom-up and top-down, were designed to achieve satisfying peptide decoration on nanocapsule surface. The coupling efficiency was measured by 1H NMR. The nanocapsule morphology was studied by CryoTEM.

Submitted by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-12-03T16:13:10Z No. of bitstreams: 1 IVAN DA SILVA KULMANN.pdf: 1348006 bytes, checksum: 0a65b00daaed554380496e8ada86e7d2 (MD5) / Approved for entry into archive by Marcos Anselmo (marcos.anselmo@unipampa.edu.br) on 2018-12-03T16:13:23Z (GMT) No. of bitstreams: 1 IVAN DA SILVA KULMANN.pdf: 1348006 bytes, checksum: 0a65b00daaed554380496e8ada86e7d2 (MD5) / Made available in DSpace on 2018-12-03T16:13:23Z (GMT). No. of bitstreams: 1 IVAN DA SILVA KULMANN.pdf: 1348006 bytes, checksum: 0a65b00daaed554380496e8ada86e7d2 (MD5) Previous issue date: 2018 / O metronidazol, denominado quimicamente por 1-(β-hidroxietil)-2-metil-5-nitroimidazol é um fármaco derivado sintético do 5-initromidazol, pertencente à classe dos imidazóis, subgrupo dos nitroimidazóis. Confere atividade antiprotozoária, antimicrobiana, sendo clinicamente eficaz na giardíase, tricomoníase e amebíase. Levando em consideração que a terapêutica destas parasitoses possui um tempo de tratamento geralmente longo, nanosistemas carreadores de fármacos tornam-se uma alternativa eficaz de auxiliar no controle de liberação do fármaco, adequar sua especificidade e melhorar a seletividade no local de ação. Dentre os componentes que podem ser utilizados como núcleo oleoso de nanocápsulas, está o óleo de melaleuca (Melaleuca Alternifolia Cheel). Este apresenta atividade antibacteriana, antifúngica e antiprotozoária bastante promissora. Desta forma o presente trabalho teve por objetivo estudar, desenvolver e caracterizar nanocápsulas poliméricas contendo metronidazol e óleo de melaleuca coencapsulados, como alternativa moderna ao tratamento de infecções envolvendo giardíase e trichomoníase. As nanocápsulas apresentaram pH ácido, diâmetro de partícula inferior a 300 nm, distribuição granulométrica estreita, potencial zeta negativo (-15,68 a -39,58 mV) e eficiência de encapsulação (EE) superior a 84%. O estudo da liberação in vitro, realizado em sacos de diálise, demonstrou que as formulações foram eficientes em controlar a liberação da metronidazol. Assim, estas nanopartículas apresentam-se como sistemas promissores para administração de metronidazol, com potencial para constituir-se em um tratamento eficaz para giardíase e tricomoníase. / Metronidazole, termed chemically by 1- (β-hydroxyethyl) -2-methyl-5-nitroimidazole is a synthetic derivative of 5-initromidazole, belonging to the class of imidazoles, subgroup of nitroimidazoles. It shows antiprotozoal activity, antimicrobial, being clinically effective in giardiasis, trichomoniasis and amebiasis. Taking into consideration that the therapy of these parasites has a generally long treatment time, drug carrier nanosystems have become an effective alternative to assist in drug release control, to tailor its specificity and to improve site selectivity of action. Among the components that can be used as an oily nucleus of nanocapsules is melaleuca oil (Melaleuca Alternifolia Cheel), due to its antibacterial, antifungal and antiprotozoal activity, it is promising. In this way the present work had the objective to study, develop and characterize polymeric nanocapsules containing metronidazole and coencapsulated melaleuca oil, as a modern alternative to the treatment of infections involving giardiasis and trichomoniasis. The nanocapsules presented acid pH, particle diameter less than 300 nm, narrow particle size distribution, negative zeta potential (-15.68 to -39.58 mV) and encapsulation efficiency of over 84%. The study of the in vitro release, carried out in dialysis bags, demonstrated that the formulations were efficient in controlling the release of metronidazole. Thus, these formulations present themselves as promising systems for administration of metronidazole, with potential to constitute an effective treatment for giardiasis and trichomoniasis.

