UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE Departamento de Farmácia Laboratório de Sistemas Dispersos (LASID) PROGRAMA DE PÓS- GRADUAÇÃO EM BIOTECNOLOGIA (RENORBIO) ÉCOLE DOCTORALE ED425 Innovation Thérapeutique : du Fondamental à l’appliqué UNIVERSITÉ PARIS-SUD Faculté de Pharmacie Centre d’Études Pharmaceutiques UMR CNRS 8612 ANNÉE 2014 - 2015 SÉRIE DOCTORAT N° 1328 THÈSE Présentée À L’UNITÉ DE FORMATION ET DE RECHERCHE FACULTÉ DE PHARMACIE DE CHÂTENAY-MALABRY UNIVERSITÉ PARIS-SUD 11 pour l’obtention du grade de DOCTEUR DE L’UNIVERSITÉ PARIS-SUD 11 Par Francisco Humberto XAVIER-JÚNIOR Titre de la thèse Systèmes dispersés pour l’administration orale du paclitaxel à base de microémulsion et de nanocapsules mucoadhésives contenant de l’huile de copaïba Directeurs de thèse : Christine VAUTHIER, Directeur de Recherche CNRS, (Université Paris-Sud) Eryvaldo Sócrates TABOSA DO EGITO, Professeur (UFRN) Composition du jury : Gilles PONCHEL, Professeur (Université Paris-Sud) Patrick SAULNIER, PU-PH (Université d’Angers) Sylvie CRAUSTE-MANCIET, MCU-PH HDR (Université de Bordeaux) « C'est le temps que tu as perdu pour ta rose qui fait ta rose si importante » Antoine de Saint-Exupéry v REMERCIEMENTS Cette thèse a été développée dans le cadre de l’accord de co-tutelle entre la France et le Brésil. En France, les études ont été réalisées à la Faculté de Pharmacie de l'Université Paris Sud à Chatenay-Malabry, au sein de l’Institut Galien Paris Sud (UMR CNRS 8612) dans l'équipe VI - Amélioration du passage des barrières par les molécules biologiquement actives. Au Brésil, les études ont été développées à la Faculté de Pharmacie de l'Universidade Federal do Rio Grande do Norte à Natal / RN, au Laboratorio de Sistemas Dispersos (LASID). Ces dernières années pendant lesquelles j’ai fait cette recherche ont été d’un chemin ardu, de formation et de gain de maturité. Le chemin n'a été ni le plus facile, ni le plus court. Cependant, maintenant avec le recul, je peux dire que ça vaut le coup de se battre pour un rêve. Cette étape a seulement été possible grâce à la présence et la contribution des nombreuses personnes que j’ai pu rencontrer sur ce chemin dans ma vie et qui ont laissé leurs marques et leurs connaissances. À ceux-ci, je suis éternellement reconnaissant pour leur soutien inconditionnel, pour les choses qu’ils m’ont apprises, les valeurs qu’ils m’ont enseignées, les encouragements qui m’ont aider à chaque étape à gagner cette victoire. Aujourd'hui, je vois dans ce document l'enregistrement de tout ce vécu au fils des ans. Et vers la phase finale de cette histoire, je tiens à exprimer mes sincères remerciements à ceux qui ont contribué directement ou indirectement à cette construction. Au départ, je remercie Dieu pour guider ma vie, donnant de la force, le courage, la sagesse et le sens pour vivre et poursuivre les rêves dont Il rêvait pour moi. Je tiens à remercier chaleureusement ma directrice de thèse en France, le Dr. Christine VAUTHIER, de m’avoir donné l’opportunité de conduire mon doctorat au sein de son laboratoire. Merci pour tous les enseignements, discussions scientifiques, attentions et préoccupation pour ma formation. Merci aussi pour faire de son laboratoire ma maison et l'équipe de travail ma famille durant les 18 mois passés sur le sol français. Je tiens également à remercier grandement mon directeur de thèse brésilien, Prof. Dr Sócrates EGITO, pour m’avoir diriger et encadrer durant mes études dans son laboratoire au cours de ces années de doctorat. Merci pour la patience, les encouragements, la confiance et les précieux enseignements. Je vous remercie également d'avoir permis mon entrée dans cette grande "aventure". En France: Au Professeur Gilles PONCHEL, le responsable de l’équipe VI, je tiens à exprimer ma gratitude pour l’accueil, le support et pour les discussions scientifiques. Merci pour ta simplicité et la joie contagieuse, ainsi d’être un grand Professeur et Chercheur et aussi d’avoir été mon meilleur «étudiant» en cours de portugais. « Obrigado !» Aux membres du jury pour corriger et enrichir cette thèse avec leurs réflexions et leurs conseils; vii Au professeur Dr. Elias FATTAL de m’avoir permis de développer ce travail au sein de l’Institut Galien Paris Sud. À Helene CHACUN de l’Institut Galien Paris Sud pour son énorme gentillesse et sa disponibilité sur les manipulations de produits cytotoxiques et de substances radioactives; Au Dr. Claire GUEUTIN de l’UMR CNRS 8612, je vous remercie de votre contribution exceptionnelle sur les analyses de la chromatographie en phase liquide à haute performance; Au Dr. Alexandre MACIUK du laboratoire de pharmacognosie de l'UMR CNRS 8076, merci pour toutes les discussions scientifiques et l'assistance concernant la caractérisation des produits naturels; Au Dr. Kawthar BOUCHEMAL pour toute l'aide et l'encouragement de la recherche pendant cette thèse. Merci; Dr Nicolas HUANG de l’UMR CNRS 8612 pour l’enseignement de la rhéologie; À Stephanie NICOLAY et Audrey SOLGADI du Service d'Analyse des Médicaments et Métabolites (SAMM), et Claire BOULOGNE et Cynthia GILLET de la plateforme Imagif de microscopie électronique, pour toutes les aides sur caractérisation des systèmes développés; Je remercie Mesdames Patricia LIVET, Dominique MARTIN et Lucie LANDRY pour leur gentillesse et efficacité dans le traitement de mes documents administratifs; Je voudrais laisser exprimer ici mes profonds remerciements aux membres de l'équipe VI pour leur soutien durant cette étape. Claudio PALAZZO merci pour l’accueil initial et les moments importants partagés avec moi pendant cette expérience à Paris "Grazie Mille". Bénédicte SACKO-PRADINES, tu es fantastique, j'admire grandement ta volonté et ta détermination, merci pour toute l'aide que tu m’as apponté pour la réalisation de cette thèse "Muito Obrigado". À mes amis de «sourire vert-jaune» de cette équipe qui ont partages les jours de travail, Elquio ELEAMEN et André SILVA, merci de les moments de détente et réflexion sur la vie. Fanny BUHLER VARENNE je suis très heureux d'avoir partagé le bureau avec toi, merci pour la conversation et les goodies. Any TAYLOR merci pour ta joie dans cette dernière étape "Muchísimas gracias". Nick FRAZIER merci pour l’amitié « Thanks a lot ». À mes amies italiennes Martina BOMBARDI, Valeria CANDIOLI et Erika SPECOGNA je vous remercier pour tous les moments passés ensemble à l'intérieur et à l'extérieur du laboratoire. Toujours envie d'exprimer ma gratitude à Christelle ZANDANEL, Godefroy MAMADOU, Aurélia NEMO, Laura DE MIGUEL, Christine CHARRUEAU, Cristina PUIGVERT, Iuliana POPA et Florian VANNESTE pour l’aide à l'utilisation des appareils de laboratoire et pour l’amitié. Je tiens également à remercier mes amis de l’Institut Galien Paris Sud qui ont rendu mon séjour plus agréable à l'intérieur et à l'extérieur du laboratoire. Pour tous ceux qui ont eu le plaisir de travailler ensemble et m'avoir beaucoup appris, je vous remercie de ix ce faire de cette réalisation. En particulier, je remercie les Brésiliens à Chatenay- Malabry pour votre précieux soutien: Andreza ROCHELLE, Eloisa BERBEL, Thaís LEITE, Letícia SANTIAGO, Acarilia SILVA et Rachel OLIVEIRA. À mes amis français, merci pour l'enseignement culturel et faciliter mon intégration dans ce magnifique pays: Nadège GABROWSKI, Nadia ABED, Hubert CHAPUIS, Ludivine MOUSNIER, Tanguy BOISSENOT, Félix, Leila ZERKOUNE et Alice GAUDIN (Merci Beaucoup!). Dans la commission des amis italiens, je tiens à remercier la vraie amitié et disponibilité (grazie mille per tutto) de Giovanna GIACALONE, Valentina AGOSTONI, Dario CARRADONI (amige), Donato COSCO et Sabrina VALETTI. Pour mes amis dans de nombreux autres pays (Espagne, Liban, Allemagne, Serbie, l'Inde, le Danemark, Viêtnam, Algérie, Tunisie ...) Patricia CALLEJA, Chantal SABBAGH, Dunja SOBOT, Naila ELKECHAI, Chau TRAN LETUYET, Agnes GALOU (cœur brésilien), Ahmet AYKAÇ, Adam BOHR, Christian RUGE ,Gopan GOPALAKRISHNAN, Alain N'GUESSAN, Moritz BROICHSITTER et tous les autres que j’ai oublié de mentionner ici, merci beaucoup d’avoir partager chaque moment important avec moi. À mes amis de la Maison du Brésil à la Cité Universitaire Internationale de Paris pour les beaux et inoubliables moments de détente de ce parcours. En particulier, merci beaucoup à Julliane TAMARA, Kellen TJIOE, Barbara FONSECA, Monize MOURA, Jane BARBOSA, Raíssa MUSARRA, Karen FUKUSHIMA, Célio COUTINHO, Kata JIN, Elena BARBON, Alessandro BENFENATI, Geovana ELEAMEN, Evandro LEONARDI, Giovana LEONARDI, vous n’avez pas idée de l'importance de chacun dans ma vie, merci pour l’affection. Aux autres compagnons non moins importants, merci pour tout: Felipe CUNHA, Hudson POLONINI, Hind BENMOUSSA, Francisco CORTEZZI, Julio MACHADO, João CARNEIRO, Tania BUEHLER, Michelle MUNK, Larissa WARNAVIN, Val BARROS, Carolina MAFRA, Anamaria DINIZ, Magno KLEIN, Marcela FRANÇA, Rodrigo COUTO, Sheyla DINIZ, Ivanete AMARAL, Cauê de SOUZA, Fábio de OLIVEIRA, Sol DOMINGUEZ, Adrienne POLICHT, Hugo PENACK, Maria THEREZA, William HERRERA, Meiry MEZARI et Augusto CAPISTRANO. Ici, je laisse un grand merci à mes amis qui m'accueillirent dans leurs maisons et m'ont fait sentir la chaleur de ma famille proche. Carol CABÉ " minha filha" je vous remercie beaucoup pour votre amitié, pour l'accueil, pour partager les joies et les tristesses de cette période, pour me faire famille dans leur maison avec leurs fils Pedro e Vitória MORAES. Merci aussi à Amanda BRUN " fofinha" pour les attentions de chaque instant, je vous remercie énormément. Au Brésil Je tiens à remercier les amis du Laboratório de Sistemas Dipersos (LASID) en se faisant toujours présent dans ma vie tout au long de ce parcours. Gyselle HOLANDA, Nednaldo DANTAS, Lourena MAFRA, Miguel ADELINO, Érica LIRA, Scheyla DANIELA, Laura FERREIRA, Walteça SILVEIRA, Izabel LARISSA, Sarah RAFAELLY, Thales PONTES, Julieta GENRE, Francine AZEREDO, Rosilene xi SANTIAGO, Bartolomeu SANTOS, Henrique MARCELINO, Cybelle HOLANDA, Christian ASSUNÇÃO, Alexandrino JUNIOR, Karol NASCIMENTO, Priscila RIBEIRO et tous les autres « débutants » et « plus experimentés », je vous remercie beaucoup pour les discussions scientifiques et la convivialité au fils des ans. Je tiens également à exprimer mon énorme gratitude à chaque membre de mon équipe de travail (MECOP). Chacun, merci pour la confiance, de rêver et vibrer avec moi, pour tout ce que nous avions passé ensemble au fil des ans ... Il en valait la peine! Et s’il y avait des difficultés dans le parcours, elles ont été adoucies grâce à votre soutien et votre présence à mes côtés. J’en suis fier car nous avons grandi ensemble. À Andreza ROCHELLE, Éverton ALENCAR, Lucas MACHADO, Renata RUTCKEVISKI, Christian MELO e Teresa FERNANDES, je tiens à vous remercier tout particulièrement pour notre amitié. Donc, mes chers amis pour l'encouragement constant et positif foulent. En particulier, les " Étoiles" (Layany MOURÃO, Alice RODRIGUES, Daiane SOARES, Lílian SOLON, Beatriz BEZERRA, Carol CABE vous êtes top), Elizabeth SANTOS, Michelly SALES, Adriele LINS, Mara LALINE, Pryscila ARAUJO, Rita ARAUJO, Natalia RAFAELA, Kleybson FERNANDES, Mayara BASTOS, Samaria ARAUJO, Karla OLIVEIRA, Regildo GOMES, Bruno CAETANO, Glauber e Thais FERNANDES, Flavia ALINE, Silvaneide ROCHA, Gilton PEDRO, la famille de mon cœur (Altamira, João, Alexandre, Léa, Jane et Yasmin MARTINS), mes nombreux collègues de la Communauté Catholique Shalom, à ceux pas énumérés ici, mais présent dans le cœur, je vous remercie infiniment pour vos prières et votre rire qui m'a fortifié tout au long du chemin. Je voudrais aussi remercier à Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES) pour le financement à la science fourni pour l'élaboration de ce travail. Je remercie également aux Professeurs et employés administratives de la Faculté de Pharmacie de l’UFRN, la Rectrice et pro-Recteur de l’école de Doctarale de l’UFRN, à l’école de Doctorat en Biotechnologie (RENORBIO), en particulier la Professeur, Dr Lucymara AGNEZ et leurs collaborateurs d’avoir permis la réalisation de ce stage doctoral à l'étranger, étant toujours d'une grande attention. Et comme on le dit si bien, gardons le meilleur pour la fin. Voici, je remercie grandement tous les membres de ma famille, mais en particulier Francisca PEREIRA (Mainha) et Humberto XAVIER (Painho) par le don de la vie et de la motivation aux études. Pour les encouragements constants et leur compréhension pour mon absence de la maison. Sans vous près de moi, rien servirait tout cela parce que je suis cette personne grâce à votre support inconditionnel. Je vous remercie beaucoup. À mon frère et ma sœur que j’aime tant, Maxweel e Annice XAVIER, merci pour la motivation, l'enthousiasme et nos franches rigolades. Je remercie ma tante, Rosilene XAVIER, et sa famille: Lamberto, Yago e Ygor pour le support pendent ces années. Pour mes grands- parents (Nicinha et Joaquim Xavier, et Ana et Chiquito Pereira), oncles, cousins, je suis éternellement reconnaissant de croire que mon rêve était possible et d'être toujours à mes côtés. MERCI ! xiii AGRADECIMENTOS Esta tese foi desenvolvida pelo acordo de co-tutela entre a França e o Brasil. Na França, os estudos foram realizados na Faculdade de Farmácia da Université Paris Sud 11 em Châtenay-Malabry, dentro do Institut Galien Paris-Sud (UMR CNRS 8612) ao seio da equipe VI – Melhoramento de passagem das barreiras através de moléculas biologicamente ativas. No Brasil, os estudos foram conduzidos na Faculdade de Farmácia da Universidade Federal do Rio Grande do Norte em Natal/RN, no Laboratório de Sistemas Dispersos (LASID). Estes últimos anos em que fiz esta pesquisa foram de uma árdua jornada, de construção e amadurecimento. O caminho não foi um dos mais fáceis e nem um dos mais curtos, mas hoje ao olhar tudo apreendido posso dizer valeu a pena lutar por um sonho. Mas esta etapa só foi possível graças à presença e contribuição de inúmeras pessoas que passaram pela minha vida e deixaram suas marcas e conhecimentos. A estes, eu sou eternamente grato pelo apoio incondicional, pelas coisas que apreendi, pelos valores que guardei, pelas motivações impulsionadoras e por cada degrau conquistado desta vitória. Hoje vejo neste documento o registro de cada coisa vivida ao longo destes anos. E rumo a etapa final desta história, aqui deixo expresso a minha gratidão em palavras àqueles que direta ou indiretamente contribuíram com a sua construção. Portanto, a estes gostaria de estender meus sinceros agradecimentos. Inicialmente, agradeço a Deus por guiar minha vida, dando força, coragem, sabedoria e sentido para viver e buscar os sonhos que Ele sonhou para mim. Gostaria de agradecer enormemente a minha diretora francesa de tese, Dra. Christine VAUTHIER, por ter me dado à oportunidade de conduzir este doutorado ao seio de seu laboratório. Obrigado por todos os ensinamentos, discussões científicas, dedicação e preocupação com a minha formação. Obrigado ainda por fazer do laboratório a minha casa e da equipe a minha família durante estes 18 meses em solo Francês. Gostaria igualmente de agradecer enormemente ao meu diretor brasileiro de tese, Prof. Dr. Sócrates EGITO, por guiar meus estudos em seu laboratório durante estes anos de doutorado. Muito obrigado por tudo, pela paciência, pelo estimulo, pela confiança e pelos preciosos ensinamentos. Agradeço ainda por ter possibilitado meu ingresso nesta grande « aventura ». Na França: Ao professor Dr. Gilles PONCHEL, responsável pela equipe VI, expresso a minha gratidão pelo acolhimento, pelo apoio e pelas discussões científicas. Obrigado pela sua simplicidade e alegria contagiante, pois além de um grande professor e pesquisador foi o meu melhor “aluno” nas aulas de português. Obrigado! A banca examinadora por corrigir e enriquecer esta tese com suas reflexões e conselhos; Ao professor Elias FATTAL por me permitir desenvolver este trabalho dentro da UMR CNRS 8612; xv A Dra. Helene CHACUN da UMR CNRS 8612 por sua enorme gentileza e disponibilidade na manipulação de substâncias citotóxicas e radioativas; A Dra. Claire GUEUTIN da UMR CNRS 8612, obrigado por sua enorme contribuição nas dosagens por cromatografia líquida de alta eficiência; Ao Dr. Alexandre MACIUK do Laboratório de Farmacognosia da UMR CNRS 8076, agradeço a todas as discussões científicas e ajudas relativas às caracterizações dos produtos naturais; A Dr. Kawthar BOUCHEMAL por todas as ajudas e encorajamento nas pesquisas ao longo desta tese. Obrigado; Ao Dr. Nicolas HUANG da UMR CNRS 8612 por toda paciência e ensinamento sobre reologia; A Stéphanie NICOLAŸ e Audrey SOLGADI da Plataforma de Análise de Medicamentos e Metabólitos (SAMM), e a Claire BOULOGNE e Cynthia GILLET da plataforma IMAGIF de Microscopia Eletrônica pelas ajudas nas caracterizações dos sistemas desenvolvidos; Agradeço à Patricia LIVET, Dominique MARTIN e Lucie LANDRY por toda gentileza e eficácia no tratamento dos meus documentos administrativos; Gostaria de deixar expresso aqui o meu enorme agradecimento aos membros da equipe VI por toda à motivação nesta etapa. Cláudio PALAZZO muito obrigado pelo acolhimento inicial e por dividir comigo importantes momentos desta experiência em Paris « Grazie Mille ». Bénédicte SACKO-PRADINES, você é fantástica, admiro muito sua força de vontade e determinação, obrigado por toda ajuda nesta tese, obrigado pela sua disponibilidade « Merci beaucoup ». Aos meus amigos de sorriso Verde-Amarelo aos quais dividi dias de trabalho nesta equipe, Elquio ELEAMEN e André SILVA, obrigado pelos momentos de descontração e reflexão sobre a vida. Fanny BUHLER VARENNE sou muito feliz por ter dividido a sala de trabalho contigo, obrigado pelas conversas e pelas guloseimas. Any TAYLOR muito obrigado pela sua alegria nesta etapa final « Muchísimas gracias ». Nick FRAZIER obrigado pela sua amizade « Thanks a lot ». A minhas amigas italianas Martina BOMBARDI, Valeria CANDIOLI e Erika SPECOGNA muito obrigado por todos os momentos passados juntos dentro e fora do laboratório. Ainda quero expressar minha gratidão à Christelle ZANDANEL, Godefroy MAMADOU, Aurélia NEMO, Laura DE MIGUEL, Christine CHARRUEAU, Cristina PUIGVERT, Iuliana POPA e Florian VANNESTE pelas ajudas no manuseio dos aparelhos no laboratório e pela amizade. Gostaria também de agradecer aos meus amigos da UMR CNRS 8612 que tornaram a minha estadia mais agradável dentro e fora do laboratório. A todos estes que tive o prazer de trabalhar junto e que muito me ensinaram, obrigado por se fazerem presente nesta conquista. Em especial, agradeço a ala brasileira em Chatenay-Malabry por todo o apoio: Andreza ROCHELLE, Eloisa BERBEL, Thaís LEITE, Letícia SANTIAGO, Acarilia SILVA e Rachel OLIVEIRA. Aos meus amigos franceses, muito obrigado por cada ensinamento cultural e por facilitar a minha inserção dentro deste belo país: xvii Nadège GABROWSKI, Nadia ABED, Hubert CHAPUIS, Ludivine MOUSNIER, Tanguy BOISSENOT, Félix, Leila ZERKOUNE e Alice GAUDIN (Merci Beaucoup!). Na comissão dos amigos Italianos quero muito agradecer pela verdadeira amizade e disponibilidade (grazie mille per tutto) de Giovanna GIACALONE, Valentina AGOSTONI, Dario CARRADONI (amige), Donato COSCO e Sabrina VALETTI. Aos meus amigos de inúmeras outras nacionalidades (Espanha, Líbano, Alemanha, Servia, Índia, Dinamarca, Vietnã, Argélia, Tunísia…): Patrícia CALLEJA, Chantal SABBAGH, Dunja SOBOT, Naila ELKECHAI, Chau TRAN LETUYET, Agnes GALOU (coração brasileiro), Ahmet AYKAÇ, Adam BOHR, Christian RUGE ,Gopan GOPALAKRISHNAN, Alain N'GUESSAN, Moritz BROICHSITTER e a todos os outros que eu esqueci de citar aqui, muito obrigado pelos momentos importantes divididos. Aos meus amigos de residência pelos belos e inesquecíveis momentos de descontração nesta jornada. Em especial, muito obrigado a Julliane TAMARA, Kellen TJIOE, Barbara FONSECA, Monize MOURA, Jane BARBOSA, Raíssa MUSARRA, Karen FUKUSHIMA, Célio COUTINHO, Kata JIN, Elena BARBON, Alessandro BENFENATI, Geovana ELEAMEN, Evandro LEONARDI, Giovana LEONARDI, vocês não tem dimensão da importância de cada uma na minha vida, obrigado pelo carinho. Aos demais e não menos importantes companheiros, obrigado por tudo: Felipe CUNHA, Hudson POLONINI, Hind BENMOUSSA, Francisco CORTEZZI, Julio MACHADO, João CARNEIRO, Tania BUEHLER, Michelle MUNK, Larissa WARNAVIN, Val BARROS, Carolina MAFRA, Anamaria DINIZ, Magno KLEIN, Marcela FRANÇA, Rodrigo COUTO, Sheyla DINIZ, Ivanete AMARAL, Cauê de SOUZA, Fábio de OLIVEIRA, Sol DOMINGUEZ, Adrienne POLICHT, Hugo PENACK, Maria THEREZA, William HERRERA, Meiry MEZARI e Augusto CAPISTRANO. Aqui deixo um especial agradecimento as minhas amigas que me acolheram em seus lares e me fizeram sentir o calor da minha família tão mais próximo. Carol CABÉ « minha filha » muitíssimo obrigado pela sua amizade, pelo acolhimento, por dividir as alegrias e tristezas deste período, por me fazer família em sua casa junto aos seus filhos Pedro e Vitória MORAES. Agradeço também a Amanda BRUN « fofinha » pela torcida em cada momento, obrigado enormemente. No Brasil Quero agradecer aos amigos do Laboratório de Sistemas Dipersos (LASID) por se fazerem sempre presentes em minha vida ao longo desta longa caminhada. Gyselle HOLANDA, Nednaldo DANTAS, Lourena MAFRA, Miguel ADELINO, Érica LIRA, Scheyla DANIELA, Laura FERREIRA, Walteça SILVEIRA, Izabel LARISSA, Sarah RAFAELLY, Thales PONTES, Julieta GENRE, Francine AZEREDO, Rosilene SANTIAGO, Bartolomeu SANTOS, Henrique MARCELINO, Cybelle HOLANDA, Christian ASSUNÇÃO, Alexandrino JUNIOR, Karol NASCIMENTO, Priscila RIBEIRO e a todos os outros novatos e antigos, meu muito obrigado pelas discussões científicas e momentos de descontração ao longo destes anos. xix Quero ainda expressar minha enorme gratidão a cada pessoa da minha equipe de trabalho (MECop). A cada um, obrigado pela confiança, por sonharem e torcerem comigo, por cada coisa que passamos juntos ao longo destes anos... Valeu a Pena! E se nesta etapa houveram dificuldades, estas só não foram maiores, pois tive o apoio de vocês ao meu lado e disto muito me orgulho por crescermos juntos. À Andreza ROCHELLE, Éverton ALENCAR, Lucas MACHADO, Renata RUTCKEVISKI, Christian MELO e Teresa FERNANDES obrigado antes de tudo pela amizade. Aos meus tão caros amigos pelo incentivo constante e a torcida positiva. Em especial as « Estrelas » (Layany MOURÃO, Alice RODRIGUES, Daiane SOARES, Lílian SOLON, Beatriz BEZERRA, Carol CABE vocês são demais), Elizabeth SANTOS, Michelly SALES, Adriele LINS, Mara LALINE, Pryscila ARAUJO, Rita ARAUJO, Natalia RAFAELA, Kleybson FERNANDES, Mayara BASTOS, Samaria ARAUJO, Karla OLIVEIRA, Regildo GOMES, Bruno CAETANO, Glauber e Thais FERNANDES, Flavia ALINE, Silvaneide ROCHA, Gilton PEDRO, à família de coração (Altamira, João, Alexandre, Léa, Jane e Yasmim MARTINS), aos inúmeros amigos de partilhas da Comunidade Católica Shalom, aos não citados aqui, mas presentes no coração, a todos vocês muitíssimo obrigado pelas orações e risos que me fortaleceram no percurso. Agradeço ainda à Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES) pelo auxílio financeiro à Ciência concedido para o desenvolvimento desse trabalho. Agradeço também aos professores e funcionários do Departamento de Farmácia da UFRN, à Pró-reitora de Pós-graduação da UFRN, ao Programa de Pós- graduação em Biotecnologia (RENORBIO), em especial à Profa. Dra. Lucymara AGNEZ e seus funcionários por permitirem realizar o estágio doutoral no exterior, sendo sempre de grande atenção e presteza. E como bem se diz, melhor está guardado para o final. Eis que eu agradeço enormemente a todos os membros de minha família, mas em particular Francisca PEREIRA (Mainha) e Humberto XAVIER (Painho) pelo dom da vida e motivação nos estudos. Pela torcida sempre constante e entender as minhas ausências em casa. Sem vocês por perto de nada adiantaria tudo isso, pois me tornei esta pessoa graças ao apoio incondicional de vocês. Portanto, muitíssimo obrigado. Aos meus irmãos que tanto amo, Maxweel e Annice XAVIER, obrigado pela torcida, motivação e boas gargalhadas. A minha tia, Rosilene XAVIER, e sua família: Lamberto, Yago e Ygor por todo o suporte ao longo destes anos. Aos meus avós (Nicinha e Joaquim XAVIER, e Ana e Chiquito PEREIRA), tios, primos, sou eternamente grato por acreditaram que meu sonho era possível e por estarem sempre ao meu lado. Obrigado! xxi ABSTRACT The oral route encourages a progressive interest in anticancer drug administration, currently, mainly administered by parenteral route. However, that route is limited by problems related to the physicochemical properties of drugs, physiological factors and dosage forms that reduce the overall bioavailability of the drug. To overcome limitations, systems based on lipids and polymeric nanoparticles have been used. Therefore, the aim of this project was to develop dispersed systems for oral route containing copaiba oil in their internal phase as vehicle for an anticancer drug, paclitaxel. Researches were developed in two directions aiming to formulate suitable microemulsions and nanocapsules. In the first part of the project, copaiba oil was analyzed; a method of paclitaxel dosage in this oil was developed and validated. Then formulation of the copaiba oil/water microemulsion was assessed taking into account solubility parameter of both oil components and surfactants. This led to the obtaining of stable microemulsion with the copaiba essential oil having remarkably high volume fraction of dispersed phase (19.6%) at low surfactant concentration (13.7%). It was demonstrated that paclitaxel could be incorporated in the microemulsion without disturbing the characteristics of the system. The second system developed in this work consisted mucoadhesive nanocapsules encapsulating copaiba oil. The formulation was based on an experimental design approach which one was also used during encapsulation of the paclitaxel. The development of this system included its labeling with a fluorescent probe and incorporating radiolabeled paclitaxel. Stability of the nanocapsules in simulated gastrointestinal medium was investigated and mucoadhesion on gut mucosa was evaluated. This work has proposed two formulations of paclitaxel in nanosystems which are ready for an evaluation for their capacity to deliver this anticancer drug by the oral route. KEYWORDS: Oral route, copaiba oil, paclitaxel, microemulsion, nanocapsules, hydrophilic-lipophilic balance, chitosan, mucoadhesion. xxiii RÉSUMÉ La voie orale suscite un intérêt pour l'administration des médicaments anticancéreux, qui sont encore administrés essentiellement par voie parentérale. Cette voie est limitée par les problèmes liés aux propriétés physico-chimiques des principes actifs, aux facteurs physiologiques qui limitent fortement leur biodisponibilité orale. Pour surmonter certaines limitations, l'utilisation de systèmes à base de lipides et de nanoparticules polymères peuvent se montrer très performants. L'objectif de ce travail a consisté à développer des systèmes dispersés pour la voie orale contenant dans leur phase interne de l'huile de copaïba servant de véhicules à des médicaments anticancéreux comme le paclitaxel. Ce travail de recherche a été mené selon deux grands axes: l'un orienté vers les systèmes de type microémulsion et l'autre vers les nanocapsules. Dans la première partie du travail, l'huile de copaïba a été analysée et une méthode de dosage du paclitaxel dans l'huile de copaïba a été développée et validée. Dans la suite du travail, des microémulsions d'huile de copaïba/eau ont été formulée suivant une approche basée sur les paramètres de solubilité des composés de l'huile et des tensioactifs. Ce travail a permis l'obtention de microémulsion contenant des fractions volumiques importantes de l'huile essentielle de copaïba (19.6%) tout en maintenant les concentrations en tensioactifs faible (13.7%). Du paclitaxel a pu être incorporé dans les microémulsions sans perturber notablement les caractéristiques du system. Le deuxième système développé dans ce travail a été des nanocapsules mucoadhésives contenant de l'huile de copaïba. La formulation a été réalisée en mettant en œuvre un plan d'expérience à 2 niveaux avec trois facteurs. Des nanocapsules incorporant du paclitaxel et marquée par une sonde fluorescente et du paclitaxel radiomarqué ont également été développées. La stabilité de ces nanocapsules a été étudiée dans des milieux gastrique et intestinaux simulés. Leur mucoadhésion a été évaluée sur des fragments de muqueuse intestinale prélevés chez le rat. Les résultats de ces travaux ont conduit au développement de deux formulations de paclitaxel dans des nanosystèmes originaux qui pourront par la suite être évalués pour en étudier leur capacité à délivrer l'agent anticancéreux par voie orale. MOTS CLES : voie orale, huile de copaïba, paclitaxel, microémulsion, nanocapsules, équilibre hydrophile-lipophile, chitosane, mucoadhésion. xxv RESUMO A via oral suscita um interesse crescente para a administração de medicamentos anticancerígenos, os quais, na atualidade, ainda são administrados essencialmente pela via parenteral. No entanto, essa via é limitada por problemas relacionados às propriedades físico-químicas do fármaco, fatores fisiológicos e as formas farmacêuticas que reduzem a biodisponibilidade oral do medicamento. Para superar essas limitações, sistemas lipídicos e poliméricos nanoparticulados têm sido utilizados. O objetivo deste trabalho foi desenvolver sistemas dispersos para via oral, contendo na fase interna óleo de copaíba servindo de veículos para fármacos anticancerígenos, como paclitaxel. Os estudos foram desenvolvidos em dois sentidos com o objetivo de formular adequadas microemulsões e nanocápsulas mucoadesivas. Na primeira parte do projeto, o óleo de copaíba foi analisado e um método de dosagem de paclitaxel neste óleo foi desenvolvido e validado. Posteriormente, microemulsão de óleo de copaíba em água foi desenvolvida em função dos cálculos dos parâmetros de solubilidade entre os componentes do óleo de copaíba e os surfactantes. Tal processo levou à obtenção de microemulsão estável com o óleo essencial de copaíba cotendo elevada fração volumétrica da fase dispersa (19,6%) e uma baixa concentração de surfactante (13,7%). O Paclitaxel foi incorporado na microemulsão sem causar perturbação nas características do sistema. O segundo sistema desenvolvido neste trabalho consistiu de nanocápsulas mucoadesivas para encapsulação do óleo de copaíba. A formulação foi baseada na abordagem de planejamento experimental, o qual também foi usado durante a encapsulação do paclitaxel. O desenvolvimento deste sistema ainda incluiu a marcação com uma sonda fluorescente e incorporação de paclitaxel radioativo. A estabilidade das nanocápsulas foi investigada em meio gastrointestinal simulado. A mucoadesão foi avaliada em mucosa intestinal de ratos. Os resultados deste trabalho conduziram ao desenvolvimento de duas formulações de paclitaxel em nanosistemas originais que estão prontos para avaliação da sua capacidade de entregar de fármaco anticancerígeno pela via oral. PALAVRAS-CHAVES: Via oral, óleo de copaíba, paclitaxel, microemulsão, nanocápsulas, equilíbrio hidrófilo-lipofílico, quitosana, mucoadesão. TABLE DES MATIERES Remerciements ................................................................................................................ v Agradecimentos ........................................................................................................... xiii Abstract ........................................................................................................................ xxi Résumé ........................................................................................................................ xxiii Resumo ........................................................................................................................ xxv Abbreviations / abreviations ........................................................................................ 31 INTRODUCTION GÉNÉRALE ................................................................................. 35 SECTION I- Développement des méthodes .............................................................. 47 Chapter I-Development of gas-chromatography method for the analysis of copaiba oil........................................................................................... 49 Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients ........................................................ 85 SECTION II-Les systèmes d'administration de médicaments à base de lipides . 111 Chapter III-Prospective study for the development of emulsion systems containing natural oil products.............................................................. 113 Chapter IV- Microemulsion-based drug delivery systems containing natural oils ............................................................................................. 123 Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content ........ 179 Chapter VI-Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. . 221 SECTION III- Les systèmes d'administration de médicaments à base des polymères ..................................................................................................................... 251 Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil ...................................................................... 253 Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core-shell nanocapsules and evaluation of their mucoadhesion by in vitro methods ....................................................... 289 DISCUSSION GÉNÉRALE ...................................................................................... 331 CONCLUSION ET PERSPECTIVE ........................................................................ 347 RÉFÉRENCES ........................................................................................................... 353 ANNEXE ..................................................................................................................... 397 CURRICULUM VITAE ............................................................................................ 409 31 ABBREVIATIONS / ABREVIATIONS %CI Creaming index ANOVA Analysis of variance BSTFA N,O-Bis(trimethylsilyl) trifluoroacetamide CEO Copaiba essential oil Ch Chitosan Cop Copaiba oil CRO Copaiba resin oil CS Clinical strain DL Drug loading DLS Dynamic light scattering DMAPP Dimethylallyl pyrophosphate DMSO Dimethyl sulfoxide EE Entrapment efficiency Eh Hydrogen bonding forces ELISA Enzyme-linked immunosorbent assay Fd London dispersion forces Fp Keesom dipolar force GC-FID Gas chromatography–flame ionization detector GC-MS Gas chromatography–mass spectrometry HLB Hydrophilic-lipophilic balance HLB0 Hydrophilic-lipophilic balance requis HPLC High-performance liquid chromatography IBCA Isobutyl cyanoacrylate IPP Isopentenyl pyrophosphate LOD Limit of detection LOQ Limit of quantification ME Microemulsion 33 MECop Copaiba oil-loaded microemulsion MECop Ptx Paclitaxel into copaiba oil-loaded microemulsion MIC Minimum inhibitory concentration MWCO Molecular weight cut off NC Nanocapsule NCC Copaiba oil-loaded chitosan-poly (isobutyl cyanocrylate) core-shell nanocapsules NCC [H³]-Ptx Radioactive paclitaxel encapsulated into copaiba oil-loaded chitosan-poly (isobutyl cyanocrylate) core-shell nanocapsules NCC Ptx Paclitaxel encapsulated into copaiba oil-loaded chitosan-poly (isobutyl cyanocrylate) core-shell nanocapsules NCCdry Ptx Paclitaxel encapsulated into copaiba oil-loaded chitosan-poly (isobutyl cyanocrylate) core-shell nanocapsules after drying process NCCfluo Ptx Polyfluor ® 570 labeled in paclitaxel encapsulated into copaiba oil-loaded chitosan-poly (isobutyl cyanocrylate) core-shell nanocapsules O/W Oil-in-water OD Optical density PdI Polydispersity index PIT Phase inversion technique Ptx Paclitaxel RSD Relative standard deviation RT Retention time SD Standard deviation TEM Transmission electron microscopy TLC Thin layer chromatography TTC Tetrazolium chloride W/O Water-in-oil Introduction Générale Introduction Générale 37 Le cancer est une des maladies les plus importantes dans le monde aussi bien par l’augmentation de son incidence, sa prévalence et de sa mortalité. Il s'agit d'un problème de santé prioritaire qui est motivé par le fait que le cancer est une cause de décès majeure dans la plupart des pays dans monde (Fang et al., 2011; Siegel et al., 2014). Le paclitaxel est l’un des plus puissants agents anti-cancéreux actuellement utilisé pour le traitement des cancers du poumon et du sein, de la leucémie aiguë, ainsi que des cancers de l'ovaire avancé, du cerveau et des carcinomes du col de l’utérus (Forastiere, 1994; Rowinsky et al., 1995; Weaver, 2014). C’est un pseudoalkaloide qui présente une structure diterpénoïde. Cette molécule qui a été initialement isolée de Taxus brevifolia (Fang et al., 2005) fonctionne en favorisant la stabilisation des microtubules inhibant ainsi la prolifération cellulaire et finalement l'induction de l'apoptose (Schiff et al., 1979; Hamel et al., 1981; Horwitz, 1992). Comme de nombreux agents anti-cancéreux, le paclitaxel est administrés par voie intraveineuse, car il présente une faible biodisponibilité orale (Weingart et al., 2008). Cependant, l'administration par la voie intraveineuse présente de nombreux inconvénients comme l'extravasation du médicament ou du sang au site d'injection, l’infection de cathéter, et la thrombose qui peuvent être évités par l'administration du médicament par voie orale (Roger et al., 2011). L’administration des traitements de chimiothérapie par voie orale présente plusieurs intérêts majeurs outre celui d'améliorer le confort du patient au moment de l'administration. Elle permet d'améliorer l'observance du traitement par sa simplicité de mise en œuvre, de diminuer le coût du traitement et d'améliorer la sécurité du traitement pour le patient (Borner et al., 2001; Batlle et al., 2004). Malgré ces avantages potentiels, l'administration orale du paclitaxel est limitée par les propriétés physico-chimiques du médicament qui sont inappropriées pour permettre une Introduction Générale 38 bonne biodisponibilité par cette voie d'administration (solubilité, lipophilie, pKa). Elle est également limitée par des facteurs physiologiques (temps de transit intestinal, pH gastro-intestinal, mécanismes d'absorption, métabolisme rapide dans les entérocytes de l'épithélium digestif) et à d'autres aspects liés aux formes galéniques (faible perméabilité, instabilité). L'ensemble de ces facteurs ne permettent pas d'obtenir une biodisponibilité satisfaisante de la molécule par voie orale (Prabhu et al., 2005; Ensign et al., 2012). Il existe plusieurs facteurs qui contribuent à l'efficacité et à l'utilité des préparations pharmaceutiques en tant que supports pour la délivrance orale de molécules insolubles dans l’eau. Il s'agit notamment de la capacité des médicaments à se dissoudre, de la vitesse du transit intestinal et de l'absorption des médicaments par la muqueuse intestinale après administration orale (Liu et al., 2011). Donc, l’amélioration de la biodisponibilité orale de tels médicaments permettrait d’améliorer encore l'efficacité thérapeutique des molécules et la compliance du patient (Singh et al., 2009). Au cours des dernières années, divers formes galéniques ont été proposés en vue d'améliorer la délivrance orale de molécules difficiles à administrer par cette voie d'administration. Certaines font appel à des nanomédecines comme des nanoparticules, des nanocapsules, des micelles, des microémulsions, des liposomes, des matériaux nanoporeux et des multicouches polymères. Parmi eux, les systèmes à base de lipides et les nanoparticules polymères sont apparus utiles pour surmonter certaines limitations. Par exemple, au cours de la dernière décennie, il a été montré que les formulations à base de lipides présentent un meilleur potentiel pour solubiliser des molécules difficilement solubles dans des milieux aqueux et notamment les molécules liposolubles. Simultanément, ils améliorent la stabilité et protègent les molécules contre une dégradation. L'amélioration de la solubilité et de la stabilité des molécules actives s'accompagne généralement d'une augmentation de leur biodisponibilité orale (Shah et Introduction Générale 39 al., 1994; Constantinides, 1995; Zhao et al., 2005; Poullain-Termeau et al., 2008; Kalepu et al., 2013). Les nanocapsules biodégradables sont des systèmes vésiculaires dans lesquels un principe actif est confiné dans une cavité constituée d'un cœur liquide, elles ont été proposées pour contrôler la libération de principes actifs et le cibler vers les sites d'absorption. Quelque soit le système retenu, son utilisation est de délivrer de manière contrôlée et la plus efficace possible le principe actif. Pour cela, le système doit être conçu de manière à intégrer les différentes fonctionnalités qui lui permettront au final d'améliorer la biodisponibilité de la molécule pharmacologiquement active transportée (Li et al., 2001; Tong et al., 2008; Mora-Huertas et al., 2010; Perrier et al., 2010; Roger et al., 2010a; Groo et al., 2013). Les huiles végétales entrent dans la composition de nombreux systèmes d'administration orale de principes actifs (Lee et al., 1995; Dantas et al., 2010; Attaphong et al., 2012). Certaines huiles végétales ont des propriétés pharmacologiques utilisées en médecine traditionnelle. Par exemple, l’huile de Copaïba, Copaifera langsdorffi, produite au Brésil est largement utilisés en médecine traditionnelle pour le traitement de maladies inflammatoires, microbiologiques et cancéreuse (Gomes et al., 2007; Mendonça et al.,2009a; Comelli-Júnior et al., 2010; Souza et al., 2011). Son activité pharmacologique est liée à sa composition en composés diterpéniques et sesquiterpéniques (Veiga-Junior et al., 2002; Gomes et al., 2008). Il pourrait être proposé que de telles huiles deviennent un constituant majeur d'un système d'administration de médicament anticancéreux et pourrait ainsi éventuellement potentialiser l'activité biologique d'un principe actif associé au vecteur. L'objectif de notre travail de thèse a été de développer des systèmes d'administration des médicaments anticancéreux pour la voie orale à base d'huile végétale thérapeutique. Introduction Générale 40 L'huile thérapeutique sélectionnée pour ce travail a été l'huile de copaïba et le principe actif anticancéreux retenu a été le paclitaxel. Les travaux ont été menés en trois parties. La première partie a été consacrée au développement de méthodes destinées à analyser et doser l'huile de copaïba (chapitre I) et à doser le paclitaxel dans l'huile de copaïba pour en déterminer sa solubilité dans l'huile de copaïba et son coefficient de partage (chapitre II). La deuxième partie du travail qui regroupe les chapitres III à VI, a été consacrée à des travaux de formulation du paclitaxel dans des microémulsions et d'en évaluer la capacité à promouvoir l'attachement du paclitaxel sur du tissu intestinal ex-vivo. Ainsi, le chapitre III de la thèse propose une revue de l’état de l’art du développement de micro- émulsion avec des huiles naturelles. Le chapitre IV présente les résultats d'un travail initial qui avait pour objectif de mettre en place de l'huile de copaïba dans des systèmes émulsionnés. Il est intitulé « Prospective study for the development of emulsion systems containing natural oil products». Le chapitre V présente la formulation et la caractérisation d'une microémulsion préparée avec de l'huile de copaïba. La stratégie de formulation s'est appuyée sur l'utilisation des paramètres de solubilité destinée à optimiser la miscibilité des parties lipophiles des tensioactifs dans les constituants majeurs de l'huile de copaïba. L'incorporation du paclitaxel dans la microémulsion et l'évaluation de la mucoadhésion est décrite dans le chapitre VI. La troisième partie de nos travaux consignée dans les chapitres VII à VIII a été consacrée à l'étude de formulations de nanocapsules polymères. 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Introduction Générale 45 Section I Développement des méthodes Chapter I Development of gas-chromatography method for the analysis of copaiba oil. Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 51 Le travail présenté dans ce premier chapitre avait pour objectif de développer une méthode rapide, simple et précise pour la quantification des huiles copaïba. Les méthodes a été développées et validées en utilisant une technique chromatographie en phase gazeuse pour être appliquée à l'analyse de l'huile résine et de l'huile essentielle de copaïba. L’huile essentielle de copaïba a été efficacement extraite par la méthode de l'hydrodistillation à partir de l'huile de résine de copaïba. La réaction de dérivatisation de l’huile résine a été effectuée et confirmée par technique de chromatographie sur couche mince qui a permis l'identification des composés diterpéniques. Les analyses en chromatographie en phase gazeuse couplée à la spectrométrie de masse ont été mises au point et en œuvre pour déterminer la composition des huiles copaïba et identifier la position des pics de ces composants. Les principaux composés identifiés dans l'huile essentielle de copaïba ont été le β-bisabolène (23,6%), le β-caryophyllène (21,7%) et l'α-bergamotène (20,5%). Les principaux composant identifiés dans l’huile résine de copaïba méthylée ont été l'acide copalique (15,6%), le β-bisabolène (12,3%), le β- caryophyllène (7,9%), l'α-bergamotène (7,1%) et le l'acide Labd-8 (20) -ène-15,18- dioïque (6,7%). Une bonne corrélation entre les chromatographies en phase gazeuse en utilisant les détecteurs à ionisation de flamme et de spectrométrie de masse ont été obtenus au cours de la transposition de la méthode d’analyse. La méthode a démontré une haute performance pour les paramètres de validation pris en compte incluant la sensibilité, la spécificité, la linéarité, la précision, l'exactitude et les limites de détection et de quantification pour les analyses du β-caryophyllène, α- humulène et l'oxyde de caryophyllène dans les huiles de copaïba. Ce travail a été adapté à la quantification fiable dans le contrôle de la qualité de l'huile de copaïba et peut également être utilisé pour mesurer l’huile de copaïba lorsqu'il est chargé dans les formulations pharmaceutiques ou cosmétiques. Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 52 Mots-clés: Huile de copaïba, composition chimique, chromatographie en phase gazeuse à haute résolution, validation, β-caryophyllène, α-humulène, oxyde de caryophyllene Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 53 DEVELOPMENT OF GAS-CHROMATOGRAPHY METHOD FOR THE ANALYSIS OF COPAIBA OIL. Xavier-Junior, F.H. 1, 2 , Maciuk, A. 3 , Morais, A.R.V. 1, 2 , Alencar, E.N.¹, Rehder, V. L. G. 4 , Egito, E.S.T. 1 , Vauthier, C.²*, 1 Universidade Federal do Rio Grande do Norte, Centro de Ciências da Saúde, Departamento de Farmácia, Laboratório de Sistemas Dispersos (LaSiD). Av. Gal. Gustavo Cordeiro de Farias, S/N, Petrópolis, 59010-180, Natal-RN-Brazil. 2 Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. ³ Université Paris Sud, Laboratoire de Pharmacognosie - UMR CNRS 8076 BioCIS - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. 