Orientadores: Maria Cristina Volpato, Eneida de Paula, Francisco Carlos Groppo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-15T12:59:46Z (GMT). No. of bitstreams: 1 Zago_PatriciaMariaWiziack1982-_M.pdf: 1548841 bytes, checksum: 532fa0a5d09fdd83740f529142e2e993 (MD5) Previous issue date: 2010 / Resumo: Estudos em animais têm demonstrado que a encapsulação lipossomal da prilocaína aumenta sua eficácia anestésica em tecidos moles. Este estudo randomizado, cruzado e cego teve por objetivo avaliar a eficácia anestésica da formulação lipossomal de prilocaína 3% comparada à prilocaína 3% sem aditivos e prilocaina 3% com felipressina 0,03UI/mL, aplicadas por técnica infiltrativa na região vestibular do canino superior direito em 32 voluntários. As formulações foram aplicadas em 3 sessões, com ordem aleatória de aplicação e intervalo mínimo de 1 semana. O sucesso, a latência e a duração da anestesia pulpar foram avaliados com aplicação de estímulo elétrico no incisivo lateral, canino e primeiro pré-molar superiores; a latência e duração da anestesia em tecidos moles foram avaliadas por pressão com instrumento rombo na gengiva inserida da região vestibular do canino superior direito e a dor à injeção por meio da escala analógica visual (EAV). Considerou-se como sucesso anestésico quando a latência foi menor ou igual a 10 minutos com duração mínima de 10 minutos. Os voluntários e o pesquisador que avaliou as anestesias não tinham conhecimento da formulação aplicada. Os resultados foram submetidos aos testes Kruskal-Wallis (latência e duração da anestesia pulpar), Tuckey (EAV), Friedman (duração da anestesia gengival), Log Rank e McNemar (sucesso). A formulação lipossomal apresentou resultados semelhantes à formulação sem aditivos (p>0,05) e estatisticamente inferiores à prilocaína com felipressina (p<0,05), com relação à duração de anestesia gengival e sucesso e duração de anestesia pulpar para o canino e pré-molar. Com relação a latência e sucesso da anestesia no incisivo lateral, a prilocaína lipossomal não diferiu das demais formulações (p>0,05) e a prilocaina sem aditivos apresentou menor sucesso e maior latência (p<0,05) que a prilocaína com felipressina. As formulações não diferiram quanto à duração de anestesia pulpar para o incisivo lateral, dor à injeção e latência anestésica para canino, pré-molar e gengiva (p>0,05). Conclui-se que a prilocaína lipossomal apresenta eficácia anestésica semelhante à solução sem aditivos e menor eficácia do que a solução de prilocaína com felipressina em infiltração na maxila, não havendo, portanto, vantagem no seu uso / Abstract: Animal studies have shown that liposome encapsulation increases prilocaine anesthetic efficacy in soft tissues. This randomized, blind, crossover, three period study evaluated the anesthetic efficacy of liposome-encapsulated 3% prilocaine compared to 3% plain prilocaine and 3% prilocaine with 0.03IU/mL felypressin, after 1.8mL infiltration in the buccal sulcus of the maxillary right canine, in 32 volunteers. The formulations were randomly applied in three sessions, spaced one week apart. Anesthesia success and onset and duration of pulpal anesthesia in the lateral incisor, canine and first premolar were evaluated by using an electric pulp tester; onset and duration of soft tissue anesthesia were evaluated by pinprick test in the buccal attached gingiva of maxillary right canine. Injection pain was analyzed through Visual Analogue Scale (VAS). Anesthesia was considered successful when the onset time was less than 10 minutes and the duration was at least 10 minutes. The volunteers and the researcher who evaluated anesthesia parameters were blind to the formulations injected. Results were submitted to Kruskal-Wallis (onset and duration of pulpal anesthesia), Tuckey (VAS), Friedman (duration of gingival anesthesia), Log-Rank and McNemar tests (anesthesia success), (?