Made available in DSpace on 2017-07-21T14:13:03Z (GMT). No. of bitstreams: 1 Daiane Cristine Mirante.pdf: 2726451 bytes, checksum: 032734e58cd5a2dc0e010068ceda3d38 (MD5) Previous issue date: 2015-02-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The development of antimicrobial agents is one of the most important advances in the therapeutic area due to microbial resistance, causing problems to public health, with serious economic, social and political implications. Thus, this study aimed to the technological development of microparticles of biopolymer poly-L-lysine (PLL) and polymeric nanocapsules containing essential oil of Melaleuca alternifolia Cheel uncoated or coated with PLL. The microparticles were obtained by the spray drying technique with a yield of 42.4%. The nanocapsules were prepared by interfacial deposition technique of a preformed polymer. The nanocapsules showed average nanometer diameter (160-200 nm), polydispersity index of less than 0.25 and negative zeta potential for nanocapsules poly(caprolactone) (PCL) (-39.8 mV) and positive for the nanocapsules of PLL (+17, 75 mV), indicating that there was an electrostatic adsorption interaction between PCL and PLL. An analytical method was developed and validated by UV-vis pectrophotometry in order to enable a reliable determination of encapsulation efficiency (EE). This method showed specificity, linearity, precision, detection and quantification satisfactory limits according to current recommendations. The EE was 99.86%. The antimicrobial activity of micro systems and nanocapsules was evaluated, containing PLL against the microorganisms, E. coli S. aureus, S. pyogenes, P. aeruginosa and C. albicans. The results demonstrate that the incorporation of the essential oil of M. alternifolia in the nanostructured system developed has been able to increase their antibacterial activity. The microparticles and nanocapsules, can, in the future, be promising antimicrobial agents, promoting the development of an effective therapeutic alternative and more effective at lower doses. / O desenvolvimento de agentes antimicrobianos representa um dos mais importantes avanços na área terapêutica, devido à resistência microbiana acarretar problemas para a saúde pública, com sérias implicações econômicas, sociais e políticas. Visto isso, este trabalho teve como objetivo o desenvolvimento tecnológico de micropartículas do biopolímero -poli-L-Lisina (PLL) e de nanocápsulas poliméricas contendo óleo essencial de Melaleuca alternifolia Cheel, revestidas ou não -PLL. As micropartículas foram obtidas pela técnica de secagem por aspersão com rendimento de 42,4 %. As nanocápsulas foram preparadas pela técnica de deposição interfacial de um polímero pré-formado. As nanocápsulas apresentaram diâmetro médio nanométrico (160-200 nm), índice de polidispersão inferior a 0,25 e potencial zeta negativo para as nanocápsulas de poli(-caprolactona) (PCL) (-39,8 mV) e positivo para as nanocápsulas de PLL (+17,75 mV), demonstrando que houve o processo de adsorção por interação eletrostática entre a PCL e a PLL. Foi desenvolvido e validado um método analítico por espectrofotometria de UV-Vis com a finalidade de permitir uma determinação confiável da eficiência de encapsulação (EE). Este método apresentou especificidade, linearidade, precisão, limites de detecção e quantificação satisfatórios de acordo com as recomendações vigentes. A EE foi de 99,86%. Foi avaliada a atividade antimicrobiana dos sistemas micro e nanoencapsulados contendo PLL contra os micro-organismos E. coli, S. aureus, S. pyogenes, P. aeruginosa e C.albicans. Os resultados demonstram que a incorporação do óleo essencial de M. alternifolia no sistema nanoestruturado desenvolvido foi capaz de aumentar a sua atividade antibacteriana. As micropartículas e as nanocápsulas, podem futuramente ser agentes antimicrobianos promissores, promovendo o desenvolvimento de uma alternativa terapêutica eficaz com doses menores e mais efetivas.