4 Universidade Estadual de Campinas (UNICAMP) – Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas. Rua Alexandre Cazelatto, 999, Vila Betel, Paulínia – SP. *Corresponding author: Christine Vauthier Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. Christine.vauthier@u-psud.fr Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 54 ABSTRACT A rapid, simple and precise method for the quantification of copaiba oils (Copaifera langsdorffii) have been developed and validated using gas chromatography analyses. Copaiba essential oil was efficiently extracted by hydrodistillation method from the copaiba resin oil. Oil derivatization was performed and confirmed by Thin Layer Chromatography technique which allowed the identification of diterpenes compounds. Gas chromatography coupled to mass spectrometry analyses was effectively developed to determine the composition of the copaiba oils. The main compounds identified in the copaiba essential oil were β-Bisabolene (23.6%), β-caryophyllene (21.7%) and α- bergamotene (20.5%). On the other hand, from the methylated copaiba resin oil were copalic acid (15.6%), β-Bisabolene (12.3%), β-caryophyllene (7.9%), α-bergamotene (7.1%) and Labd-8(20)-ene-15,18-dioic acid (6.7%) were found. A good correlation between the gas chromatography interfaced with flame ionization and mass spectrometry detectors were obtained favoring the transposition of the methodology analyses. The method showed a high performance concerning sensitivity, specificity, linearity, precision, accuracy, limits of detection and quantification parameters for the β- caryophyllene, α- humulene and caryophyllene oxide analyses in the copaiba oils. This work should be suitable to the reliable quantification in the quality control of copaiba oil and can also be used to copaiba oil quantification when loaded in pharmaceutical or cosmetic formulations. Keywords: Copaiba oil, Copaifera langsdorffii, Chemical composition, high resolution gas chromatography, Validation, β- caryophyllene, α- humulene, caryophyllene oxide Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 55 1.0. INTRODUCTION The significant use and development of pharmaceuticals originated from synthetic and natural sources have taken place along with the analytical methods responsible for determining, identifying and quantifying those products (Aturki et al., 2014). Several methods are responsible for analyses of drugs, impurities, intermediates, degradation products, mixtures of compounds, phytoextracts etc (Maggio et al., 2014). Among these methods, such as, potentiometric, spectroscopic and microbiological, the chromatographic ones stand out due to the useful property of separation and its powerful performance for the analysis of complex products such as natural oils, which can be further enhanced interestingly along with other possible features. Concerning the use for pharmaceutical application, the most known chromatographic methods include high performance liquid chromatography, liquid chromatography coupled to mass spectrometer and gas chromatography coupled to different detectors, besides the traditional techniques such as thin layer chromatography, especially concerning screening in complex mixtures. As in any chromatographic method, the methods might allow to access separation, identification and quantification. Accordingly, gas chromatography is well used in several fields of science (Skoog et al., 2007; Attimarad et al., 2011; Marriott et al., 2012). Gas chromatography technique is one of the most efficient concerning separations of complex mixtures such as natural products in which compounds can be volatilized. Natural products have been widely studied regarding development of novel pharmaceutical compounds due to their pharmacological activities and their renewable character. The active compounds obtained from vegetable sources are usually complex mixtures of plant’s secondary compounds known by their protective activity against Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 56 predators. However, on the human body, these compounds are pharmacologically active, and they are likely to have evolved in order to interact with cell membranes and interact with specific target proteins (Stone & Williams, 1992; Saleem et al., 2010). Copaiba oil is extracted from trees of the genus Copaifera. The trees from this genus are distributed amongst South America, Central America and Africa. However, the greater number of species is located in South America, specifically, in Brazil (Veiga Junior & Pinto, 2002). The oil extracted from the trunks of the trees has been used in folk medicine since ancient times. A complex mixture of diterpenes and sesquiterpenes comprises the oil (Gramosa & Silveira, 2005; Sousa, J. P. B. et al., 2011; Gelmini et al., 2013; Alencar, É. N. et al., 2015). Besides the pharmacological activities that these compounds provide to the copaiba oil, they can be useful to identify and quantify copaiba oil species in pharmaceutical systems. Amongst the currently studied pharmacological activities of copaiba oil, its antibacterial, antifungal, anti- inflammatory, healing, anti-Leishmania and anti-cancer activities have attracted the attention of many researchers (Vieira et al., 2009; Deus et al., 2011; Santos et al., 2011; Santos et al., 2013; Sousa et al., 2013). Over the last years, biological studies on copaiba oil justified its vast use in folk medicine and its importance for the development of new natural products (Xavier- Júnior et al., 2012b). Therefore, the development of a validated analytical method to accurately quantify these compounds became mandatory. A method validation is performed in order to standardize the process and the use of the instrumentation aiming to minimize random error and ensure that the method may be trusted and be used in different location. Moreover, the development and the determination of many experimental parameters such as, accuracy, linearity, sensitivity, selectivity, precision Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 57 and the knowledge of the limits of quantification and detection is necessary to ensure that the method is validated (González et al., 2014; Mujawar et al., 2014; Nikolaou et al., 2015). Therefore, the aim of this work was to develop a method of validation for copaiba oil by gas chromatography using the β-caryophyllene, α- humulene and caryophyllene oxide as standards. In addition, this work established a correlation between gas chromatography interfaced with flame ionization and mass spectrometry analyses promoting an exhaustive study on the chemical composition of copaiba essential and resin oil constituents. 2.0 MATERIALS AND METHODS 2.1. Materials Copaiba resin oil (Copaifera langsdorffii) was obtained from Flores & Ervas (Piracicaba, SP, Brazil). N,O-Bis(trimethylsilyl) trifluoroacetamide (BSTFA), diazomethane, β-caryophyllene, α- humulene and caryophyllene oxide were provided by Sigma-Aldrich (Saint-Quentin Fallavier, France). Hexane and ethyl acetate were purchased from Fisher Scientific (Pittsburgh, PA, EUA). Ultrapure water was obtained from a Millipore purification system (Milli-Q ® plus, Millipore, St Quentin en Yvelines, France). All chemicals were of reagent grade and used as received. 2.2. Copaiba essential oil extraction Copaiba essential oil was produced by the hydrodistillation method. 400 mL of copaiba resin oil with four times the volume of ultrapure water were placed in the Clevenger- Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 58 type apparatus for 3 h to essential oil extraction. Posteriorly, the essential oil extracted was dried with sodium sulphate, filtered through 0.22 μm cellulose membrane (Merck Millipore, Billerica, MA, USA) and stored in borosilicate glass vial at −20 °C until further use. 2.3. Copaiba resin oil derivatization Copaiba resin oil was submitted to a derivatization reaction before gas chromatography analyses. A methylation reaction was achieved by diluting 20-30 mg of copaiba resin oil with 2 mL of ethyl acetate. This mixture was placed in an ice bath and 2 mL of diazomethane was slowly added. After reaction, the solvent was completely evaporated under air flow. Silylation derivatization was performed using BSTFA. Five mg of copaiba resin oil was diluted in 0.5 mL of ethyl acetate and an excess of silylating reagent was added. This solution was heated at 60 °C for 30 minutes. For both derivatization reactions, the blank reagent was prepared and the volume was adjusted to 1.5 mL in ethyl acetate prior gas chromatography analysis. 2.4. Copaiba oil analysis 2.4.1. Thin Layer Chromatography Samples were spotted on pre-coated thin layer chromatography plates (silica gel 60 F254, 10 x 20cm, 0.25mm layer thickness, Merck Millipore, SP, Brazil) in order to indentify the oil profiles and to confirm the derivatization of copaiba resin oil before gas chromatography analyses. Samples were diluted in ethyl acetate and applied in the plate. Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 59 Mobile phase consisted of hexane/ethyl acetate (9:1) solution. After elution the samples were analyzed in ultraviolet-visible at 254 nm, following anisaldehyde solution application and drying at 105 °C for 5 min. Retention factor (Rf) was calculated by the ratio of migration distance of substance present in copaiba oils and the migration distance of solvent front. 2.4.2. Gas-Chromatography Mass Spectrometry Identification of copaiba resin and essential oil constituent were performed by gas chromatography coupled to mass spectrometry (GC-MS) using Hewlett-Packard 6890 gas chromatograph with HP-5975 mass selective detector. The column used was a HP- 5MS cross-linked fused silica capillary column (30 m× 0.25 mm × 0.25 µm). (Agilent J&W, Santa Clara, CA, USA). Chromatographic parameters to copaiba resin and essential oils analysis are described in Table 1. The injected volume for all samples was 1µL. The split ratio was 1:25 and the electron ionization system was set at 70 eV. Helium was the carrier gas at a flow rate of 1 mL.min -1 . Data acquisition and integration were carried out using the MSD ChemStation software. Copaiba oils components were identified by comparing their mass fragmentation with both the National Institute of Standards and Technology (NIST) mass spectral library data and the published data elsewhere. β- caryophyllene, α- humulene and caryophyllene oxide injected as standards were identified by comparison of its retention time and mass spectrum with the ones found on the NIST library. Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 60 Table 1- Chromatographic parameters to copaiba resin and essential oil analysis by GC- MS Parameters Copaiba Resin oil Copaiba essential oil Initial oven temperature: 110 °C 60 °C Initial rate: 5 °C.min -1 3 °C.min -1 Final temperature: 280 °C (final hold time of 26.0 min at 300°C) 240 °C (final hold time of 7.0 min at 250 °C) Injector temperature 250 °C 220 °C Detector temperature 300 °C 250 °C 2.4.3. Gas chromatography – Flame Ionization Detector The quantification of volatile constituents were performed using a PR2100 Gas- Chromatography (Alpha MOS, Toulouse, France) interfaced with a Flame Ionization Detector (GC-FID). A fused silica capillary column (25 m × 0.32 mm i.d., 0.5 µm) film thickness coated with cross-linked 5% Phenyl Polysilphenylene-siloxane (SGE Analytical Science Pty Ltd, Victoria, Australia) was used. The work temperatures for the copaiba oils were as follows: oven temperature started at 90 °C, isothermal, then heating 2 °C.min -1 to 150 °C, and after, isothermally heating 20 °C.min -1 to 300 °C. The injector temperature was 250 °C and the detector temperature was 300 °C. The volume injected for all samples was 1 µL. The split ratio was 1:80. The nitrogen was the carrier gas at a flow rate of 1 mL.min -1 . Data acquisition and integration were carried out using Winilab 3 software. β -caryophyllene, α- humulene and caryophyllene oxide were Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 61 selected as the standard for the quantification of the main components presented in the copaiba oils. 2.5. Method validation for β- caryophyllene, α- humulene and caryophyllene oxide The validation procedures for β- caryophyllene, α- humulene and caryophyllene oxide were performed following the international conference on harmonization (ICH) (Validation of analytical procedures: Text and Methodology, ICH‐Q2 (R1), 2005) and the Food and Drug Administration (Food and Drug Administration, Guidance for Industry. Bioanalytical Method Validation 2001) guidelines. The validation procedures followed the good manufacturing practices. All equipments and volumetric glassware were evaluated and calibrated before analysis. The balance (Sartorius MSA-224S-000- DU Cubis Analytical Balance, Elk Grove, USA) was calibrated to minimal measures of 0.1mg. Specificity, selectivity, linearity range, accuracy, precision, detection and quantification limits were evaluated using the GC-FID. 2.5.1. Preparation of stock solutions Three individual stock solutions of β- caryophyllene, α- humulene and caryophyllene oxide were prepared in ethyl acetate at 1 mg.mL -1 , placed in an amber vial hermetically sealed and kep at -20 °C until use. These stock solutions were diluted to obtain the concentrations required for preparation of standard working solutions. For all substances, working solutions ranged from 40 to 160 µg.mL -1 (40, 70, 100, 130 and 160 µg.mL -1 ) were prepared in 1 mL of ethyl acetate and used for the validation analyzes. Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 62 2.5.2. Specificity and selectivity The specificity/selectivity of the analytical method was confirmed by the analyses of solutions containing 100% of the normal working concentration of β- caryophyllene, α- humulene and caryophyllene oxide. The ability to separate all the compounds (related substances, degradation products and excipients) from standard samples was analyzed. 2.5.3. Linearity Standard calibration curves of individual stock solutions of β-caryophyllene, α- humulene and caryophyllene oxide were prepared a concentration ranged from 40 to 160 µg.mL -1 (40, 70, 100, 130 and 160 µg.mL -1 ). Peak area ratios of the standards were individually plotted against the analyte concentrations. Standard calibration curves of the compounds were developed by calculation of the regression line using the least squares method. Linearity curves were performed on 3 different days. 2.5.4. Determination of the limit of detection and quantification The Limit of Detection (LOD) was determined based on the ratio between the standard deviation of the response and the slope estimated from the calibration curve of the standards multiplied by 3.3. The Limit of Quantitation (LOQ) was determined as the lowest amount of analyte that was reproducibly quantified. This parameter was calculated by the ratio of the standard deviation of the response and the slope of the calibration curve of the standards multiplied by 10. Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 63 2.5.5. Accuracy To determine the accuracy of the proposed method, recovery studies were carried out by adding different amounts (80, 100 and 120 %) of bulk samples of β- caryophyllene, α- humulene and caryophyllene oxide along with the linearity range taken in triplicate. Then percentage recovery values were determined by the absolute percentage deviation at each concentration of the standard solutions. 2.5.6. Precision Precision was estimated by: intra-day (repeatability) and inter-day precision. Intra-day precision was investigated by injecting triplicate samples of β- caryophyllene, α- humulene and caryophyllene oxide solutions of three different concentrations (40, 100 and 160 µg.mL -1 ). Inter-day precision was assessed by injecting the same three samples over three consecutive days. Inter- and intra-day precisions were expressed as the relative standard deviation (RSD). 2.6. Determination of the β- caryophyllene, α- humulene and caryophyllene oxide in copaiba oils by GC-FID Stock solutions (10 mg.mL -1 ) of copaiba essential and resin oils were prepared in triplicate with ethyl acetate. These stock solutions were injected in GC-FID in the same conditions of the validation studies, as described above. The dosages of β- caryophyllene, α- humulene and caryophyllene oxide were performed based in the standard calibration curves of individual compound. Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 64 2.7. Statistical analyses All the experiments were conducted in triplicates. All values are expressed as their mean and standard deviation. Means of two groups were compared using non-paired Student’s t-tests. When comparing multiple groups, one way analysis of variance (ANOVA) was applied with the Tukey multiple comparison procedure. The statistical data were considered significant at p < 0.05 3.0 RESULTS AND DISCUSSION Copaiba essential oil was obtained by hydrodistillation from the copaiba resin oil using Clevenger apparatus to separate the colorless volatile fraction of the viscous residue. The yield of this extraction was calculated according to the ratio (w/w) of the volatile oil obtained and the resin oil material used initially for extraction. Thus, the copaiba essential oil yield was of 11 ± 0.8 %. Gelmini et al obtained a yield of 22.5 % for C. langsdorffii using steam distillation as the extraction method (Gelmini et al., 2013). Although the yield has been lower in this work, this method was considered of high performance since the amount of essential oil extracted was elevated (44 mL) and there were minimal loss of volatile substances due the operation in a closed circuit with cohobation of the water. In addition, there was no step using chemical solvents and the extraction time was considered ideal to prevent degradation of chemical compounds. Derivatization reactions were performed in order to identify the diterpenoic acids and derivative components presented in copaiba resin oil but normally no detectable in gas chromatography. This method modifies an analyte’s functionality in order to enable chromatographic separations (Orata, 2012). The reaction of diazomethane (CH2N2) with Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 65 a carboxylic acid formed instantly methyl esters compounds. The derivative shows lower polarity, relative to the parent substance due the replacement of the hydrogen by an alkyl group (Figure 1A). Silylation is the introduction of a silyl group into a molecule, usually in substitution for active hydrogen. The reaction is a nucleophilic attack upon the silicon atom of the silyl donor. Replacement of the active hydrogen by a silyl group reduces the polarity of the compound (Figure 1B). Figure 1: Derivatization reactions of carboxylic acid (i.e. copalic acid). The Figure A and B show the methylation and silylation reactions, respectively. Thin layer chromatography was used to identify the profile of copaiba oils and to confirm the derivatization reactions (Figure 2). It was observed that after derivatization, two initial groups formed by a large compound mixtures at retention factor (Rf) of 0.1 and 0.47, respectively, from the copaiba resin oil became the eluted in four compounds at Rf of 0.25, 0.37, 0.59 and 0.71, respectively, for the methylation and three compounds at Rf of 0.25, 0.59 and 0.71, respectively, for the silylation reaction. These results showed indications that the derivatization reactions were successful performed due to the conversion of polar compounds (as diterpenic acids) in derivative ones with lower polarity, which can be observed after the elution of the mobile phase. Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 66 Derivatization reactions did not change the Rf profile of the compounds present in the copaiba essential oil on the plate in the thin layer chromatography analysis (data not shown). Figure 2- Thin layer chromatography profile of copaiba oils. Where RO is the copaiba resin oil, mRO is the copaiba resin oil after methylation, sRO copaiba resin oil after silylation and EO is the copaiba essential oil. In order to analyze the compounds presented in the copaiba resin and essential oils, the GC-MS method was developed. Figure 3 shows the chromatographic profile of copaiba oils. The chromatogram showed a series of peaks indicating a good separation between the compounds during elution. It can be observed that the derivatization reaction preserved the sesquiterpenes compounds profile. However it was possible to identify a large amount of compounds which were eluted at high intensity in typical region of Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 67 identification of the diterpenes (Alencar, É. N. et al., 2015). After derivatization by silylation and methylation reactions, same chromatographic profiles were also obtained. Figure 3- Gas chromatography profile of copaiba oils. Where RO is the copaiba resin oil, mRO is the copaiba resin oil after methylation and EO is the copaiba essential oil. The residue fraction obtained after extraction of the essential oil possessed a lower amount of sesquiterpenes (less that 40 ± 3%), indicating that sesquiterpenes compounds were extracted by hydrodistillation and concentrated in the essential oil (Figure 4). In the same way, it was observed an increase in the concentration of diterpenes compounds in the methylated residue fraction (Figure 4 B). Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 68 Figure 4- Difference between the methylated copaiba resin oil (A) before start the hydrodistillation process and methylated residue fractions (B) obtained after extraction of the copaiba essential oil (C). The gray color represents the precision of the method to determination of copaiba oil compounds Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 69 The parameters of the chromatographic copaiba essential oil analysis were different in comparison with to the ones of copaiba resin oil. The low rate of heating of the copaiba essential oil was performed, in order to better separate the compounds and to preserve the chemical structure of possible unstable molecules from their degradation. Therefore, the retention times of the sesquiterpenes compounds were different, but the chromatographic profile of the essential oil possessed the same sequence of the elution of the fraction from the copaiba resin oil. The chemical composition of copaiba essential and resin oils were obtained by GC-MS (Table 2). All chromatograms showed small peaks but only components giving a peak area greater than 0.1 % of all peak areas detected on the chromatograms were further considered in this work. The assays were able to detected 38 components from the non-derivatized copaiba resin oil, corresponding to 98.1 % of all compounds presented in the oil of which 95.9 % were sesquiterpenes. After the methylation reaction, it was possible to identify 52 compounds in the copaiba oil, in which 44.8 % of the relative area corresponded to sesquiterpenes and 48.2 % to diterpenes, totaling 93 % of detected compounds. In the copaiba essential oil, only sesquiterpenes were detected, corresponding to 97.5 % of all peak areas on the chromatograms. Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 70 Table 2- GC–MS analyses of C. langsdorffii. Peak identification, retention time (RT, min) and relative area percentage of RO: Copaiba resin oil, mRO: Methylated copaiba resin oil, EO: Copaiba essential oil. Compounds MW RT (min) RO Area (%) mROArea (%) RT (min) EO Area (%) δ-Elemene 204 6.90 0.4 0.2 21.17 0.6 α-Cubebene 204 7.13 0.1 0.1 21.67 0.2 (+)-Cyclosativene 204 7.53 0.5 0.2 22.31 0.7 α-Copaene 204 7.66 1.0 0.4 22.77 1.4 trans-α-Bergamotene 204 7.81 0.3 0.1 23.39 0.3 (-)-β-Elemene 204 7.92 1.5 0.6 23.46 2.0 δ-Selinene 204 8.19 0.4 0.1 23.74 0.5 (Z,β)-Farnesene 204 8.33 0.2 - - - β-Caryophyllene 204 8.57 18.7 7.9 24.63 21.7 α-Bergamotene 204 8.75 16.0 7.1 25.29 20.5 α-Guaiene 204 8.84 1.2 0.5 25.38 0.9 Aromadendrene 204 8.95 0.3 0.1 25.54 0.4 α-Humulene 204 9.04 2.9 1.3 25.96 2.9 β-Farnesene 204 9.23 1.6 0.8 26.11 1.7 (+)-Valencene 204 - - - 26.81 0.3 τ-Muurolene 204 9.60 0.8 0.4 26.89 0.5 β-Cubebene 204 9.75 4.8 2.2 27.06 1.7 β-Selinene 204 9.88 4.4 2.0 27.27 6.1 α-Selinene 204 - - - 27.62 2.3 Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 71 τ-Gurjunene 204 - - - 27.79 0.5 β-Chamigrene 204 10.04 5.7 2.7 27.95 0.9 β-Bisabolene 204 10.21 25.2 12.3 28.24 23.6 γ-Cadinene 204 10.40 0.3 0.1 - - δ-Cadinene 204 10.51 2.4 1.2 28.76 1.4 α-Bisabolene 204 10.86 2.7 1.4 - - ni 204 11.34 0.3 0.2 29.50 1.2 Caryophyllene oxide 220 11.89 0.4 0.2 31.04 4.1 ni 220 12.30 0.3 0.2 - - ni 220 12.72 0.2 0.1 - - ni 220 12.86 0.1 - - - ni 204 13.03 1.8 1.4 33.40 0.4 Aromadendrane 206 13.21 0.7 0.4 - - ni 204 - - - 33.72 0.5 α-Cadinol 222 13.31 0.2 0.2 - - α-Bisabolol 222 13.84 0.5 0.4 35.11 0.2 Hexadecanoic methyl ester 270 18.58 - 0.2 - - Kaur-16-ene 272 20.99 0.4 0.3 - - ni 272 21.45 0.3 - - - ni 286 21.66 0.3 0.2 - - Linoleic acid methyl ester 294 21.75 - 0.5 - - ni 296 21.82 0.2 - - - ni 286 22.45 0.3 0.2 - - ni 286 24.22 - 0.5 - - ni 320 24.28 - 1.9 - - Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 72 ni 320 24.44 - 0.8 - - ni 320 24.77 - 3.3 - - ni 286 24.95 0.5 0.3 - - Kaur-16-en-18-oic acid methyl ester 318 25.38 - 2.4 - - Methyl copalate 318 25.59 - 3.5 - - Kauran-19-oic acid methyl ester 318 25.67 - 1.9 - - ni 318 26.38 - 3.8 - - Copalic acid methyl ester 330 26.51 - 15.6 - - ni 332 26.79 - 0.4 - - ni 318 26.94 - 0.2 - - ni 332 27.26 - 0.5 - - ni 330 27.66 - 2.3 - - ni 336 28.11 - 0.4 - - Labd-8(20)-ene-15,18-dioic acid methyl ester 364 28.58 - 6.7 - - ni 364 28.76 - 0.2 - - ni 362 29.40 - 0.6 - - ni 376 30.55 0.2 0.1 - - ni 376 31.06 - 1.4 - - Total of Sesquiterpenes 95.9 44.8 97.5 Total of Diterpenes 2.2 48.2 0.0 Total detected 98.1 93.0 97.5 MW = molecular weight; ni = not identified; - absent Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 73 The major compounds identified in the copaiba essential oil were β-caryophyllene (21.7 %), α-bergamotene (20.5 %) and β-Bisabolene (23.6 %). From the copaiba resin oil, the major compounds were β-caryophyllene (18.7 %), α-bergamotene (16 %) and β- Bisabolene (25.2 %). In the methylated copaiba resin oil were β-caryophyllene (7.9 %), α-bergamotene (7.1 %), β-Bisabolene (12.3 %), copalic acid methyl ester (15.6 %) and Labd-8(20)-ene-15,18-dioic acid methyl ester (6.7 %) were the major compounds. Gramosa et al. reported that the major component in the copaiba essential oil was β- caryophyllene (53%) (Gramosa & Silveira, 2005). Soares et al. also detected the high level of β-caryophyllene (42.3%) in the volatile fraction rich in sesquiterpenes. On the other hand, the nonvolatile fraction consisted of a higher amount of copalic acid content (49.9%) (Soares et al., 2013). Other studies reveal the presence of large amounts of α- bergamotene and copalic acid about 48 and 22 %, respectively for the copaiba essential oil and for the copaiba resin oil (Gelmini et al., 2013). It is noteworthy this amount variation of the main compound found in the copaiba essential oil and the copaiba resin oil reported by different authors. However, differences between the major compounds of the natural oils from different studies in the literature are well recognized, since it is well known that the chemical composition of natural oils is not constant. The variation may be attributed to several factors, which influence their composition, such as: the geographical origin of the plants, the environmental factors such as light, temperature, soil composition and season, the period and harvest time, as well as the plant organ, age and stage in the vegetative cycle (Raileanu et al., 2013). To perform the validation studies, the same analytical profile of the copaiba essential oil and the copaiba resin oil was developed using GC-FID. The goal was to find the Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 74 correlation between the two methods of analysis for further development of the dosages of the components presented in copaiba oil samples. Although mass spectrometers are sometimes considered the most powerful detectors for chromatographic methods, the flame ionization is the most used detector in gas chromatography because it has adequate sensitivity, large linear response range and low noise for most needed analysis, becoming, therefore, less expensive than mass spectrometers (Skoog et al., 2007; Marriott et al., 2012; Yuan et al., 2015). Figure 5 presented the difference between all peaks areas detected on the chromatograms analyzed by GC-MS and GC-FID of copaiba resin oil (A) and copaiba essential (B) oil, respectively. There were no major changes in the areas of the compounds analyzed by both methods (maximum variation of 4%). The differences between the two methods were not statistically significant (p>0.05). In addition, the main compounds detected by GC-MS were also identified by the GC-FID in the same retention time, indicating correspondence between both methods. Figure 5- The main difference between all peaks areas detected on the chromatograms analyzed by GC-MS and GC-FID of copaiba resin oil (A) and copaiba essential (B) oil, respectively. The results were calculated based on the percentage area (%) difference Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 75 between the compounds. The gray color represents the precision of the method to determination of copaiba oil compounds GC-FID methods of copaiba oils were initially performed, aiming to increase the resolution of the peaks and to reduce the analysis time between the samples. This process did not alter the sequence and neither the area percentage of the eluted compounds in the copaiba oil. This small adjustment in the methodology presented only a slightly change in the retention time. However, the analysis quality rest unchanged. Therefore, in order to quantify the compounds presented in the copaiba oil, the validation procedure was performed using β- caryophyllene, α- humulene and caryophyllene oxide as reference substances. These compounds, which represent an important group of sesquiterpenes, were selected due to their presence in all copaiba oil samples previously analyzed. The studied standards showed good resolution peaks, indicating high specificity and selectivity (Figure 6). This method was specific for the standards with no interference of the peaks at the retention time. The purity of the peaks was confirmed by mass fragmentation in the GC-MS. The retention times measured by GC-MS analyses for β- caryophyllene, α- humulene and caryophyllene oxide were 13.15, 14.87 and 21.52 minutes, respectively. The examination of the chromatogram in Figure 6A revealed the presence of impurities that was eluted after β- caryophyllene (peak at 14.9 minutes). However, this fact was already expected due the quality of the sample. This small peak can be α- humulene, which has the same retention time. Therefore, the proposed method was considered adequate for β- caryophyllene, α- humulene and caryophyllene oxide Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 76 assay because the peak of the standards were well separated from each other compounds and no peaks interfered with the observed analyte peaks. Figure 6- Representative GC-FID chromatograms of the β- caryophyllene (A), α- humulene (B) and caryophyllene oxide (C) standards at concentration of 160, 130 and 130 µg.mL -1 , respectively. A linear range equation was judged to produce the best fit of the concentration / response relationship. Linearity of the analytical procedure was evaluated by plotting detector response (peak area) against analyzed concentration. Calibration plots were constructed after analysis of β- caryophyllene, α- humulene and caryophyllene oxide solutions at concentrations of 40, 70, 100, 130 and 160 µg.mL -1 . Each level was injected Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 77 in triplicate and the goodness to fit for concentrations were consistently greater than 0.99 during the course of the validation and study period. The regression equation was showed in the Table 3. Correlation coefficients for the method were 0.999, 0.997 and 0.998 for β- caryophyllene, α- humulene and caryophyllene oxide, respectively. The intercept was very small and the correlation coefficient closes to the unity for all standards. All these results indicated that the method was linear over the range of 40– 160 μg.mL-1. Table 3- Validation parameters of β- caryophyllene, α- humulene and caryophyllene oxide Parameters β-Caryophyllene α-Humulene Caryophyllene oxide Retention Time (min) 13.15 ± 0.02 14.87 ± 0.02 21.52 ± 0.04 Linearity a (slope) 0.204 ± 0.007 0.202 ± 0.001 0.227 ± 0.005 b (intercept) -1.332 ± 0.395 - 0.5044 ± 0.255 - 2.354 ± 0.038 Correlation coefficient(R²) 0.999 0.997 0.998 Detection limit (µg.mL -1 ) 6.38 4.16 0.55 Quantitation limit (µg.mL -1 ) 19.36 12.62 1.67 Accuracy (%RSD) 3.21 3.46 2.24 LOD was identified as the lowest concentration of an analyte that the assay can reliably differentiate from the background noise (Validation of analytical procedures: Text and Methodology, ICH‐Q2 (R1), 2005). β- caryophyllene, α- humulene and caryophyllene Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 78 oxide showed a LOD of 6.38, 4.16 and 0.55 µg.mL -1 , respectively (Table 3). LOQ was identified as the lowest concentration of an analyte in a sample that could be determined with acceptable precision and accuracy under the stated experimental conditions for this method (Food and Drug Administration, Guidance for Industry. Bioanalytical Method Validation 2001)). LOQ for β- caryophyllene, α- humulene and caryophyllene oxide was 19.36, 12.62 and 1.67 µg.mL -1 , respectively (Table 3). The accuracy of the assay was defined as the absolute value of the ratio between the calculated mean values of the quality control samples and their normal values. Standards accuracy was determined on the range of 80–120% of the analytical working concentration by calculating recovery. The accuracy of the β- caryophyllene, α- humulene and caryophyllene oxide ranged from 98.81 to 102.51 %, 99.87 to 103.66 %, and 98.61 to 101.11 %, respectively. These results values showed a RSD lower than 3.21, 3.46 and 2.24 % for β- caryophyllene, α- humulene and caryophyllene oxide, respectively (Table 3). Thus, it can be inferred that the method demonstrated a good correlation between the theoretical and the practical values, satisfying the drug quality research requirements. Precision was estimated by the intra-day (repeatability) and the inter-day precision. Intra-day precision was investigated by injecting triplicate samples of β- caryophyllene, α- humulene and caryophyllene oxide solutions at three different concentrations (40, 100 and 160 µg.mL -1 ). Inter-day precision was determined by evaluating the repeatability of the analytical procedure, if re-produced in the same laboratory, but under the analysis carried out on another day. The results obtained for the inter- and intra-day precision studies are presented in Table 4. Based on these results, these Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 79 methods were considered satisfactory, presenting lower random errors (p <0.05) and representing a true measure of the obtained analytical results proximity. Table 4- Intra and inter-day variations of β- caryophyllene, α- humulene and caryophyllene oxide Spiked Concentration (µg.mL -1 ) Measured Concentration β- caryophyllene α- humulene Caryophyllene oxide Mean (µg.mL -1 ) SD RSD (%) Mean (µg.mL -1 ) SD RSD (%) Mean (µg.mL -1 ) SD RSD (%) Intra-day variation 40 41.4 1.0 2.3 40.3 1.2 3.0 41.1 1.7 4.2 100 99.4 2.4 2.4 103.5 2.0 1.9 100.5 3.9 3.9 160 162.3 3.2 2.0 161.3 1.9 1.2 161.1 2.5 1.5 Inter-day variation 40 40.8 1.2 2.9 41.5 1.8 4.3 41.5 1.8 4.3 100 98.8 4.3 4.4 105.5 2.5 2.4 105.5 2.0 1.9 160 159.1 4.9 3.1 162.1 2.1 1.3 162.1 1.9 1.2 Values are for n= 3 observations; S.D., standard deviation and R.S.D., relative standard deviation. Finally, this validated analytical method was used to determine the amount of the compounds in the copaiba resin oil and copaiba essential oil by GC-FID. The stock solution at 10 mg.mL -1 of copaiba essential oil contained 1982 ± 13, 279 ± 25 and 24 ± 0.9 µg.mL -1 of β- caryophyllene, α- humulene and caryophyllene oxide, respectively; In Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 80 contrast, the copaiba resin oil showed standards dosages of 808 ± 25, 97 ± 6 and 16 ± 0.6 µg.mL -1 for β- caryophyllene, α- humulene and caryophyllene oxide, respectively. As chromatograms always show the same ratio between the peaks, these ratios can be used to determine the concentration of other components from the copaiba oil samples. The results of the ratio among β- caryophyllene and others compounds identified in the copaiba oils were shown in Figure 7. Figure 7- Ratio among the peaks areas of β- caryophyllene (BCF) and the others peak area compounds detected on the chromatograms from the copaiba essential oil (A) and the copaiba resin oil (B). 4.0 CONCLUSION Compounds presented in the copaiba oils were effectively identified by GC-MS analysis. Furthermore, the derivatization of the copaiba resin oil was performed promoting the identification of diterpenes compounds. A good correlation between the GC-FID and GC-MS analysis were obtained, favoring the transposition of the Chapter I- Development of gas-chromatography method for the analysis of copaiba oil 81 methodology analysis. A rapid, simple, accurate and precise GC-FID method for the quantitation of β- caryophyllene, α- humulene and caryophyllene oxide in copaiba oil samples was developed and validated. Acceptable values were obtained for the following validation parameters, such as: Linearity, LOD, LOQ, precision and accuracy. The methods described in this work were successfully used for quantifying the β- caryophyllene, α- humulene and caryophyllene oxide in the copaiba oils samples. This work should be suitable to the reliable quantification for quality control of different copaiba oil species and can also be used for copaiba oil quantitation when loaded in pharmaceutical or cosmetic formulations. ACKNOWLEDGEMENTS The authors would like to thank the financial support from “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior- CAPES” through the COFECUB 721/11 projet for Xavier-Junior, F.H. fellowship. REFERENCES ALENCAR, É. N., XAVIER-JÚNIOR, F. H., MORAIS, A. R. V., DANTAS, T. R. F., DANTAS-SANTOS, N., VERISSIMO, L. M., REHDER, V. L. G., CHAVES, G. M., OLIVEIRA, A. G. & EGITO, E. S. T. 2015. Chemical Characterization and Antimicrobial Activity Evaluation of Natural Oil Nanostructured Emulsions. J Nanosci Nanotechnol, 15, 1, 880-888. ATTIMARAD, M., MUEEN AHMED, K. K., ALDHUBAIB, B. 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YUAN, C., CHEN, D. & WANG, S. 2015. Drug confirmation by mass spectrometry: Identification criteria and complicating factors. Clin Chim Acta, 438, 0, 119-125. Chapter II HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 87 Le deuxième chapitre de ce mémoire de thèse présente une méthode très sensible, simple, rapide, innovante et économique a été développée et validée pour la quantification de paclitaxel dans l'huile de copaïba utilisant la chromatographie en phase liquide à haute performance couplée à une détection par absorption dans l'ultraviolet. La séparation chromatographique a été réalisée par sur une colonne de type Uptisphere Strategy 100A en phase inverse C-18 (150 mm x 3 µm x 3 mm). La phase mobile était constituée d'acétonitrile et d'eau (50:50). Le débit était de 0,4 mL.min -1 et la détection a été effectuée à 228 nm. Aucun pic d'interférence a été observés lors de l'élution de paclitaxel sur un temps d'analyse totale de 15 min. La courbe d’étalonnage du paclitaxel dans un milieu contenant 10 µg.mL -1 d'huiles résine ou d'huile essentielles de copaïba était linéaire sur la gamme de concentration de 50 à 2000 ng.mL -1 avec un coefficient de détermination de 0,999 et des résidus de régression faibles avec une dispersion homoscédastique. Les limites de quantification et de détection étaient basse à 21,03 et 6,31 ng.mL -1 , respectivement. L'exactitude et la précision des déterminations étaient inférieures ou égales à 0,77 et 0,65%, respectivement. La méthode développée a été appliquée avec succès pour l'évaluation de la solubilité du paclitaxel dans des échantillons de l'huile de copaïba par des études de coefficient de partage. Le paclitaxel a montré une caractéristique lipophile (log P> 1) et une plus grande solubilité dans les huiles résine et essentielle de copaïba par rapport à l’eau. En conclusion, la méthode a montré une sensibilité, linéarité, précision, exactitude et spécificité nécessaires pour le succès de la quantification du paclitaxel dans des échantillons d'huile de copaïba. Ces méthodes sont adaptées pour une application aux analyses quantitatives de paclitaxel incorporé dans les systèmes d'administration de médicaments contenant de l'huile de copaïba . Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 88 Mots-clés: paclitaxel, huile de copaïba, validation, chromatographie en phase liquide à haute performance, solubilité, coefficient de partage Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 89 HPLC METHOD FOR THE DOSAGE OF PACLITAXEL IN COPAIBA OIL. DEVELOPMENT, VALIDATION, APPLICATION TO THE DETERMINATION OF THE SOLUBILITY AND PARTITION COEFFICIENTS. Xavier-Junior, F. H. ¹ , ², Gueutin, C.², Morais, A. R. V. 1,2 , Alencar, E. N.¹, Egito, E. S. T. 1 , Vauthier, C.² * 1 Universidade Federal do Rio Grande do Norte, Centro de Ciências da Saúde, Departamento de Farmácia, Laboratório de Sistemas Dispersos (LaSiD). Av. Gal. Gustavo Cordeiro de Farias, S/N, Petrópolis, 59010-180, Natal-RN-Brazil. 2 Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. Corresponding author Christine Vauthier, Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. Christine.vauthier@u-psud.fr Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 90 ABSTRACT A highly sensitive, simple, rapid, innovative and economic method has been developed and validated for the quantification of paclitaxel in copaiba oil using High-Performance Liquid Chromatography (HPLC) with UV detection. Chromatographic separation was performed by Uptisphere Strategy 100A reversed-phase C-18 (150 mm x 3 µm x 3 mm) column. The mobile phase was constituted of acetonitrile and water (50:50). The flow rate was 0.4 mL.min -1 and the detection was performed at 228 nm. No interfering peaks were observed during the paclitaxel elution at the total run time of 15 min. Standard curves of paclitaxel containing 10 mg of copaiba resin oil was linear over the concentration range from 50 to 2000 ng.mL -1 with a determination coefficient of 0.999 and lower regression residues with a homoscedastic dispersion. The lower quantification and detection limits were 21.03 and 6.31 ng.mL -1 , respectively. The accuracy and precision determinations were less or equal to 0.77 and 0.65 %, respectively. The method developed was successfully applied to the evaluation of paclitaxel in copaiba oil samples by solubility and partition coefficient studies. Paclitaxel showed a lipophilic characteristic (logP>1) and a higher solubility in copaiba resin oil and copaiba essential oil. In conclusion, the method showed sensitivity, linearity, precision, accuracy and specificity necessary for successfully quantification of the paclitaxel in copaiba oil samples. This analytical method can be, therefore, also applied to quantify paclitaxel in drug delivery systems in which copaiba oil is associated, such as lipid and polymer systems. Keywords: Paclitaxel, Copaiba Oil, Validation, HPLC, Solubility, Partition Coefficient Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 91 1.0 INTRODUCTION Paclitaxel (C47H51NO14) is a pseudoalkaloid with a diterpenoid structure having unique tri- or tetracyclic 20 carbon skeletons and a molecular weight of 853 Da (Fang & Liang, 2005). Originally, it was isolated in early 1960s from the bark of Pacific Yew (Taxus brevifolia; family Taxaceae) (Wani et al., 1971). Paclitaxel has been the most effective antitumor agent developed in the past three decades (Kim et al., 2005). However, the production of paclitaxel in large-scale has been limited due to the low abundance and slow growth of Taxus trees, combined to a low concentration of the drug in the trees (Frense, 2007). Nowadays, the paclitaxel has been produced by semi-synthesis reaction from 10-deacetylbaccatin III, a renewable precursor found in the needles of the European yew tree (Taxus baccata) (Gueritte-Voegelein et al., 1994) or through of the suspension culture of Taxus cells (Kajani et al., 2012), as promising alternative less expensive, less difficult and nondestructive methods to the Taxus species. Paclitaxel is an antineoplastic agent used effectively to treatment of a variety of cancers including refractory ovarian, breast, small and non-small cell lung, head and neck carcinoma and AIDS-related Kaposi’s sarcoma (Wani et al., 1971; Rowinsky et al., 1990; Rowinsky et al., 1992; Huizing et al., 1995; Singla et al., 2002). Paclitaxel has a unique mechanism of action. It disrupts the dynamic equilibrium within the microtubule system promoting the hyper-stabilization of the cellular microtubules, forming an incomplete metaphase plate of chromosomes and an abnormal organization of spindle microtubules (Horwitz, 1992; Rao et al., 1994). Therefore, it blocks cells in the late G2 phase and M phase of the cell cycle, thereby, inhibiting cell replication and promoting the cellular apoptosis (Schiff et al., 1979; Panchagnula, 1998). Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 92 The diterpenoid pseudoalkaloid drug paclitaxel is insoluble in aqueous media and show a poor permeability across biological membranes that complicates its application in therapeutic formulations (Singla et al., 2002). Furthermore, paclitaxel is a substrate for P-glycoprotein, a membrane transporter that serves as a drug efflux pump and can alter paclitaxel pharmacokinetics and sensitivity to tumor cells (Guo et al., 2003). To overcome this mechanism of cancer drug resistance, the use of additional substances in the formulation is required. In this sense, copaiba oil is a natural oil, rich in caryophyllene compounds, that can be used to increase the uptake of paclitaxel by inhibition of the P-glycoprotein efflux transporters (Legault & Pichette, 2007). The copaiba oil consists of a mixture of sesquiterpenes and diterpenes hydrocarbons (Sousa, J. P. et al., 2011; Alencar, E. N. et al., 2015; Xavier-Junior, Chapter I, 2015a). This oil has been traditionally used in folk medicine due its therapeutic properties as anticancer, anti-inflammatory, antioxidant, antimicrobial, antitetanus, and antiseptic among others (Gomes, N. M. et al., 2007; Santos, A. O. et al., 2008; Leandro et al., 2012). In order to associate the anticancer activity of paclitaxel and copaiba oil in a single nanomedicine formulation, a method capable to dose paclitaxel in a medium that contained copaiba resin oil and copaiba essential oils were of grand interest. Such a method would be also needed to determine the solubility of paclitaxel in the copaiba oil and to evaluate its partition coefficient, which would be good predictor characteristics for the success of a nanomedicine formulation containing it. In fact, the majority of methods used to produce nanomedicines involve dispersions of an organic phase into an aqueous phase. Over the course of the current analysis, the complex components mixture from copaiba oil can hamper the quantification of paclitaxel when associated with these formulations. Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 93 Accordingly, the need for an accurate and precise analysis method of paclitaxel in copaiba oil is mandatory for quality control and drug development. Several High- Performance Liquid Chromatography (HPLC) methods for the separation and determination of paclitaxel from biological samples were reported in the literature (Song & Au, 1995; Supko et al., 1999). However, there are only few studies that have reported the analysis of this drug in highly complex matrices such as vegetable oils. Most of the proposed methods displayed high sensitivity including HPLC- MS and immunoassays, but their high cost prevents their application on a routine analytical use (Wang et al., 2003). Moreover, HPLC- UV, a simple and efficient method, can be a suitable analytical tool to perform paclitaxel analyzes at a low cost with high reproducibility. Thus, the aim of the present work was to develop and validated a simple, fast, specific and sensitive HPLC-UV method for the quantification of paclitaxel associate with copaiba oil. The method was investigated to be applied on the determination of the solubility of paclitaxel in the copaiba oils and to evaluate its partition coefficient. 2.0 MATERIALS AND METHODS 2.1 Materials Copaiba oil (Copaifera langsdorffii) was purchased from Flores & Ervas (Piracicaba, SP, Brazil). Paclitaxel was obtained from CHEMOS GmbH (Regenstauf, Germany). Methanol, ethanol, n-octanol and acetonitrile were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Ultrapure water was obtained from a Millipore purification system (Milli-Q ® plus, Millipore, St Quentin en Yvelines, France). Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 94 2.2. Copaiba essential oil extraction Copaiba essential oil was obtained from 400 mL of copaiba resin oil by hydro- distillation using a Clevenger-type apparatus for 3 h. The extract was dried with sodium sulphate, filtered and stored at −20°C. 2.3 Chromatographic equipment and conditions A Waters HPLC system equipped with a Waters 515 pump, a Waters 717 plus autosampler, and a Waters 486- Tunable Absorbance detector (Waters Corp., Milford, MA) were used. Chromatographic separations were achieved using a Uptisphere Strategy 100A reversed-phase C-18 (150 mm x 3 µm x 3 mm) column and a Uptisphere Strategy C18-2 (10 mm x 3 µm x 4 mm) guard column (Interchim SA, Montluçon, France). The detection wavelength was set at 228 nm, which was the maximum absorbance level observed for paclitaxel. The mobile phase consisting of acetonitrile: water (50:50) was pumped through the column at a flow rate of 0.4 mL.min -1 at 30°C. 25 µL samples were introduced onto the HPLC system every 15 min. The mobile phase and the samples were filtered through a 0.20 µm hydrophilic nylon membrane filter (Merck Millipore, Billerica, MA, EUA) prior to use. Chromatographic data were monitored and analyzed using Azur software (Datalys, France) 2.4. Validation methods The validation of the chromatographic method was performed according to the ICH validation guidelines (Validation of analytical procedures: Text and Methodology, ICH- Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 95 Q2 (R1), 2005) for specificity/selectivity, linearity, precision, accuracy, and limit of detection (LOD) and limit of quantification (LOQ). Validation tests followed the good manufacturing practices and all equipments and volumetric glassware were evaluated and calibrated before analysis. Three individual stock solutions of paclitaxel at 1 mg.mL -1 were prepared in ethanol, placed into a hermetically sealed amber vial and stored at -20 °C until use. The analytical standards at concentrations ranging from 50 to 2000 ng.mL -1 (50, 200, 500, 800, 1100, 1400, 1700 and 2000 ng.mL -1 ) were prepared in the mobile phase and used for the validation analyzes. 2.4.1 Specificity/selectivity The specificity/selectivity of the analytical method was confirmed by analyzes of the paclitaxel analytical standard solutions at concentration of 200 ng.mL -1 prepared with either 1 mL of mobile phase, or 1 mL of mobile phase with 10 mg of copaiba resin or copaiba essential oils. These analyses were repeated six times. The ability to separate all compounds demonstrating the lack of chromatographic interference from standard samples was analyzed. 2.4.2. Linearity Paclitaxel stock solution was diluted to give a series of sub-stock solutions with concentrations ranging from 50 to 2000 ng.mL -1 (50, 200, 500, 800, 1100, 1400, 1700 and 2000 ng.mL -1 ). Sub-stock solutions were prepared by appropriate dilution of paclitaxel stock solutions to a final volume of 1 mL of the mobile phase containing 10 Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 96 mg of copaiba resin oil. Calibration curves were obtained by least-squares linear regression, weighted by the reciprocal of the concentration, using the peak area of the drug. Linearity curves were performed on 3 different days. 2.4.3. Detection and quantification limits The LOD was defined as the lowest concentration of paclitaxel resulting in a peak area greater or equal to three times from the background noise (S/N ≥ 3). The LOQ was determined based on the standard deviation of the response and the slope with peak area greater or equal to ten times from the background noise (S/N ≥ 10). LOD and LOQ were estimated from the calibration curve of the paclitaxel concentrations ranging from 50 to 2000 ng.mL -1 in 1mL of mobile phase containing 10 mg of copaiba resin oil. 2.4.4. Accuracy The accuracy of the HPLC method was demonstrated by the percentage of deviation. Accuracy was determined by six replicates of paclitaxel concentrations (ranged from 50 to 2000 ng.mL -1 ) in 1 mL of mobile phase with 10 mg of copaiba resin oil. The concentrations found were obtained by refitting peak response ratios from paclitaxel solutions of concentrations added into a derived regression equation. The found and added concentrations were, then, used to determine the absolute percentage of deviation at each paclitaxel concentration containing copaiba resin oil. Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 97 2.4.5. Precision The precision of the method was assessed by analyzing the intra- and inter-day variability of paclitaxel samples. The intra-day precision was determined by quantification of paclitaxel samples at three distinct concentrations (800, 1400 and 2000 ng.mL -1 ) in 1 mL of mobile phase with 10 mg of copaiba resin oil. The samples were injected six times and prepared within a day. The inter-day precision was assessed separately from the obtained peak areas by injecting the same three drug concentrations in distinct days. Posteriorly, concentrations were calculated by refitting peak response ratios into a derived regression equation from the calibration curve and the relative standard deviation (%RSD) were calculated for the intra- and inter-day precision. 2.5. Solubility evaluation of paclitaxel Paclitaxel solubility was determined in milli-Q ® water, copaiba essential oil and copaiba resin oil by adding a surplus of drug substance in a glass vial with 1 mL solvent. The vials were sealed and shaken at room temperature for 24 h to assure saturation. After equilibration, samples were removed and centrifuged at 15,000 rpm for 15 min (Eppendorf centrifuge 5418, Rotor FA-45-18-11, Hamburg, Germany) to remove insoluble crystals of paclitaxel. The supernatants were filtered through 0.20 µm nylon filter (Merck Millipore, Billerica, MA, EUA). The filtrates were diluted as required with the mobile phase and stirred in an ultrasound bath (Elma Elmasonic S10H, Elma Hans Schmidbauer GmbH & Co. KG, Singen, Germany) for 5 minutes. The resulting solutions were analyzed by the HPLC method as previously described. Paclitaxel Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 98 concentrations in the samples were obtained using the linear regression equation from the calibration curve. 2.6. Partition coefficient of paclitaxel The partition coefficient of paclitaxel between n-octanol/water, copaiba resin oil/water and copaiba essential oil/water were determined at 25 °C. One mL of each lipophilic substance and 1 mL of milli-Q ® water were placed separately in glass stoppered flasks. These flasks were previously shaken by magnetic stirrer (C-MAG HS 7 IKA, Staufen, Germany) for 24 hours at 25 ± 1°C. Thus, 1 mg of the drug was added, following by magnetic stirrer for another 24 hours at 25 ± 1°C. To promote the separation of the aqueous and oily phases, the samples were centrifuged for 5 min at 3,000 rpm (Eppendorf centrifuge 5418, Rotor FA-45-18-11, Hamburg, Germany). Posteriorly, the aqueous (Milli-Q ® water) and oily (n-octanol, copaiba essential oil and copaiba resin oil) phases were carefully separated and centrifuged at 15,000 rpm for 15 min to precipitate insoluble crystals of paclitaxel. The supernatant from each separated systems (n-octanol/water, copaiba resin oil/ water and copaiba essential oil/ water) were filtered through a 0.20 µm nylon filter and diluted if required in 1 mL of the mobile phase using ultrasound bath for 5 min. The resulting solutions were analyzed by the HPLC method as previously described. The partition coefficient of the paclitaxel was taken as the logarithm ratio between the drug concentration (w/v) solubilized in each lipophilic and hydrophilic phases (logP). Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 99 2.7 Statistical analyzes All the experiments were conducted in triplicates. All values are expressed as their mean ± standard deviation (SD). Means of two groups were compared using non-paired Student’s t-tests. When comparing multiple groups, one way analyzes of variance (ANOVA) was applied. The statistical data were considered significant at p < 0.05 3.0 RESULTS AND DISCUSSION The development and validation of a HPLC-UV method for the quantification of paclitaxel in natural oils from Copaifera langsdorffii was performed. Chromatographic conditions were developed to achieve a routine analysis for paclitaxel in copaiba oils, with high reproducibility and sensibility, combined to a simple, rapid and economic way. The copaiba essential oil, obtained from the copaiba resin oil by the hydro- distillation method with yield of 11 ± 0.8%, was transparent. Previous studies showed that copaiba essential oil is constituted by a mixture of sesquiterpenes compounds, which the major identified compounds were β-caryophyllene (21.7 %), α-bergamotene (20.5 %) and β-bisabolene (23.6 %). However, the copaiba resin oil showed 64 different compounds, among them, 47.7 % of diterpenes, especially copalic acid (15.6 %) and labd-8(20)-ene-15,18-dioic acid (6.7 %), besides sesquiterpene compounds as β- caryophyllene (7.9 %), α-bergamotene (7.1 %) and β-bisabolene (12.3 %) (Xavier- Junior, Chapter I, 2015a). The complex mixtures of copaiba oil compounds make difficult the identification and quantification of lipophilic molecules when incorporated into this oil because of the presence of innumerable potential interfering compounds. Therefore, the development Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 100 of an effective methodology able to identify the paclitaxel when incorporated in copaiba oil samples was required. The specificity/selectivity of the analytical method was confirmed by analyzing solutions containing 200 ng.mL -1 of the working drug and known amount of copaiba oils (Figure 1). The chromatograms showed a good resolution and separation of paclitaxel from other peaks attributed to compounds presented in the copaiba oil. No statistically significant difference was observed between the chromatogram areas of samples containing copaiba resin oil or copaiba essential oils in comparison with the chromatogram obtained only with the mobile phase. Peak purity values for paclitaxel in the chromatograms were in the range from 0.996 to 1.0. These results indicated that the peaks were homogenous with a retention time at 9.7 ± 0.2 minutes and showed a significant ability to separate the paclitaxel from the complex mixture of copaiba oil. Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 101 Figure 1- Chromatograms showing the elution peak of paclitaxel spiked at the concentration of 200 ng.mL -1 in the mobile phase only (A), in the mobile phase containing 10 mg of copaiba oil resin (B) and in the mobile phase containing 10 mg.mL - 1 of copaiba essential oil (C). Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 102 The calibration curve of the HPLC-UV assay demonstrated that the method was linear (r 2 = 0.999) in the range from 50 to 2000 ng.mL -1 . The standard deviation of the curve of the analyzed drug was not significant. The straight-line equation was y = 0.198x (± 0.0003) + 4.401 (±0.395) (Where, x is the concentration of paclitaxel (ng.mL -1 ) and y the peak area (mAu s) (Figure 2A). Through the ANOVA analyzes, it was possible to perform the significance test for linear regression in order to determine if the regression model was suitable to generate a linear relation between the response variable y and some of the regression variables x. Thus, as Fcalculated= 706741> F tabulated = 5.99 for α=0.05 for three measures from the calibration curve containing 10 mg of copaiba resin oil, the proposed model was considered adequate to describe the linear regression. In addition, the Figure 2B showed homoscedasticity of the residues, indicating a higher normal distribution of values found around the regression line of the model. Figure 2- Calibration curve (A) and residual plots (B) of paclitaxel in the mobile phase containing 10 mg.mL -1 of copaiba resin oil analyzed by HPLC-UV. The LOQ was determined based on the standard deviation values of the response and the slope produced from the calibration curve data. The LOQ to produce the requisite precision and accuracy for this method was 21.03 ng.mL -1 , under the stated experimental conditions. The LOD was determined based on the signal-to-noise ratio using an analytical response of three times the background noise. Thus, the LOD of Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 103 paclitaxel was 6.31 ng.mL -1 . The sensitivity of this method was comparable with most other HPLC–UV methods developed for paclitaxel dosages using complex compound mixtures (Wang et al., 2003; Kim et al., 2005; Mohammadi et al., 2009; Choudhury et al., 2014). Accuracy was determined by quantifying six samples of paclitaxel at all concentrations of the calibration curve (range from 50 to 2000 ng.mL -1 ) in copaiba resin oil (Table 1). The recoveries ranged from 97.1 to 102.7 % and the RSD were less than 0.77 %. These results satisfied the drug quality research requirements. Table 1- Accuracy of paclitaxel assay in mobile phase containing 10 mg.mL -1 of copaiba resin oil Concentration (ng.mL -1 ) Concentration found (ng.mL -1 )* SD Accuracy (%) RSD (%) 50 48.6 0.4 97.1 0.77 200 205.4 0.9 102.7 0.46 500 498.5 1.3 99.7 0.27 800 805.5 3.9 100.7 0.48 1100 1117.7 7.8 101.6 0.70 1400 1392.3 4.2 99.4 0.30 1700 1731.8 12.4 101.9 0.72 2000 2009.3 7.8 100.5 0.39 *n=6, SD= standard deviation, RSD= relative standard deviation Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 104 Precision was estimated by the intra-day (repeatability) and the inter-day precision. Intra-day precision was investigated by the quantitation of triplicate samples of paclitaxel solutions at three different concentrations (800, 1400 and 2000 ng.mL -1 ). Inter-day precision was assessed by quantifying the same three samples over three consecutive days (Table 2). RSD for intra- and inter-day were less or equal to 0.65 %, which indicates that the method possessed low values of random errors. In other words, the method does not suffer significant changes between analyzes. Table 2- Intra-day and inter-day precision analyzes of paclitaxel. Parameters Concentration (ng.mL -1 ) Concentration found (ng.mL -1 )* SD RSD (%) Intra-day precision 800 802.9 3.9 0.49 1400 1390.0 3.7 0.27 2000 2009.1 9.6 0.48 Inter-day precision 800 796.5 3.1 0.39 1400 1401.3 8.1 0.57 2000 2003.6 13.1 0.65 *n=3, SD= standard deviation, RSD= relative standard deviation To investigate the suitability of this analytical method, the solubility and partition coefficient studies of paclitaxel were performed. Paclitaxel solubility was determined in water and in both copaiba essential oil and copaiba resin oil. The paclitaxel solubility in Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 105 water was lower than 0.4 µg.mL -1 , which is in agreement to the data of the literature (Konno et al., 2003; Zhao et al., 2010). Paclitaxel solubility in copaiba essential oil and copaiba resin oil were 52.9 (± 8.7) and 797.2 (± 79.1) µg.mL -1 , respectively, which were respectively 132 and 2,000 times higher compared to the solubility in water. This considerable increase of solubility may be explained by the lipophilic character of the drug molecule. The evaluation of the partition coefficient is an important step prior studies of the physicochemical characteristics of drugs for the development of liquid formulations (Silva et al., 2006). In fact, the distribution of the compound in the multi-phase system is directly related to its partitioning behavior (Balbach & Korn, 2004). Consistently with the results of solubility determined earlier in the present work and with the data of the literature (Lee et al., 2006; Surapaneni et al., 2012; Zabaleta et al., 2012), the partition of paclitaxel in n-octanol/water showed a logP of 3.89 (Table 3). When the lipophilic phase was copaiba essential oil and copaiba resin oil, the paclitaxel partition coefficients were 3.21 and 2.63, respectively (Table 3). These quite high values of paclitaxel partition coefficients explain the high solubility of paclitaxel in the oils and its extremely low solubility in water. Moreover, the high oil solubility and partition coefficient (lipophilicity) of the paclitaxel are favorable to achieve a higher drug entrapment in the internal phase of drug delivery systems. Therefore, copaiba oil can be selected as the oil phase for the development of drug delivery systems in which the combination of the anticancer activity of the oil and paclitaxel is desired in a single formulation. Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 106 Table 3- Partition coefficients of paclitaxel in, n-octanol, copaiba essential oil and copaiba resin oil. Samples * Compounds Concentration (µg.mL -1 ) SD P value (Oil/Water) LogP 1 n-Octanol 1207.5 24.3 7803.4 3.89 Water 0.2 0.1 2 Copaiba resin oil 645.8 37.9 1634.6 3.21 Water 0.4 0.1 3 Copaiba essential oil 162.4 14.3 423.3 2.63 Water 0.4 0.1 *n=3, SD= standard deviation, 4.0 CONCLUSION A rapid, simple, specific and sensitive HPLC method for the quantification of paclitaxel in samples containing copaiba oils was developed and validated. This method was highly sensitive for paclitaxel separation from a mixture of natural compounds found in copaiba oil and its essential oil extract. Calibration curves were reproducible. No significant differences were shown between the slope and the y-intercept and the residues were homoscedastic. This method showed a low LOD and LOQ levels, besides small coefficient of variation at 1% for accuracy and precision, satisfying the drug quality research requirements. It is also noteworthy that the method can be applied in all lab equipped with a basic HPLC system due to the simplicity of the material used while the cost of the analysis will remain low. The method was successful applied to the Chapter II- HPLC method for the dosage of paclitaxel in copaiba oil. Development, validation, application to the determination of the solubility and partition coefficients. 107 evaluation of concentrations of paclitaxel in various samples. Solubility characteristics of paclitaxel in copaiba oil and corresponding partition coefficients deduced from those experiments are encouraging to achieve the development of a drug delivery system that will combine the oil and paclitaxel in a single nanomedicine with the aim to potentiate their anticancer activity and enhanced efficacy of existing treatments. Acknowledgments The authors would like to thank the financial support from “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior- CAPES” through the COFECUB 721/11 project for Xavier-Junior, F.H. fellowship. REFERENCES ALENCAR, E. N., XAVIER-JUNIOR, F. H., MORAIS, A. R. V., DANTAS, T. R. F., DANTAS-SANTOS, N., VERISSIMO, L. M., REHDER, V. L. G., CHAVES, G. M., OLIVEIRA, A. G. & EGITO, E. S. T. 2015. Chemical characterization and antimicrobial activity evaluation of natural oil nanostructured emulsions. J Nanosci Nanotechnol, 15, 880-888. BALBACH, S. & KORN, C. 2004. 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Section II Les systèmes d'administration de médicaments à base de lipides Chapter III Prospective study for the development of emulsion systems containing natural oil products Chapter III- Prospective study for the development of emulsion systems containing natural oil products 115 Le troisième chapitre de cette thèse présente un article intitulé « Prospective study for the development of emulsion systems containing natural oil products ». Il a été publié dans le « Journal of Drug Delivery Science and Technology» le 19 Janvier 2012. L'étude consignée dans cet article marque le début des travaux sur les systèmes dispersés de l'huile de copaïba par notre groupe de recherche. Le choix de l'huile de copaiba avait été motivé par plusieurs raisons. Les huiles extraites des plantes ont un certain nombre d’avantages par rapport aux huiles minérales. Elles sont moins toxiques, biodégradables et renouvelables. Au cours de ces dernières années, les huiles végétales sont devenues plus attrayantes en raison de leurs avantages économiques et de leur renouvellement. En particulier, l’huile de copaïba, présente un riche mélange de composés de diterpènes et sesquiterpènes, largement utilisé dans la médecine populaire pour leurs propriétés anti-inflammatoires, anti-infectieuses et anticancéreuses. L’objective de notre étude menée sur l'huile de copaiba a été le développement et la caractérisation de systèmes émulsionnés avec cette l'huile. La première étape de notre travail a été de déterminer l'équilibre hydrophile-lipophile requis (HLB) de l'huile de copaïba sur une base expérimetale . Ensuite, des diagrammes de phases pseudo-ternaires ont été réalisés pour vérifier la formation de systèmes lipidiques différentes en changeant les concentrations des composants. Enfin, une étude de la stabilité et les méthodes de production des systèmes d'intérêt ont été réalisées. Les échantillons ont été préparés en utilisant différents mélanges binaires de tensioactifs obtenir une série de valeurs de HLB compris entre 4,5 et 16,5. Le HLB requis de l'huile de copaïba a été determiné pour une valeur de 14,8. L'émulsion obtenue a été montré une très bonne stabilité sur une période d'une année. . Les diagrammes de phases pseudo-ternaires ont été utiles pour décrire les proportions des mélanges des composants idéales pour la formation de différents systèmes disperses. Ces résultats indiquent que l'émulsion basée Chapter III- Prospective study for the development of emulsion systems containing natural oil products 116 de l'huile de copaïba pourrait être un moyen prometteur pour l'administration de médicaments. Mots-clés: Émulsion, huile de copaïba, équilibre hydrophile-lipophile, diagramme de phase pseudo-ternaire, stabilité. 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Chapter III- Prospective study for the development of emulsion systems containing natural oil products 117 Chapter III- Prospective study for the development of emulsion systems containing natural oil products 118 Chapter III- Prospective study for the development of emulsion systems containing natural oil products 119 Chapter III- Prospective study for the development of emulsion systems containing natural oil products 120 Chapter III- Prospective study for the development of emulsion systems containing natural oil products 121 Chapter III- Prospective study for the development of emulsion systems containing natural oil products 122 Chapter IV Microemulsion-based drug delivery systems containing natural oils Chapter IV- Microemulsion-based drug delivery systems containing natural oils 125 Le chapitre IV de ce manuscrit consiste en une revue de l’état de l’art de la production de microémulsions avec des huiles naturelles utilisés comme systemes pour la délivrance de principes actifs. Les produits naturels sont des mélanges très complexes contenants des composés de nature chimique différente. Certains ont des activités physiologiques ou thérapeutiques qui peuvent agir seules ou en synergie. Pour cette raison, plusieurs de ces huiles naturelles sont utilisées dans l'industrie pharmaceutique, agronomique, alimentaire, sanitaire et cosmétique. Aujourd'hui, leur intérêt est tourné vers leur immense potentiel pour prévenir et traiter de nombreuses maladies humaines. La formulation en microémulsions est apparue particulièrement appropriée pour améliorer les propriétés pharmaceutiques et biopharmaceutiques de ces huiles. Les microémulsions sont des dispersions thermodynamiquement stable, transparentes et isotropes constitué d'un mélange d'huile et d'eau stabilisé par un film interfacial de tensioactifs, typiquement en combinaison avec un co-tensioactif. Ces dispersions peuvent protéger des composés labiles d'une dégradation prématurée, contrôler la libération d'une molécule active, augmenter la solubilité d'un composé actif et par conséquent d'améliorer la biodisponibilité d'un médicament faiblement biodisponible. L'objectif de ce travail de revue bibliographique a été d'examiner les divers avantages des produits naturels formulés dans les systèmes de microémulsion à être utilisés comme des systèmes de livraison de composés bioactifs. Mots-clés: microémulsion, produit naturel, huile naturel, huiles végétales, système de livraison, effet synergique Chapter IV- Microemulsion-based drug delivery systems containing natural oils 127 MICROEMULSION-BASED DRUG DELIVERY SYSTEMS CONTAINING NATURAL OILS Xavier-Junior, F.H. 1,2 , Vauthier, C.², Morais, A.R.V.¹, Alencar, E.N.¹, Egito, E.S.T. 1 * 1 Universidade Federal do Rio Grande do Norte, Centro de Ciências da Saúde, Departamento de Farmácia, Laboratório de Sistemas Dispersos (LaSiD). Av. Gal. Gustavo Cordeiro de Farias, S/N, Petrópolis, 59010-180, Natal-RN-Brazil. 2 Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. Corresponding author Christine Vauthier, Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. Christine.vauthier@u-psud.fr Chapter IV- Microemulsion-based drug delivery systems containing natural oils 128 ABSTRACT Natural products are extremely complex mixtures containing compounds of different chemical nature. Some have physiological or therapeutical activities that may act either alone or in synergy. So many of them are used in the pharmaceutical, agronomic, food, sanitary and cosmetic industries. Today, their interest is growing toward their immense potential to prevent and treat numerous human diseases. Formulation in microemulsions appeared suitable to improve pharmaceutical and biopharmaceutical properties. Microemulsions are thermodynamically stable, transparent and isotropic dispersions consisting of oil and water stabilized by an interfacial film of surfactants, typically in combination with a cosurfactant. They can protect labile compounds from premature degradation, control drug release, increase drug solubility hence enhance the bioavailability of a poorly bioavailable drug. This paper was aimed to review the various advantages of natural products formulated in microemulsion systems to be used as delivery systems for bioactive compounds. The state of the art of parameters involved in the microemulsion formation is summarized as well. Keywords: Microemulsion, natural product, natural oil, plant oils, delivery systems, synergic effect Chapter IV- Microemulsion-based drug delivery systems containing natural oils 129 INTRODUCTION Microemulsion (ME) has attracted much interest for several years in terms of their delivery and target potentials (Tenjarla, 1999; Gupta & Moulik, 2008; Muzaffar et al., 2013). Microemulsions (MEs) are thermodynamically stable-phase transition systems, transparent, optically isotropic, which possess low surface tension and small droplet size (Danielsson, Ingvar & Lindman, Björn, 1981; Hegde et al., 2013). These systems are formed by two immiscible liquids (water and oil) mixed to form a single phase stabilized by an interfacial film of alternating surfactant and cosurfactant molecules (Singh et al., 2011; Lawrence & Rees, 2012). It is well established today that ME can appear in at least three major microstructures: swollen micellar (oil-in-water, O/W), reverse micellar (water-in-oil, W/O) and bicontinuous structures (Ghosh & Murthy, 2006; Lakshmi et al., 2013). These structures can be formed depending on the concentrations, nature, and arrangements of the molecules present in the formulation (Mcclements, 2012). The structure of micelles containing solubilized oil molecules may be spheroids (e.g., micelles or reverse micelles), cylinder-like structure (such as rod-micelles or reverse micelles), plane-like structure (e.g., lamellar structures) or sponge-like structures (e.g., bicontinuous) (Jonsson et al., 1998). MEs have many advantages as drug delivery systems, including improved appearance, high stability, easiness of preparation and small droplet size, resulting in large surface area from which the active substance can partition and be absorbed or permeate through membranes (Tenjarla, 1999; Lawrence & Rees, 2012; Ritika et al., 2012; Lakshmi et al., 2013). Also, these systems possess the ability to enhance the bioavailability of poorly soluble drugs by maintaining them in molecular dispersion, consequently Chapter IV- Microemulsion-based drug delivery systems containing natural oils 130 allowing for controlled or sustained release of the active agent (Schmalfub et al., 1997; Tenjarla, 1999). They form spontaneously (zero energy input). Therefore, they are ease of manufacture (no process dependent) and scale-up (Al-Adham et al., 2000; Vandamme, 2002; Ghosh & Murthy, 2006). These special properties of the ME offer a high potential for numerous practical applications, including enhanced oil recovery, pharmaceutical and cosmetic formulations, edible coatings for food, and others industrial applications (Singh et al., 2011). Recently, natural products MEs have been of increasing interest to researchers and have shown great potential in industrial applications. ME utility lies from the fact that they can incorporate a large amount of active natural products in the continuous or disperse phase which are otherwise difficult to formulate (Gupta et al., 2006; Biruss et al., 2007; Safaei-Ghomi & Ahd, 2010; Lawrence & Rees, 2012). Natural products are extremely complex mixtures containing compounds of various chemical natures which are widely exploited by industries around the world. Chemical constituents of natural oils act either alone or in synergy with other compounds giving a global therapeutic activity when incorporated in formulations (Guenther, 1972; Cowan, 1999; Bakkali et al., 2008; Vigan, 2010; Bilia et al., 2014). Natural products and/or their volatile constituents are used widely to prevent and treat human disease. The possible role and mode of action of these natural products is discussed with regard to the prevention and treatment of very severe disease including cancer, Alzheimer’s and cardiovascular diseases, as well as their bioactivity as spasmolythic, revulsive, anti-inflammatory, analgesic and acaricide, antibacterial, antiviral, antipsoriatic, antioxidants and antidiabetic agents (Buchbauer, 2004; Ali et al., Chapter IV- Microemulsion-based drug delivery systems containing natural oils 131 2008; Adorjan & Buchbauer, 2010; Astani et al., 2010; Chaiyana et al., 2010; Safaei- Ghomi & Ahd, 2010; Bassole & Juliani, 2012). Due to the therapeutic advantages and the complex composition of the natural products, various formulation approaches including carrier technology as ME offer an intelligent approach for the in vivo delivery of several molecules. The present review is divided into two sections. The first section covers the basic concepts of the physicochemical formulation of ME systems, main aspects and energy responsible for their formations. The second section describes the natural products uses and reviews their recent applications in ME systems. BACKGROUND The term “microemulsion” was first introduced in 1943 by Hoar and Schulman. It is characterized as a self-assembled homogeneous isotropic system, with thermodynamic stability and it contains extremely high oil/water interfacial areas, offering ultra-low interfacial tension (less than 0.1 mN/m) and viscosity. This system is comprised of two immiscible liquids, such as oil and water, which are stabilized by surfactants and co- surfactants (Hoar, T. P. & Schulman, J. H. , 1943). The dispersed phase occurred as very small droplets (10 - 100 nm), therefore which hardly scattered light in the visible wavelength domain explaining their tendency to appear as transparent. ME usage may result in high drug absorption and permeation, and hence, strong possibility of drug delivery. The molecular structures of the surfactants consist of an apolar tail region and a polar head group. The surfactants decrease the surface tension of the oil-water interface and Chapter IV- Microemulsion-based drug delivery systems containing natural oils 132 change the entropy of the system. However, it is required a relatively large amount of surfactant in order to stabilize the large interfacial area of ME (Jha, S. K. et al., 2011). The low interfacial tension compensates the dispersion entropy, hence the system is thermodynamically stable (Tenjarla, 1999) and form spontaneously under a specific set of composition and environmental conditions (Rao & Mcclements, 2011). When dispersed in water or in non-aqueous solvents, the surfactants self-associate into a variety of equilibrium phases. Once the surfactants is an amphiphilic molecule (posses within their structure a part that has an affinity for oil and a part that has affinity for water), during the mixing, the surfactants molecules migrate to the oil-water interface to form a film. The monolayer of surfactant in the interface can exert a two-dimensional surface pressure due to the expansion of the film until the pressure at both sides of the interface becomes constant. After the surfactants occupy the entire interface, the addition of more surfactant will result in micelle formation (Chen et al., 1986; Tenjarla, 1999). Depending on the structure of the surfactants present (greater affinity for water or for oil), it will be determined which type of the system will be formed, once a surfactant more soluble in water than in oil will influence the direction of the ME oil in water (o/ w ME), vice-versa for a more soluble surfactant in the oil than in water (w /o ME) or bi-continuous ME. Various structures may be formed when the surfactants are combined with water, oil or both, such as spherical micelle, reverse micelle, rod shaped micelle, hexagonal phase, lamellar phase and reverse hexagonal phase (Figure 1) (Lawrence & Rees, 2012). In addition, the required concentration of surfactants in the systems will depend of its structure, since a lower concentration of surfactant that strongly favors orientation at the oil/water interface is required in comparison with a surfactant that partitions strongly into either the oil or water phase (Bagwe et al., 2001). Concerning the concentration and the several geometry from the surfactants structures, Chapter IV- Microemulsion-based drug delivery systems containing natural oils 133 it is reasonable that the surfactants film in ME may have different shapes (Soderman & Nydén, 1999). It is possible to verify the formation of elongated, rod-like micelles and W/O spherical droplets at low water content. Whereas, at high water concentration, the most frequent form observed is O/W droplets. Additionally, bicontinuous structures may be formed in ME with similar contents of waters and oil (Podlogar et al., 2005). Figure 1: A predictive pseudo-ternary phase diagram with the existence fields of different systems: conventional micelles, reverse micelles, W/O ME, O/W ME and bi- continuous ME and of the Winsor systems (white is the water-excess region, black is the oil-excess region and gray is the microemulsion) (adapted from (Lawrence & Rees, 2012) Chapter IV- Microemulsion-based drug delivery systems containing natural oils 134 At low surfactant concentration, there are four types of ME phases that exist in equilibrium; these phases are commonly referred as Winsor phases (Figure 1) (Winsor, 1954). They are, Winsor I: With two phases, a lower O/W ME phase in equilibrium with the upper excess oil. Winsor II: with two phases, an upper (W/O) ME phase in equilibrium with the water excess. Winsor III: With three phases, a middle ME phase (O/W plus W/O, called bi-continuous) in equilibrium with the upper oil excess and lower excess water. Winsor IV: In single phase, where both oil and water are completely dispersed in the surfactant ME phase. Inter-conversion among the above- mentioned phases can be achieved by adjusting proportions of the constituents. Attempts have been made to rationalize surfactant behavior in ME formation. These approaches are fairly empirical but can be a useful guide to surfactant selection. In this context, the hydrophilic –lipophilic balance (HLB), the critical packing parameter and the solubility parameters approach were proposed in order support surfactant selection for ME application. The HLB takes into account the relative contribution of hydrophilic and hydrophobic fragments of the surfactant molecule. The W/O ME are formed through the surfactants high dispersion rates in oil, beyond the limit to form reverse micelles (Griffin, W. C., 1949b; Bagwe et al., 2001). Surfactants used to produce this type of ME have a HLB ranging from 3 to 8. On the other hand, to produce W/O ME, the oil is dispersed using surfactants having HLB ranging from 8 to 18. The formation of bicontinuous ME (HLB ≈ 10) is explained by various models, such as the Scriven model, the Random-lattice model, the Cubic random-cell model and the disordered open-connected model (outside the scope of this review) (Tenjarla, 1999). In contrast, the critical packing parameter relates the ability of surfactants to form particular aggregates to the geometry of the molecule itself. This parameter measures Chapter IV- Microemulsion-based drug delivery systems containing natural oils 135 the preferred geometry adopted by the surfactant, and consequently, it is predictive of the type of aggregate that is likely to form (Lawrence & Rees, 2012). The geometric position of the surfactant at the interface can be another factor influencing the ME structure (Ho et al., 1996). The size and shape of the ME are mainly governed by the curvature free energy of the interface between water ad oil and are determined by the bending elastic constant and curvature of the surfactant film. The elasticity of the film depends on the type of surfactant and on the thermodynamic conditions, but also on the presence of additives like alcohols, electrolytes, block copolymers, and polyelectrolytes. Co-surfactant such as short chain alcohols can improve the film's flexibility (Komesvarakul et al., 2006). The solubility parameter theory is based on the premise that when the solubility parameters of two chemical compounds are equal, the compounds are infinitely soluble (Hildebrand, 1916; Hildebrand & Scott, 1950; Burke, 1984). The intermolecular forces that cause chemical species to dissolve are the same forces that prevent those materials from boiling away until a specific temperature is reached (Vaughan, 1985). Hansen et al. included molecules interacting by dipolar and hydrogen bonding forces (as well as dispersion forces) on this theory, by making the assumption that the solubility parameter could be represented by an additive function of three components (Hansen, C. M., 1967). In this theory, for complete miscibility, two liquids need each of these parameters to be similar. The solubility parameter offers a far more comprehensive system than the HLB system concept. The HLB does not take onto account the chemical match between the surfactant and the phase components of the ME. In other words, it did not take into account the miscibility properties of the surfactants with solvents composing each phase of the ME However, solubility parameter has the disadvantage of being very complex with several alternative expressions available (Vaughan, 1993). Chapter IV- Microemulsion-based drug delivery systems containing natural oils 136 ME droplets have a larger effective interaction volume for the type O/W than W/O, which is due to a strong repulsive term introduced by the presence of an electrical double layer at the surface of the O/W droplet when the ionic surfactants are used. However, when a non-surfactant is used to stabilize O/W ME, the predominant repulsive factor might be attributed to steric interactions, although the polar head groups produce hydration shell. Additionally, the preparation process of W/O ME is easier then O/W ME, since its interfacial tension tends to be lower due to the easier surfactant arrangement at an interface with high curvature, given that the surfactant tails extend outwards into a continuous oil phase, which is entropically more favorable as the hydrocarbon tails have more directional freedom (Lawrence & Rees, 2012). THEORY OF MICROEMULSION FORMATION The formation and as well as stability of ME can be affected by various factors such as nature of surfactant, molecular weight of surfactant, alcohol chain length, temperature etc. The reduction of the interfacial free energy to a very low value is of prime importance in the ME formation. Accordingly, the ME formation has been explained by the following three approaches: interfacial or mixed film theory, solubilization theory and thermodynamic theory (Paul & Moulik, 1997; Mehta & Kaur, 2011; Lawrence & Rees, 2012). Chapter IV- Microemulsion-based drug delivery systems containing natural oils 137 Interfacial or mixed film theory Postulated by Bowcott & Schulman in 1955, this theory describes that the interfacial film is considered to be a duplex in nature (region bounded by water on one side and oil on the other), with an inner and an outer interfacial tension acting independently. (Bowcott & Schulman, 1955). Such a specialized liquid has been based on the assumption that interactions in the interface and reducing the original O/W interfacial tension to zero are capable to form a ME spontaneously. Nevertheless, the ME formation is not ensure by zero interfacial tension although the interfacial tension is generally extremely low, but it depends on the kind of molecular interactions in the liquid interface. Based on it, Robbins et al. developed the theory of MEs phase behavior which discuss that the changes on the direction and extent of curvature are due to the interactions in a mixed film, which can estimate the type and size of the ME droplets (Robbins, 1976). Furthermore, the differential tendency of water to swell the heads and oil to swell the tails of the surfactants impose the ideal kind and degree of curvature of the surfactant film molecules included in the interface to ME formation. Solubilization theory Since the 70’s it is possible to explain that ME are swollen micelles in which either the water is solubilized in reverse micelles, or the oil is solubilized in normal micelles (Shinoda & Friberg, 1975). A model of this theory was presented by Adamson et al reporting that the W/O emulsion is formed because of the balance achieved in the Laplace and osmotic pressure and that the electrical double layer system with internal Chapter IV- Microemulsion-based drug delivery systems containing natural oils 138 aqueous phase is partially responsible for the interfacial energy, which presented positive free energy, contradicting the concept of negative interfacial tension (Adamson, 1969). Thermodynamic theory Concerning the thermodynamic theory, it is important to consider that the free energy must be negative to form thermodynamically stable MEs and the ME formation depends on the reduction of the surface tension of the oil – water interface by the surfactants and the change in entropy of the system (Ruckenstein & Krishnan, 1980). Schulman explained that the formation of a thermodynamically stable ME occurs with a very low interfacial tension of the order of 10 -4 to 10 -5 dynes/cm. Moreover, the interfacial charge is responsible for controlling the phase continuity, once the thermodynamic approach accounts for the free energy of the electric double layer along with the van der Waals and the electrical double layer interaction potentials among the droplets (Hoar, T. P. & Schulman, J. H. , 1943; Mehta & Kaur, 2011). However, a significant favorable entropic change should be accompanied by large reductions in surface tension in order to achieve a negative free energy of formation, resulting in spontaneous microemulsification and in a thermodynamically stable system. This entropic change arises from monomer-micelle surfactant exchange, surfactant diffusion in the interfacial layer and the mixing of one phase in the other in the form of large numbers of small droplets (Lawrence & Rees, 2012). Chapter IV- Microemulsion-based drug delivery systems containing natural oils 139 METHOD OF PREPARATION Although MEs may form spontaneously, external factors can be used to overcome kinetic barriers hence reducing time to obtain the formation of this system. Some factors that can accelerate and facilitate the formation of the ME system can be the order of component addition, the application of a mechanical agitation, the use of ultrasounds or of heat for instance. Therefore, in order to accelerate their formation on a kinetic standpoint, two different methods were proposed to accelerate their formation. These include a phase inversion and a phase titration methods. The change in spontaneous curvature of the surfactant is used by the phase inversion method. The phase inversion may occur in response to temperature or upon dilution of excess of dispersed system inducing drastic physical changes as changes in particle size that can affect drug release both in vivo and in vitro (Jha, S. K. et al., 2011). The concept of phase inversion temperature (PIT) was introduced by Shinoda and Arai showing the importance of temperature on surfactant properties (particularly nonionic surfactants) (Shinoda & Arai, 1964; Shinoda & Arai, 1967). During the PIT the interfacial properties of the system are balanced and the very small droplet sizes are produced. The nature of the emulsified oils as well as the HLB and concentration of surfactants are important parameters for the PIT (Venkatesh et al., 2014). Additionally, changing the water volume fraction can induce a transition in the spontaneous radius of curvature (Jha, S. K. et al., 2011). The phase titration method uses the spontaneous diffusion of surfactant or solvent molecules into the continuous phase due to ultra low interfacial tension. The use of diagrams is a useful tool to understand the complex series of interactions that can occur when different components are mixed together. Pseudoternary phase diagram is often Chapter IV- Microemulsion-based drug delivery systems containing natural oils 140 constructed when there are 4 components in the formulation, wherein one corner is the mix of surfactants and the others are the oil and the water. To construct the phase diagram all the components of formulation are mixed in proportions varying from 0 to 100%. Subsequently, each system was characterized and demarcated the phase boundaries formed (Shinoda & Lindman, 1987; Hegde et al., 2013; Muzaffar et al., 2013; Venkatesh et al., 2014). USES AND APPLICATIONS OF MICROEMULSION SYSTEMS MEs have been used in a variety of chemical and industrial processes, such as in enhanced oil recovery, as fuels, as coatings and textile finishing, as lubricants, cutting oils and corrosion inhibitors, in detergency, cosmetics, agrochemicals, food, biotechnology, environmental remediation and detoxification, in analytical applications, microporous media synthesis, in pharmaceuticals and as liquid membranes (Paul & Moulik, 2001; Kartsev et al., 2009). Pharmaceutical preparations such as liquid crystals, micelles and emulsion forming systems have been studied by several authors as a method to solubilize drugs, once the solubilization using cosolvents was the conventional approach. However, the use of cosolvents cannot be employed for parenteral administration for several drugs, furthermore, other disadvantages such as precipitation of the drug on dilution, severe pain at injection site and hemolysis are related to the use of cosolvents (Date & Nagarsenker, 2008). Nevertheless, the instability of emulsions and low solubilization capacity of micelles are disadvantageous. ME is a better proposition over other compartmentalized systems due to their thermodynamic stability, minimum energy Chapter IV- Microemulsion-based drug delivery systems containing natural oils 141 necessary for formation, easiness of preparation, long- term shelf life, low viscosity, surfactant-provoked permeability and reduction of various diffusion barriers by acting as penetration enhancer, protecting against enzymatic degradation, improving drug stability and solubilization capacity which allow a large amount of drug to be incorporated. However, MEs show the disadvantage of having a high concentration of surfactants, which can be toxic to cells depending on their nature (Rozman et al., 2009; Saha et al., 2012). These structures have been investigated as drug delivery systems for the purpose of drug targeting and controlled release. They improve the bioavailability of poorly soluble drugs due to the capacity of solubilizing both lipophilic or hydrophilic drugs, and partitioning them between the dispersed and the continuous phases, or even administering them together in the same preparation (Nazar et al., 2009). Another important factor is their small droplet size which results in large surface area from which the drug can partition hence it improves problems linked with the dissolution of drugs. A that can be better absorbed or permeate through biological membranes (Gupta & Moulik, 2008). Accordingly, the enhancement of the bioavailability of the drug can reduce the dose required to provide the same pharmacological action and hence reduce associated side-effects associated (Paul & Moulik, 2001). In addition to these advantages, concerning the parenteral delivery systems, the ME improves the drug residence in the blood circulation and reduces the drug irritation (Ren et al., 2012). Furthermore, MEs cause minimum immune reactions or fat embolism in contrast to emulsions. MEs have been used to sustain or control drug release for percutaneous, peroral, topical, transdermal, ocular and parenteral administration, enhancing absorption of drugs, Chapter IV- Microemulsion-based drug delivery systems containing natural oils 142 modulating the kinetics of the drug release and decreasing the toxicity (Paul & Moulik, 2001; Singh et al., 2011). However, the uses of MEs may be limited due to the maintenance of thermodynamic stability in the temperature range between 0° and 40 °C, constant pressure during storage, low solubilizing capacity for high molecular weight drugs, toxicity and possible incompatibility of surfactants and cosurfactants, requirement at high concentrations for formulations (Paul & Moulik, 2001). NATURAL PRODUCTS Mother nature has been a source of medicinal agents for thousands of years. Human started to use plant as medicine since 60,000 years ago, approximately, and today 65% of the world’s population relies on plant for their primary health care (Cowan, 1999). Various medicinal plants have been used for years in daily life to treat disease all over the world (Bown, 2003; Nair et al., 2005). Oldest forms of healthcare include the use of leaves, flowers, stem, berries and root of herbs because of their therapeutic or medicinal value (Joseph et al., 2013). Today, it’s estimated that there are 250,000 to 500,000 plant species identified so far, about 35,000 are used worldwide for medicinal purposes (Borris, 1996; Moerman, 1996). Natural medicine is based on the premise that plants contain substances that can promote health and alleviate illness, usually with minimal toxic side effects (Chahlia, 2009; Balakumar et al., 2011; Rajan et al., 2011; Bilia et al., 2014). Focus on plant research especially medicinal plants used in traditional systems has increased all over the world (Dahanukar & Kulkarni, 2000; Biswas & Mukherjee, 2003). Ethnobotanical information has contributed to health care worldwide through the isolation of bioactive Chapter IV- Microemulsion-based drug delivery systems containing natural oils 143 compounds for direct use in medicines (Soejarto et al., 2012; Ntie-Kang et al., 2013; Ezuruike & Prieto, 2014). Indeed, plant extracts represent excellent renewable resources for human applications. The plants have an almost limitless ability to synthesize aromatic substances, most of which are phenols or their oxygen-substituted derivatives (Geissman, 1963). The most important of these biologically active constituents are alkaloids, flavonoids, tannins and phenolic compounds (Sukumaran et al., 2011). These secondary metabolites in the plants have a prominent function of protection as antibacterials, antivirals, anti-fungals, insecticides and also against herbivores by reducing their appetite for such plants (Cowan, 1999). It is believed that most of the 100,000 known secondary metabolites are involved in plant chemical defense systems, however, only 12,000 have been isolated, a number estimated to be less than 10% of the total (Schultes, 1978; Wink, 1999). Some metabolites are also involved in defense mechanisms against abiotic stress (e.g., UV-B exposure) and are important in the interaction of plants with other organisms (e.g., attraction of pollinators) (Schafer & Wink, 2009; Bassole & Juliani, 2012). Some, such as terpenoids, give plants their odors and flavor (e.g., the capsaicin from chili peppers); others (quinones and tannins) are responsible for plant pigment; and more some of the herbs and spices used by humans to season food (Cowan, 1999). In human, these natural compounds are predominantly used as antioxidant, antibacterial, anti- inflammatory, antiallergic, hepatoprotective, antithrombotic, antiviral, anticarcinogenic, and they even have vasodilatory and neuroprotective properties (Martino et al., 2009; Rasooli, 2011; Leandro et al., 2012). Regarding natural oils studies, the composition of most seed oils are made up of a wide range of fatty acids with six dominating fatty acids: palmitic, stearic, oleic, linoleic, Chapter IV- Microemulsion-based drug delivery systems containing natural oils 144 linolenic and lauric acids. Such fatty acids include those with chain lengths between 8 and 24 carbon atoms, containing varying numbers of double bonds, conjugated systems or functional groups such as acetylenic bond (triple bond), epoxy group (oxygen containing) and hydroxy group (Carlsson, 2009). The advantage about plant oil uses is the excellent renewable sources for industrial usage and further they are structurally similar to the long-chained hydrocarbons derived from petroleum (Wagner et al., 2001). Essential oil is one of the main classes of secondary metabolites. These oils are volatile, natural, highly enriched in isoprene structure characterized by a strong odor (Cowan, 