=5%). Liposomal prilocaine showed similar results in comparison to plain prilocaine (p>0.05), and lower anesthesia success and duration for canine and premolar and for duration of gingival anesthesia (p<0.05) than prilocaine with felypressin. Liposomal prilocaine did not differ from the other formulations concerning onset and anesthesia success for the lateral incisor (p>0.05); plain prilocaine presented lower success rate and slower onset of anesthesia for this tooth in comparison to prilocaine with felypressin (p<0.05). No differences were observed among the formulations in relation to duration of anesthesia for lateral incisor, VAS scores and onset of gingival and pulpal anesthesia for canine and premolar (p>0.05). In conclusion, liposomal prilocaine presents similar anesthetic efficacy in relation to plain prilocaine and lower efficacy in comparison to prilocaine with felypressin in maxillary infiltration. Therefore, there is no advantage in the use of this formulation / Mestrado / Farmacologia, Anestesiologia e Terapeutica / Mestre em Odontologia

O principal problema no desenvolvimento de vacinas de segunda geração é a baixa resposta contra os antígenos, geralmente extremamente purificados. Isto tem levado à busca de novos adjuvantes que sejam mais eficientes e menos tóxicos. Este trabalho objetivava o estudo de uma proteína de choque térmico de Mycobacterium leprae - 18kDa-hsp - recombinante, produzida em levedura, veiculada em lipossomos, como modelo de proteína carregadora para antígenos fracos. Além de um estudo de estabilização de lipossomos por liofilização. A 18kDa-hsp apresenta certas características peculiares que levaram ao desenvolvimento de um método de dosagem próprio e extensível a outras proteínas com baixo teor em aminoácido cromóforos. Tendo uso potencial como proteína carregadora, a 18kDa-hsp é estável, suportando bem aquecimento, liofilização, acilação e associação com lipossomos de fosfolípides, sem perda significativa de sua atividade. Estudos paralelos de estabilização de lipossomos unilamelares mostraram que se pode usar trealose como crioprotetora no processo de liofilização-reidratação, sem perdas significativas do conteúdo dos lipossomos e com manutenção das estruturas das vesículas. Finalmente, o sistema lipossomos-18kDa-hsp mostra-se promissor como um sistema estável para veiculação de imunógenos fracos. / The major problem on development of second generation vaccines is the poor response against some immunogens. It has leading an intense research to find other substances non toxic and more efficient on enhancement of the immunological response. The aim of this work was the study of a heat-shock protein from Mycobacterium leprae - 18kDa-hsp - produced in yeast and vehiculated in liposomes, as a protein carrier model for weak immunogens. Furthermore, there was a study on stabilization of liposomes by lyophilization. The peculiar protperties of the 18kDa-hsp lead us to develop a new method for quantification that can be extended to other proteins with low chromophore content. Potentially a carrier protein, the 18kDa-hsp is very stab1e: it has no significant loss of its antigenic reactivity when heated, lyophilized, acylated and associated to liposomes. Parallel studies on liposomes stabilisation show trehalose as a good cryoprotector. The sugar can avoid the leakage of the entrapped marker (protein) and maintain the vesicles structures on the lyophilisation-rehydration processo Finally, the system liposomes-18kDa-hsp is promising for vehiculation of weak immunogens.

