O objetivo da presente tese foi desenvolver um adesivo com nanocápsulas (NCs), contendo indometacina, e um sistema adesivo com nanocápsulas (NC), contendo indometacina e triclosan, e avaliar suas propriedades. As NCs foram produzidas por meio do método de deposição de polímero, secas e caracterizadas quanto ao tamanho de partículas, à forma, quantidade de fármaco encapsulado e citotoxicidade. Uma resina adesiva foi formulada. Foram adicionadas ao adesivo 1%, 2%, 5% e 10% de NCs em massa, e um grupo permaneceu sem NC. As NCs, contendo indometacina e triclosan, foram também incorporadas no primer comercial a 2% em peso e um grupo permaneceu sem NCs. Os adesivos foram avaliados quanto ao GC imediato e tardio, à degradação em solvente, liberação dos fármacos, difusão de indometacina pela dentina e resistência de união. O adesivo com 10% de NCs, contendo indometacina, foi também avaliado quanto à ação anti-inflamatória em modelo animal. O primer e o adesivo com as diferentes concentrações de NCs, contendo indometacina e triclosan, foram avaliados quanto à liberação dos fármacos, difusão de indometacina pela dentina, ao efeito antimicrobiano, grau de conversão (GC) in situ, ângulo de contato e à resistência de união à microtração. Os dados foram analisados por ANOVA, Tukey e teste t. As NCs apresentaram forma esférica e viabilidade celular acima de 80%. As NCs, contendo indometacina, apresentaram diâmetro médio de 165 nm e as NCs, contendo indometacina e triclosan, 159 nm. O adesivo, contendo 10% de NCs com indometacina, apresentou efeito anti-inflamatório. A incorporação de NCs não alterou o GC, que variou de 63,63 ± 1,01% a 70,50 ± 2,08%. A degradação em solvente não foi alterada com 2% de NCs. Tanto os adesivos quanto o primer apresentaram liberação controlada. A indometacina permeou através da dentina. O adesivo e primer também apresentaram efeito antimicrobiano. A incorporação de NCs no primer e no adesivo não influenciou o GC in situ nem a resistência de união imediata, em comparação aos materiais sem NCs. O uso concomitante do primer e adesivo com NCs aumentou o ângulo de contato e diminuiu a resistência de união longitudinal. Conclui-se que o uso do adesivo com a incorporação de NCs tem potencial para proporcionar ações terapêuticas à adesão dentinária. / The aim of the present thesis was to develop an adhesive with nanocapsules (NCs) containing indomethacin and an adhesive system with nanocapsules containing indomethacin and triclosan and to evaluate their properties. NCs were prepared by the interfacial deposition of preformed polymer technique, dried and characterized regarding particle size, encapsulated drug content and cytotoxicity. Adhesive resin was produced. Concentrations of 1%, 2%, 5% and 10% of NCs were added in the adhesive and a group was maintained with no NCs. Indomethacin and triclosan-loaded NCs were also incorporated into a commercial primer in a concentration of 2% and a group was maintained with no NCs. Adhesives were evaluated regarding immediate and late degree of conversion (DC), degradation in solvent, drug release, indomethacin diffusion through dentin and bond strength. The adhesive with 10% of NCs containing indomethacin was also evaluated regarding the anti-inflammatory effect in an animal model. Primer and adhesive with different concentrations of NCs containing indomethacin and triclosan were evaluated regarding drug release, indomethacin diffusion through dentin, antimicrobial effect, in situ degree of conversion, contact angle and bond strength. Data were analyzed through ANOVA, Tukey post-hoc and t-test.NCs presented a spherical shape and cell viability higher than 80%. NCs containing indomethacin presented an averaged size of 165 nm and NCs containing indomethacin and triclosan, 159 nm. The adhesive with 10% of NCs containing indomethacin presented anti-inflammatory effect. The incorporation of NCs presented no alteration of DC, varying from 63.63 ± 1.01% a 70.50 ± 2.08%. Degradation in solvent suffers no influence of NCs with 2% of NCs. Adhesives and primer presented controlled drug release. Indomethacin diffused through dentin. Adhesive and primer also presented an antimicrobial effect. The incorporation of NCs in adhesive and primer showed no influence on in situ DC and immediate bond strength compared to materials with no NCs. The use in combination of primer and adhesive with NCs resulted in higher contact angle and lower longitudinal bond strength. It is possible to conclude that the use adhesive with incorporation of NCs has potential to provide therapeutic effects on dentin adhesion.