1999; Bakkali et al., 2008). Essential oils are liquid at room temperature, though a few of them are solid or resinous, limpid, rarely colored, hydrophobic, generally they have a lower density than that of water, they are soluble in lipids and in organic solvents (Balz, 1999; Bakkali et al., 2008). They can be synthesized by all plant organs, i.e. buds, flowers, leaves, stems, twigs, seeds, fruits, roots, wood or bark, and are stored in secretory cells, cavities, canals, epidermic cells or glandular trichomes. Essential oils are extracted from various aromatic plants generally localized in temperate to warm countries like Mediterranean and Tropical countries where they represent an important part of the traditional pharmacopoeia (Bakkali et al., 2008). It is known that the percentage of the components of essentials oils varies amongst species, plants parts, age and stage in the vegetative cycle, as well as according to environmental factors such as light, temperature, soil composition and season, the geographical origin of the plants and the period of harvest (Angioni et al., 2006; Raileanu et al., 2013). Complex mixtures of volatile compounds come from two groups of distinct biosynthetic pathways. The main group is composed of terpenes and terpenoids and the other of aromatic and aliphatic constituents, all characterized by low molecular weight (Croteau, Chapter IV- Microemulsion-based drug delivery systems containing natural oils 145 1987; Croteau et al., 2000; Betts, 2001; Pichersky et al., 2006). Terpenes are made from combinations of several isoprene precursors: isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). The mevalonate route operates in the cytoplasm and mitochondria, and the deoxyxylulose pathway in the plastids (Figure 2). After isoprene precursor’s formation, the terpenes biosynthesis consists of repetitive addition of IPPs and DMAPPs molecules and modification by terpene specific synthetases to form the terpene skeleton (Geranyl pyrophosphate). Finally, secondary enzymatic modification of the skeleton occurs to attribute functional properties to the different terpenes (Pichersky et al., 2006; Bakkali et al., 2008). A monoterpene have a general chemical structure of C10H16 and they occur as diterpenes, triterpenes, and tetraterpenes (C 20, C 30 and C 40), as well as hemiterpenes (C 5) and sesquiterpenes (C 15). When the compounds contain additional elements, usually oxygen, they are termed terpenoids (Mcgarvey & Croteau, 1995; Cowan, 1999). These compounds are largely synthesized from acetate units, and despite sharing their origins with fatty acids, they differ of these because they contain extensive branching and are cyclized. Aromatic compounds occur less frequently than terpenes and they are generated from phenylpropane (C6-C3) derivatives (Cowan, 1999; Vigan, 2010). Chapter IV- Microemulsion-based drug delivery systems containing natural oils 146 Figure 2: The terpenes biosynthesis by deoxyxylulose and mevalonate pathways Chapter IV- Microemulsion-based drug delivery systems containing natural oils 147 Regarding their biological properties, it has to be kept in mind that essential oils are complex mixtures of numerous molecules that have different mechanisms of action (Guenther, 1972; Koul et al., 2008).It is likely that several components of the essential oils play a role in cell penetration and distribution, due the lipophilic or hydrophilic attraction and fixation on cell walls and membranes (Cal, 2006; Bakkali et al., 2008). In general, the terpenes compounds including limonene (Clarys et al., 1998; Lim et al., 2006), menthol, terpineol, menthone, pulegone, carvone (Jain et al., 2002), thymol, carvacrol, trans- anethole, linalool (Kararli et al., 1995), 1,8-cineole (Williams & Barry, 1991), geraniol (Arellano et al., 1996; Hanif et al., 1998) show a low systemic toxicity and skin irritancy in addition to having good penetration enhancing abilities (Cornwell et al., 1996). This feature is very important because the distribution of the oil in the cell determines the different types of produced radical reactions, depending on their compartmentalization in the cell (Hansen et al., 2006). Studies also showed that antioxidant activity may be attributed primarily to the high content of phenolic components of the essential oil (Kahkonen et al., 1999; Dorman & Deans, 2000). In general, these compounds can provoke depolarization of the mitochondrial membranes by decreasing the membrane potential, affecting ionic Ca ++ cycling and other ionic channels, reducing the pH gradient, collapsing of the proton pump and depletion of the ATP pool (Richter & Schlegel, 1993; Sikkema et al., 1994; Vercesi et al., 1997; Turina et al., 2006; Pasqua et al., 2007). They may change the fluidity of membranes, which might become abnormally permeable, resulting in leakage of radicals, cytochrome C, calcium ions and proteins, as in the case of oxidative stress and bioenergetic failure, which may explain their pharmacological and possible toxic effects (Armstrong, 2006; Lorenzi et al., 2009; Devi et al., 2010). Others studies indicated that plant extracts anti-mutagenic properties may be due to inhibition of Chapter IV- Microemulsion-based drug delivery systems containing natural oils 148 penetration of the mutagens into the cells, inactivation of the mutagens by direct scavenging, inhibition of metabolic conversion by P450 of promutagens into mutagens, or activation of enzymatic detoxification (Bakkali et al., 2008). The major anti- mutagenic compounds related are tannic acid, apigenine (Kuo et al., 1992), α-bisabolol (Gomes-Carneiro et al., 2005), thuyone, 1,8-cineole, camphor, limonene (Vukovic- Gacic et al., 2006), (-)-Menthol, (-)-α-pinene, (+)-α-pinene, β-ionone, α- terpinene, α- terpineol, citronellal, and others (Gomes-Carneiro et al., 1998; Oliveira et al., 1999). Essential oils possess antibacterial, anti-fungal (Safaei-Ghomi & Ahd, 2010; Bassole & Juliani, 2012) and antiviral (Astani et al., 2010) properties. It may be used for prevention and treatment of cancer (Bhalla et al., 2013; Bayala et al., 2014), cardiovascular diseases including atherosclerosis and thrombosis (Lahlou et al., 2005; Santos, M. R. et al., 2007). They might also be used as analgesic (Sousa, 2011), sedative (Buchbauer, 2004), anti-inflammatory (Hajhashemi et al., 2003; Silva et al., 2003), spasmolytic (Magalhaes et al., 2004), local anesthetic, antipyretic activities (Chakraborty et al., 2010), food preservative (Tiwari et al., 2009) and their fragrance can be used in cosmetic applications (Buchbauer, 2004; Adorjan & Buchbauer, 2010). Several techniques can be used to extract essential oils from different parts of the aromatic plant, including solvent extraction, expression under pressure, enfleurage, and distillation extractions, but hydro or steam distillation are the most commonly used method (Figure 3) (Stashenko et al., 1999; Bassole & Juliani, 2012). The steam distillation separation process based on the difference in composition between a liquid mixture and the vapor formed from it. The mechanical process is used exclusively for citrus fruit: their essential oils are contained in microvesicles located in the peel and may be extracted by pressure or friction. Dry distillation, without addition of water Chapter IV- Microemulsion-based drug delivery systems containing natural oils 149 vapour, is used for wood, bark and roots (Vigan, 2010). For perfume uses, extractions with lipophilic solvents and sometimes with supercritical carbon dioxide are desired. Thus, the chemical profile of the essential oil products differs not only in the number of molecules but also in the stereochemical types of molecules extracted, according to the type of extraction, thereby, the type of extraction is chosen according to the purpose of the use (Bakkali et al., 2008). Their composition can also change after extraction. Depending on the storage conditions, they can quickly become oxidized, and this oxidation is responsible in some cases for variation on the pharmacological activities (Karlberg & Dooms-Goossens, 1997). To monitor these phenomena, most of the commercialized plant extracts are chemotyped by gas chromatography and mass spectrometry analysis (Stashenko et al., 1999). Analytical monographs have been published in the pharmacopoeia from different country to ensure good quality of essential oils (Smith et al., 2005). Figure 3- Main extraction process to essential oils from fresh or dried aromatic plant parts Chapter IV- Microemulsion-based drug delivery systems containing natural oils 150 Among these secondary metabolites, it is estimated that over 3,000 essential oils are known, of which about 300 are commercially important especially for the pharmaceutical, agronomic, food, sanitary, cosmetic and perfume industries (Van De Braak & Leijten, 1999; Bakkali et al., 2008). Some essential oils appear to exhibit particular medicinal properties that have been claimed to cure one or another organ dysfunction or systemic disorder (Hajhashemi et al., 2003; Silva et al., 2003). The new attraction for natural products as essential oils is important due some of them constitute being an effective or complements alternatives to synthetic compounds used in chemical industry, without showing the same secondary effects and protecting the ecological equilibrium (Carson & Riley, 2003; Bakkali et al., 2008). In some cases, the bioactivities of essential oil are closely related with the activity of the main components of the oils (Juliani et al., 2002). However, some studies have demonstrated that whole essential oil usually have higher activity than the mixtures of their major components, suggesting that the minor components are critical to the synergistic activity, though antagonistic and additive effects have also been observed (Figure 4) (Hammer et al., 1999; Dorman & Deans, 2000; Santana-Rios et al., 2001; Gill et al., 2002; Yoon et al., 2011). An additive effect is observed when the combined effect is equal to the sum of the individual effects. Antagonism is observed when the effect of one or both compounds is less expressive when they are applied together than when individually applied. Synergism is observed when the effect of the combined substances is greater than the sum of the individual effects (Burt, 2004; Bassole & Juliani, 2012). Thus, the uses of these combinations are strategies to potential synergic effects. In that sense, for ME application, it is more relevant to study whole oil rather than some of its components because the concept of synergism appears to be more Chapter IV- Microemulsion-based drug delivery systems containing natural oils 151 meaningful, and the addition effect of the disperse system and the natural product can be expected. Figure 4: Schema of synergistic, antagonistic and additive effects between components in essential oil (adapted from Chait et al (Chait et al., 2007)). MICROEMULSION BASED ON NATURAL PRODUCTS Development of ME including natural products have been of increasing interest to researchers and have shown great potential in industrial applications (Guenther, 1972; Cowan, 1999; Bakkali et al., 2008; Vigan, 2010; Bilia et al., 2014). The association of the intrinsic advantages from ME may be able to act in synergy with the natural compounds (Gupta et al., 2006; Biruss et al., 2007; Lawrence & Rees, 2012). For the purposes of this review, recent developments will for the most part constitute an Chapter IV- Microemulsion-based drug delivery systems containing natural oils 152 evaluation of the literature in the area of ME with natural products. Table 1 show the main works of ME containing natural oils and its respective therapeutic application. Table 1- Microemulsion containing natural oils Oil Source Data about therapeutic applications Reference Babchi oil Psoralea corylifolia Psoriasis (Ali et al., 2008) Cassia oil Cinnamomum cassia Antifungal activity against Geotrichum citri-aurantii (Xu et al., 2012) Cinnamon oil Fungicide (Wang et al., 2014) Citrus oil (Fanun, 2010b) Clove oil (Gupta et al., 2006) Clove oil Syzygium aromaticum Leishmaniasis (Gupta et al., 2005) Coconut oil (Rukmini et al., 2012) Copaiba oil Copaifera Langsdorffii (Xavier-Junior, Chapter V, 2015) Corn oil (Gupta et al., 2006) Cottonseed oil (Gupta et al., 2006) Davana oil Artemisia pallens Topical delivery (Salunkhe et al., 2013) Eucalyptus oil Eucalyptus Spp Absorption promoter to transdermal drug (Majhi & Moulik, 1999; Maghraby, 2008) Lemon grass Cymbopogon citratus Alzheimer's Chaiyanaetal., 2010 Lemon oil (Rao & Mcclements, 2011) Linseed oil (Mitra et al., 1994) Chapter IV- Microemulsion-based drug delivery systems containing natural oils 153 Lizard tail Houttuynia Cordra (Yi et al., 2010) Makrut lime Citrus hystrix Alzheimer's (Chaiyana et al., 2010) Neem oil Azadirachta indica Acaricidal activity against Sarcoptes scabiei var. cuniculi larvae (Xu et al., 2010) Orange oil Absorption promoter to transdermal drug (Yotsawimonwat et al., 2006) Orange oil (Gupta et al., 2006) Palm oil (Mitra et al., 1994) Peppermint oil Mentha piperita L. (Fanun, 2010a) Peppermint oil (Gupta et al., 2006) Plai oil Zingiber cassumunar Alzheimer's (Okonogi & Chaiyana, 2012) Ricebran oil (Mitra et al., 1994) Saffola oil (Mitra et al., 1994) Sesame oil (Mitra et al., 1994) Soyabean oil (Mitra et al., 1994) Tea tree oil Melaleuca Alternifolia Psoriasis (Khokhra & Diwan, 2011) Numerous studies have considered the development of MEs incorporating natural products. Recently, the O/W ME including a high volume fraction of a natural oil (copaiba oil) (19.6%) has been developed (Xavier-Junior, Chapter V, 2015). The formulation approach was based on the chemical match between components of the oil and the lipophilic part of surfactants according to Hansen approach. This ME showed a reduced concentration of surfactant and high values of the oil/surfactant ratio (1.43). Chapter IV- Microemulsion-based drug delivery systems containing natural oils 154 ME for oral route was obtained using copaiba essential oil at final composition of 19.6 %, Pluronic F-68 ® 0.15 %, Brij O10 ® 13.55 %, and milli-Q ® water 66.7% (w/w). This system showed an incorporation of 3.8 mg.mL -1 of β-caryophyllene from copaiba oil. Gupta et al studied the phase diagram of a new pseudoternary system consisting of clove oil (Syzygium aromaticum)/ Tween ® 20/water at ratio of 5/30/65. Several compositions from the single-phase region were selected and their stability toward time, temperature, and electrolytes were examined (Gupta et al., 2005). Others authors have observed in detail the development in the phase behavior of Eucalyptus oil/ Tween 20/ Butanol/ Water and Eucalyptus oil/ Tween ® 20/ Cinnamic Alcohol/ Water systems. Triangular and tetrahedral representations have been considered to understand the topological nature of the multicomponent mixtures between the compound mixtures (Majhi & Moulik, 1999). Yotsawimonwat et al investigate the formation of ME considering orange oil in ME. Pseudoternary phase diagrams of orange oil, ethyloleate or a 1:1 mixture (w/w) of orange oil and ethyloleate as oil components, and 6:4 (w/w) mixture of polyoxyethylene 20 sorbitan monooleate and sorbitan monolaurate as surfactant components and water or propylene glycol as hydrophilic components were investigated. The authors observed a smaller ME region on the phase diagram when orange oil was used as a substitute for ethyloleate. In addition, the dimension of solution-type ME areas in the phase diagrams was likely to depend on the miscibility of components and larger ME areas were found when ethyloleate was used instead of orange oil and propylene glycol was used instead of water. Moreover, orange oil incorporation as a penetration enhancer into a topical ME affects its physical characteristics; this, in turn, may lead to instability of the ME and/or can influence the release patterns of drugs (Yotsawimonwat et al., 2006). Chapter IV- Microemulsion-based drug delivery systems containing natural oils 155 Water/propylene glycol/sucrose laurate/ethoxylated mono-di-glyceride/citrus oil ME systems were formulated. In this system, the free energy of solubilization decreased with water content in the water-in-oil ME region and increased in the oil-in-water region. Futhermore, the free energy of solubilization decreased with increasing ethoxylated mono-di-glyceride content in the mixed surfactants. The authors also observed that the hydrodynamic diameter of the diluted ME decreases with the increase in temperature (Fanun, 2010b). Microemulsification of vegetable oils (ricebran, saffola, soyabean, sesame, palm and linseed) with water using aerosol-OT and cinnamic alcohol as mixed amphiphiles have been studied. The biological formed MEs covered on the average approximately 27% of single phase area in the triangular phase diagram. Saffola oil ME at a reasonable water/aerosol mole ratio presented a moderate increase in conductance with temperature. Amongst the studied systems (sesame, saffola and ricebran), the viscosity of the first two decreased with the rate of shear whereas the ricebran’s viscosity increased. When cinnamic alcohol was used as the oil, the trend of viscosity was similar to that of sesame and saffola (Mitra et al., 1994). Another study developed a topical davana oil (Artemisia pallens) ME formulation and the same results were observed. The authors found that with the increase of Tween ® 80 concentration, the solubilization capacity of davana oil into the ME system was also increased leading to enhancement in ME region. However, transcutol P decreasing interfacial free energy and reducing surface tension promoting the homogenized droplets formation. Optimized formulation was prepared using Artemisia pallens based oil (15% w/v), tween 80 (15% w/v), transcutol P (5% w/v), and water (65% w/v) (Salunkhe et al., 2013). Chapter IV- Microemulsion-based drug delivery systems containing natural oils 156 Gupta et al. studied microemulsification of various combinations of water with corn oil, cottonseed oil, clove oil, orange oil, and peppermint oil using several non-ionic surfactants (Tween ® 20, Brij ® 30, and Brij ® 92) and cosurfactants (ethanol and isopropanol). Both ternary (oil/surfactant/water) and pseudoternary (oil/surfactant + co- surfactant/water) phase diagrams were constructed. The ternary systems produced larger ME forming zones than pseudoternary systems. Interestingly, the peppermint oil/ isopropyl alcohol /water and 1:1 (v/v) peppermint oil + isopropyl myristate / isopropyl alcohol /water combinations were used to form the proportion of single-phase of the majority ME. All systems showed excellent stability within 1 year and they withstood to temperature variations (Gupta et al., 2006). Fanum et al. also developed a peppermint oil-containing ME. The author observed O/W ME formation with droplets of up to 12 nm diameter. The solubilization capacity of water in the oil is dependent on the surfactants and ethanol/oil mixing ratios (w/w). In addition, a progressive transformation of the W/O to bicontinuous and inversion to O/W ME occurred upon dilution with water (Figure 5). The diffusion coefficients of the surfactants at the interface increased while increasing the water volume fraction (Fanun, 2010a). Chapter IV- Microemulsion-based drug delivery systems containing natural oils 157 Figure 5- Schematic presentation (not for scale) of the structural transitions along the N60 dilution line in the pseudo-ternary phase diagram. Pseudoternary phase behavior of water (W) / propylene glycol (PG)/ sucrose laurate (L1695) / ethoxylated mono-di- glyceride (EMDG)/ peppermint oil (MNT)/ethanol (EtOH) system at 25 ˚C. The mixing ratios (w/w) were mixed surfactants (L1695/EMDG) =1/1, W/PG=2/1 and MNT/EtOH=1/1 (Fanun, 2010a). A stable coconut oil-containing ME was prepared based on the HLB concept from a mixture of three surfactants using a ternary mixture of non-ionic surfactants. The W/O ME was successfully formulated with a surfactant blend composed of 16.6% of Tween ® 20, 15.0% of Span ® 20, and 68.4% of Span ® 80. Transparent ME could only be formed when the ratio of water and surfactants was at least 1:4.5 and the ratio of water/surfactants and coconut oil was kept bellow 1:3.5. These ME remained stable during storage for up to 2 months, even after centrifugation, but they were not stable Chapter IV- Microemulsion-based drug delivery systems containing natural oils 158 when subjected to heating at 70 ºC or higher (Rukmini et al., 2012). Rao et al. established conditions to fabricate stable ME from a nonionic surfactant (Tween ® 80) and flavor oil (lemon oil). The stable lemon oil-containing ME was produced only by heating the colloidal dispersion containing high surfactant-to-oil ratio. The authors suggested that there was a kinetic barrier at ambient temperature that prevented the system from reaching its most kinetically or thermodynamically stable state. In addition, the application of heating appeared to be much more effective than the application of mechanical energy at overcoming this kinetic barrier. In this study when higher temperatures (from 62 to 90°C) were applied at high surfactant concentration (20 % Tween ® 80 and 10% of lemon oil) the system was not transparent by turbidity analyses, while upon cooling back to ambient temperature, the turbidity of the system decreased and remained low at ambient temperature (Rao & Mcclements, 2011). Xu et al. developed a food-grade water-dilutable ME containing cassia oil (Cinnamomum cassia) as oil, ethanol as cosurfactant, Tween ® 20 as surfactant and water. Antifungal activity in vitro and in vivo against Geotrichum citri-aurantii was assessed. According to the authors, the phase diagram confirmed the feasibility of formulating such a ME including cassia oil. The ME was composed from cassia oil/ethanol/Tween 20 at a weight ratio of 1:3:6 (w/w/w) for each of these ingredients respectively. The average droplet size was 6.3 nm. The in vitro antifungal experiments showed that the ME inhibited fungal growth on solid medium and prevented arthroconidium germination in liquid medium. Cassia oil had a stronger activity when encapsulated in the ME. The in vivo antifungal experiments indicated that the water- dilutable ME was effective in preventing postharvest diseases of citrus fruits caused by G. citri-aurantii (Xu et al., 2012).Yifei et al. also developed the ME as a potential alternative to chemical fungicides, but their system was composed of cinnamon Chapter IV- Microemulsion-based drug delivery systems containing natural oils 159 essential oil. The ME reduced significantly the decay incidence by 18.7% of postharvest gray mold of pears (Pyrus pyrifolia) in comparison to that non-ME after 4 days storage at 20 °C. In the vapor phase, the cinnamon ME with the lowest concentration had the best control for decay incidence and lesion diameter. The authors concluded that ME may be an alternative way to control the gray mold of pears without a negative influence on its qualities (Wang et al., 2014). Preparation of neem (Azadirachta indica) oil containing ME was investigated as well as the acaricidal activity in vitro. In this systems, the mixture of Tween ® 80 and the sodium dodecyl benzene sulfonate (4:1) w/w was used as surfactants; the mixture of surfactants and hexyl alcohol (cosurfactant) (4:1) w/w was used as emulsifiers. The ME was composed of a mixture of neem oil, emulsifiers and water (1:3.5:5.5) w/w. The ME formed after stirring at 800 rpm for 15 min at 40 °C. It showed globular and uniform droplets, and have a viscosity of 9.96 mPa s at 25 °C. The ME was still clear after 6 months at room temperature. The lethal time was used to evaluate the acaricidal activity in vitro using Sarcoptes scabiei var. cuniculi larvae. The ME containing 10% neem oil showed a median lethal time value (LC50) was 81.75 min. against Sarcoptes scabiei var. cuniculi larvae. These results acknowledged an effective anti parasite activity for the neem oil containing ME (Wang et al., 2014). Natural product/oil containing ME has been developed to topical treat of the psoriasis. Ali et al. have investigated and evaluated a ME gel-based system of babchi oil (Psoralea corylifolia) for the treatments of psoriasis. Babchi oil is used because its chief constituent psoralen, this action inhibits DNA synthesis and causes decrease in cell proliferation. Thus, the authors suggested that a ME gel could be a potential vehicle for improved topical delivery of psoralen hence it could be a potential vehicle to improved Chapter IV- Microemulsion-based drug delivery systems containing natural oils 160 topical delivery of babchi oil in psoriasis lesions (Ali et al., 2008). The ME was prepared by titration of the aqueous phase into the mixture from oil and surfactant. It consisted of 1.67% v/v of babchi oil, 8.33% v/v of oleic acid, 55% v/v of Tween ® 80/ Transcutol-P (S/Co ratio 1:1) and 35% v/v of distilled water. The ME gel was a potential vehicle for improved topical delivery of psoralen and showed a potential vehicles for improved topical delivery of babchi oil. A ME formulated with tea tree oil, another natural oil containing active molecules against psoriasis including terpinin-4-ol was proposed by Khokhra et al. The ME was formulated with 5% tea tree oil, different concentrations of polysorbate 80 as surfactant and isopropyl Myristate and isopropyl alcohol as cosurfactants . The tea tree oil-containing ME showed droplets of spherical shape with a size ranging between 84 and 115 nm. These MEs showed a low viscosity . The maximum terpinen-4-ol compound content observed was 1.68 µg/mg of ME. The release profile of terpinen-4-ol from ME depicted that there was a total of 14.5% release through the excised skin from Wistar rats using Franz-type diffusion cells after 24 hours and this ME showed no signs of erythema and skin irritation (Khokhra & Diwan, 2011). Essential oils of three edible Thai plants, Cymbopogon citratus (Gramineae), Citrus hystrix (Rutaceae) and Zingiber cassumunar (Zingiberaceae) were comparatively tested for acetylcholinesterase and butyrylcholinesterase inhibitory activities in order to for enhancing the acetylcholine levels in Alzheimer's patients. Among the three oils, the C. citratus oil exhibited the highest cholinesterase inhibitory activity. Brij ® 97, Triton X ® - 114, Tween ® 20 and Tween ® 85 were employed as surfactant whereas ethanol and hexanol were used as cosurfactants in the formulation of the ME. Formulating the C. citratus oil containing ME, results revealed that the type and concentration of surfactant and co-surfactant exhibited different characteristics than influence in the formation of a large ME region in the pseudoternary phase diagram. Tween 20 ® was used in Chapter IV- Microemulsion-based drug delivery systems containing natural oils 161 combination with ethanol rather than hexanol, to be more suitable as co-surfactant to ME formation. The mixture Tween ® 20/ethanol was hardly influenced by both the pH and the ionic strength of the aqueous phase regarding the formation of the ME. The inhibitory activities of the ME based on water/C. citratus oil/Tween ® 20/ethanol was significantly greater when compared with the native oil (Chaiyana et al., 2010). The acetylcholinesterase and butyrylcholinesterase inhibitory activities of Zingiber cassumunar oil was enhanced by 20 to 25 times respectively while formulating in a ME compared with the activity reported for the non-formulated native oil (Okonogi & Chaiyana, 2012). Chaiyana et al interestingly developed a system with the alkaloidal extract from Tabernaemontana divaricata loaded in the Zingiber cassumunar oil (Plai oil) containing ME. This system was composed for Plai oil, Triton X ® -114, ethanol and water with the oil:surfactant ratios of 1:5 and 2:5. A reverse micellar phase, W/O MEs, liquid crystallines systems and coarse emulsions structures were formed along the aqueous dilution line at both oil:surfactant ratios. Formulations with the oil:surfactant ratio of 1:5 containing 0.1 µg/mL extract from T.divaricata showed a significantly higher acetylcholinesterase inhibition than those with the oil:surfactant ratio of 2:5. The ME system containing oil:surfactant ratio of 1:5 significantly increased the transdermal delivery of the T.divaricata extract within 24 h (Chaiyana et al., 2013). Some authors have also studied MEs base formulations for solubilization of volatile oils. Yi et al. studied the solubilization of volatile oil from Houttuynia Cordra in O/W ME. The ME was developed by titration method, but the varieties and amount of surfactant and co-surfactants had effects on solubilization for volatile oil. The ME was composed by medium-chain triglycerides as oil phase, polyoxyethylene castor oil EL-35 as surfactant and propylene glycol as cosurfactant (at ratio of 2). This system was Chapter IV- Microemulsion-based drug delivery systems containing natural oils 162 capable to solubilize the volatile oil from Houttuynia Cordra and this system has a broad range of therapeutic activities (Yi et al., 2010. A number of reports detail ME formulations designed to potentiate topical or transdermal permeability of drugs. Maghraby has analyzed the effects of cosurfactants on the transdermal delivery of hydrocortisone from eucalyptus oil containing ME. Pseudoternary phase diagrams were constructed in the presence and absence of cosurfactants. ME formulations containing 20% eucalyptus oil, 20% water and 60% of either Tween ® 80 or 1:1 surfactant/cosurfactant mixture were compared. On most cases during this study, the incorporation of cosurfactants expanded the ME zone. The cosurfactant free ME was viscous and showed pseudo-plastic flow. In contrast, the ME prepared with a cosurfactant was less viscous and showed a Newtonian flow. In this study, the hydrocortisone was used as model drug. The drug loading and release rate were increased in the presence of cosurfactants (ethanol being the most efficient among the tested cosurfactants) with the release depending on the viscosity, affecting the phase behavior and the transdermal delivery potential of ME (Maghraby, 2008). CONCLUSION Natural products have a promising potential in maintaining and promoting health, as well as preventing and potentially treating some diseases. The natural products are extremely complex mixtures of different functional-group classes. Drug delivery systems of natural products, as ME, represent a promising strategy for overcoming the limitations of these products, as low solubility, biodisponibility and efficacy, degradation of the active components in the presence of air, light, moisture and Chapter IV- Microemulsion-based drug delivery systems containing natural oils 163 temperatures. Several studies have been developed on the last years concerning the formulation of new ME systems containing natural products, such as extracts and oils, those studies are discovering that these systems are promising and are also an innovative approach that has potential applications in medicinal and health research, which can result in decrease of the dose, long-term safety increasing, absorption and biodisponibility enhancing, despite reducing systemic side effects. MEs have been shown to be able to protect labile drug, control drug release, improving water solubility of hydrophobic ingredients, enhance the efficacy and reduce patient variability. Furthermore, these systems can promote the retention of ingredients in the internal phase, mask the taste and reduce toxic side effects. 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YOTSAWIMONWAT, S., OKONOKI, S., KRAUEL, K., SIRITHUNYALUG, J., SIRITHUNYALUG, B. & RADES, T. 2006. Characterisation of microemulsions containing orange oil with water and propylene glycol as hydrophilic components. Pharmazie, 61, 11, 920-6. Chapter V Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 181 Le but du travail présenté dans le chapitre V était de développer une microémulsion huile dans eau comprenant une fraction de haut volume d'une huile naturelle (huile de copaïba) tandis que la concentration en tensioactif sera maintenu à un niveau faible. L'approche de formulation a été basée sur l'appariement chimique entre les composants de l'huile et la partie lipophile de tensioactifs en appliquant une approche basée sur l'analyse des paramètres de solubilité des différents composés développé par Hansen. Les tensioactifs montrant le meilleur appariement de leurs paramètres de solubilité avec ceux des principaux composantes de l'huile de copaïba ont été sélectionnés pour préparer des mélanges dans des différentes proportions pour être ajustées au HLB requis de l'huile puis ces mélanges ont été utilisés pour étudier deleur effect sur la formation de microémulsions. La concentration minimale du mélange de tensioactif nécessaire pour obtenir une microémulsion a ensuite été recherchée par titrage d'un mélange contenant 75% de l'eau et 25% de l'huile de copaïba à 25 °C. Les microémulsions ont été obtenues à la composition finale de l'huile essentielle à 19,6%, le Pluronic F-68 ® 0,15%, Brij- O10 ® 13,55% et eau milli-Q ® 66,7%. Les microémulsions sont apparus isotrope par l'observation en lumière polarisée. Ils ont été caractérisés par une taille de gouttelettes de 42 ± 0,5 nm avec une distribution unimodale. La microémulsion a permis de disperser 94% des composés trouvés dans l'huiles essentielles de copaïba, ce-qui correspondant à une concentration de 3,8 mg.mL -1 de β-caryophyllène. L'approche basée sur l'analyse des paramètres de solubilité des composants de l'huile et de la fraction lipophile des tensioactifs a été utile pour préparer des microémulsions caractérisés par des valeurs élevées du rapport huile / tensioactif (1,43) et la de fraction volumique d'huile (19,6%) par rapport aux microémulsions décrites dans la littérature. Comme la réduction de la concentration de l'agent tensioactif et l'augmentation de la fraction volumique de la phase dispersée est une préoccupation générale lors de la Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 182 formulation de microémulsions pour l'administration de médicaments, l'approche proposée dans ce travail est apparue intéressante. Elle a permis de nouveaux progrès vers le développement de microémulsions qui pourront être proposées pour la formulation de médicaments anticancéreux qui ont une mauvaise biodisponibilité raison d'une faible solubilité aqueuse destinés à la voie orale. Mots-clés: paramètres de solubilité, microémulsion, équilibre hydrophile-lipophile, huile de copaïba, voie orale. Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 183 MATCH OF SOLUBILITY PARAMETERS BETWEEN OIL AND SURFACTANTS AS A RATIONAL APPROACH FOR THE FORMULATION OF O/W MICROEMULSION WITH HIGH DISPERSED VOLUME OF COPAIBA OIL AND LOW SURFACTANT CONTENT. Xavier-Junior, F.H. 1,2 , Huang, N. 2 , Vachon, J. J. 2 , Rehder, V. L. G. 3 , Maciuk, A. 4 , Egito, E.S.T. 1 , Vauthier, C. 2 * 1 Universidade Federal do Rio Grande do Norte, Centro de Ciências da Saúde, Departamento de Farmácia, Laboratório de Sistemas Dispersos (LaSiD). Av. Gal. Gustavo Cordeiro de Farias, S/N, Petrópolis, 59010-180, Natal-RN-Brazil. 2 Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. 3 Universidade Estadual de Campinas (UNICAMP) – Centro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrícolas. Rua Alexandre Cazelatto, 999, Vila Betel, Paulínia – SP. 4 Université Paris Sud, Laboratoire de Pharmacognosie - UMR CNRS 8076 BioCIS - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. *Corresponding author: Christine Vauthier Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. christine.vauthier@u-psud.fr Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 184 ABSTRACT The aim of this work was to develop an O/W microemulsion including a high volume fraction of a natural oil (copaiba oil) while the concentration in surfactant will be kept at a low level. The formulation approach was based on the chemical match between components of the oil and the lipophilic part of surfactants according to Hansen approach. Surfactants showing the best match on their solubility parameters with that of the main complements of copaiba oil were selected to prepare blends at different proportions and to investigate the formation of microemulsions. The minimum concentration of the blend of surfactant required to obtain a microemulsion was then searched by titration of a mixture containing 75% water and 25 % copaiba oil at 25°C. Microemulsions were obtained with essential oil at final composition of 19.6 %, Pluronic F-68 ® 0.15 %, Brij O10 ® 13.55 %, and milli-Q ® water 66.7% (w/w). The microemulsions appeared isotropic by observation under polarized light by optical microscopy. They were characterized by a droplet size of 42 ± 0.5 nm with unimodal distribution. The microemulsion showed an incorporation of 94% of the mother copaiba essential oil compounds, corresponding at concentration of 3.8 mg.mL -1 of β- caryophyllene. The approach based on the analysis of solubility parameters of oil components and lipophilic fraction from surfactant was useful to prepare microemulsions characterized by high values of the oil/surfactant ratio (1.43) and oil volume fraction (19.6 %) as compared with microemulsions of the literature. As reducing the concentration of surfactant and increasing volume fraction of the disperse phase is a general concern while formulating microemulsions for drug delivery, the approach proposed in this work appeared interesting to further progress toward the development of suitable microemulsion as formulation for oral delivery of anticancer drugs that have a poor biodisponibility because of a low aqueous solubility. Keywords: Microemulsion, copaiba oil, solubility parameters, oral route. Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 185 1 INTRODUCTION Microemulsions are colloidal system which have a great potential in pharmacy to improve delivery of drugs (Lawrence & Rees, 2012; Muzaffar et al., 2013). These systems are isotropic, thermodynamically stable and single-phase liquid solution formed by mixing oil, water, and surfactants (Hoar, T. P. & Schulman, J. H. , 1943; Danielsson, Ingvar & Lindman, Björn, 1981; Mcclements, 2012). Microemulsions can increase the solubility of poorly water-soluble compounds and improve their penetration through biological membranes, thereby enhancing oral bioavailability (Araya et al., 2005; Gibaud & Attivi, 2012). Copaiba oil (Copaifera langsdorffii) has been utilized in folk medicine. Phytochemical properties of diterpenes and sesquiterpenes hydrocarbons are attributed various therapeutic effects including anti-inflammatory, antitumoral, antimicrobial, antitetanus, antiblenorrhagea, antileishmania and expectorant activities (Gomes, Niele Matos et al., 2007; Gomes N et al., 2008; Mendonça & Onofre, 2009b; Comelli Júnior et al., 2010; Souza, Martins, Souza, Furtado, Heleno, De Sousa, et al., 2011). However, the lipophilic nature of copaiba oil renders its use difficult in the folk medicine therapy due the low solubility, absorption and bioavailability by oral route. Microemulsion systems containing vegetable oils are a suitable alternative to enhance therapeutic effects, improving pharmacological activities and reduce toxicity of active compounds (Lee et al., 1995; Dantas, T. N. C. et al., 2010; Attaphong et al., 2012). Models for predicting solubility of substances in solvent mixtures have an important application in drug formulation (Barton, 1983; Thimmasetty et al., 2009). For microemulsion development, solubility study were also found as an essential approach to optimize energy of mixing oil compounds in surfactant blends which range from non- Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 186 polar to highly polar substances. Solubility parameters are an intrinsic physicochemical property of a substance expresses as its square root of the cohesive energy density. The cohesive energy density itself is defined as the ratio of the vaporization energy to the molar volume at the same temperature (Hildebrand et al., 1970). In 1967, Hansen suggested the splitting of the “global” Hildebrand solubility parameter into three parts derived from different types of cohesive forces including disperse, polar and hydrogen bond forces (Hansen, C.M. , 1967; Hildebrand et al., 1970). Formulating microemulsions, choice of solvents and surfactants are often based on empirical data rather than on a rational approach. However, an initial estimate based on oil- surfactant/ co-surfactant solubility calculations could help optimizing the formulation of microemulsion minimizing experimental expenditure. The aim of the present study was to develop and characterize a copaiba oil/ water microemulsion with a high volume fraction of the oil and a low concentration of surfactant from a rational approach based on the use of solubility parameters. Microemulsions are interesting systems because their intrinsic thermodynamic stabilities and small size which can promote the effective delivery of large amounts of copaiba oil. However, in general, microemulsions require the incorporation of a large amount of surfactants for stabilizing droplets, which can often cause toxicity (He et al., 2010; Sapra et al., 2013). It was then postulated that a rational approach of the formulation taking into account solubility parameters of the different components entering the composition of the microemulsion could improve the performance reducing the amount of surfactant while allowing the incorporation of a still interesting volume fraction of the dispersed phase. Such an approach was applied with success to the formulation of W/O microemulsions but to our knowledge it was not used to formulate O/W microemulsions containing natural oil so far. Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 187 2 MATERIALS AND METHODS 2.1 Materials Copaiba oil was purchased from Flores & Ervas (Piracicaba, SP, Brazil). Polyoxyethylene (10) oleyl ether (Brij ® O10), Polyethylene glycol sorbitan monolaurate (Tween ® 20), Polyoxyethylene-polyoxypropylene block copolymer (Pluronic ® F-68), and β-caryophyllene were provided by Sigma-Aldrich (Saint-Quentin Fallavier, France). Ultrapure water was obtained from a Millipore purification system (Milli-Q plus, Millipore, St Quentin en Yvelines, France). Ethyl acetate and Ethanol were purchased from Fisher Scientific (Illkirch, France). All chemicals reagent grade were used as received. 2.2. Copaiba essential oil extraction Copaiba essential oil was obtained from 400 mL of copaiba resin oil by hydrodistillation using a Clevenger-type apparatus for 3 h. The extract was dried with sodium sulphate, filtered and stored at -20 °C. Aliquots (10-20 mg) of the oil were dissolved in ethyl acetate (1 mL) and analyzed by a validated gas chromatography method (Xavier-Junior, Chapter I, 2015b). Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 188 2.3. Gas chromatography – Flame Ionization Detector and mass spectrometry analyses Analysis of the composition of copaiba oil was performed by gas chromatography- flame ionization detector and mass spectrometry as described by Xavier-Junior et al. (Xavier-Junior, Chapter I, 2015b). Briefly, the essential compounds showed in copaiba oil-loaded microemulsion and copaiba oil alone were determined. The apparatus used was a PR2100 gas chromatography (Alpha MOS, Toulouse, France) interfaced with a Flame Ionization Detector (GC-FID) and Hewlett-Packard 6890 gas chromatography (Agilent Technologies, Santa Clara, CA, EUA) with HP-5975 mass selective detector (GC-MS). A fused silica capillary column (25 m × 0.32 mm i.d., 0.5 µm) film thickness coated with cross-linked 5% phenyl polysilphenylene-siloxane (SGE Analytical Science Pty Ltd, Victoria, Australia) was used as the separation capillary. The injector and detector temperatures were set at 250 and 300 °C, respectively. The start of column heating was set at 90 °C, with heating ramp of 2 C.min −1 to 150 °C, then isothermally heating 20 °C min −1 to 300 °C. Helium was used as carrier gas at 1 mL.min −1 . The GC- MS electron ionization system was set at 70 eV. The volume injected for all samples was 1 µL. The oil components were identified by comparing their mass fragmentation with data from the electronic library from the Wiley 6, aro_cnrs, F&F_Lib_Argeville, MainLib and Aromes libraries and published data elsewhere. The β-caryophyllene was selected as the standard for the studies of copaiba oil quantification. 2.4 Solubility Parameters: method of calculation Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 189 The group contribution method was used to calculate the solubility parameters of the main compounds composing copaiba oil and surfactants knowing their molecular structure. In this study, the theoretical determination of solubility parameters were provided to main compounds presented in the copaiba oils and the lipophilic chain to the main surfactants used for oral route based in the partial chemical group contribution in the Table 1. The Hansen approach, which is one of the most common methods by which each solubility parameter contribution can be estimated was used using the equations (1 and 2) , while chemical group contribution were taken from tables given in the literature (Van Krevelen & Hoftyzer, 1976). Table 1- Chemical group contributions to the dispersion partial solubility parameter using the group contribution method of van Krevelen and Hoftyzer (Van Krevelen & Hoftyzer, 1976) Chemical group V (cm 3 mol -1 ) Fdi (cal 1/2 cm 3/2 mol -1 ) Fpi (cal 1/2 cm 3/2 mol -1 ) Ehi (calmol -1 ) -CH2 16.1 132.0 0.0 0.0 -CH3 33.5 205.0 0.0 0.0 -CH= 13.5 98.0 0.0 0.0 -C -19.2 -34.2 0.0 0.0 -CH -1.0 39.0 0.0 0.0 -C(CH3)2 49.0 308.0 0.0 0.0 -COO- 18.0 191.0 240.0 1675.0 -O- 3.8 49.0 196.0 1467.0 -COOH 20.5 259.2 205.4 4889.9 Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 190 Where: Fdi, Fpi and Ehi are the molar attraction constants due to dispersion, polar and hydrogen bonding energy of the partial group, respectively, and V is the group contribution to molar volume. d= Fdi V p= Fpi 2 V h= Ehi V Where: d, p, and h are the dispersive, polar and hydrogen bonding solubility parameter components, respectively. 2.5 Calculation of the HLB0 of copaiba oil and of surfactants The required Hydrophilic-Lipophilic Balance (HLB0) of copaiba oils main compounds were determined following equations (3): HLB0= 20 1 K/ d 2 0.25 p 2 0.25 h 2 L (Eq3) Where: the K value can assume the value of 39 to be applied to the formulation of an O/W emulsion (Beerbower & Hill, 1971). HLB of non-ionic surfactants were calculated from the molecular weight ratio between their hydrophilic moieties and entire molecule (equation 4). The determination of the Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 191 HLB of surfactant blends and HLB0 total of the copaiba oils were calculated according the equation 5 (Griffin, W. C., 1949a; Griffin, 1954). HLBvalue=20. Mw hydrophilic potion Mw entire molecule (Eq4) HLBblend= WAHLBA WBHLBB WA WB (Eq5) Where WA and WB are the amount (weight) of the first and second surfactants/compound used, respectively, the HLBA and HLBB are the assigned HLB values for surfactants/compound A and B, respectively. The best surfactant blends were selected based in the closer solubility parameters among the surfactants and copaiba oils compounds. This approach was expected to provide stability of the disperse system at lower levels of surfactant(s) at the same HLB0 from copaiba oils. Tween 20 ® , Brij O10 ® and PluronicF-68 ® were used to produce microemulsion loaded copaiba oil. Surfactant blends were Tween 20 ® :Brij O10 ® (w/w) (4.2:95.8 and 13.4:86.6 ratios) and Pluronic F-68 ® : Brij O10 ® (w/w) (1.1:98.9 and 3.4:96.6 ratios). 2.6 Preparation of the microemulsion Copaiba oils and milli-Q water were weighed at 25:75 and 15:85 (w/w) ratios of 1 g per batch. Thereafter, surfactant either pure or in blends Tween 20 ® :Brij O10 ® (w/w) or Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 192 PluronicF-68 ® : Brij O10 ® (w/w) were sequentially added to the copaiba oil/milli-Q ® water mixtures. After each addition of 50 mg of surfactant, the system was sonicated (Misonix XL 2020 sonicator, Farmingdale, NY, U.S.A) at 40 % amplitude for 60 seconds followed by an ultrasonic bath for 10 minutes (Elma Elmasonic S10H, Elma Hans Schmidbauer GmbH & Co. KG, Singen, Germany) and the turbidity was monitored out on the UV-VIS-Fibre Optics Spectrometer AVS-S2000 with DH-2000 deuterium Halogen light source and AvaSoft software package (Avantes, Apeldoorn, Netherlands). The addition of surfactant was pursued until a clear and transparent system was obtained. 2.7 Characterization of the microemulsion 2.7.1 Transmittance measurements Temperature-scanning transmittance measurements were used to obtain information about potential changes in the microstructure of the samples during heating. Temperature versus transmittance scans were then performed on the samples using a UV-VIS-Fibre Optics Spectrometer. The line emission spectra were observed between 400 and 800 nm wavelength and 650 nm to analysis. The samples were heated from 25 to 45 °C a rate of 2.5 °C.min -1 . 2.7.2 Polarized light microscopy In order to determine optical isotropy, copaiba oil/ water microemulsion was examined under polarized light microscopy, with a Nikon E600 Eclipse direct microscope Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 193 (Champigny/Marne, France). The microscope was equipped with a long focus objective (LWD 40 x 0.55; 0-2mm) and a Nikon Coolpix 950 camera was used to record the images with a resolution of 1600 x 1200 pixels. 2.7.3 pH analysis The pH values of the microemulsions loaded copaiba oils were measured by a pH meter (model HI 8417, Hanna Instruments Inc., Woonsocket, USA), at 20 ± 2 ◦ C. 2.7.4 Size measurement The hydrodynamic mean diameter and the size distribution of the microemulsion were determined by dynamic light scattering, using a He-Ne laser (wavelength of 633 nm) and a detector angle of 90° in a Malvern Zetasizer (NANO ZS90, Malvern Instruments Limited, UK). For size distribution measurements, a dispersion of diluted microemulsion in milli-Q ® water (1:100) was analyzed at 25 °C in a polystyrene cell. Cumulates analysis provides the characterization of a sample through the mean value (z- average) for the droplet size and polydispersity index (PdI). 