Orientador: Maria Helena Andrade Santana / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-23T12:48:43Z (GMT). No. of bitstreams: 1 Quinalia_MarianaBeatriz_M.pdf: 3702329 bytes, checksum: 7ed3bbb46f028abad9a9d677dbda482d (MD5) Previous issue date: 2013 / Resumo: Processos de alto cisalhamento têm sido utilizados para redução e homogeneização da distribuição de tamanhos de lipossomas em sua produção escalonável. No entanto, a aplicação do cisalhamento pode modificar estruturalmente essas nanopartículas, influenciando a incorporação de fármacos hidrofóbicos. Neste estudo, a chalcona sintética 3-nitro-2-hidroxi- 4,6-dimetoxichalcona (CH8), fármaco altamente hidrofóbico indicado para tratamento de leishmaniose cutânea, foi incorporada em lipossomas convencionais produzidos por processos escalonáveis de alto cisalhamento. Os lipossomas foram compostos por fosfatidilcolina de ovo e produzidos por injeção de etanol. Foram utilizados agitador mecânico com impelidor Cowles, Ultra Turrax® e microfluidizador para reduzir e homogeneizar o tamanho das partículas. A influência da taxa de cisalhamento foi controlada pelo método de Bangham seguido por extrusão em membranas. Espectros de espalhamento de raios-X a baixo ângulo mostraram que o cisalhamento leva a uma redução na distância interlamelar dos lipossomas, quando comparados ao controle. A distância interlamelar não variou com a intensidade do cisalhamento na faixa de agitação obtida com o impelidor Cowles, mas diminuiu com o aumento do cisalhamento provocado pelo Ultra Turrax®. A constante elástica, relacionada com a rigidez da bicamada e dependente da estrutura dos lipossomas, aumentou proporcionalmente com a taxa de cisalhamento para os dois agitadores. O cisalhamento também diminuiu o diâmetro médio dos lipossomas e a distribuição de tamanhos. Inicialmente, CH8 foi incorporada em lipossomas pré-formados. Os resultados mostraram que a expulsão e precipitação do fármaco incorporado foram proporcionais à intensidade do cisalhamento aplicado. Essa limitação foi contornada pela incorporação de uma baixa concentração de CH8 durante a formação dos lipossomas. A taxa de cisalhamento produzida pelo impelidor Cowles favoreceu a elasticidade da bicamada lipossomal, proporcionando alta eficiência de incorporação e capacidade de carregamento da CH8. Assim, a elasticidade da bicamada é fator determinante para a capacidade de incorporação de fármacos hidrofóbicos em lipossomas. Estas descobertas constituem uma melhoria nos processos de alto cisalhamento utilizados para carreamento de CH8 e contribuem para o desenvolvimento de formas farmacêuticas mais eficazes para o tratamento de leishmaniose cutânea / Abstract: High shear processes have been used to reduce the size and homogenize the size distribution of liposomes in their scalable production. However, high shear rates modify the liposome structure which may affect the loading of hydrophobic drugs. In this study, synthetic chalcone 3-nitro-2-hydroxy-4,6-dimethoxychalcone (CH8), a highly hydrophobic drug indicated to treat cutaneous leishmaniasis, was incorporated in conventional liposomes produced by scalable high shear processes. Liposomes were composed by egg phosphatidylcholine and produced by ethanol injection. Cowles impeller, Ultra Turrax® stirrers and microchannel microfluidizer were used to reduce and homogenize the size of the liposomes. The shear rate was controlled by Bangham's method followed by membrane extrusion. Small angle X-ray scattering spectra showed that the shearing reduced the interlamellar distance of the liposomes compared to the control. The interlamellar distance did not change with the shearing intensity in the range of Cowles stirring, but it decreased for higher shearing provided by the Ultra Turrax®. The elastic constant, which is related to the stiffness and depends on the structure of the liposomes, increased with increasing shearing intensity for both stirrers. The shearing also reduced the mean diameter of the liposomes and the size distributions. Initially, CH8 was incorporated into pre-formed liposomes. The results showed the expulsion and precipitation of loaded CH8 was proportional to the intensity of the applied shear rate. This limitation was circumvented by incorporating a low concentration of CH8 (0.2 mol/m³) during the formation of liposomes. The shear rate provided by the Cowles stirrer favored the elasticity of the bilayer and yielded high incorporation efficiency and CH8-loading. Therefore, the elasticity of the bilayer is a determining factor for the loading of hydrophobic drugs in liposomes. These findings constitute an improvement in the high shear process used to load CH8 and to the development of most effective pharmaceutical forms for the treatment of cutaneous leishmaniasis / Mestrado / Engenharia Química / Mestra em Engenharia Química