O objetivo da presente tese foi desenvolver um adesivo com nanocápsulas (NCs), contendo indometacina, e um sistema adesivo com nanocápsulas (NC), contendo indometacina e triclosan, e avaliar suas propriedades. As NCs foram produzidas por meio do método de deposição de polímero, secas e caracterizadas quanto ao tamanho de partículas, à forma, quantidade de fármaco encapsulado e citotoxicidade. Uma resina adesiva foi formulada. Foram adicionadas ao adesivo 1%, 2%, 5% e 10% de NCs em massa, e um grupo permaneceu sem NC. As NCs, contendo indometacina e triclosan, foram também incorporadas no primer comercial a 2% em peso e um grupo permaneceu sem NCs. Os adesivos foram avaliados quanto ao GC imediato e tardio, à degradação em solvente, liberação dos fármacos, difusão de indometacina pela dentina e resistência de união. O adesivo com 10% de NCs, contendo indometacina, foi também avaliado quanto à ação anti-inflamatória em modelo animal. O primer e o adesivo com as diferentes concentrações de NCs, contendo indometacina e triclosan, foram avaliados quanto à liberação dos fármacos, difusão de indometacina pela dentina, ao efeito antimicrobiano, grau de conversão (GC) in situ, ângulo de contato e à resistência de união à microtração. Os dados foram analisados por ANOVA, Tukey e teste t. As NCs apresentaram forma esférica e viabilidade celular acima de 80%. As NCs, contendo indometacina, apresentaram diâmetro médio de 165 nm e as NCs, contendo indometacina e triclosan, 159 nm. O adesivo, contendo 10% de NCs com indometacina, apresentou efeito anti-inflamatório. A incorporação de NCs não alterou o GC, que variou de 63,63 ± 1,01% a 70,50 ± 2,08%. A degradação em solvente não foi alterada com 2% de NCs. Tanto os adesivos quanto o primer apresentaram liberação controlada. A indometacina permeou através da dentina. O adesivo e primer também apresentaram efeito antimicrobiano. A incorporação de NCs no primer e no adesivo não influenciou o GC in situ nem a resistência de união imediata, em comparação aos materiais sem NCs. O uso concomitante do primer e adesivo com NCs aumentou o ângulo de contato e diminuiu a resistência de união longitudinal. Conclui-se que o uso do adesivo com a incorporação de NCs tem potencial para proporcionar ações terapêuticas à adesão dentinária. / The aim of the present thesis was to develop an adhesive with nanocapsules (NCs) containing indomethacin and an adhesive system with nanocapsules containing indomethacin and triclosan and to evaluate their properties. NCs were prepared by the interfacial deposition of preformed polymer technique, dried and characterized regarding particle size, encapsulated drug content and cytotoxicity. Adhesive resin was produced. Concentrations of 1%, 2%, 5% and 10% of NCs were added in the adhesive and a group was maintained with no NCs. Indomethacin and triclosan-loaded NCs were also incorporated into a commercial primer in a concentration of 2% and a group was maintained with no NCs. Adhesives were evaluated regarding immediate and late degree of conversion (DC), degradation in solvent, drug release, indomethacin diffusion through dentin and bond strength. The adhesive with 10% of NCs containing indomethacin was also evaluated regarding the anti-inflammatory effect in an animal model. Primer and adhesive with different concentrations of NCs containing indomethacin and triclosan were evaluated regarding drug release, indomethacin diffusion through dentin, antimicrobial effect, in situ degree of conversion, contact angle and bond strength. Data were analyzed through ANOVA, Tukey post-hoc and t-test.NCs presented a spherical shape and cell viability higher than 80%. NCs containing indomethacin presented an averaged size of 165 nm and NCs containing indomethacin and triclosan, 159 nm. The adhesive with 10% of NCs containing indomethacin presented anti-inflammatory effect. The incorporation of NCs presented no alteration of DC, varying from 63.63 ± 1.01% a 70.50 ± 2.08%. Degradation in solvent suffers no influence of NCs with 2% of NCs. Adhesives and primer presented controlled drug release. Indomethacin diffused through dentin. Adhesive and primer also presented an antimicrobial effect. The incorporation of NCs in adhesive and primer showed no influence on in situ DC and immediate bond strength compared to materials with no NCs. The use in combination of primer and adhesive with NCs resulted in higher contact angle and lower longitudinal bond strength. It is possible to conclude that the use adhesive with incorporation of NCs has potential to provide therapeutic effects on dentin adhesion.