2.7.5 Morphology of the microemulsion Droplets morphology of selected copaiba oil/water-microemulsion was observed by transmission electron microscopy (TEM) using an electron microscope JEOL 1400 (JEOL Ltd, Tokyo, Japan), equipped with a high resolution CCD Gatan digital camera Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 194 (SC1000 Orius, France) and operated at 80kV. One drop of the diluted microemulsion in milli-Q ® water (1:100) was placed on a carbon coated copper grid. One drop of 2% phosphotungstic acid was added to it. The superfluous marker on sample was wiped off by filter paper. Finally, the sample was air dried and observed in TEM at ambient temperature. 2.7.6 Rheological behavior Rheological properties of the microemulsion were determined using a rotational rheometer AR-G2 (TA instruments, New Castle, USA). Measurements were performed with an aluminum cone/plate geometry with a diameter of 40 mm, an angle of 1° and a truncation gap of 28 µm. Samples were maintained at 37ºC using a Peltier plate. Analyses were carried out by gradually increasing the shear rate from 10 -1 to 10 3 s − 1 , after 5 minutes of equilibrium time. Measurements were performed in triplicate. 2.8 Determination of copaiba oil content in the microemulsion The copaiba oil content in the microemulsion was determined as follows. Briefly, 1 mL of the microemulsion loaded copaiba essential oil was centrifugated at speed of 8500 x g for 15 min (Eppendorf centrifuge 5418, Rotor FA-45-18-11, Hamburg, Germany) to eliminate the titanium residues that may have been released from the ultrasound tip. The supernatant was used to determination of the drug content in the microemulsion formulation. Thus, 20 µL were solubilized in 1 mL of ethyl acetate to extraction using ultrasonic bath (Elma Elmasonic S10H, Elma Hans Schmidbauer GmbH & Co. KG, Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 195 Singen, Germany) for 15 minutes. The solution was filtered using a 0.1 µm teflon filter (Merck Millipore, Billerica, MA, EUA). GC-FID method was used to measure the content of copaiba oil, in particular, β-Caryophyllene according the previous validation studies as described above (Xavier-Junior, Chapter I, 2015b). 2.9 Statistical analysis All the experiments were conducted in triplicates. Means of two groups were compared using non-paired Student’s t-tests. All values are expressed as their mean ± S.D. When comparing multiple groups, one way analysis of variance (ANOVA) was applied with the Tukey multiple comparison procedure. The statistical data were considered significant at p < 0.05 3. RESULTS AND DISCUSSION 3.1 Copaiba oil characterization Figure 1 shows chromatograms given from the analysis of the resin and essential oil from Copaifera langsdorffii (Alencar, É. N. et al., 2015; Xavier-Junior, Chapter I, 2015b). In agreement with previous work, the assays allowed to identify 20 components in the copaiba resin oil, which 10 were sesquiterpenes and the other half were diterpenes compounds. Copaiba essential oil showed 15 sesquiterpenes compounds. In this work, components with concentrations greater than 6.7 % were further considered as they appeared as the main components of the resin and essential oil which composition are Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 196 given in Table 2. Partial solubility parameters were calculated for these compounds as they were the main components composing copaiba oil. Figure 1- The copaiba resin oil chromatogram, with a typical sesquiterpenes and diterpenes compounds region. * Diterpenes compounds were obtained after methylation derivatization reaction. Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 197 Table 2 – Percentage of major compounds from Copaiba (Copaifera langsdorffii) resin and essential oils Chemical Compound* RT (min) Resin oil (%) Essential oil (%) β-Caryophyllene 8.57 7.9 21.7 α-Bergamotene 8.75 7.1 20.5 β-Bisabolene 10.21 12.3 23.6 Copalic acid 26.51 15.6 - Labd-8(20)-ene-15,18-dioic acid 28.58 6.7 - Compounds concentration (<6.7 %) 43.3 32.0 Not detected compounds (%) 7.1 2.2 Total identified (%) 93.0 97.5 * compounds which composition were above 6.7 % of total components. RT (min): Retention time; (-) No detected. 3.2 Calculation of solubility parameters of oil components and of lipophilic parts of surfactants Solubility parameters can be used to predict interactions between molecules and their miscibility and solubility (Long et al., 2006). This parameter can be calculated from the contribution of cohesive energy of different chemical groups composing the chemical structure of a component (Hansen, C.M. , 1967). Regarding miscibility and solubility of Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 198 chemicals, a general principle based on like dissolves in like can be used to identify pairs of compounds that are miscible or appropriate solvents to dissolve a define molecule. Basis of this approach which is widely used in formulation is to match solubility parameters shown by each component (Greenhalgh et al., 1999; Verheyen et al., 2001). Formulating O/W-microemulsions including copaiba oil as the dispersed phase, the aim of our work was to incorporate a high volume fraction of copaiba oil using a minimum amount of surfactant. Success in formulation of inverse microemulsions (W/O) with high volume ratio (up to 40%) of the dispersed phase and stabilized with only few percent of surfactant (5%) were obtained following a rational choice of the nature of the surfactants based on their miscibility predicted from their solubility parameters . While successful to formulate W/O microemulsion but not yet applied to the formulation of O/W-microemulsion to our knowledge, the approach was applied to the formulation of O/W-microemulsions in the present work. Thus, Figure 2 showed the chemical structure of surfactants and main components found in copaiba resin and essential oils. The molecules were shown highlighting their lipophilic and hydrophilic parts respectively. The composition in chemical groups of the lipophilic part of each molecule was summarized in Table 3 where results of the calculation of partial ( p, d, h) and total ( t) solubility parameters of the main copaiba oils components and of the lipophilic part of the surfactants were reported. Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 199 Figure 2- Chemical structure of the main compounds found in copaiba resin and essential oils showing their lipophilic and hydrophilic parts. Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 200 Table 3- Solubility parameters (cal 1/2 cm -3/2 ) and required Hydrophilic-Lipophilic Balance (HLB0) of the main compounds from copaiba resin and essential oils and lipophilic chains of surfactants Compound (cal 1/2 /cm 3/2 ) Chemical contribution δd δp δh δt HLB0 HLB α-Bergamotene 6(-CH2) + 1(-CH3) + 1(-CH=) + 2(-C) + 2(-CH) +1 (C(CH3)2) 9.3 0.0 0.0 9.3 13.8 β-Caryophyllene 6(-CH2) + 3(-CH3) + 1(-CH=) + 3(-C) + 2(-CH) 9.8 0.0 0.0 9.8 14.2 β-Bisabolene 7(-CH2) + 2(-CH3) + 3(-CH=) + 1 (-C(CH3)2) 7.2 0.0 0.0 7.2 11.4 Copalic acid 8(-CH2) + 2(-CH3) + 1(-CH=)+ 3(-C) + 2(-CH) +1 C(CH3)2 + 1(-COOH) 9.6 0.9 4.7 10.7 14.3 Labd-8(20)-ene- 15,18-dioic acid 9(-CH2) + 3(-CH3) + 2(-C) + 3(-CH) + 2(-COO-) 9.3 1.4 3.7 10.1 14.0 Tween 20 ® 10(-CH2) + 1(-CH3) + 1(-COO-) 8.1 1.1 2.8 8.6 17.0 Brij-O10 ® 15(-CH2) + 1(-CH3) + 2(-C=) + 1 (-O-) 8.1 0.3 1.1 8.2 12.9 Pluronic- F68 ® 30(-CH2) + 30(-CH3) + 30(-CH) + 30(-O-) 8.1 0.7 3.7 8.9 29.0* * Result from the literature (Prakash, 2010) Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 201 Solubility parameters of the lipophilic parts of the surfactants shown in the Table 3 were quite well match with values calculated for the main components of copaiba oil. Therefore, the similar calculated total and partial solubility parameters between lipophilic chains of the surfactants and copaiba oil compounds were closely related. Total solubility parameters were calculated according to the percentage contribution of main compounds in the complex mixture from copaiba oils. Copaiba essential and resin oils showed the calculated solubility parameters of 8.7 and 9.4 cal 1/2 /cm 3/2 , respectively. A low calculated values of solubility parameters were obtained, due these samples presented a rich mixture of sesquiterpenes and diterpenes hydrocarbons. The HLB0 of the main compounds presents in the copaiba oils were calculated based in the equation 3. All compounds blended in the copaiba essential and resin oil showed a HLB0 of 13.1 and 13.5, respectively as calculated from equation 5. The HLB0 calculated through solubility parameters approach was very close to HLB0 experimental found to copaiba resin oil (14.8) (Xavier-Júnior et al., 2012a). The solubility parameters of the lipophilic chains of each surfactant used to oral application were calculated. Tween 20 ® , Brij O10 ® and Pluronic F-68 ® showed the total solubility parameter calculated of 8.6, 8.2 and 8.9 cal 1/2 /cm 3/2 , respectively. These surfactants showed the best chemical match with the oil component comparing each partial solubility parameters. Indeed, the chemical similarity determined between the lipophilic tail of the surfactant and the terpenes hydrocarbon group on the copaiba oil was taken as the key factor for the formation of optimized microemulsions optimizing the miscibility of the components. The HLB of the Tween 20 ® and Brij O10 ® were calculated based in the equation 4, being quite consistent with the results provided by the supplier. The HLB of the Pluronic F-68 ® was provided from the literature, due the Griffin’s equation is applied to surfactant which HLB less than 20. To produce the Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 202 microemulsion, the surfactants were mixed together at a composition where the HLB of the surfactant blend where identical then the required HLB of copaiba oil. The corresponding composition of the surfactants blends was calculated from equation 5 using the HLB of the each surfactant and the HLB0 value found for copaiba oils. Thus, the selects blends of surfactant were composed of Tween 20 ® :Brij O10 ® (w/w) (4.2:95.8) and PluronicF-68 ® : Brij O10 ® (w/w) (1.1:98.9) to copaiba essential oil; and Tween 20 ® :Brij O10 ® (w/w) (13.4:86.6) and PluronicF-68 ® : Brij O10 ® (w/w) (3.4:96.6) to copaiba resin oil. Solubility parameters and HLB0 determinations were assumed to show the best compromise to stabilize droplets of copaiba oil in the internal phase of the O/W- microemulsions. 3.3 Development of microemulsions A high volume fraction of copaiba oil and a low concentration of surfactant were determined as the goal of this work. Therefore, the copaiba oils and milli-Q ® water were placed in test tube at 25:75(w/w) or 15:85 (w/w) ratios corresponding as starting compositions. These mixtures were then titrated with surfactant blends and with pure surfactants, sonicated and analyzed. The maximal concentration of surfactant added in the system corresponded to 13.7 % (i.e. oil/surfactant ratio 1.43) as above such a quite high concentration of surfactant it was considered that the formulations will be out of the range of the acceptable specifications defined for this work. Figure 3 showed regions (in grey) where microemulsions with copaiba essential and resin oils were formed for different concentrations of surfactants. The copaiba resin oil was not able to form stable microemulsion at high volume fraction of the oil and with a low concentration of surfactant. The presence of high molecular weight acid resinous Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 203 compounds in the complex mixture of the raw copaiba oil may interfere with surfactants to form a stable interface allowing the obtaining of microemulsion with a reasonable concentration of surfactant. In contrast, the copaiba essential oil formed microemulsions over a large range of surfactant concentrations and considering the two blends of surfactant selected above. Microemulsions could form with concentrations of surfactant as low as 13.7% while the content in oil in the microemulsion remained high 19.6 %. Main compounds found in the essential oils were purified from heavy resinous components. The approach applied to select surfactants based on the chemical pairing between surfactants and oil component led to formulations which compositions complied with our specifications. It can be assumed that the purity of the sesquiterpenes in the essential oil contributed to the success of the method of chemical paring based on the match of the solubility parameters between the oil components and surfactants. Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 204 Composition of the starting system (Oil/water Ratio) Copaiba resin oil/surfactant Ratio 1.5 1.3 1.0 0.7 0.6 0.5 0.4 Surfactant blend 15:85 Brij O10 15:85 Tween 20: Brij O10 (13.4:86.6) 25:75 Tween 20: Brij O10 (13.4:86.6) 15:85 Tween 20 15:85 Pluronic F-68: Brij O10 (3.4:96.6) 25:75 Pluronic F-68: Brij O10 (3.4:96.6) 15:85 Pluronic F-68 Composition of the starting system (Oil/water Ratio) Copaiba essential oil/surfactant Ratio 1.5 1.3 1.0 0.7 0.6 0.5 0.4 Surfactant blend 15:85 Brij O10 15:85 Tween 20: Brij O10 (4.2:95.8) 25:75 Tween 20: Brij O10 (4.2:95.8) 15:85 Tween 20 15:85 Pluronic F-68: Brij O10 (1.1:98.9) 25:75 Pluronic F-68: Brij O10 (1.1:98.9) 15:85 Pluronic F-68 Figure 3- Formations of microemulsion systems with copaiba resin and essential oil at different surfactants blends ratios and copaiba oil/water ratios. Grey indicated the obtaining of transparent system. Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 205 Figure 4 show a macroscopic view of the microemulsions formulated with copaiba essential oil and blends of either Pluronic F-68 ® : Brij O10 ® and Tween 20 ® : Brij O10 ® . They appeared homogeneous and showed the characteristic tyndall effect of colloidal dispersions. They were stable and isotropic when observed under polarized light microscopy. The true microemulsion are isotropic materials, i.e, the sample does not rotate the plane of light polarization, because different crystallographic axes of the material have constant refractive indices; therefore, the light ray propagates with the same speed in all directions (Boonme et al., 2006b; Polizelli et al., 2006). Figure 4: Macroscopic aspect of microemulsions formulated with copaiba essential oil and a blend of Pluronic F-68 ® : Brij O10 ® at 1.1:98.9 ratio (A) and with a blend of Tween 20 ® : Brij O10 ® at 4.2:95.8 ratio (B). The oil content and surfactant content of the both microemulsions were 19.6 and 13.7 %, respectively. At 25°C, the light transmittance of the two microemulsions was 72 and 55 % for the microemulsion Pluronic F-68 ® : Brij O10 ® (1.1:98.9) and Tween 20 ® : Brij O10 ® (4.2:95.8), respectively consistently with their tyndall effect. By heating the microemulsions up to 45 °C, they became totally transparent above 40 °C (Figure 5). The systems remained within the microemulsion domain by increasing the temperature. Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 206 The fact the microemulsions became more transparent can be explained by a decrease in size of the microemulsion droplets thanks to an improvement of the structure of the interface where the lipophilic moiety of the surfactant interact with the components of the oil at the oil droplet surface. Figure 5: Temperature effect on the light transmittance of the microemulsion composed of copaiba essential oil 19.6 %, Pluronic F-68 ® : Brij O10 ® mixtures at 1.1:98.9 ratio 13.7 %, 66.7% of water (A), and copaiba essential oil 19.6 %; Tween 20 ® : Brij O10 ® mixtures at 4.2:95.8 ratio 13.7%, Water of 66.7 % (B). The results of droplet size measurement, PdI and pH of the formulations were summarized in the Table 5. Microemulsion formulated with Pluronic F-68 ® : Brij O10 ® mixtures showed lower mean diameter and PdI than the formulation with Tween 20 ® : Brij O10 ® . The size distribution with a PdI of less than 0.25 indicates a narrow size distribution of the microemulsion and consequently a homogenous monomodal Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 207 distribution. After one week, the size of the microemulsion formulated with the Tween 20 ® : Brij O10 ® was increased. Table 4- The droplet size, PdI and pH of the microemulsions formulated with copaiba essential oil and a blend of Pluronic F-68 ® : Brij O10 ® at 1.1:98.9 ratio (A) and with a blend of Tween 20 ® : Brij O10 ® at 4.2:95.8 ratio (B). The oil content and surfactant content of the both microemulsions were 19.6 and 13.7 %, respectively. Microemulsion Characteristic Size (nm) PdI pH A Translucent or tyndall effect, homogeneous and isotropic Translucent or tyndall effect, homogeneous and isotropic 42 ± 0.5 0.13 ± 0.01 6.5 ± 0.4 B 95 ± 10 0.31 ± 0.03 6.0 ± 0.5 TEM analysis was performed in order to determine the microstructure of microemulsion loaded copaiba essential oil formulated with Pluronic F-68 ® : Brij O10 ® (Figure 6). The microemulsion showed a spherical shape and uniform droplet size with droplets size about 45 nm that confirm the similar size obtained by dynamic light scattering analysis. This results corroborates with others studies than identified disperses and spherical micro-droplets using TEM analysis (Poullain-Termeau et al., 2008; Hu et al., 2011; Tian et al., 2012). Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 208 Figure 6-TEM images of the microemulsion loaded copaiba essential oil formulated with Pluronic F-68 ® : Brij O10 ® (scale bar= 200 and 50 nm, respectively) Rheology behavior is a fundamental approach to provide useful information about the microemulsion structure and stability (Formariz et al., 2010; Pal, 2011). The Figure 7 show the rheology behavior of the copaiba essential oil containing microemulsion formulated with Pluronic F-68 ® : Brij O10 ® at 1.1:98.9 ratio (A) and with Tween 20 ® : Brij O10 ® at 4.2:95.8 ratio (B) surfactants blends. Both formulations (A) and (B) were shear-thinning. Formulation (A) was shear-thinning from 1 to about 500 s -1 , and seems to reach the second Newtonian plateau above 500 s -1 . In contrast, formulation (B) was shear-thinning on the whole studied shear rate range. Between 0.1 and 10 s -1 , both formulations exhibited similar viscosity values. Above 10 s -1 , formulations (B) had higher viscosities values than formulation (A). Thus, the relatively low viscosity values may indicate that the microemulsion was composed of individual spherical droplets and non-anisometric aggregates (Moulik & Paul, 1998; Djordjevic et al., 2004; Acharya & Hartley, 2012), confirming the spherical droplet obtained by TEM analysis. Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 209 Figure 7- Flow curves of microemulsions formulated with copaiba essential oil and with a blend of Pluronic F-68 ® : Brij O10 ® at 1.1:98.9 ratio (A) and with a blend of Tween 20 ® : Brij O10 ® at 4.2:95.8 ratio (B). The oil content and surfactant content of both microemulsions were 19.6 and 13.7 %, respectively. Figure 8 showed the comparison of gas chromatograms obtained after analysis of the copaiba essential oil and the microemulsion. The data indicated that there was no significant difference in the composition and compound concentration between the copaiba oil included in the microemulsion and copaiba essential oil itself (p>0.05). The precision of the method to quantify the copaiba oil was 2.4%, the differences in the area of the peaks were less than 2 %, indicating no significant differences between the copaiba essential oil and microemulsion dosages. It was also possible to determine that 94% of the compounds found in copaiba essential oil were also present in the microemulsion; the others 6% represented minor compounds not identified. The small Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 210 loss of compounds in the copaiba essential oil and the microemulsion was the same, indicating that the loss was due to the production process but not to the oil volatilization. The copaiba oil has a rich mixture of components which gives numerous therapeutic activities. One of the most important, whether in relation to its high concentration or interesting biological activities, is the β-caryophyllene. This sesquiterpenes showed synergistic effect in the anticancer therapy, including the membrane permeabilization, block of P-glycoprotein in drug-resistant cancer cells and lipid peroxidation activities increasing the anticancer activity of the drug. From the quantitative analysis of the chromatograms, it could be determined that the copaiba essential oil-loaded microemulsion which contain the highest volume fraction for the lower concentration in surfactant (13.7 % of Pluronic F-68 ® : Brij O10 ® blend) included a concentration of 3.8 mg.mL -1 of β-caryophyllene. Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 211 Figure 8- Comparison of chromatograms obtained by gas chromatography analysis of copaiba essential oil (A) and microemulsion prepared with copaiba essential oil (B) by GC-FID analysis. C is the percentage difference between A and B. The gray color represents the precision of the method to determination of copaiba oil compounds Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 212 The use de microemulsion has been growing in recent years. To develop such systems, infinity of different ratios, components and methods can be combined to formation of the O/W-microemulsion. In this work, it was possible to obtain a microemulsion containing a high volume fraction of natural oil and a low surfactant concentration compared with previous microemulsion developed in the literature (Figure 9). The major systems produced showed lower oil concentration dispersed in the microemulsion, and, in general, this oil is only used as promoter of the drug solubility (Figure 9A). Associated this fact, the high concentration of surfactant is utilized to stabilizer the dispersion of the droplets in the aqueous medium. Thus, a large part of the work produced in the literature presenting oil / surfactant ratio of less than 1, corresponding at large amount of surfactant required to stabilize the oil droplets (Figure 9B). In the present work, microemulsion with a oil/surfactant ratio above 1 could be formulated using a rational approach for the selection of surfactants which presented high chemical compatibility with the oil based on the analysis of solubility parameters of the different components. The microemulsions formulated in this work have an increased amount of natural therapeutic oil compared to the O/W-microemulsion described in the literature. Additionally, their surfactant contents was reduced which is very interesting because high concentrations of surfactants in microemulsions limits their in vivo use being generally responsible for toxicity. Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 213 Figure 9- Comparison among the microemulsion containing copaiba oil obtained by solubility parameter approach (MECop) with other different microemulsion from the literature. A- represent the ratio of the aqueous phase/surfactant+cosurfactant/Oil phase composing the microemulsion, where medium gray, light gray and black colors represent the oil phase, the surfactant (and/or cosurfactant) and aqueous phase contributions (%) to microemulsion composition, respectively. B- show the ratio between the oil and surfactant (and/or cosurfactant) compounds, gray columns showed the data from the literature and the dark column denoted MECop showed the corresponding composition of the microemulsion produced in the present work ussing Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 214 Pluronic F-68 ® : Brij O10 ® at 1.1:98.9 ratio. 1 (Ghosh et al., 2013), 2 (Teixeira et al., 2007), 3 (Hamed et al., 2012), 4 (Zhang et al., 2008), 5 (Djordjevic et al., 2004), 6 (Boonme et al., 2006b), 7 (Surjyanarayan et al., 2009), 8 (Borhade et al., 2008), 9 (Hu et al., 2011), 10 (Dantas, T. N. C. et al., 2010), 11 (Yi et al., 2012), 12 (Spernath et al., 2002), 13 (Rao & Mcclements, 2011), 14 (Jha, S.K. et al., 2011), 15 (Polizelli et al., 2006), 16 (Tian et al., 2012), 17 (Biresh & Shiv, 2011), 18 (Mrestani et al., 2010), 19 (Solanki et al., 2012), 20 (Pestana et al., 2008b), 21 (Gao et al., 1998), 22 (Acharya et al., 2001), 23 (Agatonovic-Kustrin et al., 2003), 24 (Zeng et al., 2010). 4. CONCLUSION The determination of the solubility parameter approach and HLB required were used to select surfactants blends which showed lipophilic portions of good chemical compatibility with the majority copaiba oil compounds. The use of mixtures of these surfactants allowed the microemulsions formulation with high volume fraction and low surfactant concentrations using the essential oil which is the purified fraction. This work valid the application of this predictive approach to development for directs O/W microemulsions formulations. The composition of the oil recovered in the microemulsion was identical to the starting oil. The pharmacological properties of the copaiba oil should be preserved in the microemulsion and may be used in synergy with other active ingredients incorporated in the microemulsions to anticancer activity for oral route. Chapter V- Match of solubility parameters between oil and surfactants as a rational approach for the formulation of O/W microemulsion with high dispersed volume of copaiba oil and low surfactant content 215 ACKNOWLEDGMENTS This work was financially supported by the “coordenação de aperfeiçoamento de pessoal de nível superior” CAPES COFECUB 721/11. The authors would like to acknowledge Imagif Cell Biology Unit of the Gif campus (www.imagif.cnrs.fr) which is supported by the “Conseil Général de l'Essonne”. REFERENCES ACHARYA, A., SANYAL, S. K. & MOULIK, S. P. 2001. 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Chapter VI Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 223 Le but de ce travail était de préparer une microémulsion chargée en paclitaxel et d'en évaluer le potentiel comme les systèmes de délivrance de ce principe actif anticancéreux pour son administration orale . Les formulations de microémulsion ont été élaborés sur la base des études précédentes en utilisant les approches des paramètres de solubilité pour produire une microémulsion avec un volume élevé d’huile dispersé pour une faible concentration des tensioactifs. Le paclitaxel a été incorporé dans l’huile copaïba / eau microémulsion par sonication à 25 °C. L’évaluation de l'efficacité d'encapsulation et le taux de la charge de médicament ont été évaluées en utilisant la méthode HPLC validée au cours d'un travail précédent (Chapitre 2). La composition de la microémulsion retenue est donnée dans le rapport huile essentielle de copaïba/ Pluronic F-68 ® / Brij- O10 ® / eau milli-Q ® de 19,6: 0,15: 13,55: 66,7, respectivement. Le systeme d'apparence homogène et transparent, présentait des caractéristiques isotropes. La taille des gouttelettes était de 51 ± 1,2 nm, avec une distribution unimodale alors que la structure était inchangée par rapport à des taux de dilution de 0,4 à 50 %. La microémulsion ont été rhéofluidifiantes. L'incorporation maximale du paclitaxel dans le système était de 0,37 mg.mL -1 correspondant à 1,89 mg de paclitaxel par g de l'huile de copaïba, ce qui est équivalent une augmentation de 36 fois par rapport à la solubilité du paclitaxel dans l’huile essentiel de copaïba. Une microémulsion radiomarqué a été préparée en utilisant du [3H] -paclitaxel marqué au tritium en vue d'une étude visant à déterminer des propriétés de mucoadhésion. L'incorporation du paclitaxel radiomarqué dans la microémulsion n'a pas modifié les caractéristiques physico-chimiques de la microémulsion. La microémulsion a été marqué avec une radioactivité spécifique de 232 kBq.mL -1 de microémulsion telle que déterminée par scintillation liquide. Le test de mucoadhésion ex-vivo a été réalisée sur de la muqueuse intestinale de rat fraichement prélevée et montée dans des chambres d'Ussing. L'association maximale du paclitaxel Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 224 retrouvé sur la muqueuse a été de 4,4 ± 0,7% de la quantité initiale apportée par la microémulsion radioactive, ce qui correspondant à une quantité de 9,25 µg de paclitaxel qui se fixe par cm² de muqueuse intestinale. Mots-clés: Microémulsion, paclitaxel, huile de copaïba, mucoadhésion, administration de médicaments par voie orale. Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 225 PACLITAXEL-LOADED COPAIBA OIL IN WATER MICROEMULSION AS ORAL DRUG DELIVERY SYSTEMS: PREPARATION AND EVALUATION OF MUCOADHESION. Xavier-Junior, F.H. 1,2 , Huang N.², Morais, A.R.V. 1,2 , Alencar, E.N. 1 , Gueutin, C. 1 , Chacun, H. 2 , Egito, E.S.T. 1 , Vauthier, C. 2 * 1 UFRN, DFAR, Laboratório de Sistemas Dispersos (LaSiD), Natal – RN, Brazil. 2 Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. *Corresponding author: Christine Vauthier Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. christine.vauthier@u-psud.fr Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 226 ABSTRACT The aim of this work was to prepare and evaluate paclitaxel-loaded copaiba oil in water microemulsion as delivery systems for oral administration of this anticancer drug. The microemulsion formulations were developed based on previous studies using solubility parameters approach to produce a microemulsion with a high oil volume and using a low concentration in surfactant. Paclitaxel was incorporated in the copaiba oil/water microemulsion by sonication at 25 °C. Evaluation of the encapsulation efficiency and drug loading was assessed using a validated HPLC method. A microemulsion having a composition of copaiba essential oil, Pluronic F-68 ® , Brij O10 ® and milli-Q ® water at 19.6: 0.15: 13:55: 66.7(w/w) ratios, respectively was, homogeneous, transparent and showed isotropic characteristics. The droplet size was 51 ± 1.2 nm with unimodal distribution and the structure was unchanged from dilution ratios ranging from 0.4 to 50%. The systems were shear-thinning. The maximum incorporation of the paclitaxel in the system was 0.37 mg.mL -1 corresponding to 1.89 mg of paclitaxel per g of copaiba oil. Radiolabeled microemulsion was developed using [3H]-paclitaxel to mucoadhesion evaluation. The microemulsion showed the similar characteristics of the nonradioactive system with specific radioactivity of 232 kBq per mL of microemulsion as determined by liquid scintillation. Ex-vivo mucoadhesion test was performed in excised rat intestinal mucosa mounted in Ussing Chambers. The maximum association was 4.4 ± 0.7 % of the initial amount of [3H]-paclitaxel-loaded microemulsion, corresponding at 92.5 mg of paclitaxel by m² of the intestinal mucosa. Keywords: Microemulsion, paclitaxel, copaiba oil, solubility parameters, mucoadhesion, oral drug delivery. Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 227 1. INTRODUCTION Nanomedicine has become one of the most promising pathways for developing effective targeted therapies with particular impact in oncology (Duncan, 2004; Engineering., 2004; Kateb et al., 2011). In recent years, several efforts have been made in order to develop anticancer drug delivery systems for oral intake. Paclitaxel has been used as an anticancer agent due to its inhibitory effect of cellular growth by stabilizing the microtubule assembly, causing the death of the cell by disrupting the normal tubule dynamics required for cell division and vital interphase process (Schiff et al., 1979; Hamel, Del Campo, et al., 1981; Horwitz, 1992). This drug is particularly active against primary epithelial ovarian carcinoma, breast cancer, colon, head, non-small cell lung cancer, and AIDS related Kaposi’s sarcoma (Forastiere, 1994; Rowinsky & Donehower, 1995). However, it has a poor oral bioavailability partly due to its poor solubility in aqueous medium and to its high metabolization in the gastrointestinal epithelial cells (Adams et al., 1993; Singla et al., 2002; Kasim et al., 2004). In order to overcome this problem, the reformulation of the paclitaxel in systems that can enhance its solubility and permeability and being able to decrease its adverse effects, has been the goal of the new drug-based anticancer therapy (Kawasaki & Player, 2005). Amongst various drug delivery systems, microemulsions are considered as ideal alternatives for the oral delivery of low water solubility compounds (Constantinides, 1995; Kawakami, Yoshikawa, Hayashi, et al., 2002; Kawakami, Yoshikawa, Moroto, et al., 2002; Takahashi et al., 2002; Araya et al., 2005). Microemulsions are clear, thermodynamically stable, isotropic liquid mixtures of oil, water and surfactant, normally used in combination with a co-surfactant. Microemulsions are also characterized by a low viscosity and ultralow interfacial Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 228 tension. They may form a number of different structures (oil-in-water (O/W), water-in- oil (W/O) droplets and bicontinuous) (Bhargava et al., 1987). For microemulsions occurring as dispersions of droplets dispersed in a continuous phase, droplet size generally ranged between 20 and 200 nm (Hoar, T. P. & Schulman, J.H., 1943; Schulman et al., 1959; Danielsson, I. & Lindman, B., 1981; Lawrence & Rees, 2000; Talegaonkar et al., 2008). The major advantages of these systems include high potential of drug solubilization, thermodynamic stability, protection against degradation, improved dissolution of lipophilic molecules and surfactant-induced permeability enhancement (Shah et al., 1994; Constantinides, 1995; Zhao et al., 2005). It is noteworthy that microemulsion formulated with oils extracted from vegetal has increasing interest in technological applications over the last two decades (Lee et al., 1995; Dantas, T. N. C. et al., 2010; Attaphong et al., 2012; Xavier-Junior, Chapter IV, 2015) In a previous work, microemulsion composed of a high content of natural oil dispersed in an aqueous phase was formulated with a low concentration of surfactant from the application of a rational approach considering the miscibility of the oil components with surfactants based on the match of their solubility parameters (Xavier-Junior, Chapter V, 2015). The microemulsion incorporated an important amount of copaiba oil (Copaifera langsdorffii) (94 %), an oil that contains sesquiterpenes and diterpenes having interesting therapeutic activity which include among others antitumor, anti- inflammatory, antimicrobial, antileishmanial, expectorant, diuretic, antinociceptive activities (Veiga-Junior & Pinto, 2002; Gomes, Niele Matos et al., 2007; Gomes N et al., 2008; Mendonça & Onofre, 2009b; Comelli Júnior et al., 2010; Souza, Martins, Souza, Furtado, Heleno, De Sousa, et al., 2011). For instance, the composition of the essential oil is β-Bisabolene (23.6%), β-caryophyllene (21.7%) and α-bergamotene Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 229 (20.5%) The aim of the present study was to formulate a microemulsion incorporating paclitaxel and copaiba oil in a single formulation designed for oral administration. The rational behind this work was to design a new anticancer drug formulation combining the activity of paclitaxel with that of compounds founds in copaiba oil. Incorporation of paclitaxel in the copaiba oil containing microemulsion was developed and the physicochemical characteristics were determined. The amount of paclitaxel-loaded microemulsion was determined by a validated high-performance liquid chromatography (HPLC) method. As the microemulsion was designed for oral administration, the retention of [3H]-paclitaxel at the level of rat intestinal mucosa mounted in Ussing chamber was evaluated as an indicator of the mucoadhesion promoter. 2 MATERIALS AND METHODS 2.1 Materials Copaiba oil (Copaifera langsdorffii) was purchased from Flores & Ervas (Piracicaba, SP, Brazil). Paclitaxel was obtained from CHEMOS GmbH (Regenstauf, Germany). [3H]-paclitaxel (3 Ci.mmol -1 ) was acquired from Isobio (Fleurus, Belgium). Hionic- Fluor ® and Ultima-Gold ® (Packard, Rungis, France) were used as scintillating cocktails for radioactive analyses. Soluene-350 ® used to dissolve biological samples was obtained from Perkin-Elmer (Courtaboeuf, France). Polyoxyethylene (10) oleyl ether (Brij O10 ® ) and polyoxyethylene-polyoxypropylene block copolymer (Pluronic F-68 ® ) were provided by Sigma-Aldrich (Saint-Quentin Fallavier, France). Ethanol, acetonitrile and sodium sulphate were purchased from Fisher Scientific (Illkirch, France). Ultrapure Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 230 water was obtained from a Millipore purification system (Milli-Q plus, Millipore, St Quentin en Yvelines, France). 2.2. Copaiba essential oil extraction Hydrodistillation extraction using a Clevenger-type apparatus was performed in order to obtain the copaiba essential oil from 400 mL of copaiba resin oil. The system was operated by 3 h. The extract obtained was dried with sodium sulphate, filtered and stored at −20°C. 2.3 Preparation of the microemulsion Surfactants were selected to obtain optimal miscibility of the lipophilic moiety in the oil phase using an approach based on the match of their solubility parameters. O/W- microemulsion was the prepared by a titration method as described by Xavier Junior et al. (Xavier-Junior, Chapter V, 2015). The microemulsion was prepared with copaiba essential oil: Pluronic F-68 ® : Brij O10 ® : milli Q ® water at a weight ratio of 19.6: 0.15: 13.55: 66.7, respectively. The compounds were weighted to total volume of 1 g and sonicated (Misonix XL 2020 sonicator, Farmingdale, NY, U.S.A) at 40 % amplitude for 60 seconds followed by an ultrasonic bath for 10 minutes (Elma Elmasonic S10H, Elma Hans Schmidbauer GmbH & Co. KG, Singen, Germany). 1 mg of paclitaxel was added in the dispersion, thus resonicated at 20 % amplitude for 40 seconds. The system was filtrated using a 0.22 µm teflon filter (Merck Millipore, Billerica, MA, EUA) to remove Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 231 the excess of paclitaxel which remained undissolved in the microemulsion and was not incorporated in the dispersion. For mucoadhesion studies, the microemulsion were labeled with [3H]-paclitaxel. The microemulsions were prepared following the protocol described above with minor changes. [3H]-paclitaxel initially solubilized in ethanol was dried in nitrogen gas atmosphere under constant pressure to eliminate the organic phase. Thus, to produce radiolabeled microemulsion, 1g of microemulsion was added in this bottle with cold paclitaxel (1 mg), in order to obtain a final radioactivity of 370 kBq of [3H]-paclitaxel per mL of microemulsion. 2.4 Characterization of paclitaxel-loaded microemulsion 2.4.1 Transmittance measurements Temperature-scan and transmittance measurements were used to obtain information about potential changes in the microstructure of the samples during heating. The transmittance of the samples was monitored using a UV-VIS-Fibre Optics Spectrometer AVS-S2000 with DH-2000 deuterium Halogen light source and AvaSoft software package (Avantes, Apeldoorn, Netherlands) while the temperature of the sample varied from 25 to 45°C at an increment rate of 2.5 °C.min -1 . The fiber optics spectrometer was working in the wavelength range between 400 and 800 nm. The wavelength of 650 nm was retained to monitor the transmittance of the sample. Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 232 2.4.2 Polarized light microscopy Paclitaxel-loaded microemulsion was examined by polarized light microscopy using a Nikon E600 Eclipse direct microscope (Champigny/Marne, France). The microscope was equipped with a long focus objective (LWD 40 x 0.55; 0-2mm) and a Nikon Coolpix 950 camera was used to record the images with a resolution of 1600 x 1200 pixels. 2.4.3 Size measurement The hydrodynamic mean diameter and the size distribution of the microemulsion were determined by dynamic light scattering, using a He-Ne laser (wavelength of 633 nm) and a detector angle of 90° in a Malvern Zetasizer, NANO ZS90 (Malvern Instruments Limited, UK). For size distribution measurements, the dilution curve at concentrations ranging from 0.2 to 50% of microemulsion in milli-Q ® water was developed and analyzed in a polystyrene cell at 25 °C. Cumulates analysis provides the characterization of a sample through the mean value (z-average) for the droplets size, and a width parameter known as polydispersity index (PdI). 2.4.4 Morphology study Transmission electron microscopy (TEM) images were obtained on a JEOL 1400 microscope (JEOL Ltd, Tokyo, Japan), equipped with a high resolution CCD Gatan digital camera (SC1000 Orius, France) and operated at 80kV as the acceleration voltage. For TEM analyses, one drop of the diluted sample (1:100) was placed on a carbon- Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 233 formvar coated copper grid and then a drop of 1% phosphotungstic acid covered on the microemulsion. The superfluous of phosphotungstic acid on the sample was wiped off with a filter paper. 2.4.5 Rheological behavior Rheological properties of paclitaxel-loaded and unloaded microemulsion were determined using a rotational rheometer AR-G2 (TA instruments, New Castle, USA) . Measurements were performed with an aluminum cone/plate geometry with a diameter of 40 mm, an angle of 1° and a truncation gap of 28 µm. Samples were maintained at 37ºC using a Peltier plate. Analyses were carried out by gradually increasing the shear rate from 10 -1 to 10 3 s − 1 , after 5 minutes of equilibrium time. Measurements were performed in triplicate. 2.5 Maximum incorporation of paclitaxel-loaded microemulsion Maximum incorporation of paclitaxel-loaded microemulsion was determined by a centrifugation method. Briefly, 1 mL of sample was centrifuged at 8500 × g (Eppendorf centrifuge 5418, Rotor FA-45-18-11, Hamburg, Germany) for 15 minutes to remove the drug excess. The supernatant was recovered and carefully filtered using a 0.22 µm membrane. Thus, the filtrate was diluted in the mobile phase or scintillating cocktail and it was placed in ultrasound bath for 15 minutes. The quantitative analysis of the paclitaxel-loaded microemulsion was performed using HPLC and liquid scintillation methods. Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 234 A previous HPLC method was developed and validated to quantification of paclitaxel in copaiba oil (Xavier-Junior, Chapter II, 2015). The chromatographic system used was a Waters series, equipped with a Waters 515 pump, a Waters 717 plus autosampler and a Waters 486- Tunable Absorbance detector (Waters Corp., Milford, MA). The separation of paclitaxel was carried out at 30 °C using a Uptisphere Strategy 100A reversed-phase C-18 (150 mm x 3 µm x 3 mm) column and a Uptisphere Strategy C18-2 guard column (10 mm x 3 µm x 4 mm) (Interchim SA, Montluçon, France). The mobile phase, pumped at 0.4 mL.min -1 , was acetonitrile: water (50:50) and effluent was monitored with UV detection at 228 nm. 25 µL samples were introduced onto the HPLC system every 15 min. Chromatographic data were monitored and analyzed using Azur software (Datalys, France). Under these conditions, the paclitaxel was eluted at 9.7 minutes and microemulsion adjuvants were not able to change the specificity of drug identification. The calibration curves were designed over the range from 50 to 2000 ng.mL -1 (r²=0.999), the accuracy of the method was less or equal to 0.77 %, and relative standard deviation for intra- and inter-day precision were less or equal to 0.65 %. The limit of quantification and detection were calculated to be 21.03 and 6.31 ng.mL -1 , respectively. Radiolabeled [3H]-paclitaxel loaded microemulsion was preformatted in liquid scintillation counter (Model LS 6000 TA, Beckman, France). The samples were mixed with 10 mL of a scintillating cocktail and analyzed. For tissue digestion, 2 mL of Soluene-350 ® at 65 °C was used overnight. Posteriorly, 10 mL of scintillating cocktail was added to measure the [3H]-paclitaxel radioactivity. Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 235 2.6 Evaluation of mucoadhesion The studies were performed according to the recommendations of the ethical committee of the French Ministry of Higher Education and Research, project 2003-055 regarding the care and use of animals for experimental procedures. Male Wistar rats (200–250 g) (Charles River, Paris) were used for the mucoadhesion ex vivo assays. Animals were euthanized with an overdose of pentobarbital by intraperitoneal injection. Jejunum from fresh small intestine of sacrificed rats were excised, rinsed with chilled physiological saline solution (0.9 %) and cut into segments of 2–3 cm length. After visual examination of the tissue, sections containing Peyer’s Patches were discarded. Jejunum portions were mounted in Ussing chambers (the intestinal surface tested was 1 cm²) and bathed with ringer buffer at pH 7.4. The system was maintained at constant temperature (37 °C) and continuously oxygenated with O2 /CO2 (95 % / 5 %). After equilibration at the temperature of the experiment for 30 min, the transport buffer was removed and 50 µL of radiolabeled microemulsion was added to the donor compartment. Each compartment of the Ussing chamber was filled with 3 mL of ringer buffer. The experiment was performed over a period of 2 hours to insure the attachment equilibrium. Over incubation time, microemulsion dispersion was removed. Tissue was rinsed three times with 2 mL of ringer buffer, to eliminate non-attached microemulsion. Subsequently, the tissue with attached microemulsion was digested overnight in 2 mL of Soluene-350 ® at 65°C. Then, 10 mL of scintillating liquid were added and finally samples were analyzed by liquid scintillation to determine the amount of [3H]- paclitaxel which associated with the mucosa. Each sample was tested in three different rats in duplicate. Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 236 2.7 Statistical analysis Means of two groups were compared using non-paired Student’s t-tests. All values are expressed as their mean ± S.D. When comparing multiple groups, one way analysis of variance (ANOVA) was applied with the Tukey multiple comparison procedure. The statistical data were considered significant at p < 0.05 3. RESULTS AND DISCUSSION The aim of the work was to produce paclitaxel-loaded copaiba essential oil/water- microemulsion and to evaluate their mucoadhesive properties on intestinal fragments. The system was developed based on previous studies which were based on the match of solubility parameter approach to select suitable surfactants to formulate a microemulsion incorporating copaiba oil (Xavier-Junior, Chapter V, 2015). A microemulsion with a high volume fraction of copaiba essential oil ad a relatively low content of surfactant could be formulated. The essential oil from Copaifera langsdorffii provided a transparent oil rich in sesquiterpenes hydrocarbons compounds, including β- Bisabolene (23.6%), β-caryophyllene (21.7%) and α-bergamotene (20.5%) as main compounds (Xavier-Junior, Chapter I, 2015b). These sesquiterpenes present interesting therapeutic properties, highlighting the increase the drug anticancer activity, as paclitaxel, facilitating the passage of drug through the membrane and thus potentiates its therapeutic activity (Legault & Pichette, 2007). Copaiba essential oil: Pluronic F-68 ® : Brij O10 ® : Milli-Q water were blended at 19.6: 0.15: 13.55: 66.7 ratios to prepare the microemulsion. Paclitaxel was added to the prepared microemulsion. The paclitaxel-loaded microemulsion appeared homogeneous Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 237 and transparent (Figure 1). The formulation prepared several times displayed the same characteristics acknowledging the reproducibility and stability of the microemulsion. The paclitaxel-loaded microemulsion appeared isotropic by analysis through polarized light microscopy indicating that it was constituted by a dispersion of droplets in a continuous phase (Danielsson, I. & Lindman, B., 1981; Djordjevic et al., 2004). Figure 1: Macroscopic aspect of the paclitaxel unloaded (A) and loaded (B) microemulsions Paclitaxel-loaded microemulsion showed the maximum transmittance at temperatures ranging from 20 to 45 °C A significant difference in the variation of transmittance with the temperature was observed comparing the paclitaxel-loaded microemulsion and the corresponding non loaded microemulsion. In microemulsion apparently the paclitaxel was placed in the interface, reducing the surface tension and enhance the formation of more stable system. To evaluate the diameter of the microemulsion droplets by DLS, the microemulsion needed to be diluted. As shown on Figure 2, Diluting the microemulsion provided a curve with a constant diameter of the droplets (p>0.05) at the highest volume fraction of Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 238 the dispersed phase ranging from 1 to 50% At volume fractions below 0.4%, the diameter of the droplets increased dramatically indicating a loss of physical integrity of microemulsion (Li et al., 2005; Borhade et al., 2008) (p>0.05). After 24 hours, the diluted microemulsions did not show any phase separation, modification of droplet size and drug precipitation. This effect was related by Mohsin et al., where was presented the schematic diagram of the possible phases formed on dilution, the excess of the water can promotes the changes in the interface systems and formation of bicontinuous system (Mohsin et al., 2009). For further analysis, the dilution of the mother microemulsion dispersion at 1% was used as it permitted to keep the initial characteristics of the droplets. Figure 2- Dilution effect in the paclitaxel-loaded microemulsion analyzed by DLS at concentration ranged from 0.1 to 50 %. *= p<0.05 Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 239 Results of droplet size, PdI, pH, maximum incorporation of the paclitaxel-loaded and unloaded microemulsions were summarized in Table 1. Incorporation of paclitaxel in the microemulsion increased slightly the diameter of the microemulsion while the PDI remained low indicating a monomodal and a homogeneous distribution of the size of the droplets in both cases (Figure 3A). The microemulsions were composed of copaiba oil droplets of very small size hence provided with a large interfacial surface area, drug diffusion and kept the drug in solution throughout its passage through the gastrointestinal tract (Pouton, 2000; Nicolaos et al., 2003; Constantinides & Wasan, 2007). Table 1- The droplet size, PDI, pH, maximum incorporation of the paclitaxel-loaded microemulsion (MECop Ptx) in comparison with the system without the drug (MECop) Samples Characteristic Size (nm) PdI pH Incorporation (mg.mL -1 ) MECop Translucent or tyndall effect, homogeneous and isotropic 42 ± 0.5 0.13 ± 0.01 6.5 ± 0.4 MECop Ptx Translucent , homogeneous and isotropic 51 ± 1.2 0.21 ± 0.03 6.1 ± 0.3 0.37 ± 0.03 The pH of paclitaxel-loaded microemulsion was of 6.1 ± 0.3 that was suitable for the application of the microemulsion as a delivery system for the oral administration of drugs. The slight pH difference between paclitaxel-loaded and unloaded microemulsion was not statistically significant. Paclitaxel-loaded microemulsion was also characterized Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 240 by TEM (Figure 3B). The microstructure analyses showed a spherical shape and uniform droplet size distribution consistently with the results of size measurement performed by DLS. Figure 3- Size and morphological characteristics of the paclitaxel-loaded microemulsion. Typical droplet size distribution obtained from measurements performed by DLS (A) and aspect of the microemulsion observed by TEM. Scale Bar = 100 nm.