Orientador: Lucimara Gaziola de la Torre / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-23T18:43:23Z (GMT). No. of bitstreams: 1 Zompero_RafaelHenriquedeFreitas_M.pdf: 2571362 bytes, checksum: c3df7d704fb350757e99b3e400dee050 (MD5) Previous issue date: 2013 / Resumo: Este trabalho teve como objetivo principal o desenvolvimento e otimização de um processo escalonável de produção de lipossomas contendo ?-caroteno, visando sua posterior aplicação em produtos de interesse da indústria alimentícia. Para tanto, os efeitos das variáveis que influenciam o processo foram analisados, proporcionando um maior conhecimento a respeito da fenomenologia envolvida na produção dos nanoagregados e maior controle sobre as respostas produzidas pelo sistema. O ?-caroteno é um antioxidante natural, pró-vitamínico e que pode ser empregado como corante natural em formulações alimentícias. Porém sua elevada hidrofobicidade dificulta a aplicação em alimentos de base aquosa. Dentre as metodologias disponíveis para produção de lipossomas, o método de injeção de etanol apresenta-se como o mais facilmente adaptável as necessidades da indústria, possuindo baixo custo de implantação e operação. Na primeira etapa deste trabalho o método de injeção de etanol foi investigado visando a otimização dos parâmetros operacionais para a produção de nanoagregados tendo como objetivo obter propriedades físico químicas tais como diâmetro médio e polidispersidade de forma. Análises estatísticas dos resultados foram realizadas para determinação dos efeitos de cada variável e modelos foram desenvolvidos para predição do comportamento do sistema em diferentes condições de processo. Em seguida, a incorporação de ?-caroteno nos lipossomas obtidos na melhor condição foi avaliada em razões ?-caroteno/lipídio pré-definidas. Ensaios de incorporação de ?-caroteno aos lipossomas revelaram que proporções molares ?-caroteno/lipídio de até 0,5% mostram-se estáveis e solúveis em meio aquoso, resultado confirmado pelos ensaios de monocamadas de Langmuir realizados. A formulação otimizada foi submetida a testes de estabilidade em condições controladas de stress e, em um segundo momento, incorporada a nanofibras de álcool polivinílico e óxido de polietileno através de processo de electrospinning, conferindo proteção extra ao ?-caroteno internalizado. Estas nanofibras produzidas foram caracterizadas quanto a morfologia, diâmetro, presença de fosfolipídios, homogeneidade da distribuição de ?-caroteno e estabilidade a exposição a luz ultravioleta. Testes de rehidratação destas nanofibras foram conduzidos, verificando através de microscopia eletrônica de transmissão a liberação de lipossomas na fase aquosa. Dessa forma, a partir dos resultados obtidos conclui-se que o método de injeção de etanol foi otimizado, sendo que os efeitos de cada uma das variáveis de processo foram elucidados, contribuindo para o desenvolvimento tecnológico da técnica. Os ensaios de aplicação de condições de stress mostram que existe uma barreira a ser vencida no que diz respeito a estabilidade de lipossomas em formulações alimentícias complexas, principalmente para situações de elevada concentração de sacarose e altos teores de NaCl. Por fim, os testes de incorporação em nanofibras mostraram-se bastante promissores, mostrando a viabilidade e benefícios que podem ser agregados ao sistema através da utilização de técnica de eletrofiação contribuindo no desenvolvimento de novos materiais e produtos a serem utilizados pelo setor industrial alimentício / Abstract: The main goal of the present work was the development and optimization of a scalable process for production of ?