O objetivo da presente tese foi desenvolver um adesivo com nanocápsulas (NCs), contendo indometacina, e um sistema adesivo com nanocápsulas (NC), contendo indometacina e triclosan, e avaliar suas propriedades. As NCs foram produzidas por meio do método de deposição de polímero, secas e caracterizadas quanto ao tamanho de partículas, à forma, quantidade de fármaco encapsulado e citotoxicidade. Uma resina adesiva foi formulada. Foram adicionadas ao adesivo 1%, 2%, 5% e 10% de NCs em massa, e um grupo permaneceu sem NC. As NCs, contendo indometacina e triclosan, foram também incorporadas no primer comercial a 2% em peso e um grupo permaneceu sem NCs. Os adesivos foram avaliados quanto ao GC imediato e tardio, à degradação em solvente, liberação dos fármacos, difusão de indometacina pela dentina e resistência de união. O adesivo com 10% de NCs, contendo indometacina, foi também avaliado quanto à ação anti-inflamatória em modelo animal. O primer e o adesivo com as diferentes concentrações de NCs, contendo indometacina e triclosan, foram avaliados quanto à liberação dos fármacos, difusão de indometacina pela dentina, ao efeito antimicrobiano, grau de conversão (GC) in situ, ângulo de contato e à resistência de união à microtração. Os dados foram analisados por ANOVA, Tukey e teste t. As NCs apresentaram forma esférica e viabilidade celular acima de 80%. As NCs, contendo indometacina, apresentaram diâmetro médio de 165 nm e as NCs, contendo indometacina e triclosan, 159 nm. O adesivo, contendo 10% de NCs com indometacina, apresentou efeito anti-inflamatório. A incorporação de NCs não alterou o GC, que variou de 63,63 ± 1,01% a 70,50 ± 2,08%. A degradação em solvente não foi alterada com 2% de NCs. Tanto os adesivos quanto o primer apresentaram liberação controlada. A indometacina permeou através da dentina. O adesivo e primer também apresentaram efeito antimicrobiano. A incorporação de NCs no primer e no adesivo não influenciou o GC in situ nem a resistência de união imediata, em comparação aos materiais sem NCs. O uso concomitante do primer e adesivo com NCs aumentou o ângulo de contato e diminuiu a resistência de união longitudinal. Conclui-se que o uso do adesivo com a incorporação de NCs tem potencial para proporcionar ações terapêuticas à adesão dentinária. / The aim of the present thesis was to develop an adhesive with nanocapsules (NCs) containing indomethacin and an adhesive system with nanocapsules containing indomethacin and triclosan and to evaluate their properties. NCs were prepared by the interfacial deposition of preformed polymer technique, dried and characterized regarding particle size, encapsulated drug content and cytotoxicity. Adhesive resin was produced. Concentrations of 1%, 2%, 5% and 10% of NCs were added in the adhesive and a group was maintained with no NCs. Indomethacin and triclosan-loaded NCs were also incorporated into a commercial primer in a concentration of 2% and a group was maintained with no NCs. Adhesives were evaluated regarding immediate and late degree of conversion (DC), degradation in solvent, drug release, indomethacin diffusion through dentin and bond strength. The adhesive with 10% of NCs containing indomethacin was also evaluated regarding the anti-inflammatory effect in an animal model. Primer and adhesive with different concentrations of NCs containing indomethacin and triclosan were evaluated regarding drug release, indomethacin diffusion through dentin, antimicrobial effect, in situ degree of conversion, contact angle and bond strength. Data were analyzed through ANOVA, Tukey post-hoc and t-test.NCs presented a spherical shape and cell viability higher than 80%. NCs containing indomethacin presented an averaged size of 165 nm and NCs containing indomethacin and triclosan, 159 nm. The adhesive with 10% of NCs containing indomethacin presented anti-inflammatory effect. The incorporation of NCs presented no alteration of DC, varying from 63.63 ± 1.01% a 70.50 ± 2.08%. Degradation in solvent suffers no influence of NCs with 2% of NCs. Adhesives and primer presented controlled drug release. Indomethacin diffused through dentin. Adhesive and primer also presented an antimicrobial effect. The incorporation of NCs in adhesive and primer showed no influence on in situ DC and immediate bond strength compared to materials with no NCs. The use in combination of primer and adhesive with NCs resulted in higher contact angle and lower longitudinal bond strength. It is possible to conclude that the use adhesive with incorporation of NCs has potential to provide therapeutic effects on dentin adhesion.