(B) Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 241 The maximum incorporation of paclitaxel in the microemulsion was 0.37 mg of paclitaxel per mL of microemulsion. This corresponded to an incorporation of 1.89 mg of paclitaxel per g of copaiba oil. This incorporation rate corresponds to 37% of the initial amount of the drug added in the microemulsion. The high association of the paclitaxel into microemulsion system can be related at its high partition coefficient in copaiba oils (Xavier-Junior, Chapter II, 2015). Compared with other formulations reported in the literature that were considering lipid formulations, microemulsions or self emulsifying systems, the present formulation incorporated much higher amount of paclitaxel while at the same time, concentrations in surfactant and oil were considerably reduced (Nornoo et al., 2008; Wang et al., 2011; Li et al., 2012). Radiolabeled microemulsion was effectively developed with [3H]-paclitaxel. This system showed the similar characteristics with the nonradioactive microemulsion. The maximum radioactivity of the [3H]-paclitaxel was 232 kBq per mL of microemulsion. Rheology of microemulsion can provide useful information about their structure. Regarding the rheology, in microemulsion, formation of liquid crystalline stage coincides with formation of non-spherical aggregates (cylindrical or lamellar aggregates), which obstructs the flow in the dispersion medium (Ambade et al., 2008). The flow curves presented in the Figure 4 revealed that the microemulsions were shear- thinning. The microemulsion containing copaiba oil was shear-thinning from 1 to about 500 s -1 , and seemed to reach the second Newtonian plateau above 500 s-1. These results indicate that the systems developed were isotropic and contained spherical dispersed droplets which offer a low resistance to flow besides the fact that they exhibited low viscosity characteristics to microemulsions (Ambade et al., 2008). The rheological characteristic property of microemulsions is interesting in order to can facilitate Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 242 microemulsion preparation but is also to achieve drug administration suitable for oral delivery. In addition, the more ordered system in the dispersion may be responsible for prolonging or modifying the drug release profile (Lapasin et al., 2001; Krishnan et al., 2002; Pestana et al., 2008a). Figure 4- Rheological behavior of the paclitaxel -loaded (MECop Ptx) (open squares) and unloaded microemulsion (MECop) (full squares). The use of mucoadhesive molecules can be an important strategy to retain the preparation at the action site and to direct the drug to a specific site or tissue, decreasing the drug administration frequency and increasing the patient compliance to the therapy (Huang et al., 2000; Woodley, 2001). The mechanisms of mucoadhesion involve the contact and the consolidation stages of the ligation between the system and the mucus. Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 243 This mechanism can be affected by different factors as the molecular weight, flexibility, cross-linking density, hydrogen bonding capacity, hydration, charge and concentration of the molecules in the formulation and the mucus (Boddupalli et al., 2010). The mucus contains glycoproteins, lipids, inorganic salts and 95% water, which mucin is the most important glycoprotein of mucus and is responsible for its structure. The mucoadhesiveness of the paclitaxel-loaded microemulsion containing copaiba oil was evaluated directly on rat intestinal mucosa mounted in Ussing Chambers using [3H]- paclitaxel-loaded microemulsion. Thus, only the radioactivity associated with paclitaxel incorporated in the microemulsion could be evaluated by this method. The percentage of the association of the radioactivity of paclitaxel with the rat intestine mucosa was 4.4 ± 0.7 % of the initial dose introduced in the Ussing Chamber. Based on these results, mucoadhesion 1 mL of the paclitaxel-loaded microemulsion when in administration for oral route will need 4x10 -3 m² of the intestinal area to total formulation association. The mucoadhesion of the paclitaxel-loaded microemulsion was calculated at 92.5 mg of paclitaxel per m² of the intestinal mucosa. In the clinic, to obtained an ideal anticancer therapy to adult patients receiving chemotherapy, it is required a total paclitaxel amount of 313.3 mg to achieve the commonly dose prescription (175 mg/m² of body surface area) (Van Den Bongard et al., 2004; Sacco et al., 2010). Taking into account the large surface area of the human intestine ( about 30 m² (Helander & Fandriks, 2014)), this formulation when applied to human can associate itself with the mucus at total drug association area of 3.4 m². This association is important to oral drug delivery because it could be related to increase bioavailability of the drug. For the microemulsion developed this interaction can be related to the mucoadhesive effect of poly(oxyethylene) substances presents in the Pluronic F-68 ® and Brij O10 ® used to Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 244 produce the microemulsion developed in this work (Tiwari, Goldman, Sause, et al., 1999; Tiwari, Goldman, Town, et al., 1999; Singh & Ahuja, 2002). 4. CONCLUSION Paclitaxel, a widely used anticancer agent could be incorporated in an O/W microemulsion characterized by a low amount of surfactant and a large volume fraction of dispersed copaiba essential oil without disturbing too much the physicochemical characteristic of the original microemulsion. The concentration of paclitaxel solubilized in the microemulsion was considerable regarding the solubility of this drug in water. The system showed a good fluidity that is important for its production and its use as a drug delivery system for oral administration. Paclitaxel contained in the microemulsion associated well with rat intestinal mucosa in experiments designed to evaluate the ex- vivo mucoadhesion. The paclitaxel-loaded microemulsion proposed in this work showed promising properties to be further developed as a drug delivery system for oral administration of the anticancer molecule, however more studies are required in order to ensure the drug therapeutic dose for oral route. It is assumed that synergetic anticancer effect could be obtained with this system which contained copaiba essential oil including various components showing an activity against cancer. ACKNOWLEDGMENTS This work was financially supported by the “coordenação de aperfeiçoamento de pessoal de nível superior” CAPES COFECUB 721/11. The authors would like to Chapter VI- Paclitaxel-loaded copaiba oil in water microemulsion as oral drug delivery systems: preparation and evaluation of mucoadhesion. 245 acknowledge Imagif Cell Biology Unit of the Gif campus (www.imagif.cnrs.fr) which is supported by the “Conseil Général de l'Essonne”. REFERENCES ADAMS, J. D., FLORA, K. P., GOLDSPIEL, B. R., WILSON, J. W., ARBUCK, S. G. & FINLEY, R. 1993. Taxol: a history of pharmaceutical development and current pharmaceutical concerns. J Natl Cancer Inst Monogr, 15, 141-7. AMBADE, K. W., JADHAV, S. L., GAMBHIRE, M. N., KURMI, S. D., KADAM, V. J. & JADHAV, K. R. 2008. 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Section III Les systèmes d'administration de médicaments à base des polymères Chapter VII Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 255 Le but du travail présenté dans ce chapitre était le développement, la caractérisation et l’optimisation de nanocapsules polymères mucoadhésives composées d’un cœur d’huile de copaïba. L’enveloppe des nanocapsules est composée de poly(cyanoacrylate d'isobutyle) recouverte de chitosane pour lui conférer des propriétés mucoadhésives. La conception de ce vecteur destiné à l’administration d’agents anticancéreux par voie orale a été abordée par une démarche faisant appel à un plan d’expérience. Les nanocapsules ont été obtenus par le développent d'une méthode originale de polymérisation interfaciale du cyanoacrylate d'isobutyle en utilisant du chitosane comme agent de stabilisation des nanocapsules et d'ajustement des propriétés de surface. Des études préliminaires ont été réalisées en utilisant différentes masses moléculaires de chitosane, différentes caractéristiques de l'huile de copaïba et en modifiant la composition de la phase organique utilisée pour préparer les nanocapsules par la méthode de polymérisation interfaciale. Ensuite, les caractéristiques de taille et potentiel zêta des nanocapsules ont été optimisées par un plan d’expérience à 2 niveaux avec trois facteurs (le pH, la température et la concentration du chitosane dans le milieu de polymériation) et des points centraux afin d’identifier les conditions de synthèse produisant des nanocapsules stables des plus petites dimensions et présentent un potentiel zêta le plus élevé de valeur positive. Les échantillons ont été observés par microscopie électronique à transmission. L'huile encapsulée dans les nanocapsules a été analysé par une méthode validée en chromatographie en phase gazeuse en utilisant le β- caryophyllène comme référence pour la caractérisation de l'huile de copaïba. Finalement, l'efficacité d'encapsulation de l’huile a été déterminée. Les résultats ont montré que la taille des nanocapsules variait de 300 à 1200 nm en fonction des différents facteurs étudiés. Des valeurs de charge de surface positive ont été obtenues dans tous les cas, témoignant de la présence du chitosane à la surface des nanoparticles. Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 256 Les plus petites nanocapsules stables qui ont été obtenues ont un diamètre de 473 nm et un potentiel zêta de 34 mV. L’efficacité d'encapsulation de l’huile de copaïba a été de 75,8%, ce qui correspond à une teneur en β-caryophyllène de 55,5 µg.mg-1 de nanocapsules. La composition de l’huile encapsulée dans les nanocapsules est identique à celle de l’huile initiale indiquant que le procédé d'encapsulation préserve la qualité des caractéristiques de l'huile de copaïba. Mots-clés: Nanocapsules, huile de copaïba, chitosane, plans d’expériences, poly(cyanoacrylate d'isobutyle), polymérisation interfaciale Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 257 EXPERIMENTAL DESIGN APPROACH APPLIED TO THE DEVELOPMENT OF CHITOSAN COATED POLY(ISOBUTYLCYANOCRYLATE) NANOCAPSULES ENCAPSULATING COPAIBA OIL Xavier-Junior, F.H. 1, 2 , Egito, E.S.T. 1 , Morais, A.R.V. 1, 2 , Alencar, E.N.¹, Maciuk, A.³, Vauthier, C.²*, 1 UFRN, DFAR, Laboratório de Sistemas Dispersos (LaSiD), Natal – RN, Brazil. 2 Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. ³ Université Paris Sud, Laboratoire de Pharmacognosie - UMR CNRS 8076 BioCIS - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. Corresponding author Christine Vauthier, Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 Chatenay-Malabry Cedex – France. Christine.vauthier@u-psud.fr Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 258 ABSTRACT The aim of this work was to develop, characterize and optimize the natural copaiba oil- loaded chitosan decorated poly(isobutylcyanocrylate) nanocapsules. These systems were obtained by developing an original method of interfacial polymerization of isobutyl cyanoacrylate using chitosan as a stabilizer for the nanocapsules. A preliminary study investigated the influence of the molecular weight of chitosan, the characteristics of the copaiba oil and of the solvent phase. This showed that nanocapsules could only be produced with copaiba resin oil, while the size varied from 300 to 1200nm. Nanocapsule size and zeta potential were then optimized by two-level three-variable full-factorial experimental design. By transmission electron microscopy, samples showed spherical objects. The composition of the oil entrapped in the nanocapsules as analyzed by a validated method of gas chromatography using β- caryophyllene with reference to copaiba oil characterization revealed that the oil encapsulated was of the same composition then the initial oil. Nanocapsules with positive zeta potential were obtained consistently with the expected distribution of chitosan on the nanocapsule surface. Optimal nanocapsules showed a diameter of 473 nm, a zeta potential of +34 mV and an encapsulation efficiency of the oil of 74 % including 55.5 µg of β- caryophyllene/mg of nanocapsules. The designed nanocapsules show valuable characteristics to be further development as oral carrier for anticancer molecules including paclitaxel to develop a synergistic effect between oil component and the chemo-therapeutic agent. Keywords: nanocapsules, copaiba oil, chitosan, poly(isobutylcyanoacrylate), interfacial polymerization Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 259 1 INTRODUCTION Nanoparticles composed of mucoadhesive polymers are promising systems for oral drug delivery applications (Ponchel & Irache, 1998; Petit et al., 2012). Generally, nanoparticles are defined as solid colloidal particles that include both nanospheres and nanocapsules (Guterres et al., 2007). The nanocapsules are vesicular systems in which the drug is confined in a liquid/solid cavity surrounded by a polymeric membrane. The upper size limit is ∼1000 nm in diameter (Fessi et al., 1989; Quintanar-Guerrero et al., 1998). In general, nanoparticulate systems show promise as drug carrier due to their capacity to modulate drug biodistribution to release the drug in a controlled manner, increase intracellular uptake and improve the stability of active substances (Cruz et al., 2006; Pinto Reis et al., 2006a; Leite et al., 2007; Anton et al., 2008). Nanoparticles made of biodegradable polymers, including poly(isobutylcyanocrylate), may provide an alternative solution for oral delivery of drugs across the gastrointestinal barrier thanks to their extremely small size. Their surface may be turned to increase mucoadhesion (Bravo-Osuna, Vauthier, et al., 2007; Bravo-Osuna et al., 2008). For instance, chitosan has been a widely used polysaccharide in formulation of mucoadhesive drug delivery systems (Thanou et al., 2001; Chen et al., 2014). This polysaccharide is biocompatible and nontoxic. Its inherent mucoadhesive properties come from its chemical structure including amino groups able to promote electrostatic interactions with sialic acid groups of the mucus (Gåserod et al., 1998; Illum et al., 2001). In addition, the positive charges of chitosan are also believed to be essential to increase permeability of the intestinal epithelium thanks to its capacity to disturb calcium concentration balance near the tight junction (Bernkop-Schnurch et al., 2006; Bravo-Osuna, Millotti, et al., 2007). Although Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 260 widely used to improve mucoadhesion of nanospheres, this polysaccharide was not yet used to improve mucoadhesion of polymeric nanocapsules which are interesting drug delivery systems for delivery of lipophilic drugs. The copaiba oil-resin (Copaifera langsdorffii) is an oily plant extract which is used in folk medicine in its in-natura form (Sousa, J. P. et al., 2011). Phytochemical studies on oil-resin reveal that it contains a complex mixture of diterpenes and sesquiterpenes hydrocarbons (Veiga Junior et al., 2007; Alencar, E. N. et al., 2015), giving this oil many interesting therapeutic activities. For instance, these include anti-inflammatory, antitumor, anti-tetanus, antimicrobial, antileishmania activities among others (Gomes, N. M. et al., 2007; Santos, A. O. et al., 2008; Leandro et al., 2012). Although used for years in folk medicine, it is believed that pharmacological activities of this oil may be increased developing appropriate formulations. Thus, the aim of this work was to develop an original oral formulation of copaiba oil by encapsulating the oil in mucoadhesive polymer nanocapsules. These systems were chosen because they appeared the more appropriate formulations for the delivery of oil, while their small size is desirable to promote mucoadhesion on the gut mucosae. The polymer composing the nanocapsule envelope was a critical choice and poly(isobutylcyanocrylate) was selected because of its capacity to formulate nanocapsule that resist well to the gastric medium and promote release in the intestinal medium (Aboubakar et al., 2000). Although the development of mucoadhesive oil containing nanocapsule of poly(isobutylcyanocrylate) was not described before, this development was based on the use of an experimental design approach that was never applied so far while developing new formulations of oil containing nanocapsules prepared by interfacial polymerization of isobutylcyanocrylate. It is noteworthy that Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 261 experimental design approach was not so much applied in development of nanocapsules while such an approach could be helpful tool to optimize formulations limiting the number of experiments to perform (Patel et al., 2013). 2 MATERIALS AND METHODS 2. 1. Materials Copaiba resin oil was purchased from Flores & Ervas (Piracicaba, SP, Brazil). Isobutyl cyanoacrylate was provided by ORAPI engineered solutions worldwide (Vaulx-en- Velin, France). Water soluble chitosan Mw 20,000 g/mol was purchased from Amicogen (Jinju, South Gyeongsang, South Korea). Ethanol, acetone, 2-propanol, sodium hydroxide, ethyl acetate, nitric acid were provided by Fisher Scientific (Pittsburgh, PA, EUA). Diazomethane and β-caryophyllene were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Ultrapure water was obtained from a Millipore purification system (Milli-Q plus, Millipore, St Quentin en Yvelines, France). All chemicals were reagent grade and used as received. 2.2 Copaiba essential oil extraction Copaiba essential oil was obtained from 400 mL of copaiba resin oil by hydrodistillation using a Clevenger-type apparatus for 3 h. The extracted essential oil was dried with sodium sulphate, filtered, stored in a refrigerator and protected from light until use. Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 262 2.3 Method of preparation of the nanocapsules Copaiba oil-loaded chitosan-decorated poly(isobutylcyanocrylate) nanocapsules were elaborated by the method of interfacial polymerization (Couvreur et al., 1979; Fallouh et al., 1986) that was adapted because of the use of chitosan. Thus, 0.25 mL of copaiba oil and 0.032mL of isobutylcyanoacrylate were solubilized in 6.25 mL of ethanol to produce the organic phase. This phase was slowly injected dropwise in 12.5 mL of chitosan solution (0.3, 0.6 or 0.9 %) (Polymerization medium) prepared at various pH (3, 6 or 9) and homogenized for 10 min at 1250 rpm (at 5, 25 or 45 °C) (Fisher- Bioblock Scientific AM 3001K, Illkirch, France). The obtained colloidal dispersion was concentrated by rotary evaporator for 20 min at 35°C / 43mBar (BÜCHI Rotavapor R- 125, Heating Bath B-491, Vacuum pump V-700, recirculating Chiller F-108, Flawil, Switzerland) to eliminate ethanol. Posteriorly, the formed nanocapsules were filtered through a 5 µm minisart NML membrane (Sartorius GmbH, Goettingen, Germany). The obtained nanocapsules dispersion were purified by dialysis (Spectra/Por Biotech membranes, cellulose ester, 100,000 g/mol molecular weight cut off (MWCO), Rancho Dominquez, CA, USA) against ultrapure water three times for 60 min and once overnight to remove non associated chitosan. After dialysis, the nanocapsules were stored at +4°C for 24 hours before characterization. 2.4 Experimental design and nanocapsules optimization process In the present study, a 2 3 full-factorial experimental design with center points leading to 11 experimental randomized runs was used to optimize formulation and process parameters for the preparation of copaiba oil-loaded chitosan decorated- Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 263 poly(isobutylcyanocrylate) nanocapsules. For the optimization of the preparation of the nanocapsules, three independent variables including, the pH of the polymerization media (x1) (3, 6 and 9), the temperature of production (x2) (5, 25, 45 °C) and the concentration of chitosan 20 kDa (x3) (0.3, 0.6, 0.9 %) were selected. Each variable was set at a low, middle and high level. The size and zeta potential of nanocapsules were chosen as the dependent output response variables. The effects of the studied variables were graphically and statistically interpreted using the Statistic software (Version 7.0, StatSoft Inc., USA) to validate the statistical design. Response surface plots were generated to visualize the simultaneous effect of each variable on each response parameter. 2.5 Characterization of the nanocapsules. Size measurement Hydrodynamic mean diameter and size distribution of the nanocapsules were determined at 25°C by quasi-elastic light scattering using a Zetasizer Nano ZS90 (Malvern Instruments Ltd, Orsay, France). The scattered angle was fixed at 90°. The samples were diluted 1:100 before analysis. Each measurement was done in triplicate, and the average effective diameter and polydispersity were recorded. Determination of the zeta potential Zeta potential of the nanocapsules was deduced from the electrophoretic mobility by Laser Doppler Electrophoresis (Zetasizer Nano ZS90 (Malvern Instruments Ltd, Orsay, Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 264 France). Nanocapsules suspensions were diluted (1:100) with NaCl at 1 mmol/L. Values are presented as mean from three replicate samples. Morphology of nanocapsules Transmission electron microscopy (TEM) analysis of copaiba oil-loaded chitosan- decorated poly(isobutylcyanocrylate) nanocapsules was performed using a JEOL 1400 apparatus (JEOL Ltd, Tokyo, Japan), and Gatan CCD digital camera (Orius SC1000) high-resolution to investigate the morphology of formed samples. Nanocapsules were directly observed at 60kV after staining with phosphotungstic acid 2% (pH 7.4) for 30 seconds. 2.6 Analysis of the encapsulated copaiba oil Copaiba oil composition was analyzed by gas chromatography- Flame Ionization Detector. PR2100 gas chromatography (Alpha MOS, Toulouse, France) equipped with 5% Phenyl Polysilphenylene-siloxane (SGE Analytical Science Pty Ltd, Victoria, Australia) non polar fused silica capillary column (25 m × 0.32 mm i.d., 0.5 µm) film thickness coated with cross-linked was used. This method was previously validated for the analysis of the composition of copaiba oil (Xavier-Junior, Chapter I, 2015b). Samples were diluted with dichloromethane and 1.0 µL was injected in the chromatograph. The operating conditions to the samples were: oven temperature program from 90 °C (2 °C min −1 ) to 150 °C, after isothermally heating 20 °C.min −1 to 300 °C, kept for 5 min at the final temperature. Split injection was 20 mL.min −1 , carrier Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 265 gas helium, flow rate 1 mL.min −1 , temperature of injector and detector fixed at 250 °C and 300 °C, respectively. Composition of the major compounds present in the copaiba oil encapsulated in the nanocapsules was analyzed and compared with that of the initial oil taken prior to encapsulation. 2.7 Determination of encapsulation efficiency, encapsulation rate and concentration in the nanocapsule dispersion For the determination of the encapsulation efficiency, the encapsulationn rate and the concentration of copaiba oil in the nanocapsules, samples were prepared as explained bellow prior to their analysis by gas chromatography . Copaiba oil-loaded chitosan-decorated poly(isobutylcyanocrylate) nanocapsules were recovered by an ultrafiltration method. The nanocapsules were centrifugated in a Microcon centrifugal filter unit (Ultracel YM-100, regenerated cellulose, Merck Millipore, Billerica, MA, USA) at a speed of 10,000 rpm for 20 min (Eppendorf centrifuge 5418, Rotor FA-45-18-11, Hamburg, Germany) to remove the dispersion phase. Copaiba oil-loaded chitosan-decorated poly(isobutylcyanocrylate) nanocapsules were separated in the different fractions. Nanocapsules dispersion, nanocapsules retained on the membrane and dispersion phase (i.e. the ultrafiltrate). These fractions were then ressupensed in 1 mL of dichloromethane, sonicated for 1 hour and filtered through a 0.22 µm millipore filter. β-Caryophyllene on trapped in copaiba oil containing nanocapsules was analyzed by gas chromatography, as previously described. The encapsulation efficiency was calculated as follows (Equation 1): Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 266 EE (%)= (total amount of copaiba oil used amount of copaiba oil unloaded) total amount of copaiba oil used x 100 (Eq1) The nanocapsule concentration in the dispersion was evaluated by gravimetry. 1g of the purified nanocapsule dispersion was freeze-dried and the dry residue was weighted to deduce the percentage of nanocapsules contained in 1g of the dispersion. The encapsulation rate was determined by the ratio between weights of the β-caryophyllene on trapped in copaiba oil loaded on the nanocapsules and the total weight of the nanocapsule analyzed by gas chromatography. 2.8 Statistical analysis The results of these experiments were compared using analysis of variance (ANOVA), which was able to determine if the variables and the interactions between variables were significant. Regression model, t-tests and F-test with a 95% confidence level (p<0.05) were performed. To statistical analysis were used the Graph Pad Prism (Version 5.0, La Jolla, CA, USA ) and Statistic software (Version 7.0, StatSoft Inc., USA). 3 RESULTS AND DISCUSSION Copaiba oil nanocapsules were obtained from a new method of interfacial polymerization of poly (isobutylcyanocrylate) performed with chitosan and in absence Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 267 of surface active compounds. Preliminary studies were performed in order to identify the best substances to nanocapsule production. In this context, chitosan of different molecular weight (20, 70 and 250 kDa) were investigated. Copaiba resin and essential oils were also used to produce nanocapsules. In addition, selection of an ideal solvent to solubilize the compounds of the organic phase was achieved based on the use of ethanol, 2-propanol and acetone. Nanocapsules obtained were characterized by measurement of the particle size and zeta potential (Figure 1). Figure 1- Particle size (A) and zeta potential (B) of copaiba oil nanocapsules with chitosan coated in the surface. Wherein RO and RE corresponds to nanocapsules produced with copaiba resin and essential oils, respectively; C20, C70 and C250 referred to the three different chitosan molecular weight of 20, 70 and 250 kDa; and E, iP and A indicated the type of organic solvent used, ethanol, 2-propanol and acetone respectively, used to nanocapsules production. Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 268 As can be seen in the Figure 1 A, the mean diameters of the nanocapsules ranged from 440 ± 8 nm for copaiba resin oil and 932 ± 28 nm for copaiba essential oil. The size increase observed in these nanocapsules probability are associated to their concentration process, since the ethanol and water evaporation at reduced pressures may promote diffusion (drag) of essential oil molecules from nanocapsule. Thus, the oil can promote a positive pressure into the capsule, and this fact may be associated to expansion and size increase of the system. Nanocapsules prepared with higher molecular weight chitosan presented a significant increase of the size, compared to those prepared with smaller molecular weight chitosan (Figure 1A). Saremi et al. suggested that this may be due to the higher viscosity of polymeric droplets of higher molecular weight chitosan (Saremi et al., 2013). Also worth noting the morphological differences observed in the nanocapsules by TEM (Figure 2). Nanocapsules obtained with 20 and 70 kDa chitosan were spherical and uniform suggesting that the oily core was surrounded by an envelope typically characteristic of these systems. However, the nanocapsule obtained with 250 kDa chitosan appeared as elliptical structure. Formation of such elliptical structure may be possible due to different spatial conformations related to the viscosity of the reaction medium (Pastoriza-Santos & Liz-Marzán, 2009). However because of the size of the particle it should not completely discarded that the nanocapsule shape was modified during drying on the grid due to the softness of its cavity filled by the oil. Although it will be necessary to confirm the shape of these nanocapsules and these objects are far too large regarding the aim of the study, these images clearly showed that large objects encapsulating copaiba oil in a polymer membrane can be produced. Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 269 Figure 2- Transmission electron microphotographs of the nanocapsules at different molecular weight chitosan. Copaiba oil-loaded poly(isobutylcyanocrylate) nanocapsules coated with Chitosan 20 (A), 70 (B) and 250 kDa (C). Scale bar A and B 200nm; and C 0.5µm Organic solvents play an important role in nanocapsules development. Solvents are responsible for the dissolution of the oil and/or drug, and rapid nanoparticle formation as a process due to differences in surface tension, wherein aqueous phase pulls more strongly on the surrounding liquid than one with a low surface tension. Consequently, turbulences and thermal inequalities at the interface of both liquids cause violent spreading of the solvent flow away from regions of low surface tension and the polymer that formed by rapid polymerization tends to aggregate on the oil surface and forms the nanocapsule envelope (Quintanar-Guerrero et al., 1998). Copaiba resin oil-loaded in nanocapsules coated with chitosan formulated with ethanol, 2-propanol and acetone as solvent phase presented average particle size of 440 ± 8, 669 ± 8 and 536 ± 7 nm, respectively (p<0.05) (Figure 1A). Concerning the electrophoretic mobility results, all nanocapsules presented positive values of zeta potential indicating the presence of chitosan on the nanocapsule surface. Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 270 The charges on the nanocapsule envelope were statistically different depending on conditions of preparation. Zeta potential was also an important index to judge for the stability of nanoparticle dispersions. Nanocapsules with high absolute value of zeta potential can be stabilized by repulsive electrostatic forces preventing aggregation of the particles in the dispersion (Zhang & Feng, 2006). Zeta potential of formulations containing copaiba essential oil was much lower although still positive (less than +12 mV) (Figure 1B). Based on results obtained from this series of preparation, optimization of the procedure of preparation of chitosan-coated nanocapsules containing copaiba oil was pursue using chitosan 20kDa, ethanol and copaiba resin oil. The optimization was carried out following a 2 3 full-factorial experimental design approach with center points. The pH of the polymerization media (x1), the temperature of production (x2) and the concentration of chitosan 20 kDa (x3) were selected as independent variables for nanocapsules optimization. The pH of the polymerization medium was considered in regard with the mechanism of polymerization of isobutylcyanoacrylate which is highly sensitive to pH and in regard with the solubility properties of chitosan which depends on hydroxyl and hydrogen ion concentrations. The amount of chitosan added in the medium was also considered important as this component was assumed to insure stability of the formed nanocapsule and was expected to confer mucoadhesive proprieties to the nanocapsules. Temperature was included as design variable because it may obviously influence interactions between components during the formation of the nanocapsules. Thus, chitosan 20 kDa in the different concentrations, temperature and the pH of the polymerization media were studied in order to perform the optimization of the preparation of the nanocapsule according to the experimental design presented in the Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 271 Table 1. Optimization was aimed to obtain small and stable nanocapsules which parameters were evaluated by measuring the size and zeta potential of each preparation. Table 1: Variables and levels chosen to define the experimental region and their corresponding coded values for nanocapsule production The mean particle hydrodynamic diameter was strongly influenced by the variables selected for the study emphasizing their relevancy. The size value varied from 200 to 1200 nm. Standardized effects of the independent variables and their interactions on the dependent variable were investigated by preparing a Pareto chart (Figure 3). The length of each bar in the chart indicated the standardized effect of the corresponding variable on the response. Negative values in the response of the standardized effects indicated unfavorable or antagonistic effect on the nanocapsule development, while positive coefficients of the response of the standardized effects showed a favorable or synergistic effect. Independent Variable Level i xi -1 0 +1 1 pH 3 6 9 2 Temperature (°C) 5 25 45 3 Chitosan 20kDa concentration (%) 0.3 0.6 0.9 Dependent Variable (yi) Desired Response 1 Mean globule size (nm) Minimize 2 Zeta potential (mV) Maximize Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 272 Figure 3: The Pareto Chart of standardized effects to size and zeta potential dependent variables (p<0.05) According to the Pareto’s chart, an increase of the pH of the polymer medium had a statistically positive effect in the increase of nanocapsule droplet size. Additionally, the interaction of this variable with the other studied variables contributed to modify the nanocapsule size. This modification of size may be due to the hydration of free-amino groups of chitosan in acid solution (pH of 3.0). With a pKa value of 6.3, chitosan is a polycation when dissolved in acid solution and its amino groups are protonated giving free –NH3 + sites (Ravikumara & Madhusudhan, 2011). The increase in particle size at pH 9 might be due to a lower degree of protonation of the amino groups of chitosan (Zhao et al., 2002). At low pH, the reaction rate is too slow to allow the formation of small particles, corroborating the findings found by McCarron et al (Mccarron et al., 1999). Although the polymerization was slow down which gave possibility of Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 273 coalescence of the emulsion droplets before polymerization of the isobutylcyanoacrylate required to form the nanocapsule envelope. While at high pH, a too fast and uncontrolled polymerization may produce polymer aggregates. The increase of the temperature contributed for a particle size increment. The concentration of chitosan used when analyzed alone did not show significant effect on the size of the nanocapsule produced. However, the high concentration of chitosan in association with temperature variable showed a positive significant effect. In the acid solution at high concentration of chitosan, the increase of the temperature from 5 to 45 °C provoked the increase of the nanocapsule size from 250 to 1070 nm, while in basic polymerization medium the temperature variation did not show significant size changes (size of 930 nm). Opposite effect in the nanocapsules size, was observed between the concentration of chitosan and the pH of the medium. The concentration of chitosan, the temperature and the pH of the polymerization medium are parameters that have an influence on the viscosity of the polymerization medium. In turn, the viscosity of the polymerization medium may be an important factor that can promote the ideal interaction among the compounds in nanocapsule production. An increase of the temperature decreases the viscosity of the polymerization medium, due to the increase of the thermal motion of the polymer with temperature (Desbrieres, 2002; El-Hefian et al., 2010). The viscosity of the polymerization medium also decreased while reducing the concentration in chitosan which is a macromolecule. It is also decreased when the pH reached acid values due to the screening effect of anionic groups of chitosan in solution (Wang et al., 1994; Martínez-Ruvalcaba et al., 2004). The significance of independent variables and their interactions were tested by analysis of variance. An alpha-level of 0.05 was used to determine the statistical significance Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 274 between all analyses. The model’s goodness of fit was checked by the coefficient of determination (R 2 ). The R 2 values provide a measure of how much variability in the observed response values can be explained by the experimental variables and their interactions (fluctuation) (Cochran & Cox, 1957). These experiments were determined statistically significant with linear relationship of R²=0.96, indicating that 96% of the variability in the response could be explained by the model. In addition, the value of the adjusted determination coefficient (Adj R 2 = 0.94) was also very important to confirm a high significance of the model. These ensured a satisfactory adjustment of the polynomial model to the experimental data (Liu et al., 2004). For this study, the adjusted R 2 was very close to the experimental R 2 value. By applying multiple regression analysis on the design matrix and analyzing the responses given in the experiments, the first-order polynomial equation given in equation 2 in the coded form was established to size droplets: Y1= 834 +214x1+58x2-104x1x2-81x1x3+174x2x3-169x1x2x3 (Eq2) Where Y1 was the predicted droplets size (nm), x1, x2 and x3 are the coded terms for three independent test variables including the pH, the temperatures and the concentration of the chitosan, respectively. According to the regression model’s ANOVA, it was possible to observe that the linear model was significant (p<0.05). This was evidenced from the Fisher's F –test which provided a F-value of the model (F model = 23.5) much greater than the tabulated F- Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 275 value (F Tab= 2.4) at the 5% level, indicating that the computed Fisher's variance ratio at this level was large enough to justify a very high degree of adequacy of the linear model and also to indicate that treatment combinations are highly significant (Liu et al., 2004; Sen & Swaminathan, 2004). Since the rapport Fmodel /F tab was about 10, the Fisher's F- test was concluded with 95% certainty that the regression model explained a significant amount of the variation in the dependent variable. The normal (percentage) probability plot of the residuals was an important diagnostic tool to detect and explain the systematic departures from the assumptions that errors were normally distributed and were independent of each other and that the error variances are homogeneous (Liu et al., 2004). In this study, a plot of normal probability of the residuals indicated almost no serious violation of the assumptions underlying the analyses (F model = 3.5). This value was found to be lower than the tabulated F- value (F Tab= 4.26) at the 5% level, indicating that the experiment exhibited predictive results (residues model F-calculated/ tabulated F- value < 1). This satisfactory normal distribution confirmed the normality assumptions previously made and the independence of the residuals. Aiming the straight forward examination of the experimental variables on the responses, the three-dimensional response surfaces were used. The Figure 4 (A) shows a three- dimensional diagram of calculated size response surface relating both pH and temperature to copaiba oil nanocapsule size. It can be observed that the decrease in pH and temperature reduced the size considerably. The linear nature of the response surface demonstrated that there were considerable interactions between each of the independent variables and the nanocapsules sizes. Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 276 Figure 4: 3D Response surface for size droplets (A) and zeta potential (B) variable dependency with temperature and pH independents variables to production of nanocapsules. Regarding the zeta potential, the copaiba oil nanocapsules showed values ranging from + 15 to + 55 mV. These positive values might be explained by the exposure of chitosan’s positive charges on the nanocapsule surface. Otherwise, poly(isobutylcyanocrylate) nanocapsules obtained by the same method but without Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 277 chitosan generally show negative values (Aboubakar, Puisieux, Couvreur, Deyme, et al., 1999; Cournarie et al., 2004). In the zeta potential analysis, the standardized effects of the independent variables and their interactions on the dependent variable were also investigated by preparing a Pareto chart. The increasing temperature and the decreasing pH enhanced positively surface charge more significantly than the chitosan concentration variable (Figure 3). Possibly, because the polymer revealed itself to be more soluble and protonated, it exposed the carbohydrate free amino groups easily, being responsible for the increasingly positive zeta potential values. The concentration of the chitosan in polymerization medium when analyzed individually showed no significant difference in the amount of surface charges in the nanocapsules. However, the interaction of chitosan concentration with the other studied variables showed significant changes in the nanocapsules charges. These experiments presented the linearity regression of R²=0.94, therefore this model explains 94% of the variability in the response. The value of the adjusted determination coefficient (Adj R 2 = 0.92) presented a high significance of the model. The F test applied in the mathematical model shows the significative (Regression model F calculated/ F tabulated > 10) and predictive results (residues model F calculated/ tabulated F value < 1) of this experiments. In addition, the full first-order response surface was plotted for analysis of the optimal zeta potential on the nanocapsules (Figure 4 B). High positive values can be achieved by maintaining a low pH and increasing the temperature of the polymerization medium. The high temperature promotes particle size increase, however the opposite does not change the charge of the zeta potential sufficiently to cause destabilization of the system. Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 278 The result of the optimization study following a 2 3 full-factorial experimental design approach allowed to identify the optimal conditions for the nanocapsules preparation that have both a small particle size and positive zeta potential as initially researched. The optimal characteristics of the nanocapsule preparation were pH 3, temperature 4 °C and with a concentration of chitosan of 0.9 % (w/v). The samples prepared under these conditions showed a droplet size of 473 ± 1 nm, the size distribution was uni-modal with a polydispersion index of 0.20 ± 0.02, and a zeta potential of +34.8 ± 0.2 mV. TEM analysis showed that the optimal nanocapsules prepared by interfacial polymerization were spherical and not aggregated (Figure 5 A). The diameter of the nanocapsules observed by microscopy agreed well with particle sizes determination by dynamic light scattering. In addition, the nanocapsules had an oily core surrounded by an envelope typically characteristic of these systems. Also in Figure 5 B, it can be observed the dark details in the nanocapsule surface around the oil core and poly(isobutylcyanocrylate) envelope decorated with chitosan, confirming effective coating of this polymer on the system developed. Figure 5: Transmission electron microscopy analysis of the optimal formulation of copaiba oil- loaded chitosan- poly (isobutylcyanocrylate) nanocapsules stained with phosphotungstic acid (2%) at 60 kV (Imagif). Scale bar of 100nm. Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 279 The gas chromatography analysis was performed to characterize the copaiba oil recovered from the nanocapsule and compared it with the chromatogram obtained from the oil before nanoencapsulation (Figure 6). The Figure 6C gives the difference in the area of the peaks between the 2 samples. All components found in the original oil were also found in the nanocapsules. The differences in concentrations of the components present in copaiba oil were less that 2% which is within the range of precision of the gas chromatography method used for the determination. In addition, these results indicated that there was no modification of the oil during the preparation of the nanocapsules. The method preserved well the oil which was encapsulated under its native form. Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 280 Figure 6: Major compounds encapsulated in copaiba resin oil-nanocapsules coated with chitosan. Figures A and B represent the copaiba resin oil chromatograms before and after recovery from the nanocapsule. Figure C gives the difference in the area of the peaks between the copaiba resin oil in native form and the oil encapsulated in the nanocapsules. The gray color represents the precision of the method to determination of copaiba oil compounds Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 281 The encapsulation efficiency of copaiba oil was high at 75.8 ± 3 %. It could be calculated that the nanocapsule dispersion contained 2.5 mg of β-caryophyllene encapsulated in the nanocapsules per mL of the dispersion while the β-caryophyllene loading efficiency was 55.5 µg /mg of nanocapsules. This high encapsulation indicated that over the course of the preparation, copaiba oil immediately diffused in the internal phase of nanocapsules, and was then encapsulated by the polymer coat after addition of the organic phase into the polymerization medium. These high values are an important parameter that was used to evaluate nanocarriers and may improve therapeutic effects and lower the dose of drug required (Zhao et al., 2013). β-Caryophyllene presented in copaiba resin oil-nanocapsules coated with chitosan show a major potential impact in therapeutic application on human health. Klauke et al. suggested an average daily β-caryophyllene intake in the range of 10- 200 mg, which corresponds to human daily dose of 0.16- 3.3 mg/kg of the β-caryophyllene for a 60 kg human (Klauke et al., 2014). Thanks to potential mucoadhesive of these nanocapsules and the high concentration of β-caryophyllene encapsulated, it can be expected to achieve a therapeutic dose even smaller and more efficient than currently suggested (0.18- 3.6 g of nanocapsules). In addition, studies carried out by Legault et al. suggest the β-caryophyllene may accumulate in the membranes of cancer cells and increase membrane permeability of bioactive compounds (Legault & Pichette, 2007). Thus, it is expected that the β-caryophyllene presented in copaiba resin oil-nanocapsules coated with chitosan may be capable to increase intracellular accumulation of lipophilic anticancer drugs by the oral route and consequently enhance potential anticancer activity of another anticancer drug associated with the nanocapsules thanks to a synergetic effect between the oil and a co-encapsulated drug in the nanocapsules. Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 282 4 CONCLUSION Poly(isobutylcyanocrylate) nanocapsules incorporating copaiba oil could be prepared by the method of interfacial polymerization while surfactant was omitted and replaced by chitosan. As assumed, chitosan associated with the nanocapsules conferring positives charges to the nanocapsule surface. Experimental design approach was useful for the optimization of the formulation to provide nanocapsules of small diameter and high positive zeta potential. This approach also highlighted the role of three important variables that may vary during the preparation of the nanocapsules and that greatly influenced the nanocapsule characteristics. Copaiba oil was encapsulated at high encapsulation efficiency and the composition of the encapsulated oil was identical to that of the native oil. These nanocapsules are expected to be mucoadhesive and suitable to serve as carrier system for lipophilic anticancer drugs by the oral route with possible synergistic effect between the oil and the drug. ACKNOWLEDGEMENTS Authors acknowledge financial support from CAPES COFECUB 721/11 and would like to thank Imagif Cell Biology Unit of the Gif campus (www.imagif.cnrs.fr) which is supported by the Conseil Général de l'Essonne for providing facilities for TEM analysis. REFERENCES ABOUBAKAR, M., COUVREUR, P., PINTO-ALPHANDARY, H., GOURITIN, B., LACOUR, B., FARINOTTI, R., PUISIEUX, F. & VAUTHIER, C. 2000. Insulin-loaded Chapter VII- Experimental design approach applied to the development of chitosan coated poly(isobutylcyanocrylate) nanocapsules encapsulating copaiba oil 283 nanocapsules for oral administration: In vitro and in vivo investigation. Drug Dev Res, 49, 2, 109-117. ABOUBAKAR, M., PUISIEUX, F., COUVREUR, P., DEYME, M. & VAUTHIER, C. 1999. Study of the mechanism of insulin encapsulation in poly(isobutylcyanoacrylate) nanocapsules obtained by interfacial polymerization. J Biomed Mater Res, 47, 4, 568- 76. ALENCAR, E. N., XAVIER-JUNIOR, F. H., MORAIS, A. R. V., DANTAS, T. R. 