Le glioblastome (GB), tumeur primitive du cerveau, la plus agressive, et la plus fréquente chez l’adulte, présente une prolifération vasculaire importante. Des agents thérapeutiques innovants ciblant à la fois l'angiogenèse et les cellules tumorales sont recherchés, ainsi que des systèmes pour augmenter leur délivrance dans la tumeur cérébrale. Un de ces agents est le sorafénib (SFN), un inhibiteur de tyrosine kinase. Sa mauvaise solubilité aqueuse et ses effets secondaires indésirables limitent son utilisation. Le premier objectif de cette thèse était d'encapsuler cet agent dans des nanocapsules lipidiques (NCL) pour contrer ces inconvénients. Nous avons développé des NCL avec une haute efficacité d'encapsulation du SFN qui inhibaient in vitro l'angiogenèse et la viabilité de la lignée de GB humain U87MG. La délivrance intratumorale de SFN-NCL chez des souris porteuses d’une tumeur intracérébrale U87MG induit une normalisation vasculaire tumorale précoce qui pourrait améliorer l'efficacité de la chimiothérapie et de la radiothérapie. Le second objectif était de définir si la délivrance intranasale de NCL pouvait constituer une voie non-invasive alternative. Nous avons étudié via le transfert d'énergie par résonance de type Förster, le devenir des NCL chargées d’un fluorochrome à travers des monocouches de cellules Calu-3, un modèle de l'épithélium nasal. L'utilisation de NCL augmente le passage du fluorochrome à travers les cellules Calu-3, mais les particules sont rapidement dégradées après leur capture. Ces données mettent en évidence que les NCL sont appropriées pour la délivrance locale du SFN mais doivent être modifiées pour une délivrance intranasale. / Glioblastoma (GB), the most aggressive, and the most frequent primary tumor of the brain in adults, present a prominent vascular proliferation. Innovative therapeutic agents targeting both angiogenesis and tumor cells are urgently required, along with competent systems for their delivery to the brain tumor. One such agent is sorafenib (SFN), a tyrosine kinase inhibitor. However, poor aqueoussolubility and undesirable side effects limit its clinical application. The first objective of this thesis was to encapsulate this drug inside lipid nanocapsules(LNCs) to overcome these drawbacks. We developed LNCs with a high SFN encapsulation efficiency (>90%) that inhibited in vitro angiogenesis and the viability of the human U87MG GB cell line. Intratumoral delivery of SFN-LNCs in mice bearing intracerebral U87MG tumors induced early tumor vascular normalization which could be used to improve the efficacy of chemotherapy and radiotherapy in the treatment of GB. The second objective was to define whether intranasal delivery of LNCs could be an alternative non-invasive route. In this regard, we investigated through Förster resonance energy transfer, the fate of dye-loaded LNCs across Calu-3 cell monolayers, a model of the nasal mucosa. We showed that employment of LNCs dramatically increased the delivery of the dye acrossCalu-3 cell monolayer but they were rapidly degraded after their uptake. These data highlight that LNCs are suitable nanocarriers for the local delivery of SFN but must be redesigned for enhancing their nose-to-brain delivery.

Ces travaux de thèse ont porté sur l'utilisation d'un nouveau vecteur particulaire lipidique (LNCs) destiné à la voie orale. Ce système devrait permettre d'encapsuler une molécule anticancéreuse et améliorer sa biodisponibilité. Notre premier axe de recherche a été d'étudier les propriétés in vitro des LNCs encapsulant du paclitaxel en vue d'une administration par voie orale. La stabilité in vitro dans les milieux gastro-intestinaux simulés a été démontrée. De plus, les études de transport à travers l'épithélium intestinal (modèle cellulaire Caco-2) ont montré que les LNCs permettaient d'augmenter le transport du paclitaxel grâce à un mécanisme d'endocytose actif clathrine- et cavéole-dépendant situé dans le microenvironnement de la P-gp. Nous avons aussi mis en évidence les interactions entre l'endocytose des nanocapsules et l'activité de la P-gp. Notre second axe de recherche a porté sur l'application des LNCs pour encapsuler un nouvel anticancéreux : le Sn38 qui est le métabolite actif de l'irinotécan. Une formulation modifiée de nanocapsules lipidiques a été obtenue en utilisant un co-tensioactif et une huile hydrophile en plus des excipients standards. Les propriétés physico-chimiques et la stabilité, l'absorption epithéliale et la toxicité in vitro de cette formulation ont ensuite été vérifiées. Enfin, une activité antitumorale de courte durée après administration orale chez la souris a été observée. Ce travail fait donc la preuve de concept de l'absorption d'anticancéreux après administration par voie orale à l'aide de vecteurs colloïdaux. Néanmoins, certaines optimisations restent encore à réaliser pour prévoir une application clinique.