Made available in DSpace on 2014-07-29T16:11:51Z (GMT). No. of bitstreams: 1 Luciano Aparecido Stecanella.pdf: 1386754 bytes, checksum: 98d171bf870e6a2ec50ece485d0e203b (MD5) Previous issue date: 2011-12-23 / Grandisin, a tetrahydrofuranic lignan produced by Virola surinamensis seeds and leaves, is popularly used to treat erysipelas, colic and dyspepsia, and is active against Schistosoma mansoni, Plasmodium falciparum, Leishmania donovani and Trypanosoma cruzi. It also antinociceptive, anti-inflamatory and potential for protective (dose dependent) activities, preventing cyclophosphamide induced chromosomal fragmentation, being considered a potencial anti-cancer candidate. The encapsulation of grandisin in polymeric nanocapsules can increase its solubility in aqueous media, allowing drug administration by intravenous route. In the presente work, polymeric nanocapsules containing grandisin were produced by nanoprecipitation method (or interfacial pre-formed polymer deposition). The average diameter of these nanocapsules were around 160 nm, PdI < 0,2, zeta potential -15,73 mV and grandisin load of 6,54%, with encapsulation efficiency of (EE%) 98%. Stability study, comprising 30 days under refrigeration (4ºC ± 1ºC), showed that physicochemical characteristics of the formulation (color, bluish translucency, etc.) were the same as compared to the nanocapsules dispersion originally produced, so there were no alterations such as deposition of residues and/or crystals, lumps formation, creamming or supernatant oil, droplets adhered to the glass, flocculation or phase separation. Regarding encapsulated grandisin, during stability test, there was a reduction of 6,4% of the total encapsulated drug. Drug release study showed quick liberation in the first 48h (30,54%) and then kept slower, because only 52% after 21 days. Polymeric nanocapsules containing grandisin, obtained in this work, showed potential to be a drug release system to administration by intravenous route. / A grandisina, uma neolignana tetraidrofurânica obtida das sementes e folhas da planta Virola surinamensis, é popularmente utilizada no tratamento de erisipelas, cólicas e dispepsias, e possui ação comprovada contra Schistosoma mansoni, Plasmodium falciparum, Leishmania donovani e Trypanosoma cruzi. Apresenta também atividade antinociceptiva, anti-inflamatória e potencial ação quimioprotetora (dose dependente), prevenindo a fragmentação cromossômica induzida pela ciclofosfamida, sendo assim considerada potencial candidata a agente anti-câncer. A encapsulação da grandisina em nanocápsulas poliméricas pode aumentar sua solubilidade em meio aquoso, consequentemente, permitindo sua administração por via intravenosa. No presente trabalho foram produzidas nanocápsulas poliméricas contendo grandisina pelo método de nanoprecipitação (ou deposição interfacial de polímero pré-formado). Estas nanocápsulas apresentaram diâmetro médio em torno de 160 nm, PdI < 0,2, potencial zeta de 15,73 mV e carga de grandisina de 6,54%, com eficiência de encapsulação (EE%) de 98%. O estudo de estabilidade, realizado por um período de 30 dias sob refrigeração (4ºC ± 1ºC), mostrou que as características físicas da formulação (coloração, translucência azulada, etc.) foram mantidas em relação à dispersão de nanocápsulas originalmente produzida, não sendo observadas alterações como deposição de resíduos e/ou cristais, formação de grumos, cremagem ou nata sobrenadante, gotículas de óleo aderidas ao vidro, floculação ou separação de fases. Com relação à grandisina encapsulada, durante o estudo de estabilidade, foi verificada uma redução de 6,4% do total encapsulado. O estudo de liberação mostrou rápida liberação nas primeiras 48h (30,45%) e posteriormente manteve-se mais lenta, com 52% após 21 dias de ensaio. As nanocápsulas poliméricas contendo grandisina, obtidas neste trabalho, mostraram ser um potencial sistema de liberação para viabilizar uma forma de administração por via parenteral para esta substância.

Le but de cette thèse de doctorat fut de développer des nanoparticules pour la délivrance de deux molécules hydrophobes de faible poids moléculaire, l’apigénine (AG) et un ferrocifène (FcTriOH), comme stratégie innovante pour le traitement du glioblastome(GBM). Dans un premier temps, différents types de nanoparticules, liposomes, nanocapsules lipidiques (LNC), et nanocapsules à base de polymères, furent formulés et comparés en termes de caractéristiques physico-chimiques, de libération en drogue ou encore de toxicité. Les LNCs furent ainsi sélectionnées. Dans un deuxième temps, les LNCs furent fonctionnalisées en surface par un peptide pénétrant (CPP). La concentration de peptide fut augmenté afin d’améliorer significativement l’internalisation des LNCsdans des cellules humaines de GBM. Les mécanismes de macropinocytose et d’endocytose dépendant de la clathrine et de la cavéoline furent observés. De plus, il fut montré que l’internalisation de ces LNCs fonctionnalisées était réduite dans les cellules saines humaines d’astrocyte. L’efficacité biologique des LNCs chargées en AG et chargées en FcTriOH fut évaluée et comparée : le résultat le plus prometteur fut obtenu avec les LNCs chargées en FcTriOH. Une administration intracérébrale des LNCs sur un modèle tumoral murin orthotopique montra une potentielle toxicité et un besoin d’optimiser la dose administrée. Pour finir, les études menées sur un modèle tumoral ectopique murin montrèrent des résultats prometteurs, après une administration parentérale des LNCs chargées en FcTriOH. Ainsi, cette dernière formulation pourrait ouvrir la voie au développement d’une stratégie thérapeutique alternative pour le traitement du GBM. / The aim of this thesis was to develop nanocarriers for efficient delivery of two low molecular weight hydrophobic drugs, apigenin (AG) and a ferrocifen-derivative(FcTriOH) to glioblastoma (GBM) as potential therapeutic strategies. Firstly, two liposomes, a lipid nanocapsule (LNC), and a polymer-based nanocapsule were develope dand compared by their physicochemical characteristics, drug loading capacity, storage stability, stability in biological serum, drug release profiles, complement consumption and toxicity. Due to various advantageous characteristics, the LNCs were selected for further optimization. Secondly, the LNCs were surface functionalized by adsorbing a GBM-targeting cellpenetratingpeptide (CPP). The CPP concentration increased to significantly enhance LNCinternalization in human GBM cells. The uptake mechanisms observed in U87MG cellswere : micropinocytosis, clathrin-dependent and caveolin-dependent endocytosis. Moreover, the optimized CPP-functionalized LNCs were internalized preferentially in theGBM cells compared to normal human astrocytes. Additionally, the in vitro efficacy of the AG-loaded and FcTriOH-loaded LNCs was evaluated. The FcTriOH-loaded LNC-CPP showed the most promising activity with a low IC50 of 0.5 μM against U87MG cells. Intracerebral administration of the LNCs in a murine orthotopic U87MG tumor modelshowed possible toxic effects and the need for dose optimization. Finally, studies inmurine ectopic U87MG tumor model showed promising activity after parenteral administration of the FcTriOH-loaded LNCs. Overall, these results exhibit the promising activity of FcTriOH-loaded LNCs as potential alternative GBM therapy strategy.