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S., ASAMI, K. & LEI, J. P. 2002. Dielectric analysis of chitosan microsphere suspensions: study on its ion adsorption. Colloid Polym Sci, 280, 11, 1038- 1044. ZHAO, Y. Q., WANG, L. P., MA, C., ZHAO, K., LIU, Y. & FENG, N. P. 2013. Preparation and characterization of tetrandrine-phospholipid complex loaded lipid nanocapsules as potential oral carriers. Int J Nanomedicine, 8, 4169-4181. Chapter VIII Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core-shell nanocapsules and evaluation of their mucoadhesion by in vitro methods Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 291 Le dernier chapitre de cette thèse présente les résultats de l'étude visant à évaluer la mucoadhésion du paclitaxel encapsulé dans les nanocapsules d'huile de copaïba développées précédemment (Chapitre VII). Ces nanocapsules de poly(cyanoacrylate d'isobutyle) enrobées de chitosane ont été développées pour l'administration de médicaments par voie orale. La formulation de nanocapsules de paclitaxel a été optimisée en faisant varier la concentration de l'huile de copaïba, du cyanoacrylate d'isobutyle utilisé comme monomère et du paclitaxel. La rhodamine B et [3H]- paclitaxel radioactif ont également été ajoutés à la formulation en vue de pouvoir suivre les nanocapsules et le paclitaxel au cours d'expériences menées in vitro et ex-vivo visant à évaluer les propriétés mucoadhésives des formulations. Toutes les nanocapsules produites ont été caractérisées par la taille des particules, le potentiel zêta et des observations en microscopie électronique à transmission. L’efficacité d’encapsulation et l'adhésion aux tissus muqueux du paclitaxel a été déterminée par HPLC en utilisant une méthode qui a déjà validée au début de nos travaux de thèse (Chapitre I) et par mesure de radioactivité en scintillation liquide. Une étude de stabilité des nanocapsules dans les fluides gastro-intestinaux simulés a été réalisée et une étude de stabilité au stockage après application d'un processus de séchage a été effectuée. In vitro, les propriétés mucoadhésives ont été explorées par la mise en œuvre d'un test d'agrégation in-vitro avec la mucine. Ces études ont été complétées par des travaux visant à evaluer la mucoadhésion du principe actif apporté sous forme de nanocapsules sur une muqueuse intestinale de rat fraîchement excisée sur un modèle d'étude ex vivo en chambre d’Ussing. Les nanocapsules chargées en paclitaxel ont montré un diamètre hydrodynamique moyen de 470 nm, un indice de polydispersité faible et une forme sphérique. L'efficacité d'encapsulation et le taux de charge en paclitaxel était de 74 ± 1% et 1,70 ± 0,02%, respectivement, ce qui correspond à une teneur 16.8 ± 0.27 µg du Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 292 paclitaxel par mg de nanocapsules. Après séchage, les nanocapsules peuvent être redispersées sans changement de leur caractéristiques de taille ni de structure. Les dispersions de nanocapsules sont apparues stables en milieu gastrique simulé sur une période de 120 minutes et après six mois de conservation à 4 °C dans l’eau milli-Q®. Le potentiel zêta était +37 mV. Cette valeur nettement positive indique la présence de chitosane sur la surface des nanocapsules. Les nanocapsules ont montré des propriétés mucoadhésives intéressantes avec les mucines. Les études menées avec le modèle d'étude de la mucoadhésion ex-vivo confirme le potentiel mucoadhésif des nanocapsules. Elles ont permis d'associées 3.4 g de [3H] -paclitaxel encapsulé par m² de muqueuse intestinale de rat démontrant leur potentiel à transporter le principe actif et à le fixer au plus proche des sites d'absorption. Mots-clés: Mucoadhésion; nanocapsules; paclitaxel; livraison orale; poly (cyanoacrylate d'isobutyle); chitosane; huile de copaïba Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 293 PREPARATION OF PACLITAXEL-LOADED CHITOSAN- POLY (ISOBUTYLCYANOACRYLATE) CORE-SHELL NANOCAPSULES AND EVALUATION OF THEIR MUCOADHESION BY IN VITRO METHODS Xavier-Junior, F.H. 1,2 , Gueutin, C. 1 , Chacun, H. 2 , Egito, E.S.T. 1 , Vauthier, C. 2 * 1 UFRN, DFAR, Laboratório de Sistemas Dispersos (LaSiD), Natal – RN, Brazil. 2 Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 chatenay-Malabry Cedex – France. *Corresponding author: Christine Vauthier Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 chatenay-Malabry Cedex – France. christine.vauthier@u-psud.fr Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 294 ABSTRACT The aim of this work was to study mucoadhesive property of paclitaxel encapsulated into copaiba oil containing-poly(isobutylcyanoacrylate) nanocapsules coated with chitosan designed for oral drug delivery. Samples were produced by interfacial polymerization. Formulations of paclitaxel containing nanocapsules were optimized by varying copaiba oil, isobutylcyanoacrylate and paclitaxel concentrations. Rhodamine B and radioactive [3H]-paclitaxel were also added in the formulation. All produced nanocapsules were characterized by particle size, zeta potential and transmission electron microscopy. Encapsulation efficiency of paclitaxel and paclitaxel adhering to mucosal tissue were determined by HPLC using a method that was previously validated and liquid scintillation analyses. Simulated gastrointestinal fluids, drying process and storage stability studies were performed. Mucoadhesion tests were performed by in- vitro aggregation test with mucin and ex-vivo in Ussing Chamber using freshly excised rat intestinal mucosa. Nanocapsule-loaded paclitaxel showed a mean hydrodynamic diameter of 470 nm, a low polydispersity index and a spherical form. The encapsulation efficiency and drug loading of paclitaxel were 74 ± 1% and 1.70 ± 0.02%, respectively. After drying, nanocapsules could be redispersed with no change of the nanocapsule structure. Dispersions of nanocapsules were stable in simulated gastric medium for 120 min and after six months at 4°C. Potential zeta was + 37 mV due to the presence of chitosan on the nanocapsule surface. The nanocapsules showed interesting mucoadhesive properties with mucins. They could promote association of 9 % of the amount of [3H]-paclitaxel encapsulated with the intestinal mucosa of the rat. Keywords: Mucoadhesion; Nanocapsules; Paclitaxel; Oral Delivery; Poly(isobutylcyanoacrylate); Chitosan; Copaiba Oil Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 295 1. INTRODUCTION Cancer remains a major cause of death in most countries in the world, and its incidence increases over the years (Fang et al., 2011). Recently, many works have been focused on the development of oral anticancer drugs to improve the ease of treatments for patients (Roger et al., 2010b; Mazzaferro et al., 2013a; b). Paclitaxel (C47H51NO14) is a pseudoalkaloid anticancer drug with a diterpenoid structure, extracted from the bark of the Pacific yew tree (Taxus brevifolia) (Wani et al., 1971; Fang & Liang, 2005). This drug’s mechanism of action is based on the inhibitory effect of cellular growth by hyperstabilizing the cellular microtubules. Hence inhibiting cell replication in the late G2 mitotic phase of the cell cycle which in turn leads to apoptosis (Schiff et al., 1979; Horwitz, 1992). Paclitaxel has a powerful antitumor ability against a wide spectrum of cancers, such as breast and lung cancers, acute leukemia, advanced ovarian, head and neck carcinomas (Horwitz, 1992; Rowinsky & Donehower, 1995; Allen, 2002; Rivkin et al., 2010). Theoretically, oral administration of paclitaxel as many other drugs are a preferable choice compared to other routes due to various advantages: higher convenience for patient hence, better compliance to treatment, lower cost and higher safety. Unfortunately, paclitaxel that is insoluble in aqueous based medium and metabolized over absorption by epithelial cells of the gut mucosa shows a limited oral bioavailability (<10%), which complicates its oral administration (Malingre et al., 2001; Peltier et al., 2006). Nowadays, strategies based on the use of nanoparticles are proposed to overcome these limitations. Indeed, it was shown that association of drugs with nanoparticles may be efficient to increase bioavailability of many drugs including paclitaxel by protecting the drug against degradation and eventually enhancing the permeability across the intestinal Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 296 epithelium (Ponchel & Irache, 1998; Brigger et al., 2002; Bae et al., 2007; Rivkin et al., 2010; Zabaleta et al., 2013). Additionally, these systems can reduce toxicity controlling the biodistribution from the blood compartment and once in tissue, it can enhance delivery of the drug to resistant cancer cells over expressing the P-glycoprotein (Verdiere et al., 1994; Sparreboom et al., 1997; Verdiere et al., 1997; Varma et al., 2003; Jabr-Milane et al., 2008). A large part of published works reported the delivery of drugs including paclitaxel after association with nanospheres. Drawbacks of these systems are their generally low payload which makes the part of the drug composing the nanoparticles only few percent of the composition of the carrier. Nanocapsules are vesicles appear more suitable systems to achieve high payload especially when the drug is soluble in the component filling out the nanocapsule cavity and have favorable partition coefficient to remain in this medium during the nanocapsule preparation (Quintanar-Guerrero et al., 1998; Buzea et al., 2007). In a previous work, we have designed new poly(isobutylcyanoacrylate) nanocapsules decorated with chitosan and filled with natural oil having different interesting biological activities including anticancer properties(Xavier-Junior, Chapter VII, 2015). They can be used as “passive tumor targeting” due to accumulation in certain solid tumors by the enhanced permeability and retention effect (Maeda et al., 2000; Arias et al., 2001; Danhier et al., 2010). These systems are attractive to enhance drug delivery by the oral route as suggested from previous work carried on insulin delivery (Damge et al., 1988). They were stable in gastric environment while they appeared to be rapidly translocated in the blood from the intestine despite observations of relative in vitro instability in simulated intestinal medium (Aboubakar et al., 2000; Pinto-Alphandary et al., 2003). Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 297 The new nanocapsule exhibiting chitosan on their surface are expected to demonstrate mucoadhesive properties which are assumed to further potentialize oral administration of the drug. Paclitaxel would be a suitable drug to incorporate in these nanocapsules as it was recently demonstrated that it is well soluble in copaiba oil, the component included in the cavity of the chitosan-decorated poly(isobutylcyanoacrylate) nanocapsules (Xavier-Junior, Chapter II, 2015). The aim of the present work was to investigate the encapsulation of paclitaxel in the copaiba oil nanocapsules decorated with chitosan and to evaluate their potential to interact with the gut mucosa of rats thanks to the presence of chitosan on their surface. Optimization of the paclitaxel-loaded nanocapsules was achieved using a statistical interaction approach considering three variable parameters including the concentration of copaiba oil, the concentration in isobutylcyanoacrylate and the concentration of paclitaxel used to prepare the nanocapsules by interfacial polymerization of isobutylcyanoacrylate. Stabilitty of paclitaxel-loaded nanocapsules were then evaluated in simulated gastrointestinal fluids, under different storage conditions and after drying. Mucoadhesive properties were evaluated based on an aggregation test with mucins and on the evaluation of their retention at the level of rat intestinal mucosa mounted in Ussing Chamber. 2 MATERIALS AND METHODS 2.1 Materials Isobutylcyanoacrylate was provided by ORAPI engineered solutions worldwide (Vaulx- en-Velin, France). Copaiba oil was purchased from Flores & Ervas (Piracicaba, SP, Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 298 Brazil). Chitosan 20,000 Da was purchased from Amicogen (Jinju, South Gyeongsang, South Korea). PolyFluor ® 570: methacryloxyethyl thiocarbamoyl rhodamine B (N-[9- (2-carboxy-x-methacryloxy-ethylthiocarbamoylphenyl)-6-diethylamino-3H-xanthen-3- ylidene]-N-ethyl-ethanaminium chloride) was provide from Biovalley Polyscience (Marne-la-Vallée, France). Paclitaxel was obtained from CHEMOS GmbH (Regenstauf, Germany). [3H]-paclitaxel (3 Ci/mmol) was purchased from Isobio (Fleurus, Belgium). Hionic-Fluor ® and Ultima-Gold ® (Packard, Rungis, France) were used as scintillating cocktails for radioactive analyses. Soluene-350 ® used to dissolve biological samples was obtained from Perkinelmer (Courtaboeuf, France). Pancreatin, pepsine, sodium chloride, hydrochloric acid, sodium hydroxide, monobasic potassium phosphate and mucin from porcine stomach were purchased from Sigma-Aldrich (Saint-Quentin Fallavier, France). Ethanol, Acetonitrile and nitric acid were provided by Fisher Scientific (Illkirch, France). Ultrapure water was obtained from a Millipore purification system (Milli-Q plus, Millipore, St Quentin en Yvelines, France). All chemicals were reagent grade and used as received. 2.2 Preparation of nanocapsules Nanocapsules were prepared by interfacial polymerization as described by Al Khouri Fallouh et al (Al Khouri Fallouh et al., 1986) following the modification introduced by Xavier-Junior et al. (Xavier-Junior, Chapter VII, 2015). Briefly, an organic phase composed of ethanol (6.25 mL), copaiba oil (0.250, 0.350 and 0.450 mL), isobutylcyanoacrylate (0.032, 0.037 and 0.042 mL) and paclitaxel (2, 6 and 10 mg) were introduced dropwise in an 5 °C aqueous medium containing 12.5 mL of chitosan (0.9 %) which pH was adjusted at 3. Stirring rate was fixed at 1,250 rpm during addition Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 299 and over the next 10 minutes. Ethanol was then evaporated using rotary evaporator at 35 °C for 20 min at 43 mBa (BÜCHI Rotavapor R-125, Heating Bath B-491, Vacuum pump V-700, recirculating Chiller F-108, Flawil, Switzerland). The obtained nanocapsule dispersions were filtered (5µm nylon membrane filter, Merck Millipore, Billerica, MA, EUA). Then, they were purified and concentrated to 2 mL in Amicon Ultra centrifugal filter, 100 kDa molecular weight cut off (Merck Millipore, Billerica, MA, USA). This system was placed in centrifuge (Eppendorf centrifuge 5804 R, Rotor S-4-72, Hamburg, Germany) under 4,000 g at 20 °C against Milli-Q ® water three times for 20 min. Optimization of the paclitaxel encapsulation was achieved using statistical interaction approach between the concentrations of copaiba oil, isobutylcyanoacrylate and paclitaxel variables. The dependent variables analyzed were particle size and zeta potential. For mucoadhesion and stability studies, the nanocapsules were labeled with PolyFluor ® 570: methacryloxyethyl thiocarbamoyl rhodamine B, and with [3H]-paclitaxel. The nanocapsules were prepared following the protocol described above with minor changes. Initially, 0.25 µL of PolyFluor ® 570 (1 mg.mL -1 in ethanol) was added in the organic phase, immediately in this phase were added copaiba oil and isobutylcyanoacrylate for fluorescent nanocapsules preparation For radiolabeled nanocapsules containing paclitaxel, 4.2 kBq of [3H]-paclitaxel per mL of final nanocapsules suspension were dissolved in organic phase before polymerization. For some experiments non chitosan-coated nanocapsules were also needed. These nanocapsules were prepared with Pluronic F-68 ® according previous work (Al Khouri Fallouh et al., 1986; Gallardo et al., 1993; Aboubakar, Puisieux, Couvreur & Vauthier, 1999). Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 300 2.3 Characterization of nanocapsules The hydrodynamic diameter and the size distribution of the nanocapsules were determined by dynamic light scattering (DLS) using a Zetasizer Nano ZS90 (Malvern Instruments Ltd, Worcestershire, UK) at 25°C. The scattered angle was fixed at 90 °. Samples were diluted at 1:100 with Milli-Q ® water before analysis. Results were expressed as the mean hydrodynamic diameter, the standard deviation of the size distribution and the polydispersity index (PdI). Zeta potential of the nanocapsules was measured by Laser Doppler Electrophoresis using Zetasizer Nano ZS90 (Malvern Instruments Ltd, Worcestershire, UK). To maintain a constant ionic strength, samples were diluted (1:100) in saline solution (NaCl) at 1 mM. All results corresponded to the average of three determinations. Transmission electron microscopy (TEM) was used to investigate the nanocapsule morphology. Observations were performed using a JEOL 1400 transmission electron microscope (JEOL Ltd, Tokyo, Japan) coupled with a Gatan CCD high-resolution digital camera (Orius SC1000). One drop of the diluted nanocapsule in Milli-Q ® water (1:100) was placed on a formvar-carbon coated copper grid for 5 minutes. Thereafter, unfixed nanocapsules were removed by filter paper and one drop of 2% phosphotungstic acid (pH 7.4) was added to it for 30 seconds. The superfluous marker on sample was wiped off by filter paper. Finally, the grid was air dried prior to its introduction in the electron microscope. Fluorescence microscopy studies were conducted to test the stability of the nanocapsules in simulated gastrointestinal fluids. They were performed using a fluorescent microscope (Leitz Diaplan, Wild Leitz GmBH, Wetzlar, Germany) equipped with a filter N 2.1 adapted to PolyFluor ® 570: methacryloxyethyl thiocarbamoyl Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 301 rhodamine B (excitation 515–561 nm, emission cutoff 580 nm). The images were captured using QED capture version 2.0.24. A control experiment was performed by diluting the nanocapsules in Milli-Q ® water (1:50) instead of using simulated gastrointestinal fluids. The same parameters observations were maintained for all samples. 2.4 Determination of paclitaxel 2.4.1 HPLC analysis The amount of paclitaxel associated with the nanocapsules was quantified by high- performance liquid chromatography (HPLC). The method was developed and validated in a previous work (Xavier-Junior, Chapter II, 2015). The chromatographic system used was a Waters 515 pump, a Waters 717 plus autosampler and a Waters 486- Tunable Absorbance detector (Waters Corp., Milford, MA). Chromatographic separations were achieved using a Uptisphere Strategy 100A reversed-phase C-18 (150 mm x 3 µm x 3 mm) column and a Uptisphere Strategy C18-2 guard column (10 mm x 3 µm x 4 mm) (Interchim SA, France). The mobile phase, pumped at 0.4 mL.min -1 , was acetonitrile: water (50:50) at 30 °C monitored with UV-detection at 228 nm. The mobile phase and the samples were filtered through a 0.20 µm hydrophilic nylon membrane filter (Merck Millipore, Billerica, MA, EUA) prior to use. Under these conditions, the run time was 15 min and the paclitaxel was eluted at retention time of 9.7 minutes. Chromatographic data were monitored and analyzed using UV Winilab3 software (Perkin Elmer, Shelton, USA). The method was validated demonstrating that it was linear (r 2 = 0.999) within the range of concentration comprised between 50 to 2000 ng.mL -1 , recovery ranged from Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 302 97.1 to 101.9 % and relative standard deviation for intra- and inter-day precision were less or equal to 0.65 %. The specificity was tested in presence of the nanocapsules adjuvant and demonstrated that these factors did not alter the paclitaxel assay. The limit of quantification and limit of detection were 21.03 and 6.31 ng.mL -1 , respectively. 2.4.2 Radioactivity analysis Radiolabeled [3H]-paclitaxel loaded copaiba oil- poly(isobutylcyanoacrylate) nanocapsules coated with chitosan was determined by liquid scintillation counter (Model LS 6000 TA, Beckman, France). Samples (40 µL) were vortexed for 1 minute with 10 mL of a scintillating cocktail and analyzed. For analysis in the rat intestinal tissue, 1 cm² from excised tissue was digested in 2 mL of Soluene-350 ® at 65 °C overnight. Then, 10 mL of scintillating cocktail was added into the bottle, vortexed for 1 minute and measured the [3H]-paclitaxel radioactivity. 2.5 Drug loading and encapsulation efficiency Free drug was determined in the clear supernatant obtained after separation of nanocapsules from aqueous dispersion medium by ultrafiltration-centrifugation technique (Microcon centrifugal filter, Ultracel YM-100, regenerated cellulose, Merck Millipore, Billerica, MA, USA). Nanocapsules were centrifugated at 10,000 rpm for 20 min (Eppendorf centrifuge 5418, Rotor FA-45-18-11, Hamburg, Germany) over the ultrafiltration unit. Drug loading (DL) and encapsulation efficiency (EE) were expressed as percentages and deduced from equations 1 and 2, respectively: Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 303 D % = Weight of nanocapsules loaded paclitaxel Total weight of nanocapsules X 100% % = Experimental drug loading Theorical drug loading X 100% ( 2) 2.6 Stability of paclitaxel loaded nanocapsules Paclitaxel loaded copaiba oil- poly(isobutylcyanoacrylate) nanocapsules coated with chitosan dispersion stability was evaluated for over a period of 6 months in terms of size and zeta potential while storage was achieved at 4 °C and 25 °C. Stability of the nanocapsules was investigated after incubation in simulated gastrointestinal fluids, according to the conditions described in United States Pharmacopoeia XXXIV (Convention, 2011). Simulated gastric fluid medium was composed by 0.2 % of sodium chloride, 8 % of hydrochloric acid (1M) and 0.32 % (w/v) of pepsin with a pH of 1.2. Simulated intestinal fluid medium was formed by 0.62 % monobasic potassium phosphate solution, 7.7 % of sodium hydroxide with pancreatin 1 % (w/v) (pH 6.8). For this study, nanocapsules labeled with PolyFluor ® 570 were added in the simulated fluid at dilution of 1:50 and incubated at 37 °C for various time over of total 120 minutes. At defined times, the samples were collected and analyzed by DLS for size measurement. Integrity of the nanocapsules was appreciated by fluorescence microscopy observations as previously described. Stability studies were also investigated after drying the nanocapsules dispersion in Eppendorf Vacufuge ® 5301 vacuum centrifuge (Eppendorf Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 304 AG, Hamburg, Germany) at ambient temperature. The dried nanocapsules were analyzed by DLS and TEM after rehydration and redispersion in the equivalent amount to recovered initial volume of Milli-Q ® water. 2.7 Evaluation of mucoadhesion 2.7.1 Aggregation of nanocapsules in presence of mucin Mucin from porcine stomach was prepared using Milli-Q ® water for 2 h at room temperature (20 °C) to obtain dispersion at 1 % (w/v). Dispersions of nanocapsules were prepared in Milli-Q ® water at different concentrations: 1.5; 2.0; 2.5; 3.0; 3.5 and 4.0 mg.mL -1 . Then, 75 µL of the nanocapsules dispersion were placed in the wells of a 96- microwell plate with polystyrene conical bottom. The absorbance initial (A0) of each suspension was evaluated at 450 nm using a microplate reader (Multiskan Anscent, Labsystems SA, Cergy-Pontoise, France). 30 µL of mucin suspension (0.25 mg.mL -1 ) was added and the plate was incubated for 1 h at 37 C. To quantify the aggregation, the absorbance A1h was evaluated after centrifugation (240 g) for 5 min at 25 °C (Eppendorf centrifuge 5804 R, Rotor A- 4- 81 with MTP/Flex carrier, Hamburg, Germany). Each experiment was repeated three times and the difference between A0 and A1h (A0 - A1h) was plotted as a function of the dilution performed for each nanocapsule dispersion. Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 305 2.7.2 Mucoadhesion assay on rat intestinal mucosa Animal experiments were carried out according to the recommendations of the ethics committee of the French Ministry of Higher Education and Research, project 2003-055 regarding the care and use of animals for experimental procedures. Male Wistar rats (200–250 g) (Charles River, Paris) were used for the mucoadhesion ex vivo assays. Rats were allowed free access to water and food, and housed under controlled environmental conditions (constant temperature, humidity, and a 12 h dark-light cycle). Animals were euthanized with an overdose of pentobarbital by intraperitoneal injection. Fresh small intestine (jejunum) portion was excised, rinsed with physiological saline (NaCl 0.9 %) and cut into small segments of 2-3 cm length. After visual examination of the tissue, sections containing Peyer’s patches were discarded. Intestinal portions were mounted in Ussing chambers with a delimited intestinal mucosa surface area (1 cm 2 ). The systems were maintained in ringer buffer at 37 °C, continuously oxygenated with O2 /CO2 95%/ 5%. After removing the transport buffer, 0.1 mL of radiolabeled nanocapsules in ringer buffer (pH 7.5) were applied to the mucosal surface. Each compartment of the Ussing chamber was filled with 3 mL of ringer solution. The experiment was performed over a period of 2 hours to insure the attachment equilibrium. After incubation for 2 hours, the nanocapsule dispersion was removed. Tissue was rinsed three times with 3 mL of ringer buffer, to eliminate non- attached nanocapsules while it was still mounted in the Ussing Chamber. Subsequently, the mucosa with the attached nanocapsules was recovered and let to dissolve in 2 mL of Soluene-350 ® at 65 °C overnight. Then, 10 mL of scintillating liquid were added and finally samples were analyzed by liquid scintillation to determine the amount of [3H]- paclitaxel which associated with the mucosa. Results were expressed as the amount of Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 306 attached nanocapsules per apparent surface (g/m 2 ) and by the number of attached nanocapsules in the tissue, as defined by the equation 3: N= mT 6 S d ( ) where N is the number of attached nanocapsules, mT the mass of attached nanocapsules (g), the nanocapsules density oil estimated (0.99 g/cm3), d the nanocapsules diameter (cm) and S the nanocapsules surface: S= 4π (d/2)². Each sample was tested in three different rats in duplicate. 2.8 Statistical analysis All experiments were conducted in triplicates. All values were expressed as their mean ± standard deviation (SD). Means of two groups were compared using non-paired Student’s t-test. When comparing multiple groups, one way analysis of variance (ANOVA) was applied with the Tukey multiple comparison procedure. The analyses were performed using the Graph Pad Prism (Version 5.0, La Jolla, CA, USA) and Statistic software (Version 7.0, StatSoft Inc., USA). The statistical data were considered significant at p < 0.05 Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 307 3.0 RESULTS AND DISCUSSION 3.1 Optimization of the preparation of paclitaxel loaded nanocapsules Paclitaxel loaded copaiba oil- poly(isobutylcyanoacrylate) nanocapsules coated with chitosan were produced by the new method of interfacial polymerization of isobutylcyanoacrylate carried out in the presence of chitosan while surfactant was absent in the polymerization medium (Xavier-Junior, Chapter VII, 2015). This method was used to obtain chitosan-coated nanocapsules encapsulating paclitaxel assuming that they will show mucoadhesive properties. Optimization of the incorporation of paclitaxel was studied considering the influence of the isobutylcyanoacrylate, copaiba oil and paclitaxel concentrations on the size and zeta potential of the produced nanocapsules (Figure 1). It was considered that nanocapsules with smaller sizes promote mucoadhesion (Ponchel et al., 1994; Ponchel & Irache, 1998; Bertholon et al., 2006). Furthermore, after reaching the blood, small nanocapsules can undergo capillary distribution and uniform perfusion. Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 308 Figure 1- Size (top line) and zeta potential (bass line) influence in paclitaxel loaded nanocapsules formed at low and high concentration of isobutylcyanoacrylate (A and D), copaiba oil (B and E) and paclitaxel (C and F). * Broad distribution size An opposite and significant effect was observed in the size when increasing concentrations in isobutylcyanoacrylate and paclitaxel in the polymerization medium (p < 0.05). Considering the concentration of monomer, a decrease of the diameter of the nanocapsules of 100 nm was observed by increasing the amount of isobutylcyanoacrylate introduced in the polymerization medium from 0.022 to 0.042 mL (Figure 1A). The nanocapsule dispersion obtained with concentration in isobutylcyanoacrylate of 0.022 mL were unstable showing aggregates while size Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 309 distribution was broad. This can be associated with the formation of a low polymer concentration, possibly insufficient to effectively cover the available surface of the copaiba oil droplets which form during dispersion of the organic phase in the polymerization medium (Douglas et al., 1985). By increasing the concentration of drug from 2 to 10 mg.mL -1 in the organic phase, the diameter of the nanocapsule which formed was decreased (Figure 1C). Analyzing the chemical structure of paclitaxel, a slight amphiphilicity can be highlighted (Figure 2). It may be enough to promote formation of emulsion droplets of lower size then those obtained in the absence of paclitaxel during dispersion of the organic phase in the polymerization medium hence of the nanocapsules. A slight increase in the nanocapsule size was observed when increased the volume of copaiba oil loaded in the systems from 0.25 to 0.45 mL, however, this difference was not statistically significant (p > 0.05) (Figure 1B). Figure 2: Chemical structure of the paclitaxel Positive zeta potential values were observed with all nanocapsules suggesting that chitosan was covering the nanocapsules surface as expected. The rather strong positive zeta potential may be explained by the presence of quaternary ammonium groups in Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 310 chitosan (Bathool et al., 2012) at the almost neutral pH of measurement. The positive zeta potential of the nanocapsules was expected to promote mucoadhesion thanks to electrostatic interactions with the mucus surface, which is negatively charged at physiological pH (Bernkop-Schnurch, 2005). Higher value of zeta potential (positive or negative), are also highly favorable to obtain stable dispersions of nanocapsules as electrostatic repulsion forces between particles having the same electric charge prevent aggregation of the dispersion (Feng & Huang, 2001). No significant difference (p > 0.05) was observed between nanocapsules prepared with two concentrations in isobutylcyanoacrylate (Figure 1D). Zeta potential was much influenced by the amount of copaiba oil introduced in the polymerization so did modification of the concentration of paclitaxel but in the opposite way (Figure 1 E and F). To explain these effect either small amounts of copaiba oil and paclitaxel could adsorbed on the nanocapsule surface or they influenced the coverage of the nanocapsule surface by the chitosan molecules leading to masking or exhibiting the positive charge of the polysaccharide (Harivardhan Reddy & Murthy, 2003; Mohammadpour Dounighi et al., 2012). In order to obtain nanocapsules of small size, but at the same time large amounts of chitosan coated on the surface, it can be deduced that optimized paclitaxel loaded nanocapsules were produced using 0.032 mL of isobutylcyanoacrylate, 0.25 mL of copaiba oil and 6 mg of paclitaxel. 3.2 Characteristics of the optimized paclitaxel loaded nanocapsules The mean particle diameter of these nanocapsules was found to be 486 ± 3 nm with a narrow PdI of 0.17 (Figure 3). Zeta potential of those nanocapsules was frankly positive Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 311 (+ 37.1 ± 0.3 mV), indicating presence of chitosan on the nanocapsule surface. Preparation of unloaded nanocapsules performed in same conditions but omitting paclitaxel provided with nanocapsule which characteristic did not differ significantly (p > 0.05) from that of the loaded nanocapsules to size, zeta potential and morphology analyses. Figure 3: Size (A) and zeta potential (B) of different nanocapsules. NCC: Copaiba oil- loaded chitosan-poly (isobutylcyanocrylate) core-shell nanocapsules; NCC PTX: Paclitaxel into NCC; NCCfluo Paclitaxel: PolyFluor ® 570 labeled NCC PTX; NCCdry PTX: NCC PTX after drying process. TEM images showed the spherical shape and surface morphology of the paclitaxel unloaded and loaded nanocapsules (Figure 4 A and B, respectively). The nanocapsules appeared well separated on all preparations. As confirmed by TEM examination, nanocapsules suggesting that they are formed by an oily cavity (copaiba oil where paclitaxel was dissolved) surrounded by a polymer membrane. The actual diameter Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 312 observed by microscopy around 475 nm which was consistent with values determined by DLS. Figure 4- TEM of copaiba oil-loaded chitosan-poly (isobutylcyanocrylate) core-shell nanocapsules (NCC) (A); Paclitaxel into NCC (NCC PTX) (B); NCC PTX after drying process (C); PolyFluor ® 570 labeled NCC PTX (D). Scale bar of 200 nm and 500 nm to isolated and grouped nanocapsules, respectively. Concerning the maximum EE and DL of paclitaxel loaded in nanocapsules, the results showed values of 74.5 ± 1.2% and 1.7 ± 0.02% (w/w), respectively. The drug concentration loaded in the formulation was 16.8 ± 0.3 µg of paclitaxel per mg of Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 313 nanocapsules, corresponding at 746 ± 12 µg.mL -1 of nanocapsules dispersion. This corresponding to an excellent association of the drug with the nanosystem compared with previously described systems. The DL showed by these nanocapsules was also relevant with a therapeutic dose when it is compared with existing injectable therapies. For instance, in the Ambraxane ® , paclitaxel which is formulated in a nanomedicine is administrated at concentration of the dispersions of 5 mg.mL -1 . Previous studies, reported 70% of paclitaxel EE into PEGylated poly (lactide-co-glycolide) nanoparticles by nanoprecipitation method (Danhier et al., 2009). Huang et al showed DL of 0.18 and 0.56 % in paclitaxel-loaded poly (butylcyanoacrylate) nanoparticle obtained by in emulsion and microemulsion polymerization methods (Huang et al., 2007). To explain the high DL obtained in the nanocapsules, it is believed that the good solubility of the paclitaxel in copaiba oil combined with its high partition coefficient in favor to copaiba oil had contributed favorably to the success of the encapsulation method developed in the present work (Xavier-Junior, Chapter II, 2015). Biopharmaceutical studies require the labeling of particles in order to localize them in vivo and/or in vitro during assays carried out under various experimental conditions (Bravo-Osuna, Ponchel, et al., 2007; Vauthier & Bouchemal, 2009). Fluorescent labeled nanocapsules were synthesized by incorporation of methacryloxyethyl thiocarbamoyl rhodamine B (Polyfluor ® 570), a fluorescent co-monomer, which reacts with isobutylcyanoacrylate during the formation of the nanocapsules. Fluorescent labeled nanocapsules did not show significant difference in size and morphology compared with the non-labeled nanocapsules. They only differed by a slight increase of zeta potential (Figure 3 and 4D). The nanocapsules appeared clearly fluorescent under observation by fluorescent microscope. Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 314 Radiolabeled nanocapsules were effectively developed with [3H]-paclitaxel. This dispersion showed a radioactive activity of 3.14 ± 0.07 kbq.mL -1 . The encapsulation efficiency was similar to that of the nanocapsules prepared with the non-radioactive drug, indicating that the use of radiolabeled drug did not modify characteristics of the nanocapsules. The non chitosan-coated nanocapsules prepared with Pluronic F-68 ® were characterized by a size of 230 ± 3.2 nm, a PdI of 0.18, a zeta potential of -21.1 mV ±0.06. These characteristics were consistent with nanocapsules synthesized in previous works. 3.3 Stability of the nanocapsules upon storage The storage of nanocapsules was investigated as dispersion at 4 and 25 °C and after elimination of the water to obtain a dried powder. Upon storage under the form of a dispersion particle size and zeta potential were evaluated over a period of 6 months. Results presented in Figure 5 showed that nanocapsule dispersion stored at 4 °C maintained the initial properties and no aggregation were observed (p > 0.05). The nanocapsule dispersion stored at 25 °C remained stable over 3 months with only slight variation of their mean diameter and zeta potential by 5 and 7 %, respectively. After 6 months, the sample presented a statistic significant increase of the size by 19 % while the zeta potential decreased by 20 % (p < 0.05). These effects were probably caused by a fusion growth process which is more pronounced at elevated temperatures. These results were consistent with Lemoine et al. and Coffin et al., where minor changes in the in nanoparticles size were observed when stored at 4 or 5 °C in contrast with higher temperatures (25 or 37 °C) (Coffin & Mcginity, 1992; Lemoine et al., 1996). Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 315 Figure 5- Size and zeta potential of paclitaxel into copaiba oil-loaded chitosan- poly(isobutylcyanocrylate) core-shell nanocapsules stability stored at 4 °C and 25° C over a period of 6 months. Storage of formulations under a dried form is generally preferable. However, one major problem with nanoparticles is that they often aggregate during the drying process and the recovery of dispersion having the same size characteristics than the parent dispersion is the main difficulty. Besides irreversible aggregation, nanocapsules can be destroyed by the drying process (Abdelwahed, Degobert & Fessi, 2006; Abdelwahed, Degobert, Stainmesse, et al., 2006; Tewa-Tagne et al., 2007). Here the nanocapsules were dried and characterized by DLS and TEM after redispersion to reconstitute the parent dispersion. Particle size (513.5 ± 5.4 nm with polydispersion index of 0.19), zeta potential (+ 37.2 ± 0.2 mV) and morphology of the nanocapsules appeared similar to the initial dispersion after drying and reconstitution in water (Figure 3 and 4 C). Comparing Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 316 the size and polydispersity index of the two nanocapsule dispersions the difference was not statistically significant (p > 0.05). The nanostructure as observed by TEM was also well preserved demonstrating that the nanocapsule can be dried without losing their physicochemical characteristics and morphology. 3.4 Stability of nanocapsules in simulated gastrointestinal fluids The size and the fluorescent image of the labeled nanocapsules were followed over a period of 120 minutes of incubation in simulated gastric and intestinal media (Figure 6 and 7). In the reconstituted gastric medium, no important increase of the size occurred after 120 minutes of incubation (p>0.05). By fluorescent microscopy, the fluorescence remains confined within the nanocapsules for at least 1 hour while released of fluorescence clearly started after 120 minutes of incubation in the gastric medium. This indicated that the nanocapsules were quite stable in the gastric environment while they start to leak after 120 minutes in this medium. Although the nanocapsules became leaky, no significant aggregation was observed. At strong acid pH, chitosan having a pKa of 6.3 is highly protonated hence positively charged which can explain that the nanocapsules remained well dispersed (Meng et al., 2010). During the first 30 min of incubation in the intestinal medium, the nanocapsules dispersion showed a dramatic increase of the size of the nanocapsule corresponding to five times more and PdI of 0.64, while observations by fluorescent microscopy revealed a marked fluorescent background. Aggregation of the fluorescent nanocapsules, only shown at 120 minutes, suggesting that this system were leaky and already started to degrade with time as well as the size measured by DLS over the time of the experiment. Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 317 In the intestinal medium, chitosan protonation is low hence the charge. Nanocapsules may tend to aggregate because the electrostatic forces contributing to the colloidal stability may be diminished. The enzymatic environment is also greatly favorable to the loss of stability of the poly(isobutylcyanoacrylate) nanocapsules. Esterase’s can degrade the polymer causing leakage as well as swelling of the polymer envelope (Lenaerts et al., 1984; Scherer et al., 1994). The present observations, we were very consistent with those reported by Aboubakar et al. on insulin nanocapsules (Aboubakar et al., 2000). Figure 6- The size of the polyFluor ® 570 labeled nanocapsules in different media simulating the pH environment in the gastrointestinal tract after two hours of incubation. Dark bars= Simulated gastric fluid, light bar= Simulated intestinal fluid and *=p<0.05 Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 318 Figure 7- Polyfluor ® 570 labeled nanocapsules stability in reconstituted gastric and intestinal fluids by 120 minutes. 3.5 Mucoadhesion studies The potential use of small mucoadhesive polymer particle formulations lies in possible prolongation of the residence time near absorption sites of the drug either through non- specific (van der Waals and/or hydrophobic interactions) or specific interactions between components of the particles and the mucus. In general, increases of the Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 319 bioavailability of the loaded drug were then obtained (Ponchel & Irache, 1998; Hagerstrom et al., 2003; Moghaddam et al., 2009a). Mucus which is composed of highly hydrated glycoprotein called mucins cover mucosa forming a continuous adherent blanket on the surface of the epithelium. While adhering to the mucosa, nanocapsules would have to interact with mucins. There are several methods that are suitable to investigate mucoadhesion of nanocapsules on the gut mucosa (Neves et al., 2011; Laffleur & Bernkop-Schnurch, 2013). For the present study, the method evaluating aggregation of nanocapsules in presence of mucin was selected as a convenient in-vitro test. Adhesion of nanocapsules was also evaluated directly on rat intestinal mucosa. Results from the aggregation of mucin are presented on Figure 8 considering unloaded and loaded-paclitaxel nanocapsules coated with chitosan. There were compared with results obtained with nanocapsules composed of poly(isobutylcyanoacrylate), copaiba oil but which were not coated with chitosan. These nanocapsules were stabilized by using Pluronic including PEG chains that are also know to display mucoadhesive properties (Kim et al., 2007; Jones et al., 2009; Gratieri et al., 2010). Different profiles of mucin-nanocapsule aggregation were obtained comparing the different types of nanocapsules. At concentrations bellow 2.5 mg.mL -1 the loaded and unloaded paclitaxel nanocapsules coated with chitosan showed a marked difference while above this concentration, the two types of nanocapsules showed a maximum of aggregation in the presence of mucins which was acknowledged by the plateau observed on the curves. Both types of nanocapsules were coated with chitosan. The occurrence of the plateau indicated occurrence of a saturation phenomenon. This was consistent considering that interactions between the nanocapsules and the mucins consisted of chemical bonds between the positively charged amino groups of chitosan and the Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 320 negatively charged sialic acid residues of mucus glycoproteins at concentrations above 2.5 mg.mL -1 in nanocapsules (Rossi et al., 2000). As can be observed in Figure 8, the presence of Pluronic F-68 ® on the nanocapsule surface did not provoke aggregation of the mucin over the range of concentrations studied. In contrast with the nanocapsules coated with chitosan, the nanocapsules coated with Pluronic did not interacted with mucins. These results showing higher interactions of chitosan-coated nanocapsules with mucins compared with Pluronic-coated nanocapsules suggested that the addition of chitosan on the nanocapsule surface should promote their mucoadhesion. Figure 8- in-vitro mucin aggregation test to evaluate mucoadhesive properties of the nanocapsules. NCC: Copaiba oil-loaded chitosan-poly (isobutylcyanocrylate) core-shell nanocapsules; NCC PTX: Paclitaxel into NCC; NC-Plu: Copaiba oil-loaded Pluronic- poly (isobutylcyanocrylate) nanocapsules. Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 321 Mucoadhesion of the nanocapsules was also evaluated on freshly excised rat intestinal mucosa while the tissue was mounted in Ussing Chambers. The nanocapsules were loaded with [3H]-paclitaxel. Thus, only mucoadhesion of the radiolabeled nanocapsules could be evaluated by this method. The nanocapsule concentration used was the same as in the plateau observed in the mucin aggregation test described above. After incubation of the nanocapsules with the mucosa in the Ussing chambers, 9 ± 1.1% of the initial amount of the radioactivity introduced with the nanocapsules was associated with the mucosa after two hours. This corresponded to a deposition and strong attachment of 6.2 x10 9 nanocapsules per square centimeter of the intestinal mucosa or to 3.4 g of nanocapsules per square meter. In the literature, Bravo-Osuna et al. have observed that the presence of chitosan on the poly(isobutylcyanoacrylate) nanoparticle surface increased dramatically the mucoadhesive behavior of the nanoparticles thanks to the formation of hydrogen and ionic bonds between the positively charged amino groups of the polysaccharide and the negatively charged sialic acid residues of mucin glycoproteins (Bravo-Osuna, Vauthier, et al., 2007). The authors have also reported a relation between the amount of attached chitosan 20 kDa-coated nanoparticles per square meter of intestinal mucosa of rat at concentration of 2 mg.mL -1 corresponding at nanoparticles adhesive interaction about of 1.5 g/m 2 . Thus, comparing the studies developed in this work with Bravo-Osuna et al., it was observed an increase by 2-folds in the nanocapsules adhesive interaction with the mucosa. Results from the studies devolopped in the present work also agreed well with those reported by Moghaddam et al. who have found approximately the same amount of nanoparticles attached on the intestinal mucosa of rat while investigating the mucoadhesion of chitosan 20kDa- pHEMA nanoparticles (Moghaddam et al., 2009b). Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 322 In the clinic, paclitaxel dose is commonly prescribed at 175 mg/m² and administered intravenously over a period of 3-24 hours (Van Den Bongard et al., 2004). Sacco et al. observed the overall mean body surface area of 1.79 m 2 (95% CI 1.78–1.80) to patients receiving chemotherapy for head and neck, ovarian, lung, upper GI/pancreas, breast or colorectal cancers (Sacco et al., 2010). Thus, to obtain an ideal anticancer therapy, a dose of 313.3 mg of paclitaxel is required. Paclitaxel loaded copaiba oil- poly(isobutylcyanoacrylate) nanocapsules coated with chitosan developed in this work show a important impact as new anticancer therapy by oral route for paclitaxel. Taking into account the ideal therapy, is required 18.6 g of nanocapsules containing paclitaxel. Although the amount administered of nanocapsules appears to be high, other factors such as the synergic effect of copaiba oil in the cancer therapy and character of the modified release have not been considered in order to reduce the traditional dose. 4 CONCLUSION New poly(isobutylcyanoacrylate) based nanocapsules were synthesized by interfacial polymerization having a surface coated with chitosan. Paclitaxel could be incorporated in the nanocapsules which cavity was filled out with copaiba oil while chitosan conferred interesting mucoadhesive properties to the new formulation. Properties of the nanocapsules agreed well with those expected for a formulation designed to enhance oral bioavailability of the associated drug. Taking together, results from the present work are encouraging to pursue the development of the chitosan-coated nanocapsules for oral delivery of paclitaxel as new treatment for cancer with possible synergetic anticancer effect with therapeutically active components found in copaiba oil. Chapter VIII- Preparation of paclitaxel-loaded chitosan- poly (isobutylcyanoacrylate) core- shell nanocapsules and evaluation of their mucoadhesion by in vitro methods 323 ACKNOWLEDGEMENTS The authors would like to thank the financial support from “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior- CAPES” through the COFECUB 721/11 projet for Xavier-Junior, F.H. fellowship The authors are also grateful to Imagif Cell Biology Unit of the Gif campus, Conseil Général de l'Essonne by images analyses (www.imagif.cnrs.fr) for the access of the TEM facility. REFERENCES ABDELWAHED, W., DEGOBERT, G. & FESSI, H. 2006. Freeze-drying of nanocapsules: impact of annealing on the drying process. 