-carotene loaded liposomes, aiming its application in the food industry. In that way, the effects of the variables that have influence on the process were analysed, providing greater information about the phenomenology evolving the production of these nano aggregates and improved control over the system responses. ?-carotene is a natural antioxidant, pro-vitaminic, and can be employed as a natural colorant in food formulations. However, its high hydrophobicity makes it difficult to be applied in water based foods. The development of feasible processes that can be implemented in the food industry for ?-carotene encapsulation is a actual research challenge. On the first step of this work the ethanol injection method was investigated aiming process parameters optimization for liposomes production with controlled size and polidispersity. Statistical analyses of the results were performed for variables effects determination and mathematical models development for system behaviour prediction. Then, ?-carotene incorporation on liposomes produced at the optimized condition was evaluated studying different ?-carotene/lipid ratios. ?-carotene incorporation experiments revealed that 0.5% ?-carotene/lipid ratios show to be stable and soluble in aqueous media, result confirmed by Langmuir monolayer experiments. The optimized formulation was submitted to stability tests under controlled stress conditions and, in a second stage, incorporated to polyvinyl alcohol and polyethylene oxide nanofibers using electrospinning technique for extra ?-carotene protection. The produced nanofibers were characterized regarding morphology, diameter, phospholipids presence, ?-carotene homogeneity and stability to UV light exposure. Nanofibers rehydration tests were conducted, verifying using transmission electron microscopy that liposomes were released in the aqueous media. In that way, from the obtained results we can conclude that the ethanol injection method was successfully optimized and the evaluation of the effects of each variable was elucidated, contributing to the technological development of the technique. Experiments regarding application of different stress conditions to liposomes formulations show that there is a barrier that must be overcome related to stability of liposomes to complex food formulations. Finally, incorporation tests on nanofibers showed to be promising, demonstrating the feasibility and benefits that can be aggregated to the system by using electrospinning, contributing to the development of new materials and products to be used by the food processing industries / Mestrado / Engenharia Química / Mestre em Engenharia Química

Orientador: Maria Cristina Volpato, Francisco Carlos Groppo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-20T01:41:18Z (GMT). No. of bitstreams: 1 Silva_CamilaBatistada_M.pdf: 1077516 bytes, checksum: e1dc439935f8cbbd32841c7929a7872f (MD5) Previous issue date: 2012 / Resumo: Este estudo avaliou a eficácia anestésica das seguintes formulações: articaína 4% encapsulada em lipossomas multilamelares - Arti-MLV, articaína 4% encapsulada em lipossomas unilamelares - Arti-LUV e solução comercial de articaína 4% com epinefrina 1:100.000 - Arti-Epi, em três modelos animais (bloqueio do nervo infraorbital - BNIO, infiltração subcutânea em ferida cirúrgica - ISFC e bloqueio do nervo alveolar inferior - BNAI). Para o BNIO, 24 ratos (8 animais/grupo) receberam 0,1 mL da preparação próximo ao forame infraorbital. Foi avaliada a duração da anestesia em tecido mole por pinçamento do lábio superior, a cada 5 minutos. Para a ISFC, 36 ratos (6 animais/grupo) receberam 0,1 mL das formulações anestésicas 24h após a realização de uma ferida cirúrgica (incisão e sutura) na superfície plantar da pata traseira direita. A anestesia foi avaliada pela aplicação de força ao lado da ferida com o analgesímetro eletrônico de von Frey. Para o BNAI, 45 ratos (15 animais/grupo) receberam 0,2 mL das formulações próximo ao forame mandibular e os parâmetros latência, duração e sucesso da anestesia pulpar foram avaliados por estímulo elétrico. Nos três modelos estudados foram utilizadas, como controle negativo, as formulações: solução de NaCl 0,9%, suspensão lipossomal multilamelar e suspensão lipossomal unilamelar. Os resultados foram submetidos a ANOVA e avaliados pelos testes de Kruskal-Wallis, Student-Newman-Keuls, Log-Rank e Exato de Fisher, com nível de