Le déficit en transporteur de la créatine est une maladie rare neurologique dans laquelle la perte de fonctionnalité du transporteur de la créatine (SLC6A8) conduit à une absence de créatine au niveau cérébral et à des retards de développement majeurs chez les enfants. A l'heure actuelle, aucune thérapie efficace n'est disponible.Une approche thérapeutique potentielle est le développement de molécules prodrogues de la créatine plus lipophiles qui franchiront les membranes cellulaires de façon passive et la recherche d'une formulation galénique susceptible d'emmener la prodrogue vers les cellules cibles d'intérêt, les neurones. Ainsi, dans cette thèse, nous proposons une nouvelle voie de synthèse originale d'esters de la créatine à longue chaîne aliphatique. Ces composés présentent des propriétés pharmacologiques intéressantes : nous montrons qu'il existe une relation de structure-activité entre la taille de la chaîne aliphatique (et donc la lipophilie) et la capacité de la molécule à être internalisée dans les cellules endothéliales cérébrales, astrocytaires et neuronales, constituant l'unité neurovasculaire. Il ressort de nos observations expérimentales que l'ester dodécylique de créatine est le meilleur candidat médicament. De plus, après avoir été internalisé dans les fibroblastes des patients présentant un déficit fonctionnel du transporteur de la créatine, l'ester dodécylique subit une conversion par les estérases cellulaires, libérant ainsi la créatine dans le compartiment intracellulaire.La formulation galénique permettant de protéger ces esters de créatine jusqu'au cerveau repose, elle, sur la nanovectorisation, par encapsulation de l'ester dodécylique de créatine dans des NanoCapsules Lipidiques. L'avantage de cette formulation est de permettre également un ciblage actif vers la Barrière Hémato-Encéphalique, obstacle majeur dans le développement de thérapies ciblant le Système Nerveux Central. Nos observations expérimentales mettent en exergue cette double stratégie thérapeutique pour le traitement du déficit en transporteur de la créatine.Ce travail a été soutenu financièrement par la Fondation Lejeune.

A epilepsia é normalmente a associação de pré-disposição genética e doença ou uma lesão cerebral. Aproximadamente 1 entre 50 a 100 pessoas apresentam essa pré-disposição à convulsões. Um dos fármacos mais prescritos e utilizados para o tratamento de convulsões é o ácido valpróico (AV), tornando-se a medicação de primeira escolha no tratamento da epilepsia infantil por apresentar um amplo espectro de ação, embora apresente efeitos colaterais bastante conhecidos como pancreatite e a hepatotoxicidade, que pode ser fatal. Sistemas nanoparticulados como nanocápsulas, obtidas a partir da utilização de polímeros biodegradáveis como o polietilenoglicol (PEG) e macromoléculas naturais como a quitosana (QS), que proporcionam hidrofilia e bioadesividade, têm sido estudadas com o objetivo de aumentar a penetração cerebral e reduzir a dosagem do fármaco. Nesse contexto, o presente trabalho teve como objetivo desenvolver e caracterizar físico-quimicamente nanocápsulas de poli(ε-caprolactona) contendo AV e revestidas com QS (NCQ) e/ou com PEG 6000 (NCP e NCQP), investigar a farmacocinética plasmática, a penetração do AV através da barreira hematoencefálica (BHE) por microdiálise e a hepatotoxicidade em ratos Wistar. Nanocápsulas revestidas com QS (NCQ) foram obtidas pelo método de nanoprecipitação do polímero pré-formado seguido do revestimento com adição de 5 mL de solução de QS a 1%. Para a preparação de NCP, foi utilizada metodologia similar adicionando 0,7% de PEG 6000 na fase aquosa. Para a preparação de NCQP, as nanocápsulas preparadas com a adição de 0,35% de PEG 6000 e revestidas posteriormente com solução de QS a 1% (2,5 mL). As formulações foram caracterizadas físico-quimicamente avaliando-se o tamanho das partículas, potencial zeta, pH e taxa de incorporação. As nanocápsulas foram visualizadas por MET e a estabilidade foi investigada por retroespalhamento de luz (Turbiscan Lab®). As formulações (AV 5 mg/mL) apresentaram um pequeno tamanho de partícula (144,2 ± 2,0 nm, 153,2 ± 1,8 nm, e 231,3 ± 15,6 nm, para NCQ, NCP e NCQP, respectivamente), com baixo índice de polidispersão, alta taxa de incorporação (95 a 98 %), pH ácido, potencial zeta positivo para NCQ (+8,7 ± 0,4 mV) e negativo para NCP (- 6,6 ± 0,8 mV) e NCQP (- 2,8 ± 1,3 mV). As fotomicrografias mostraram partículas de forma esférica e as formulações demonstraram boa estabilidade durante 24h de análise a 40°C. As concentrações plasmáticas foram investigadas em ratos Wistar (15 mg/kg via i.v. de AV) para todas as formulações e valproato sódico (grupo controle). A análise farmacocinética