Ce travail de thèse est consacré à la préparation de glycopolymères porteurs de groupements pendants mannoside d’heptyle et à l’évaluation de la capacité de ces ligands multivalents à inhiber la fixation bactérienne sur les cellules humaines. Nous avons synthétisé, par polymérisation radicalaire contrôlée, une série de glycopolymères linéaires ou en étoile présentant des masses molaires, des densités en mannoside et des microstructures modulables dans le but d’évaluer l’influence de ces paramètres sur les processus d’interactions avec diverses souches de bactéries E coli (AIEC LF82 et UTI 89). Nous avons tout d’abord mis en évidence par diffusion dynamique et statique de la lumière, la formation d’agrégats entre ces glycopolymères et FimH, la lectine à l’origine de la fixation de souches de bactéries E coli, traduisant des interactions fortes entre les motifs mannosides et les sites de reconnaissance au mannose de la lectine. Nous avons ensuite évalué l’aptitude de ces ligands multivalents à bloquer l’adhésion bactérienne d’AIEC LF82 (impliquée dans la maladie de Crohn) sur des cellules épithéliales intestinales T84. Il a été démontré en conditions in vitro que l’ajout de 10 nM ou 100 nM d’unités mannoside (respectivement en pré- ou post-incubation) réduit de moitié l’adhésion des bactéries sur les cellules épithéliales. L’effet anti-adhésif de ces glycopolymères a été confirmé par des tests ex vivo réalisés sur des intestins isolés de souris transgéniques CEABAC10. Enfin, nous avons exploité la technique de nanoprécipitation pour l’élaboration de nanocapsules de glycopolymères à cœur huileux. Le procédé développé permet la synthèse de nanocapsules de dimensions contrôlées, porteuses de groupements fonctionnels (fluorophores, ligands) ou de particules métalliques et l’encapsulation de molécules actives à cœur en une seule étape. / This PhD work focuses on the preparation of glycopolymers bearing pendent heptyl mannose groups and the evaluation of the capability of such multivalent ligands to inhibit bacterial adhesion to human cells. Aiming at understanding the impact of various structural parameters on glycopolymer/ E coli interactions (AIEC LF82 et UTI 89 strains of E. coli), a series of linear and star-shaped glycopolymers with tunable molecular weight, mannoside density and microstructure (block copolymers, gradient copolymers, random copolymers) has been constructed. The association of the glycopolymers with FimH adhesin, a lectin which possesses a mannose-specific receptor site and is responsible for recognition and binding to host cells, was first confirmed by static and dynamic light scattering experiments. The propensity of the glycopolymers to prevent attachment of E. coli (AIEC LF82 involved in Crohn’s disease) to intestinal epithelial cells (T84 cells) was further investigated through adhesion assays. It was shown that under in vitro conditions, the addition of 10 nM or 100 nM of glycopolymer on a mannose unit basis (in pre-incubation and post-incubation respectively) decreases by half the bacterial adhesion to intestinal epithelial cells. The anti-adhesive effect of these multivalent ligands was further confirmed in ex vivo conditions for colonic loops of transgenic CEABAC10 mice (Crohn’s disease model mouse). Finally we took advantage of the nanoprecipitation process to generate glyconanocapsules with oily core. The employed strategy allowed for preparing well-defined nanocapsules bearing groups of interest (tags, ligands) or metal particles within the shell and loaded with active molecules in the core in one step.