significância de 5%. No BNIO, a Arti-Epi não diferiu da Arti-MLV (p>0,05) na duração e no sucesso da anestesia, porém foi melhor que a Arti-LUV (p<0,05) na duração da anestesia. Arti-LUV proporcionou menor (p<0,05) sucesso que as demais formulações. No modelo de ISFC, a duração e sucesso da Arti-Epi foram superiores (p<0,05) aos das demais formulações. No BNAI, não houve diferença estatística entre as formulações testadas (p>0,05) com relação a latência, sucesso e duração da anestesia. Conclui-se que, as formulações lipossomais de articaína apresentaram eficácia anestésica equivalente à da solução contendo epinefrina, podendo ser opção de uso para anestesia pulpar (BNAI) e em tecido mole não inflamado (BNIO, apenas com a formulação lipossomal multilamelar). Entretanto, a solução contendo epinefrina foi mais potente em aumentar a duração e taxa de sucesso da anestesia em tecidos com hipernocicepção / Abstract: This study assessed the anesthesic efficacy of the following formulations: 4% articaine encapsulated in multilamellar liposomes (Arti-MLV), 4% articaine encapsulated in unilamellar liposomes (Arti-LUV) and 4% articaine with 1:100,000 epinephrine (Arti-Epi), in three animal models: infraorbital nerve block (IONB), subcutaneous infiltration in surgical wound (SISW) and inferior alveolar nerve block (IANB). For IONB, 24 rats (8/group) received 0.1 mL of the anesthetic formulations near the right infraorbital foramen. The duration of anesthesia was assessed by upper lip pinching every 5 minutes. For SISW, 36 animals (6/group) received 0.1 mL of the anesthesic formulations 24 hours after a surgical wound (incision and suture) was performed in the plantar region of the right hind paw. Paw anesthesia was assessed by force application at the side of the wound with electronic von Frey anesthesiometer. For IANB, 45 rats (15/group) received 0.2 mL of the anesthetic formulations near the right mandibular foramen. The success, onset and duration of pulpal anesthesia were assessed by electric stimulus. 0.9% NaCl solution, multilamellar liposomal suspension and unilamellar liposomal suspension were used as negative controls in the three models studied. Data were submitted to ANOVA, Kruskal-Wallis, Student-Newman-Keuls, Log-Rank, and Fisher's exact tests (5% significance level). For IONB no differences in duration and anesthesic success (p>0.05) were observed between Arti-Epi and Arti-MLV; Arti-Epi presented higher anesthesia duration than Arti-LUV (p<0.05). Arti-LUV provided lower (p<0.05) anesthesia success than other formulations. For SISW, Arti-Epi provided higher success and longer anesthesia duration than the other formulations (p<0.05). For BNAI no differences among the formulations were observed in relation to onset, success and anesthesia duration (p>0.05). In conclusion, the articaine liposomal formulations presented anesthetic efficacy equivalent to that showed by the solution with epinephrine and could be an option for pulpal (IANB) and soft tissue anesthesia in non-inflamed tissues (IONB - only the multilamellar liposome formulation). However, the solution with epinephrine was more potent to improve anesthetic success and duration in tissues with hipernociception / Mestrado / Farmacologia, Anestesiologia e Terapeutica / Mestre em Odontologia

Orientadores: Lucimara Gaziola de La Torre, Adriano Rodrigues Azzoni / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-20T20:39:39Z (GMT). No. of bitstreams: 1 Balbino_TiagoAlbertini_M.pdf: 2581790 bytes, checksum: 674a2d22b439ef811cbfb642774f6d4d (MD5) Previous issue date: 2012 / Resumo: Esta pesquisa teve como objetivo o desenvolvimento tecnológico de processo microfluídico para a obtenção de vetores não virais, baseados na complexação eletrostática entre lipossomas catiônicos (LC) e DNA plasmideal (pDNA) destinados à terapia e vacinação gênica. O desenvolvimento desse processo foi comparado ao processo convencional "bulk", que, a partir da simples mistura manual entre as soluções ou em sistema de vórtice, gera dificuldade no controle do tamanho destas estruturas e pode produzir variações nos resultados biológicos e na estabilidade coloidal. Já o processo microfluídico, que utiliza dispositivos que processam pequenas quantidades de fluidos (10-9 a 10-18 litros), permite a complexação