compartimental apresentou uma distribuição muito rápida para o AV em NCQ e a ASC0-∞ aproximadamente duas vezes menor em comparação à NCP, NCQP e o fármaco livre (3874 ± 1775; 8280 ± 2136; 7849 ± 1021 e 7978 ± 3622 μg/mL/min, respectivamente). O clearance do AV aumentou significativamente para NCQ (0,284 ± 0,156 L/h/kg) (α = 0,05%). A penetração do AV através da BHE foi realizada em ratos Wistar acordados por microdiálise (MD) cerebral, no córtex frontal utilizando sondas CMA/12 (3 mm). Os experimentos de MD mostraram um aumento de 5 vezes no fator de penetração cerebral após a administração de NCQ em comparação com o fármaco em solução (0,110 and 0,021, respectivamente), demonstrando a viabilidade da utilização de QS como polímero de revestimento objetivando a BHE. A NCP demonstrou um aumento de 1,7 vezes no fator de penetração cerebral e NCQP não demonstrou qualquer diferença na penetração. A investigação da hepatotoxicidade do AV foi realizada após cinco dias de tratamento (dose de 30 mg/kg q12h de AV) em solução ou em nanocápsulas (NCQ ou NCP) com grupo controle de solução salina. Os níveis séricos de asparto aminotransferase (AST), alanina aminotransferase (ALT), gama-glutamiltransferase (GGT), fosfatase alcalina (FAL), creatinina (CRE) e uréia foram determinados. Os resultados mostram a manutenção dos níveis normais de enzimas hepáticas como a ALT e FAL para NCQ (54,2 ± 11,2 UI/mL and 149 ± 26 UI/mL) demonstrando um efeito hepatoprotetor não observado para os outros grupos. Análises histológicas do fígado dos animais não apresentaram a formação de esteatose microvesicular para NCQ em comparação com a formação de esteatoses em todos os outros grupos, incluindo o grupo controle. Ao final, os resultados indicaram que NCQ possa ser uma formulação em potencial, necessitando ser investigada pelo aumento da penetração cerebral de AV e efeito hepatoprotetor observados. / Epilepsy is usually a combination of genetic pre-disposition and a disease or a brain damage. About 1 in 50 to 100 people has this genetic predisposition to seizures. One of the world’s most prescribed drugs to treat epileptic seizures is valproic acid (VA), which is the first choice drug to treat epilepsy in childhood due to its broad spectrum of action, although its well known side effects such as pancreatitis, hepatotoxicity can be fatal. Nanoparticulated systems such as nanocapsules, obtained from biodegradable polymers like polyethylene glycol and natural macromolecules like chitosan, who gives the system hidrophilicity and bioadhesivity, have been used to increase brain penetration and reduce drug doses. In this context, the present work aimed to develop and physicochemically characterize poly(ε-caprolactone) nanocapsules loaded with VA and coated with chitosan (NCQ) and/or polyethylene glycol (PEG) 6000 (NCP and NCQP), and to investigate their plasma pharmacokinetics, VA blood-brain barrier penetration (BBB) by microdialysis and hepatotoxicity in Wistar rats. Nanocapsules coated with chitosan were obtained by nanoprecipitation of preformed polymer followed by coating with 1% chitosan solution added prior to final adjustments at a volume of 5 mL. For NCP preparation, similar methodology was used adding 0.7% PEG 6000 in the aqueous phase. For NCQP preparation, the nanocapsules prepared with PEG 6000 (0.35% w/v) was further coated with chitosan 1% in solution adding 2.5 mL prior to the final adjustments. The formulations were physicochemical characterized by particle size, zeta potential, pH, incorporation efficiency. The particles were visualized by MET and the stability investigated by backscattering (Turbiscan Lab®). The formulations (VA 5 mg/mL) presented small particle sizes (144.2 ± 2.0 nm, 153.2 ± 1.8 nm, and 231.3 ± 15.6 nm, for NCQ, NCP and NCQP, respectively), with low polidispersion index, high incorporation efficiency (95 to 98 %) and acid pH. The zeta potential was positive for NCQ (+8.7 ± 0.4 mV) and negative for NCP (- 6.6 ± 0.8 mV) and NCQP (- 2.8 ± 1.3 mV). The photomicrography of all formulations showed spherically shaped particles. The formulations showed good stability during 24 hours investigation at 40 ºC. Plasma concentrations were investigated in Wisar rats after 15 mg/kg i.v. dosing of all formulations and sodium valproate solution (control group). The pharmacokinetic compartmental analysis showed a very rapid distribution of VA when incorporated in NCQ in comparison to the other formulations and the AUC0-∞ about two times lower in comparison to NCP, NCQP and the drug alone (3874 ± 1775; 8280 ±2136; 7849 ± 1021 and 7978 ± 3622 μg/mL/min, respectively). VA clearance was significantly increased after NCQ dosing (0.284 ± 0.156 L/h/kg) (α = 0.05 %). Drug