L'objectif de ce travail est de comprendre les mécanismes en jeu au plan cinétique et nanoostructural dans les procédés d'encapsulation par émulsion diffusion, et leur modélisation.<br />Dans ce travail nous avons proposé un nouveau modèle qui décrit la séparation de phases couplée au transfert de matière multiconstituant pour un système ouvert. L'approche choisie repose sur le concept d'équilibre local. Dans ce modèle, la séparation de phases est traitée localement en utilisant la méthode du plan tangent. Cette méthode permet de tester la stabilité d'un petit volume dV donné, et en cas d'instabilité, de calculer le nombre et la composition des phases dans ce volume. En ce qui concerne le transfert de matière dans chaque volume, les équations de bilan de matière, de flux de transfert et d'équilibre thermodynamique sont établies. Les flux de transfert de tous les constituants sont décrits par le modèle de diffusion développé par Fornasiero. L'interphase est représentée par un modèle de film, dans lequel l'équilibre thermodynamique est admis.

Nanopartículas poliméricas biodegradáveis têm recebido atenção como carreadores de fármacos ao longo dos últimos anos. Em muitos casos, a segurança destes nanocarreadores não foi demonstrada e pouco se sabe sobre a relação entre as suas características físico-químicas e suas propriedades toxicocinéticas e toxicodinâmicas. A nanotoxicologia está emergindo como uma especialidade importante da nanotecnologia e/ou toxicologia, e refere-se ao estudo da interação de nanoestruturas com sistemas biológicos. Nos últimos anos, a maioria das pesquisas foi centrada em estudos in vitro, entretanto, os resultados destes estudos necessitam também ser avaliados em experimentos in vivo para o avanço na utilização de nanocarreadores na área biomédica. Com isso, o objetivo deste trabalho foi avaliar a toxicidade de nanocápsulas de núcleo lipídico (LNC), de poli(-caprolactona), após administração intraperitoneal (i.p.) e intradérmica (i.d.) em ratos Wistar. Para a avaliação toxicológica aguda, foi administrada dose única em que se observaram sinais clínicos e fisiológicos, em ambas as vias. Após 14 dias, os animais foram eutanasiados e análises macroscópicas e histopatológicas foram realizadas. Além disso, sangue e urina foram coletados para análises laboratoriais e avaliação de funções teciduais. A avaliação toxicológica subcrônica foi procedida da mesma forma, exceto pela administração de doses repetidas diárias durante 28 dias. As suspensões de nanocápsulas foram preparadas pelo método de precipitação do polímero pré-formado, as quais apresentaram tamanho médio de partícula inferior a 250 nm, índice de polidispersão (IPD) < 1, potencial zeta negativo e pH em torno de 6,7. Os animais tratados pela via i.p. (n=6/grupo) receberam para avaliação da toxicidade aguda: solução salina ou polissorbato 80 (PS80) (12 ml/kg), utilizados como controles e diferentes doses de LNC (18,03, 36,06, e 72,12 × 1012 LNC/kg); no teste de toxicidade subcrônica foram utilizados os mesmos controles porém com doses de 3mL/kg e 6,01, 12,02 ou 18,03 × 1012 LNC/kg. Nos testes de toxicidade aguda, nos animais administrados pela via i.p., foi observada diminuição significativa de peso nos grupos tratados com LNC mesmo após 14 dias da administração (p<0,05). Entretanto no teste subcrônico esta alteração foi transitória, e ocorreu apenas no grupo que recebeu a maior dose até o quinto dia de administração (p<0,05). Houve aumento no peso relativo do baço nos animais que receberam a dose mais alta de LNC (p<0,05) no tratamento agudo. A análise histopatológica em ambos os tratamentos, demonstrou a presença de um granuloma de tipo corpo estranho no fígado e no baço dos animais que receberam a dose mais alta, provavelmente devido ao volume de LNC administrado. Não houve alteração nas análises bioquímicas de dano hepático, renal, dentre outros em todos os grupos tratados. Os dados hematológicos apresentaram uma leve alteração, entretanto foi demonstrada interferência metodológica, evidenciada por testes preliminares in vitro. Além disso, foram avaliados biomarcadores do estresse oxidativo (EO), marcadores inflamatórios e de genotoxicidade. Os resultados dos biomarcadores de oxidação de proteínas e lipídios não foram suficientes para iniciar um processo oxidativo, visto que não houve peroxidação lipídica. Ainda, não houve depleção de antioxidantes, dano ao DNA ou alteração nos marcadores inflamatórios. Nos ratos tratados pela via i.d., foi utilizada solução salina 1,2 ml/kg como grupo controle e uma dose de 7,2 × 1012 LNC/kg de LNC, para um estudo preliminar agudo e solução salina ou PS 80 (0,9ml/kg) e três doses de LNC (1,8, 3,6 ou 5,4 × 1012 LNC/kg) para avaliação da toxicidade subcrônica. No teste de toxicidade aguda, não houve alteração do peso corpóreo, entretanto no teste de toxicidade subcrônica houve uma diminuição reversível do peso no grupo que recebeu PS80 (p<0,05). Os dados histopatológicos não apresentaram alteração. Não houve alteração nos parâmetros bioquímicos, exceto uma leve diminuição da atividade da butirilcolinesterase no grupo que recebeu a dose mais alta (p<0,05). Por outro lado, houve