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Discussion Générale Discussion Générale 333 La voie orale est certainement la voie préférée des patients pour l’administration de médicaments, en effet c’est la meilleur voie du point de vue du leur confort, en termes d'observance, de simplicité, de coûts du traitement et également de sécurité d'emploi pour le patient (Borner et al., 2001; Batlle et al., 2004). Toutefois, elle est limitée pour certains principes actifs dont les propriétés physico-chimiques, les facteurs physiologiques et d'autres aspects liés aux formes galéniques conduisent à une dissolution et une absorption réduite, ce qui entraîne une faible biodisponibilité orale (Prabhu et al., 2005; Ensign et al., 2012). Il est donc nécessaire que la formulation permette d’obtenir un profil pharmacocinétique pertinent, caractérisé par une biodisponibilité suffisante, ce qui améliorerait encore l'efficacité thérapeutique et la compliance du patient (Singh et al., 2009). Au cours du temps, les nanomédecines sont apparus comme des outils de formulation qui peuvent aider à surmonter ces difficultés (Duncan, 2004; Engineering., 2004; Kateb et al., 2011). Au cours des dernières années, de nombreaux efforts ont été réalisés afin de développer des systèmes d'administration de médicaments anticancéreux pour la voie orale. Ces efforts de reformulations fournissent également la possibilité d'améliorer l'efficacité de la thérapie à base de médicaments anti-cancéreux (Kawasaki & Player, 2005). Les nouveaux systèmes d'administration de médicaments par voie orale s’avèrent très utiles car ils permettront d’augmenter l’observance des patients, d’améliorer la biodisponibilité, et ainsi de fournir de meilleurs effets thérapeutiques tout en augmentant la qualité de vie des patients (Verma & Garg, 2001; Roger et al., 2010b; Gibaud & Attivi, 2012; Mazzaferro et al., 2013a; b). Les systèmes d'administration modernes empruntés aux nanomédecines sont également capables d'améliorer la stabilité des molécules actives fragiles ou rapidement dégradées dans le milieu gastro- intestinale, réduire la toxicité non-spécifique et améliorer la perméabilité à travers Discussion Générale 334 l'épithélium intestinal tout en diminuant la dose de médicaments délivrés aux cellules cancéreuses (Sparreboom et al., 1997; Varma et al., 2003; Jabr-Milane et al., 2008; Muthu et al., 2009). De cette façon, l’encapsulation des molécules anticancéreuses dans des vecteurs nanothérapeutiques s’est avérée être une solution prometteuse pour le traitement du cancer. Dans cette thèse, le paclitaxel a été utilisé comme molécule anticancéreuse pour plusieurs raisons. C’est en premier lieu l’un des plus puissants agents anti-cancéreux utilisé pour le traitement des cancers et il fait partie à la classe biopharmaceutique IV (peu soluble et faiblement perméable). Ces raisons justifient de rechercher des formulations originales capables d’améliorer les caractéristiques physico-chimiques du paclitaxel pour augmenter sa biodisponibilité par voie orale (Forastiere, 1994; Rowinsky & Donehower, 1995; Weaver, 2014). Il est intéressant de noter que cette molécule est d'origine végétale extrait du Taxus brevifolia (Fang & Liang, 2005). C’est un pseudo-alcaloïde qui présente une structure diterpénoïde. Son mode d'action est defavoriser la stabilisation des microtubules dans le cytoplasme des cellules inhibant ainsi la prolifération cellulaire et induisant finalement l'apoptose (Schiff et al., 1979; Hamel, Campo, et al., 1981; Horwitz, 1992). Cette molécule anticancéreuse étant d’origine naturelle, l'encapsulation dans des nanosystèmes contenant des huiles d'origine végétale peut être éventuellement intéressant pour augmenter le taux d'encapsulation, la stabilité et favoriser un effet thérapeutique synergique. (Lee et al., 1995; Dantas, T. N. C. et al., 2010; Attaphong et al., 2012). De plus, les huiles extraites des plantes ont un certain nombre d’avantages potentiels en comparaison aux huiles minérales, elles présentent une faible toxicité, sont biodégradables et renouvelables. (Dossat et al., 2002; Aluyor et al., 2009). Dans ce Discussion Générale 335 contexte, l’huile végétale de copaïba (Copaifera langsdorffi) semble être une bonne candidate. Cette huile formée par une complexe mélange de composés diterpéniques et sesquiterpéniques permet de lutter contre maladies inflammatoires, microbiologiques et cancéreuse (Veiga-Junior & Pinto, 2002; Gomes, Niele Matos et al., 2007; Gomes N et al., 2008; Mendonça & Onofre, 2009a; Comelli-Júnior et al., 2010; Souza, Martins, Souza, Furtado, Heleno, Sousa, et al., 2011). Cette thèse avait pour de but développer des systèmes miniaturisés pour améliorer la délivrance de médicaments anticancéreux, le paclitaxel, par voie orale en utilisant l'huile végétale de copaïba qui présente elle-même des propriétés thérapeutique. Dans ce contexte, deux systèmes différents ont été proposés. L'un de nature lipidique et l'autre polymére. Les travaux expérimentaux initiaux sur l’équilibre hydrophile-lipophile et le développement d'émulsions d'huile de copaïba ont été réalisés au Laboratorio de Sistemas Dispersos (LASID) à l'Universidade Federal do Rio Grande (UFRN) à Natal, au Brésil, sous la Direction du Professeur Dr. Sócrates Egito. La deuxième partie de cette thèse concernant le développement des formulations orales à base de microémulsions et de nanocapsules de l'huile de copaïba pour l'encapsulation du paclitaxel ont été élaborées au sein de l’Institut Galien Paris-Sud (UMR 8612), dans l’Université Paris-Sud, à Châtenay Malabry, en France, sous la Direction du Dr Christine Vauthier. Ce travail pu être effectué grâce à une collaboration bilatérale développées dans le cadre d'un projet financé par la « Coordination de perfectionnement du personnel de l'enseignement supérieur du Ministère de l'Education brésilien- CAPES/MEC», le « Comité Français d’Évaluation de la Coopération Universitaire et Scientifique avec le Brésil -COFECUB » et la mise en place d'un accord de co-tutelle de thèse entre les universités brésilienne et française. Discussion Générale 336 Afin d'associer les activités anticancéreuses de l'huile de copaïba et d'un principe actif utilisé couramment en cancérologie dans une formulation de la nanomédecine unique, le développement de méthodes destinées à analyser et doser l'huile de copaïba et le paclitaxel solubilisé dans cette huile ont été développées. La validation de la méthode a été réalisée afin de normaliser le processus et l'utilisation de l'instrumentation visant à minimiser l'erreur aléatoire et s’assurer que la méthode peut être digne de confiance. La détermination de nombreux paramètres expérimentaux ont été nécessaire pour garantir que la méthode est validée (González et al., 2014; Mujawar et al., 2014; Nikolaou et al., 2015). La validation de la méthode a été réalisée par la détermination de la précision, linéarité, sensibilité, sélectivité, et des limites de détection et quantification. Les critères qui ont été retenus pour le choix des méthodes d'analyses et de dosage du paclitaxel et de l’huile de copaïba étaient basés sur la rapidité de l'analyse, la simplicité de mise en œuvre, la précision, la sensibilité et la performance économique de la mesure. Une analyse par chromatographie gazeuse a été proposée pour l'huile de copaïba. Plusieurs composants ont été identifiés et la méthode a été développée pour permettre leur quantification à l'aide d'un détecteur à ionisation de flamme. Les principaux composés identifiés dans l'huile essentielle de copaïba ont été le β- bisabolène (23,6%), le β-caryophyllène (21,7%) et l’α-bergamotène (20,5%). Les composés identifiés dans l’huile résine de copaïba sont l’acide copalic (15,6%), le β- bisabolène (12,3%), le β-caryophyllène (7,9%), l’α-bergamotène (7,1%) et l’acide Labd-8(20)-ene-15,18-dioïque (6,7%). En utilisant un détecteur à ionisation de flamme, la méthode est linéaire pour une gamme de concentration en 0,99 du 40 à 160 μg.mL-1. L'analyse quantitative peut être réalisée sur un temps d'analyse de 13,15, 14,87 et 21,52 pour le β- caryophyllène, l’α- humulène et l'oxyde caryophyllene, respectivement. La limite de détection a été déterminée à 6,38, 4,16 et 0,55 µg.mL -1 et la limite de Discussion Générale 337 quantification à été établie à 19,36, 12,62 et 1,67 µg.mL -1 pour le β- caryophyllène, l’α- humulène et l'oxyde caryophyllene, respectivement. La précision et l’exactitude de la méthode ont été inférieures à 4,4 et 3,5 %, respectivement. Le mélange de composants complexes de l'huile de copaïba peut entraver la quantification du paclitaxel lorsqu'il est associé à ses formulations. En conséquence, le besoin d'une méthode d'analyse exacte et précise de paclitaxel dans l'huile de copaïba est obligatoire pour le contrôle de qualité et le développement de médicaments. Par conséquent, l’HPLC UV a été un outil analytique approprié pour effectuer des analyses de paclitaxel à faible coût avec une haute reproductibilité et une méthode simple et efficace. Le dosage du paclitaxel a pu être réalisé par une méthode HPLC développées sur un temps d'analyse de 9,7 min. La courbe d'étalonnage obtenue est linéaire (r²=0.9998) pour une gamme de concentration du 50 à 2000 ng.mL -1 et les résidus de régression présentait une dispersion homoscédastique. Les limites de détection et de quantification ont été établies à 21.03 et 6.31 ng.mL -1 , respectivement. Les déterminations de l'exactitude et la précision ont été inférieures ou égales à 0.77 et 0.65 %, respectivement. La méthode a été appliquée à la déterminer de la solubilité du paclitaxel dans l'huile de copaïba et des coefficients de partage du paclitaxel entre différents milieux lipophiles et l'eau. Ainsi, la solubilité du paclitaxel dans l'huile de copaïba résine est intéressante 0,8 mg.mL -1 puisqu'elle est apparue 2000 fois supérieure à celle de l'eau. Les coefficients de partage déterminé pour l’huile résine et essentielle sont de 3,2 et 2,6 respectivement. La bonne solubilité du paclitaxel dans l'huile de copaïba est un paramètre favorable en vue de l'association de ce principe actif à des systèmes de délivrance de médicaments issus des nanomédecines. La détermination de l’équilibre hydrophile-lipophile (HLB) requis pour l'huile de copaïba pour être compatible avec un mélange d'agents tensio-actifs capable de Discussion Générale 338 stabiliser les émulsions du type huile dans l'eau a été développé. Le système HLB est le rapport (ou balance) entre les portions hydrophiles de l'agent tensioactif non- ionique à la partie lipophile. Des valeurs HLB des agents stabilisants affectent la formation et stabilité des systèmes lipidiques dévelopés (Griffin, W.C, 1949). Donc, différents systèmes lipidiques développés. La valeur du HLB requis pour l'huile de copaïba a été déterminé expérimentalement en préparat différents systèmes lipidiques avec des mélanges de tensio-actifs de HLB différents et en étudiant les diagrammes de phase pseudo-ternaires des mélanges de tensioactifs / co-tensioactifs / huile de copaïba / eau. Les systèmes dispersés répondant au cahier des charge fixé ont été obtenus dans la région optimale de HLB du tensio-actif de 14,8, donnant un HLB requis pour l’huile de copaïba de 14.8. Il est intéressant de noter que la dispersion de l'huile de copaïba dans les systèmes émulsionnés était stable pendant plus d'un an, et différents systèmes dispersés (microémulsions, nanoémulsions…) ont été produites en utilisant des diagrammes de phases. En utilisant les informations obtenues sur l’HLB de l’huile de copaïba, un système lipidique plus stable avec une taille de gouttelettes plus petites pouvant transporter une plus grande quantité d'huile et de medicament ont été développés. Pour se faire, nous nous sommes intéressés à la formulation de microémulsions. D'après a litérature, ces systèmes ont un fort pouvoir de solubilisation des principes actives lipophiles. De plus, ils agissent comme des promoteurs d’absorption pour les molécules de classe IV comme le paclitaxel. Les microémulsions peuvent aussi moduler le profil de libération des principes actifs encapsulés ainsi que promouvoir l’absorption des formulations administrées par voie orale par le système lymphatique évitant ainsi l'effet du premier passage hépatique (Schmalfub et al., 1997; Tenjarla, 1999; Singh et al., 2011; Lawrence & Rees, 2012; Mcclements, 2012; Ritika et al., 2012; Lakshmi et al., 2013). L'approche Discussion Générale 339 suivie pour la formulation des microémulsions a été basée sur une analyse des paramètres de solubilité des composés dans le but de privilégier un choix basé sur leur miscibilité, a priori favorable pour favoriser la stabilité des interfaces huile/eau créées lors de la préparation des microémulsions. Cette démarche a permis de formuler des microémulsions contenant des fractions volumiques importantes d'huile essentielle de copaïba (19,6%) tout en maintenant la concentration en tensioactifs faible (13.7 %). Le paclitaxel a pu être incorporé dans les microémulsions avec une efficacité d’encapsulation de 37% donnant une concentration en paclitaxel de 0,37 mg.mL-1 de microémulsion. Cette incorporation ne perturbe pas notablement le diamètre des gouttelettes de la microémulsion ni la structure. Une étude de mucoadhésion a montré que la microémulsion contenant le paclitaxel permet de concentrer ce principe actif sur la muqueuse intestinale de rat. Dans cette thèse nous avons également chercher à développer une formulation de nanocapsules polyméres mucoadhésives pour le transport simultané de l’huile de copaïba et du paclitaxel par voie orale. Les nanocapsules ont été choisi car elles présentent un noyau huileux enveloppé dans une coque de polymère et que ceses systèmes se sont déjà montrés prometteurs pour permettre d'améliorer la biodisponibilité de molécules actives administrées par la voie orale (Cruz et al., 2006; Pinto Reis et al., 2006b; Leite et al., 2007; Anton et al., 2008). L'objectif du travail a été de développer des nanocapsules contenant de l'huile de copaïba et du paclitaxel avec des propriétés mucoadhésives grâce à l'utilisation de chitosane. Des nanocapsules ayant les propriétés recherchées ont été obtenus par le développent d'une méthode originale de polymérisation interfaciale du cyanoacrylate d'isobutyle en présence de chitosane comme agent assurant la stabilité colloïdale de la dispersion et devant promouvoir la mucoadhésion des formulations. L'obtention de nanocapsules de petite dimension et de Discussion Générale 340 potentiel zêta positif de valeur absolue élevée a été optimisez par un plan d’expérience à 2 niveaux en cosidérant trois variables indépendantes (pH, température et concentration du chitosane dans le milieu). Les systèmes développés et optimisés ont montré un diamètre de 473 nm, un potentiel zêta de +34 mV et une efficacité d'encapsulation de l’huile de copaïba de 75,8% soit une association de 55,5 µg de β- caryophyllène / mg de nanocapsules. Le procédé d'encapsulation n'a pas modifié la composition de l'huile qui est demeurée inchangée après encapsulation par rapport à l'huile de départ. Les valeurs du potentiel zêta positives sont en conformité avec la distribution attendue chitosane sur la surface des nanocapsules (Thanou et al., 2001; Mao et al., 2004). Le paclitaxel incorporé n’a pas modifiée la taille, la morphologie et le potentiel zêta des nanocapsules. Le rendement d'encapsulation du paclitaxel était 75%, ce qui correspond à 17 µg.mg -1 de nanocapsules. Cette formulation était stable en milieu gastrique reconstitué pendant 120 minutes et au bout de six mois à 4 °C en suspension dans l’eau. Les études de mucoadhésion ont permis de montrer que 9% de la quantité de paclitaxel apporté sous forme de nanocapsules avaient adhéré à une surface de 1 cm² de muqueuse intestinale après 2 heures d'incubation en chambre de Ussing ce qui correspond à une quantité de 3.4 g de nanocapsules par m² de la muqueuse intestinale. Ces systèmes ont montré une bonne corrélation avec d'autres nanoparticules mucoadhésives développés dans la littérature (Moghaddam et al., 2009b) et l'augmentation de la mucoadhesion en 2 fois par rapport autres études en utilisant nanoparticules recouverte avec chitosan 20 kDa (Bravo-Osuna, Vauthier, et al., 2007). Les deux systèmes développés dans cette thèse ont présenté une efficacité de transport du paclitaxel associé avec l'huile de copaïba par voie orale. Cependant, la quantité de paclitaxel dans les nanocapsules a été deux fois plus que les microémulsions et ceux-là ont été stable à séchage. Les nanocapsules ont aussi été systèmes plus performant pour Discussion Générale 341 le transport du paclitaxel, car ils ont présenté une plus haute mucoadhésion correspondant à une plus grande association du anticancéreux dans les nanocapsules avec la muqueuse intestinale chez le rat. 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Current status of drug delivery technologies and future directions. Pharm Technol, 251 - 14. WEAVER, B. A. 2014. How Taxol/paclitaxel kills cancer cells. Mol Biol Cell, 25, 18, 2677-81. Conclusion et Perspective Conclusion et Perspective 349 Les travaux présentés dans cette thèse, s’inscrivent dans le cadre de recherches menées sur le développement d’un nouveau système galénique oral pour la libération d'agents anticancéreux tel le paclitaxel, en utilisant de l’huile végétale de copaïba. L’objectif de ces travaux était donc de mettre au point une forme galénique optimale pour l’administration par voie orale pour le traitement du cancer utilisant des huiles végétales de copaïba. Le but recherché par la nouvelle formulation galénique est d’améliorer et de permettre la réduction des effets indésirables qui compliquent le suivi du traitement anticancéreux. Les travaux expérimentaux ont été réalisés grâce à un accord de coopération international établi entre le Laboratorio de Sistemas Dispersos (LASID) à l'Universidade Federal do Rio Grande (UFRN) à Natal, au Brésil et l’Institut Galien Paris-Sud (UMR 8612), dans l’Université Paris-Sud, à Châtenay Malabry, en France et à la mise en place d'un accord de cotutelle pour la préparation de la thèse. Dans cette thèse, des méthodes analytiques ont été développés et validées pour réaliser l'analyse qualitative et quatitative de l’huile de copaïba d'une part et pour doser le paclitaxel dans des milieux contenant de l'huile de copaiba d'autre part. Les méthodes développées chromatographies gazeuses et par l’HPLC ont montré une haute performance sur les paramètres de la validation. La méthode HPLC a été appliquée pour déterminer la solubilité et les coefficient de partage du paclitaxel dans l’huile de copaïba puis pour doser le paclitaxel dans les formulations développées avec l'huile de copaiba. . La formulation des émulsions et microémulsions huile dans eau contenant de l'huile de copaiba a été basée sur l'utilisation d'approches rationelles pour le choix des tensio- actifs. Dans le cas des émulsions, le HLB requis de l'huile a été recherché avec différents mélanges de tensio-actifs puis la formulation optimale a été identifiée en étudiant les diagrammes de phase pseudoternaires. Pour aborder la formulation de Conclusion et Perspective 350 micromulsions incorporant une phase dispersées d'huile importante à faible concentration de tensio-actif une autre stratégie a été adoptée. Elle a été basée sur la recherche de tensio-actifs pour lesquels la partie lipophile présentaient la meilleure miscibilité avec les composés de l'huile de copaiba sur la base de l'analyse des paramètres de solubilité. Cette démarche a permis de proposer une formulation de microémulsion montrant des performances très au dessus des microémulsions de la litérature en terme de quantité d'huile dispersées et une concentration en tensio-actif qui reste en dessous de celle des microémulsions de la litérature. Du paclitaxel a été incorporé à la microémulsion sans changer de manière importante les propriétés et en permettant d'augmenter de manière importante la quantité solubilisée par unité de volume comparée à la solubilité en milieu aqueux qui présente une limite pour l'administration de ce principe actif. Enfin, au cours de ce travail, il a été montré que le paclitaxel incorporé dans la microémulsion pouvait être capturé par la muqueuse intestinale de rat. Au cours de cette thèse, nous nous sommes aussi intéréssés à la formulation de nanocapsules mucoadhésives. Des nanocapsules originales incorporant de l'huile de copaiba et formées d'une enveloppe de poly(cyanoacrylate d'isobutyle) recouverte de chitosane ont été développées et optimisées par l'application d'un plan d'expérience. Nous avons montré qu’il était possible d’encapsuler du paclitaxel dans le cœur d’huile de copaïba de ces nanocapsules obtenues avec un rendement de fabrication satisfaisant et une teneur élevée en paclitaxel. Ces nanocapsules chargées en paclitaxel sont apparues stables dans les milieux gastro-intestinaux et dans les conditions de conservation étudiées. Ces nanocapsules ont été marquées avec une sonde fluorescente et du [3H]-paclitaxel radiomarqué permettant de démontrer leur intérêt pour leur Conclusion et Perspective 351 capacité à associer le paclitaxel à la muqueuse intestinale de rat grâce à leur propriété mucoadhésive. Les travaux réalisés au cours de cette thèse ont apporté deux formulations de paclitaxel mucoadhésives de nature différentes incorporant de l'huile de copaiba, une huile naturelle utilisée en médecine traditionnelle pour ses propriétés anticancéreuses. Les études menée sur l'évaluation de la capacité de ces systèmes à promouvoir une association du paclitaxel avec la muqueuse digestive ont montré un transfert de concentrations intéressantes de cette molécule au tissu intestinal de rat. Cependant, les nanocapsules ont été plus efficace par rapport à les microémulsions en ce qui concerne à la mucoadhésion dans la muqueuse intestinale chez le rat. De nombreuses perspectives s'ouvrent à l'issue de ce travail et peuvent être proposées pour la suite. En effet, plusieurs études complémentaires permettraient de conforter et d’affiner certaines hypothèses et d’approfondir certains aspects qui n'ont pas encore pu être explorés. Ainsi, les travaux sur ces formulations méritent d'être poursuivis en vue d'en évaluer la capacité à améliorer la biodisponibilité du paclitaxel administré par voie orale dans un traitement aux anticancéreux et à en comprendre le mécanisme d'action. Il sera également intéressant d'entreprendre des travaux pour étudier l'hypothèse d'une synergie d'action issue de l'association du paclitaxel avec l'huile de copaiba. La synergie pourrait intervenir à différents niveaux. L'huile de copaiba pourrait jouer un rôle de promoteur d'absorption au niveau de la muqueuse intestinale permettant ainsi d'augmenter significativement la biodisponibilité orale du paclitaxel. Des études complémentaires utilisant le modèle des chambres d’Ussing pourraient permettre d'élucider cet effet avant d'étudier l'impact de la présence de l'huile de copaiba dans les formulations sur la biodisponibilité du paclitaxel administré par voie orale chez Conclusion et Perspective 352 l'animal. L'huile de copaiba contenant des composants anti-cancéreux, celle du paclitaxel pourrait être potentialisée par celle des composants de l'huile. Pour ces études, nous pourrions envisager des travaux menés sur des lignées cellulaires en culture et destinés à évaluer l'activité anticancéreuses des formulations. Ces travaux pourront ensuite être complétés par une étude de l'efficacité d'un traitement appliqué à un modèle de tumeur développé chez l'animal. Dans le cas où une synergie pourra être mise en évidence, une nouvelle étude pourrait avoir pour objectif de réduire la dose de paclitaxel administrée. Une étude similaire pourrait aussi être proposée dans le cas où les formulations modifient fortement la pharmacocinétique et la biodistribution en favorisant une distribution dans le tissue tumoral. En fonction des résultats, les études toxicologiques aiguës et chroniques seront à entreprendre rapidement. 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Annexe Annexe 399 Annexe 400 Annexe 401 Annexe 402 Annexe 403 Annexe 404 Annexe 405 Annexe 406 Annexe 407 Curriculum Vitae Curriculum Vitae 411 Francisco Humberto XAVIER JUNIOR Universidade Federal do Rio Grande do Norte, CCS, Departamento de Farmácia. Laboratório de Sistemas Dispersos (LASID). R. Gustavo Cordeiro de Farias s/n. Petrópolis. CEP: 59010780, - Natal RN – Brasil Université Paris Sud, Institut Galien Paris-Sud - UMR CNRS 8612 - Faculté de Pharmacie, 92296 chatenay-Malabry Cedex – France. Mail: ffhxjunior@yahoo.com.br Né le 01 novembre 1986 à Conceição/PB – Brasil Nationalité brésillienne Formation 2013 - ce jour Doctorante en biopharmacie et technologie pharmaceutique UMR CNRS 8612 Physico-chimie, Pharmacotechnie et Biopharmacie, Faculté de Pharmacie, Université Paris-Sud 11. Châtenay-Malabry - France. 2011 - ce jour Doctorante en biotechnologie Programa de Pós-Graduação em Biotecnologia (Renorbio). Universidade Federal do Rio Grande do Norte, Laboratório de Sistemas Dispersos, à Natal, Brésil 2011 - ce jour Pharmacien clinique dans l'étude de phase III de vaccins pour les maladies infectieuses Centro de Estudos e Pesquisas em Molestias Infecciosas LTDA, sponsor Sanofi Pasteur AS 2011-2011 Maître de conférences en pharmacognosie, contrat de courte durée Universidade Federal do Rio Grande do Norte, Laboratório de Sistemas Dispersos, à Natal, Brésil 2008 - 2011 Master en Sciences de la Santé. Programa de Pós-Graduação em Ciências da Saúde, Universidade Federal do Rio Grande do Norte, Laboratório de Sistemas Dispersos, à Natal, Brésil 2004 - 2008 Licence en pharmacie. Universidade Federal do Rio Grande do Norte Curriculum Vitae 412 Compétences Expertise scientifique : Maîtrise des stratégies de devellopment des vector pour l’amélioration du passage des barrières biologiques par des médicaments, characterization physico-chimique des systèmes lipidiques et polimeriques, contrôle de la qualité physico-chimique et microbiologique des médicaments, encapsulation d’agents anticancéreux et de traceurs fluorescents, maitrise des moyens de solubilisation de principes actifs, domaine de l'extraction, l'isolement et la caractérisation des produits naturels, maitrise du plan d'expériences pour l'optimisation des processus, essais cliniques de phase III. Expertise technique: Chromatographie en phase liquide à haute performance (HPLC), chromatographie en phase gazeuse (GC-MS, GC-FID), Rheologie, mesures du diamètre par diffusion quasi-élastique de la lumière et du potentiel zêta, Microscopie électronique à transmission, radioactivité, infrarouge, l'analyse thermique, spectrophotométrie, lyophilisation, manipulation en culture cellulaire, culture de microrganismes, études in-vivo chez les rats, mucoadhésion et étude du passage de principes actifs à travers de la Chambre d’Ussing. Savoir-faire organisationnel : Encadrement de stagiaires, services administratifs et organisationnels, recherche des collaborations scientifiques, organisation de congrès scientifiques, communication orale et écrite. Langues : Portugais (langue maternelle); Français, Anglais et Espagnol (courants) Connaissances informatiques : Microsoft Office, Windows, Statistic, GraphPad, Origin, Endnote, Prezi, Lightroom, Photoshop Production scientifique Articles 1. ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., DANTAS, T. R. F., DANTAS-SANTOS, N., VERISSIMO, L. M., REHDER, V. L. G., CHAVES, G. M., OLIVEIRA, A. G., EGITO, E. S. T. Chemical Characterization and Antimicrobial Activity Evaluation of Natural Oil Nanostructured Emulsions. Journal of Nanoscience and Nanotechnology, v.15, p.880 - 888, 2015. 2. XAVIER-JUNIOR, F. H. , SILVA, K. G. H., FARIAS, I. E. G., MORAIS, A. R. V., ALENCAR, E. N., ARAÚJO, I. B., OLIVEIRA, A. G., EGITO, E. S. T. Prospective study for the development of emulsion systems containing natural oil products. Journal of Drug Delivery Curriculum Vitae 413 Science and Technology. , v.22, p.367-372, 2012. 3. SILVA, K. G. H., XAVIER-JUNIOR, F. H. , FARIAS, I. E. G., SILVA, A. K. A., SOUZA, L. C. A., CALDAS NETO, J. A., SANTIAGO, R. R., ALEXANDRINO-JUNIOR, F., NAGASHIMA JUNIOR, T., SORES, L. A. L., MAGALHÃES, N. S. S., EGITO, E. S. T. Stationary cuvette: a new approach to obtaining analytical curves by UV-VIS spectrophotometry. Phytochemical Analysis on line. , v.20, p.265 - 271, 2009. 4. SILVA, K. G. H., XAVIER-JUNIOR, F. H. , FARIAS, I. E. G., CALDAS NETO, J. A., SILVA, A. K. A., NAGASHIMA JUNIOR, T., MEDEIROS, A. C., DIMESTEIN, EGITO, E. S. T. A new insight about pharmaceutical dosage forms for benzathine penicillin G. Revista de Ciências Farmacêuticas Básica e Aplicada. , v.27, p.21 - 26, 2006. Communications Scientifiques avec résumé étendu publiés 1. FARIA, M. A. S., ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., MACHADO, L. A., RUTCKEVISKI, R., DANTAS, T. R. F., OLIVEIRA, C. M., DANTAS- SANTOS, N., CHAVES, G. M., EGITO, E. S. T. Emulsions based on copaiba essential oil: a source of treatment for infectious diseases In: XXI International Conference on Bioencapsulation, 2013, Berlim. 2. FARIA, M. A. S., ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., MACHADO, L. A., RUTCKEVISKI, R., DANTAS, T. R. F., OLIVEIRA, C. M., DANTAS- SANTOS, N., CHAVES, G. M., EGITO, E. S. T. Evaluation of antibiofilm activity of cobaiba essential oil emulsion In: XXI International Conference on Bioencapsulation, 2013, Berlim. 3. SILVA-FILHO, M. A., MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MACHADO, L. A., OLIVEIRA, C. M., DANTAS, T. R. F., SILVEIRA, W. L. L., DANTAS- SANTOS, N., EGITO, E. S. T. Evaluation of the antifungal activity of microemulsions containing Amphotericin B after the lyophilization process In: XXI International Conference on Bioencapsulation, 2013, Berlim. 4. SILVA-FILHO, M. A., MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., RUTCKEVISKI, R., OLIVEIRA, C. M., DANTAS, T. R. F., DANTAS-SANTOS, N., SILVA- JUNIOR, A. A., EGITO, E. S. T. Preliminary design of freeze-dried microemulsion containing Amphotericin B In: XXI International Conference on Bioencapsulation, 2013, Berlim. 5. SILVA-FILHO, M. A., RUTCKEVISKI, R., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., ALENCAR, E. N., MACHADO, L. A., OLIVEIRA, C. M., DANTAS, T. R. F., DANTAS- SANTOS, N., EGITO, E. S. T. Quality control of bullfrog (Rana catesbeiana Shaw) oil: a contribuition for the extraction and chemical identification In: XXI International Conference on Bioencapsulation, 2013, Berlim. 6. SILVA, K. G. H., MARTINS, A. S., XAVIER-JUNIOR, F. H. , EGITO, E. S. T. Copaiba oil microemulsion containing ketoconazole: Assessment of the rate of encapsulation In: XVIII International Conference on Bioencapsulation, 2010, Lisboa. 7. XAVIER-JUNIOR, F. H. , SILVA, K. G. H., ALENCAR, E. N., MORAIS, A. R. V., ARAÚJO, I. B., EGITO, E. S. T. Phase diagrams of copaiba oil: comparative study on their production process In: XVIII International Conference on Bioencapsulation, 2010, Porto. 8. SILVA, K. G. H., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., ALENCAR, E. N., EGITO, E. S. T. Copaiba oil microemulsions: A new system for future use in inflammatory diseases In: XVIIth International Conference on Bioencapsulation, 2009, Groningen, Netherlands. Curriculum Vitae 414 Communications Scientifiques avec résumés publiés 1. MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MACHADO, L. A., RUTCKEVISKI, R., OLIVEIRA, C. M., DANTAS, T. R. F., BARRATT, G., EGITO, E. S. T. Preparation of freeze-dried emulsion system based on copaíba oil by design of experiment In: XIV Journée de l’école de doctorale, 2014, Paris. 2. MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MACHADO, L. A., DANTAS, T. R. F., OLIVEIRA, C. M., DANTAS-SANTOS, N., VERISSIMO, L. M., EGITO, E. S. T. Analyses of the glass transition temperature of microemulsion containing glucose and lactose by differential scanning calorimetry In: 3rd Conference on Innovation in Drug Delivery - Advances in Local Drug Delivery, 2013, Pisa. 3. ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., MACHADO, L. A., DANTAS, T. R. F., OLIVEIRA, C. M., DANTAS-SANTOS, N., CHAVES, G. M., VERISSIMO, L. M., EGITO, E. S. T. Copaiba oil-resin (Copaifera langsdorfii) emulsion as potent biofilm inhibitors In: 3rd Conference on Innovation in Drug Delivery - Advances in Local Drug Delivery, 2013, Pisa. 4. XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MORAIS, A. R. V., MACHADO, L. A., RUTCKEVISKI, R., DANTAS-SANTOS, N., EGITO, E. S. T., VAUTHIER, C. Emulsions based on natural oils as antimicrobial agents In: XIIIèmes Journées de I'École Doctorale - Innovaton Thérapeutique, 2013, Kremlin-Bicêtre. 5. MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., DANTAS, T. R. F., OLIVEIRA, C. M., NASCIMENTO, A. E. G., RUTCKEVISKI, R., DANTAS-SANTOS, N., EGITO, E. S. T. Influence of cryoprotectants on the surface tension of microemulsions In: 3° Encontro Brasileiro para Inovação Terapêutica, 2013, Jaboatão dos Guararapes. 6. MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MACHADO, L. A., OLIVEIRA, C. M., DANTAS, T. R. F., DANTAS-SANTOS, N., EGITO, E. S. T. Optimization of lyophilization process of microemulsion containing lactose and glucose by experiment design In: 3° Encontro Brasileiro para Inovação Terapêutica, 2013, Jaboatão dos Guararapes. 7. DANTAS, T. R. F., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MORAIS, A. R. V., DANTAS-SANTOS, N., EGITO, E. S. T. A influência do potencial zeta na estabilidade de sistemas dispersos contendo ativos naturais In: XIV Congresso Científico da UnP e XIII Mostra de Extensão da UnP, 2012, Natal. 8. SILVA, K. G. H., ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., DANTAS-SANTOS, N., CHAVES, G. M., EGITO, E. S. T.Antibiofilm Activity of Bullfrog (Rana Castebeiana Shaw) Oil Emulsion In: Groupe Thematique de Recherche sur la Vectorisation - GTRV, 2012, Paris. 9. ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., DANTAS, A. B., PAULA, P. R., CHAVES, G. M., DANTAS-SANTOS, N., EGITO, E. S. T.Anti-Candida activity of Copaiba Oils: A comparative study between volatile and resin oils In: IV Simpósio Nacional de Produtos Naturais, 2012, João Pessoa. 10. ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., DANTAS, T. R. F., DANTAS-SANTOS, N., EGITO, E. S. T.Atividade anti-Staphylococcus do óleo essencial e resina de Copaíba (Copaifera langsdorffii): estudo comparativo In: XIV Congresso Científico da UnP e XIII Mostra de Extensão da UnP, 2012, Natal. 11. MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., OLIVEIRA, C. M., Curriculum Vitae 415 DANTAS-SANTOS, N., EGITO, E. S. T.Caracterização de sistemas microemulsionados de óleo de copaíba a partir de estudos termoanalíticos em calorimetria exploratória diferencial In: XIV Congresso Científico da UnP e XIII Mostra de Extensão da UnP, 2012, Natal. 12. OLIVEIRA, C. M., MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., DANTAS-SANTOS, N., EGITO, E. S. T.Centrifugação analítica como método para determinação da estabilidade de sistemas emulsionados In: XIV Congresso Científico da UnP e XIII Mostra de Extensão da UnP, 2012, Natal. 13. SILVA, K. G. H., ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., DANTAS-SANTOS, N., CHAVES, G. M., EGITO, E. S. T.Copaiba Oil (Copaifera langsdorffii) Emulsions: An Alternative to Treat Cutaneous Infections In: Groupe Thematique de Recherche sur la Vectorisation - GTRV, 2012, Paris. 14. MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., PAULA, P. R., DANTAS-SANTOS, N., EGITO, E. S. T. opaiba oil emulsions: characterization of pseudoternary phase diagram by analytical centrifugation In: IV Simpósio Nacional de Produtos Naturais, 2012, João Pessoa. 15. MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., OLIVEIRA, C. M., DANTAS-SANTOS, N., EGITO, E. S. T. Crioprotetores e o seu mecanismo de ação em sistemas coloidais liofilizados In: XIV Congresso Científico da UnP e XIII Mostra de Extensão da UnP, 2012, Natal. 16. XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MORAIS, A. R. V., PAULA, P. R., OLIVEIRA, C. M., DANTAS-SANTOS, N., EGITO, E. S. T. Critical HLB of Rana catesbeiana oil emulsions: Preliminary study In: IV Simpósio Nacional de Produtos Naturais, 2012, João Pessoa. 17. ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., DANTAS, T. R. F., DANTAS-SANTOS, N., EGITO, E. S. T. Desenvolvimento de sistemas emulsionados sem tensoativos contendo Óleo de Copaíba: estudo In: XIV Congresso Científico da UnP e XIII Mostra de Extensão da UnP, 2012, Natal. 18. XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., ALENCAR, E. N., PAULA, P. R., OLIVEIRA, C. M., DANTAS-SANTOS, N., EGITO, E. S. T. Development of emulsified systems based on Bullfrog (Rana catesbeiana) oil In: IV Simpósio Nacional de Produtos Naturais, 2012, João Pessoa. 19. OLIVEIRA, C. M., MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., DANTAS-SANTOS, N., EGITO, E. S. T. Efeito da cristalização em soluções de crioprotetores para liofilização de sistemas coloidais In: XIV Congresso Científico da UnP e XIII Mostra de Extensão da UnP, 2012, Natal. 20. DANTAS-SANTOS, N., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MORAIS, A. R. V., MACHADO, L. A., EGITO, E. S. T. Estudo das propriedades terapêuticas da rã-touro (Rana catesbeiana Shaw) In: XIV Congresso Científico da UnP e XIII Mostra de Extensão da UnP, 2012, Natal. 21. XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MORAIS, A. R. V., DANTAS-SANTOS, N., REHDER, V. L. G., EGITO, E. S. T. Extraction, emulsion development and CG-MS characterization of copaiba essential oil (Copaifera langdorffii). In: Groupe Thematique de Recherche sur la Vectorisation - GTRV, 2012, Paris. 22. ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., SOUZA, E. S., Curriculum Vitae 416 PAULA, P. R., DANTAS, A. B., OLIVEIRA, C. M., DANTAS-SANTOS, N., REHDER, V. L. G., CHAVES, G. M., EGITO, E. S. T. Gas chromatography-mass spectrometryanalysis of antimicrobial compounds of bullfrog (Rana catesbeiana shaw) oil In: XXI Congresso Latinoamericano de Microbiologia (XXI ALAM), 2012, Santos 23. ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., PAULA, P. R., EGITO, E. S. T. Identification of antimicrobial compounds of Copaiba oil by Bioautography: a preliminary study In: AAPS Anual Meeting and Exposition, 2012, Chicago. 24. MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., OLIVEIRA, C. M., DANTAS-SANTOS, N., EGITO, E. S. T. Influence of Multiple Lyophilization Factors on the Microemulsion Droplet Size: An Experimental Design In: Groupe Thematique de Recherche sur la Vectorisation - GTRV, 2012, Paris. 25. GOES, P. A., XAVIER-JUNIOR, F. H. , ALVES, M.S.C.F Integralidade na Promoção da Saúde do Idoso: Relato de Experiência In: World Nutrition Rio2012, 2012, Rio de Janeiro. 26. MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., PAULA, P. R., SILVEIRA, W. L. L., OLIVEIRA, A. G., EGITO, E. S. T. Lyophilization of microemulsions: Influence of different cryoprotectants in their droplet size In: AAPS Anual Meeting and Exposition, 2012, Chicago. 27. ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., DANTAS, A. B., PAULA, P. R., DANTAS-SANTOS, N., CHAVES, G. M., EGITO, E. S. T. Natural oils as potent inhibitors of biofilm formation In: IV Simpósio Nacional de Produtos Naturais, 2012, João Pessoa. 28. DANTAS-SANTOS, N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., ALENCAR, E. N., MACHADO, L. A., EGITO, E. S. T. Obtenção de sistemas coloidais contendo Óleo de Copaíba, Tween 20, Phosal 50 e água In: XIV Congresso Científico da UnP e XIII Mostra de Extensão da UnP, 2012, Natal. 29. MACHADO, L. A., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., ALENCAR, E. N., DANTAS-SANTOS, N., EGITO, E. S. T. Perfil de prescrição de antimicrobianos no Hospital Universitário Dr. Mário Araújo de Bagé In: XIV Congresso Científico da UnP e XIII Mostra de Extensão da UnP, 2012, Natal. 30. GOES, P. A., XAVIER-JUNIOR, F. H. . Práticas educativas como promoção de saúde- Relato de experiencia em um grupo de idosos In: Simpósio Norteriograndense de Geriatria e Gerontologia, 2011, Natal. 31. DANTAS, T. R. F., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MORAIS, A. R. V., DANTAS-SANTOS, N., EGITO, E. S. T. Recentes avanços do desenvolvimento de sistemas de liberação contendo produtos naturais para tratamento de doenças infecciosas In: XIV Congresso Científico da UnP e XIII Mostra de Extensão da UnP, 2012, Natal. 32. MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., OLIVEIRA, C. M., DANTAS-SANTOS, N., EGITO, E. S. T. Study of Microemulsion Glass Trasition Temperature Containing Maltose and Mannitol by Differential Scanning Calorimetry In: Groupe Thematique de Recherche sur la Vectorisation - GTRV, 2012, Paris. 33. MACHADO, L. A., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., ALENCAR, E. N., DANTAS-SANTOS, N., EGITO, E. S. T. Uso de ativos naturais no tratamento de asma: revisão da literatura In: XIV Congresso Científico da UnP e XIII Mostra de Extensão da UnP, 2012, Natal. Curriculum Vitae 417 34. SILVA, G. H. F., MELO, M. C. B., XAVIER-JUNIOR, F. H. . Análise do indice peso/idade em crianças beneficiadas do Programa Bolsa Familia In: 11° Congresso Nacional de Nutrição Baseada em Evidencia, 2011, Fortaleza. 35. ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., SILVA, K. G. H., PAULA, P. R., EGITO, E. S. T. Antimicrobial Activity Evaluation of Copaiba Oil Microemulsion System: A Preliminary Study In: II internacional Symposium of Pharmaceutical Sciences, 2011, Natal. 36. MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., SILVA, K. G. H., PAULA, P. R., EGITO, E. S. T. Aplicação da Análise Térmica no Controle de Qualidade de Óleos Vegetais In: XIII Congresso Científico e a XII Mostra de Extensão, 2011, Natal. 37. XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MORAIS, A. R. V., SILVA, K. G. H., BARBOSA, L. M. Q., ARAÚJO, I. B., EGITO, E. S. T. Comparação de metodologias para determinação da atividade antifungica do oleo de copaiba In: I Workshop Brasileiro de Tecnologia Farmacêutica e Inovação, 2011, Aracaju. 38. FERREIRA, L. F., SILVEIRA, W. L. L., XAVIER-JUNIOR, F. H. , DAMASCENO, B. P. G. L., EGITO, E. S. T. Development and Validation of an Analytical Method for Quantification of Simvastatin in Microemulsion by High Performance Liquid Chromatography In: II International Symposium of Pharmaceutical Sciences, 2011, Natal. 39. ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., SILVA, K. G. H., PAULA, P. R., EGITO, E. S. T. Emulsões Sem Tensoativos: Recentes Avanços e Aplicabilidades In: XIII Congresso Científico e a XII Mostra de Extensão, 2011, Natal. 40. XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MORAIS, A. R. V., SILVA, K. G. H., ARAÚJO, I. B., EGITO, E. S. T. Estudo de parâmentros físico-químicos do cloranfenicol e seu doseamento em microemulsões In: I Workshop Brasileiro de Tecnologia Farmacêutica e Inovação, 2011, Aracaju. 41. SILVA, G. H. F., MELO, M. C. B., XAVIER-JUNIOR, F. H. . Indices antropométricos e o acompanhamento nutricional de crianças beneficadas do Programa Bolsa Familia In: 11° Congresso Nacional de Nutrição Baseada em Evidencia, 2011, Fortaleza. 42. XAVIER-JUNIOR, F. H. Inserção sócio-cultural utilizando o método científico In: XVII semana de ciência, tecnologia e cultura, 2011, Natal. 43. ALENCAR, E. N., XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., SILVA, K. G. H., PAULA, P. R., EGITO, E. S. T. Métodos de Análise Antimicrobiana de Óleos Vegetais In: XIII Congresso Científico e a XII Mostra de Extensão, 2011, Natal. 44. MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , ALENCAR, E. N., SILVA, K. G. H., PAULA, P. R., EGITO, E. S. T. O Processo de Liofilização nas Indústrias: Revisão Bibliografica In: XIII Congresso Científico e a XII Mostra de Extensão, 2011, Natal. 45. SILVA, G. H. F., MELO, M. C. B., XAVIER-JUNIOR, F. H. . Programa Bolsa Familia: Negligencia ao acompanhamento das medidas antropométricas de crianças beneficiadas In: 11° Congresso Nacional de Nutrição Baseada em Evidencia, 2011, Fortaleza. 46. XAVIER-JUNIOR, F. H. , SILVA, K. G. H., ALENCAR, E. N., MORAIS, A. R. V., SANTOS, E. C. G., EGITO, E. S. T. Controle de qualidade microbiológico e estudo da atividade antibacteriana de sistemas microemulsionados e do óleo de copaíba contra Curriculum Vitae 418 staphylococcus epidermidis In: 62ª Reunião Anual da Sociedade Brasileira para o Progresso da Ciência, 2010, Natal. 47. XAVIER-JUNIOR, F. H. , SILVA, K. G. H., MORAIS, A. R. V., ALENCAR, E. N., SANTOS, E. C. G., EGITO, E. S. T. Estudo da concentração inibitória mínima do óleo de copaíba em cepas bacterianas do gênero staphylococcus In: 62ª Reunião Anual da Sociedade Brasileira para o Progresso da Ciência, 2010, Natal. 48. XAVIER-JUNIOR, F. H. , SILVA, K. G. H., SANTOS, E. C. G., MORAIS, A. R. V., ALENCAR, E. N., ALEXANDRINO-JUNIOR, F., ARAÚJO, I. B., EGITO, E. S. T.Evaluation of antibacterial activity of copaiba oil colloidal systems In: 70th FIP World Congress of Pharmacy/Pharmaceutical Sciences, 2010, Lisboa. 49. XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., ALENCAR, E. N., SILVA, K. G. H., ARAÚJO, I. B., EGITO, E. S. T. Incorporação de cloranfenicol em microemulsão de óleo de copaíba In: XXI Simpósio de Plantas Medicinais do Brasil, 2010, João Pessoa. 50. XAVIER-JUNIOR, F. H. , SILVA, K. G. H., ALEXANDRINO-JUNIOR, F., SILVA, A. K. A., ALENCAR, E. N., MORAIS, A. R. V., ARAÚJO, I. B., EGITO, E. S. T. Introducing the scientific method in the high school classroom: Pharmaceutical Sciences as a background In: 70th FIP World Congress of Pharmacy/Pharmaceutical Sciences, 2010, Lisboa. 51. XAVIER-JUNIOR, F. H. , MORAIS, A. R. V., ALENCAR, E. N., SILVA, K. G. H., ARAÚJO, I. B., EGITO, E. S. T. O ensino de praticas em saúde por meio de instrumentos pedagógicos In: II Congresso Multiprofissional da Saúde, 2010, Natal. 52. XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MORAIS, A. R. V., SILVA, K. G. H., SANTOS, E. C. G., ARAÚJO, I. B., EGITO, E. S. T. Óleo de Copaíba como antifúngico natural: estudo pela técnica de difusão em ágar In: XXI Simpósio de Plantas Medicinais do Brasil, 2010, João Pessoa. 53. SILVA, K. G. H., XAVIER-JUNIOR, F. H. , FARIAS, I. E. G., SANTIAGO, R. R., ALEXANDRINO-JUNIOR, F., EGITO, E. S. T. A new tool to obtaining analytical curves by UV-Vis spectrophotometry In: 21 st International Symposium on Pharmaceutical and Biomedical Analysis, 2009, Orlando, USA. 54. XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MORAIS, A. R. V., SILVA, K. G. H., SANTOS, E. C. G., EGITO, E. S. T., MORAIS, A. R. V. Avaliação da atividade antimicrobiana do óleo de copaíba In: I Simpósio Nacional em Ciências Farmacêuticas Básicas e Aplicadas : Fronteiras do Conhecimento e Políticas de Inovação, 2009, Natal. 55. XAVIER-JUNIOR, F. H. , SILVA, K. G. H., ALENCAR, E. N., MORAIS, A. R. V., ARAÚJO, I. B., EGITO, E. S. T. Avaliação do Perfil dos Alunos de Escolas Públicas Participantes dos Cursos de Férias de Farmácia In: XI Congresso Científico: Educação, Inclusão E Sustentabilidade: Grandes Desafios Da Ciência, 2009, Natal. 56. ALENCAR, E. N., XAVIER-JUNIOR, F. H. , SILVA, K. G. H., MORAIS, A. R. V., EGITO, E. S. T. Controle de Qualidade do Óleo de Copaíba: Estudo de Pré-Formulação para o Desenvolvimento de Sistemas Emulsionados In: XX Congresso de Iniciação Científica da UFRN - CIC2009, 2009, Natal. 57. MORAIS, A. R. V., XAVIER-JUNIOR, F. H. , SILVA, K. G. H., ALENCAR, E. N., EGITO, E. S. T. Desenvolvimento de Diagramas de Fases Pseudoternário de Óleo de Copaíba/Tween 20/ Phospholipon 100H e Água In: XX Congresso de Iniciação Científica da UFRN - CIC2009, 2009, Natal. Curriculum Vitae 419 58. XAVIER-JUNIOR, F. H. , SILVA, K. G. H., FARIAS, I. E. G., ALENCAR, E. N., MORAIS, A. R. V., ARAÚJO, I. B., NAGASHIMA JUNIOR, T., EGITO, E. S. T. Development of Based Microemulsion System of Copaiba Oil In: 7th International Congress of Pharmaceutical Sciences CIFARP, 2009, Ribeirão Preto. 59. XAVIER-JUNIOR, F. H. , SILVA, K. G. H., MORAIS, A. R. V., ALENCAR, E. N., ARAÚJO, I. B., EGITO, E. S. T. Diagrama de Fases de Óleo de Copaíba: Estudo Comparativo para a Produção de Microemulsão In: XI Congresso Científico: Educação, Inclusão E Sustentabilidade: Grandes Desafios Da Ciência, 2009, Natal. 60. XAVIER-JUNIOR, F. H. , ALENCAR, E. N., MORAIS, A. R. V., SILVA, K. G. H., ARAÚJO, I. B., EGITO, E. S. T. Diagramas de Fases Pseudoternário do Óleo de Copaíba: Estudo de Regiões de Emulsões In: XI Congresso Científico: Educação, Inclusão E Sustentabilidade: Grandes Desafios Da Ciência, 2009, Natal. 61. XAVIER-JUNIOR, F. H. , SILVA, K. G. H., MORAIS, A. R. V., ALENCAR, E. N., ARAÚJO, I. B., EGITO, E. S. T. Freeze-Dried Microemulsion Copaiba Oil: Preliminary Studies In: 7th International Congress of Pharmaceutical Sciences CIFARP, 2009, Ribeirão Preto. 62. MARTINS, A. S., XAVIER-JUNIOR, F. H. , GUARDA, L. T. A., LOUSADO, E. S. Judicialização do direito a saúde: uma abordagem crítica In: XI Congresso Científico e X Mostra de Extensão da Universidade, 2009, Natal. 63. XAVIER-JUNIOR, F. H. , SILVA, K. G. H., MORAIS, A. R. V., ALENCAR, E. N., ARAÚJO, I. B., EGITO, E. S. T. Perfil dos Professores Participantes dos Cursos de Férias de Farmácia In: XI Congresso Científico: Educação, Inclusão E Sustentabilidade: Grandes Desafios Da Ciência, 2009, Natal. Résumé Des systèmes dispersés pour la voie orale contenant dans leur phase interne de l'huile de copaïba servant de véhicules pour le paclitaxel ont été développées. Des microémulsions et des nanocapsules bioadhésives ont été formulées selon deux approches originales, l’une basée sur l’appariement chimique des composés de la microémulsion, et l’autre et sur la mise en œuvre d’un plan d'expérience. Deux méthodes d’analyses originales ont été développées et validées destinées à l’analyse de la composition de l’huile de copaiba et au dosage du paclitaxel dans les formulations contenant de l’huile de copaiba. MOTS CLES : voie orale, huile de copaïba, paclitaxel, microémulsion, nanocapsules, équilibre hydrophile-lipophile, chitosane, mucoadhésion. LABORATOIRE DE RATTACHEMENT : Institut Galien Paris Sud, UMR CNRS 8612 Equipe 6 : Amélioration du passage des barrières par les molécules biologiquement actives 5, Rue Jean Baptiste Clément 92296 Châtenay-Malabry, France PÔLE : PHARMACOTECHNIE ET PHYSICO-CHIMIE PHARMACEUTIQUE UNIVERSITÉ PARIS-SUD 11 UFR «FACULTÉ DE PHARMACIE DE CHATENAY-MALABRY » 5, rue Jean Baptiste Clément 92296 CHÂTENAY-MALABRY Cedex