penetration through BBB was performed in awaken Wistar rats by brain microdialysis at the frontal cortex using CMA/12 probes (3 mm). The microdialysis experiments showed a five times increase in VA brain penetration factor after NCQ administration in comparison to drug alone (0.110 and 0.021, respectively), demonstrating the viability of chitosan as coating polymer to aim the BBB. NCP showed only a 1.7 times increase in brain penetration factor and NCQP did not showed any difference in comparison to drug alone. The investigation of drug hepatotoxicity was conducted after 5 days i.v. dosing of VA 30 mg/kg q12h as solution or nanocapsules (NCQ or NCP). A saline control group was also investigated. Serum levels of asparte aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, alkaline phosfatase, creatinin and urea were determined. The results showed the maintenance of normal levels of hepatic enzymes such as alanine aminotransferase and alkaline phosfatase for NCQ (54.2 ± 11.2 IU/mL and 149 ± 26 IU/mL) showing a hepatoprotective effect not observed in the other groups investigated. Histological analysis of animals livers showed no microvesicular steatosis formation when NCQ was administered in comparison with the formation of steatosis in all other groups including control. Overall the results indicate that NCQ is a potential formulation to be investigated for the treatment of epilepsy due to its increase in VA brain penetration and hepatoprotective effect observed.

O trabalho tem como objetivo determinar a influencia da concentração de monoestearato de sorbitano, componente do núcleo oleoso das nanocápsulas, na cinética de liberação do éster etílico de indometacina a partir de nanocápsulas de poli(ε-caprolactona) (PCL). Com este propósito o éster etílico de indometacina foi associado a cada sistema e sua hidrólise alcalina foi realizada para simular uma condição sink. A velocidade de consumo do éster etílico de indometacina foi menor conforme o aumento da concentração do monoestearato de sorbitano. O tempo de meia-vida do consumo do éster etílico de indometacina associado as nanocápsulas foi relacionado com a concentração do monoestearato de sorbitano, sendo maior, enquanto maior a concentração do monoestearato. O mecanismo de liberação foi determinado como sendo transporte anômalo. Foi observada uma relação linear direta entre o aumento da concentração do monoestearato de sorbitano e a concentração de partículas nas suspensões de nanocápsulas (R2=0,9711). Mistura de outras nanopartículas que não as nanocápsulas, foram observadas e caracterizadas. O fluxo difusional do éster a partir das nanocápsulas foi determinado e diminuiu significativamente com o aumento da concentração do monoestearato, devido a mudanças na viscosidade do núcleo das nanocápsulas com o aumento da concentração do monoestearato de sorbitano. Por fim, os resultados demonstraram que o principal fator que contribui para o retardo no tempo para o consumo do éster etílico de indometacina é a relação direta entre a concentração do monoestearato de sorbitano e a permeabilidade das nanocápsulas (R=0,9894). / The aim of this work was to evaluate the influence of the sorbitan monoestearate concentration, one of the components of the oil core of the nanocapsules, in the release kinetic of the indomethacin ethyl ester-loaded poli(ε-caprolactone) nanocapsules. In this way, the indomethacin ethyl ester was entrapped within each system and its alkaline hydrolysis was carried out to simulate a sink condition. The rate for the indomethacin ethyl ester consumption decreased with the increase in sorbitan monostearate concentrations. The indomethacin ethyl ester half-live was related to the sorbitan monostearate concentration, increasing as the sorbitan monostearate concentration increased. The drug release mechanism was determined as anomalous transport. Linear correlations were obtained between the increase in the sorbitan monostearate concentration and the particles concentration in the suspensions (R2 = 0.9711). Mixture of different nanoparticles that are not nanocapsules were observed by density gradient and characterized. The indomethacin ethyl ester fluxes from the nanocapsules were determined and presented a decrease of the flux as the sorbitan monostearate concentration increased. This result was related to changes in the oil core viscosity caused by the variation of the sorbitan monostearate concentration. Finally, the results demonstrated that the main factor that contributes for the delaying in the time for the indometahcin ethyl ester consumption was the direct relation between the sorbitan monostearate concentration and the apparent permeability of the nanocapsules (R2 = 0.9894).