aumento nos leucócitos no grupo que recebeu LNC, no teste de toxicidade aguda e nos grupos que receberam PS 80 e 5,4 × 1012 LNC/kg (p<0,05) após doses repetidas. Em relação à avaliação sanguínea e tecidual dos biomarcadores do EO e dos marcadores inflamatórios, foi observada uma indução nos marcadores de oxidação de proteínas juntamente com uma indução enzimática nos ratos que receberam a dose mais alta, além de uma diminuição dos níveis do IL-10 nos grupos que receberam PS80 e a dose mais alta (p<0.05). Pode-se concluir que nas condições dos experimentos, tanto pela via i.p. quanto pela via i.d., não foram demonstrados danos teciduais, pois os achados laboratoriais foram condizentes com os achados histopatológicos. Além disso, os mecanismos de reparo foram suficientes para contrabalançar eventuais danos oxidativos ou inflamatórios. Assim, o presente trabalho contribui para futuras avaliações toxicológicas de nanocápsulas poliméricas, visto que foram realizadas avaliações agudas e subcrônicas sistemáticas, com marcadores de dano renal precoce e possíveis mecanismos de toxicidade envolvidos após administração por ambas as vias. O aumento na utilização destas nanocápsulas e as lacunas nas informações toxicológicas fazem com que desafios importantes devam ser superados para permitir sua incorporação segura. Com isso, estudos nesta linha podem embasar a avaliação da resposta tóxica e, consequentemente, levar ao estabelecimento de regulamentações para avaliação da toxicidade da maioria das nanopartículas poliméricas biodegradáveis utilizadas como carreadoras de fármacos. / Biodegradable polymeric nanoparticles have received attention as drug carriers over the past years. In many cases, the safety of nanocarriers has not been demonstrated and little is known about the relationship of its physicochemical characteristics and their toxicokinetic and toxicodynamic properties. Nanotoxicology is emerging as an important field of nanotechnology and toxicology, and refers to the study of the interaction of nanostructures with biological systems. In recent years, most research has focused on in vitro studies; however, the results of these studies should also be evaluated trough in vivo experiments, in order to advance in biomedical application of nanocarriers. Thus, the objective of this study was to evaluate the toxicity of lipid-core nanocapsules (LNC), prepared with poly(ɛ-caprolactone), after intraperitoneal (i.p.) and intradermal (i.d.) administration in rats. For acute toxicological evaluation, it was administered a single dose, i.p. and i.d., clinical signs and physiological effects were observed. After 14 days, animals were euthanized and macroscopic and histopathological analyses were done. In addition, blood and urine were collected for laboratory analysis and evaluation of tissue functions. Subchronic toxicological evaluation was similar, except for the administration of repeated doses for 28 days. The suspension of nanocapsules were prepared by interfacial deposition of polymer, which had particle size less than 250 nm, polydispersity index (IPD) <1, negative zeta potential and pH around 6.7. Animals were treated via i.p. (N = 6/group), the doses used for acute toxicity test were: saline or polysorbate 80 (PS80) (12 ml/kg) as controls or three different doses of LNC (18.03, 36.06, e 72.12 × 1012 LNC/kg); for subchronic toxicity test, same controls were used but the doses were 3 ml/kg and 6.01, 12.02 ou 18.03 × 1012 LNC/kg administered daily for 28 days. In acute toxicity test, with i.p. administration, groups treated with LNC presented a significant reduction in relative weight even after 14 days of administration (p<0.05); however in the subchronic test, this change was transient, and occurred only in the group receiving the highest dose until the fifth day of administration (p<0.05). There was an increase in relative weight of spleen in animals that received the highest dose of LNC (p<0.05) in acute treatment. Histopathological analysis in both the treatments, showed a granulomatous foreign body reaction in liver and spleen of animals receiving the highest dose, probably because the volume of LNC administered. There were no changes in biochemical parameters of liver or kidney damage, among all treated groups. Hematological data showed a slight change; however it was demonstrated an interference of the methodology, further evidenced by preliminary in vitro tests. Furthermore, we evaluated biomarkers of oxidative stress (OS), inflammatory and genotoxicity markers. The results of the oxidation of proteins and lipids biomarker were not sufficient to initiate an oxidative process, since no lipid peroxidation occurred. Still, no depletion of antioxidants, DNA damage or change in inflammatory markers was observed. In rats treated via i.d., saline was used as control (1.2 ml/kg) and a dose of 7.2 × 1012 LNC/kg of LNC to a preliminary acute study, and saline or PS 80 (0.9ml/kg) used as controls or three doses of LNC (1.8, 3.6 ou 5.4 × 1012 LNC/kg) for subchronic toxicity evaluation. In acute toxicity test, there was no change in relative body weight, however, a decreased was found for the group receiving