Neste trabalho foi avaliada a permeação/penetração cutânea in vitro (pele suína) de propionato de clobetasol nanoencapsulado incorporado em um semissólido, empregando células de difusão de Franz. A nanoencapsulação foi capaz de reduzir a quantidade de fármaco que penetra nas camadas da pele (estrato córneo, epiderme e derme) sem alterar a forma (distribuição percentual) como o propionato de clobetasol se distribui. A adequabilidade de diferentes membranas sintéticas (acetato de celulose, policarbonato e membrana de diálise) para a avaliação da liberação in vitro, empregando células de difusão de Franz, a partir desta formulação foi também estudada. A partir da combinação de diferentes técnicas analíticas (espalhamento de luz dinâmica, microscopias eletrônicas de transmissão e varredura) foi observado que a membrana de menor tamanho de poro (membrana de diálise, 12 kDa de cut off) é a mais adequada para a condução deste tipo de avaliação, pois é a única capaz de evitar a passagem de nanocápsulas íntegras da formulação para o meio receptor das células de difusão, em detrimento das membranas de policarbonato e acetato de celulose (0,05 μm e 0,45 μm de tamanho de poro, respectivamente). Além disso, uma nova estratégia para a avaliação da liberação in vitro de fármacos associados a nanocápsulas de núcleo lipídico, combinando fluxo contínuo de meio de liberação e sacos de diálise foi proposta neste trabalho. A técnica mostrou-se adequada para a obtenção do perfil de liberação in vitro a partir de suspensões de nanocápsulas contendo diferentes fármacos modelo (prednisolona e propionato de clobetasol), possibilitando a diferenciação destes sistemas de soluções contendo os fármacos livres, graficamente e pelos valores de fluxo calculados. Adicionalmente, esta estratégia mostrou-se apropriada para a manutenção da concentração de fármaco no meio de liberação afastada da saturação, contribuindo para o atendimento da condição sink. Ainda, classificamos o sistema como um protótipo semi-automatizado para a avaliação da liberação in vitro de fármacos, capaz de gerar resultados com maior precisão em relação à diálise convencional. / The in vitro cutaneous permeation/penetration (porcine skin) of clobetasol propionate-loaded lipid-core nanocapsules incorporated into a semisolid dosage form was evaluated, using the Franz diffusion cells technique. It was shown that the nanoencapsulation was able to reduce the drug amount penetration into skin layers (stratum corneum, epidermis and dermis) without changing the way (percentual distribution) that it was distributed. The suitability of different synthetic membranes (cellulose acetate, polycarbonate, and dialysis membrane) to assess the in vitro drug release using Franz diffusion cells from this formulation was also studied. It was ascertained by combining different analytical techniques (dynamic light scattering, scanning and transmition electron microscopy) that the membrane with smaller pore size (dialysis membrane, 12 kDa cut off) is the most appropriate for conducting this kind of study, because it is the only one able of preventing the passage of intact nanocapsules from formulation to Franz diffusion cells receptor media, instead of polycarbonate and cellulose acetate membranes (0.05 and 0.45 pore size, respectively). In addition, a new strategy to assess in vitro drug release drug-loaded lipid-core nanocapsules was proposed, associating continuous flow of release media and dialysis sac. The proposed system was adequate to assess the in vitro drug release profiles from nanocapsule suspensions containing different model drugs (prednisolone and clobetasol propionate), enabling the differentiation of these systems from drug solutions, graphically and by the calculated flux values. Furthermore, this strategy was suitable to maintain the drug concentration into release media far away from saturation, contributing to the sink condition. Also, the proposed system was described as a semi-automated prototype for in vitro drug release evaluation, able to produce results with greater accuracy than conventional dialysis technique.