PS 80 in subchronic test (p <0.05). No histopathological alteration was found. Also, there was no change in biochemical parameters, except a slight decrease of butyrylcholinesterase activity in the group receiving the highest dose (p<0.05). Moreover, in acute toxicity test, it was found an increase in white blood cells in group receiving LNC; these increasing also occurred after repeate dose test, in PS 80 and 5.4 × 1012 LNC/kg of LNC groups (p <0.05). Regarding blood and tissue biomarkers of OS and inflammatory markers, an induction in protein oxidation marker along with antioxidant induction in rats which received the highest dose were observed, also reduced levels of IL-10 in rats that received the higher dose and PS80 (p <0.05). It can be concluded that, under the experimental conditions, for i.p. and i.d. administration, tissue damage was not found, since laboratorial analysis results were consistent with histopathological findings. Furthermore, mechanisms of repair were sufficient to offset oxidative damage or inflammation.Thus, this study contributes to future toxicological evaluations of polymeric nanocapsules, since a systematic acute and subchronic evaluation with early renal damage markers and possible mechanisms of toxicity involved after ip and id routes were performed. The increase in the use of these nanocapsules and the gaps in toxicological information make important to overcome these challenges in order to allow its safe incorporation. Thus, studies in this line are important to evaluate toxic response, and lead to establishing rules for evaluating the toxicity of most biodegradable polymer nanoparticles used as carrier of drugs.

Ce travail de thèse a pour objectif le traitement local des gliomes malins via l'administration de nanocapsules lipidiques (LNC) par convection enhanced delivery (CED). Deux types de complexes métalliques lipophiles aux propriétés thérapeutiques ont été encapsulés au sein des LNC. Le premier est un complexe radioactif de Rhénium-188 et le second, un agent anticancéreux dérivé du tamoxifène et du ferrocène, le ferrociphénol (Fc-diOH). Les LNC de 188Re permettent une rétention de l'émetteur β- au niveau local et une éradication complète de la tumeur est possible pour une dose de 8Gy puisque 33% des animaux sont de longs survivants. Cette dose optimisée s'est révélée être une dose efficace, intermédiaire entre des doses toxiques (10-12 Gy) ou inefficaces (3-4 Gy). Les LNC-Fc-diOH présentent des taux d'encapsulation élevés, et sont quantitativement internalisées dans les cellules 9L. De plus, l'activité du ferrociphénol est conservée après encapsulation et se révèle très efficace sur des cellules de gliome 9L (IC50=0.6μM). En revanche, l'activité est très réduite sur les astrocytes, cellules au potentiel de division quasiment nul. L'action intratumorale des LNC-Fc-diOH dans un modèle de gliome sous-cutané entraîne une réduction significative des masses et volumes tumoraux. De plus, l'association entre le ferrociphénol et les photons X est une association synergique conférant à Fc-diOH des propriétés de molécule radio-sensibilisante. La médiane de survie du groupe traité par une CED de LNC-Fc-diOH suivie d'une radiothérapie externe de 18Gy (3x6Gy) augmente de 48% par rapport au groupe contrôle avec la présence de 17% de longs survivants.

In the recent years, development of various organic and inorganic nano-sized systems has gained great interests especially for cancer diagnosis and treatment and intense researches are carried out in this area. Regarding to the recent trends for drug delivery system design, the novel approaches for drug carriers are mainly based on development of smart and nano-size drug carriers which are targeted to cancer cells. Hence, for an effective tumor-targeted delivery device, besides its chemical structure further criteria such as detection of tumor site and sensitivity to the higher temperature and lower pH of the tumor compare to rest of the body gains importance. The aim of this study is to design and prepare polysebacic anhydride (PSA) based nanocapsules (NCs) loaded with Doxorubicin (DOX) which is an anti cancer drug. In order to obtain an intelligent delivery system, drug-loaded nanocapsules were coated with pH sensitive poly (L-histidine). PSA nano-carriers were firstly loaded with DOX and then in order to introduce pH sensitivity, they were coated with poly (L-histidine). PLH-coated NCs were modified with polyethylene glycol (PEG) to prevent their macrophage uptake. Drug release profile from this system was examined in two different buffer solutions prepared as acidic (pH 4) and physiological (pH 7.4) media. The physical and chemical properties of the nano particles were characterized by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), ultraviolet and visible absorption spectroscopy (UV-VIS), and scanning electron microscopy (SEM). In vitro studies of the prepared nanocapsules were performed on MDA-MB-231 breast cancer cells by using WST Kit 8 cell viability test. In order to obtained results, pH sensitive nanocapsules with size 230 nm exhibited cellular uptake and promising intracellular release of drug.