eletrostática em regime contínuo, com o controle das condições difusionais, o que também permite melhor controle do tamanho destes complexos. Metodologicamente, o trabalho foi dividido em três principais etapas: na primeira parte, foi realizado o estudo físico-químico, estrutural e biológico dos complexos pDNA/LC obtidos por processo "bulk". Nessa etapa, verificou-se a correlação das propriedades físico-químicas e estruturais dos complexos com o processo de transfecção in vitro em células HeLa. A segunda parte do trabalho visou à otimização da produção de lipossomas catiônicos em dois dispositivos microfluídicos, com uma única e com dupla focalização hidrodinâmica, de modo a se obter lipossomas similares aos estudados na primeira parte do trabalho. Na utilização do segundo dispositivo, foi possível operar em vazões volumétricas mais altas quando comparadas ao primeiro. Por fim, na terceira parte, foi realizado o estudo da complexação entre LC e pDNA por processo microfluídico também em dois diferentes dispositivos, um similar ao utilizado na segunda parte do trabalho, com focalização hidrodinâmica única, e outro com blocos regulares nas paredes do microcanal, o que aumenta a área de contato entre os fluidos. Os complexos formados no primeiro dispositivo apresentaram melhores respostas biológicas in vitro, as quais foram similares às do processo "bulk". No segundo dispositivo, ensaios de acessibilidade de sonda de fluorescencência ao DNA indicaram alteração na associação entre LC e DNA. Dessa forma, a partir dos resultados, conclui-se que os dispositivos microfluídicos estudados são uma alternativa promissora para a formação de LC e também sua complexão com pDNA em modo contínuo, tanto pela potencialidade tecnológica quanto biológica, o que contribui para o desenvolvimento de produtos farmacêuticos que veiculam DNA e que são destinados à terapia e vacinação gênica / Abstract: This research aimed at the technological development of microfluidic process for nonviral carriers production based on the electrostatic complexation between cationic liposomes (CL) and plasmidal DNA (pDNA) for gene and vaccine therapy applications. The development of this process was compared to the conventional bulk process, in which the solutions are mixed followed by the simple hand shaking or brief vortexing, what generates difficulties on the particles sizes control and can affect the biological functionality and colloidal stability of the formulations. In contrast, microfluidic process, which uses devices that manipulate small amounts of fluids (10-9 to 10-18 liters), allows the electrostatic complexation in continuous mode, controlling diffusion conditions, which also allows the colloidal control of the obtained formulations. Furthermore, microfluidic devices have minimum dimensions and operate with low energy consumption. Methodologically, the present work was carried out in three mean steps: in the first step, the physicochemical, structural and biological characteristics of the pDNA/CL complexes obtained by the bulk process were studied. In this step, it was possible to verify the correlation of physicochemical and structural properties with the transfection phenomenon in vitro of HeLa cells. The second part of this work focused the optimization of the production of CL through two microfluidic devices, with single and double hydrodynamic focusing, to obtain similar CL to those of the first step of this work. By employing the second device, it was possible to operate at higher volumetric flow rates than the first one. Finally, in the third step, it was explored the complexation between CL and pDNA via microfluidic process also in two different microfluidic devices; the first was similar to that employed in the second part of this work, with a single hydrodynamic focusing, and a second one with patterned microchannel walls, which increase the surface contact area between the fluids. The complexes formed in the first device showed better biological results in vitro, which were similar to the complexes formed in the bulk complexation method. In the patterned device, the experiments of the DNA accessibility to fluorescent probe pointed out modifications between the pDNA and CL association in the complexes. In conclusion, we showed that the studied microfluidic devices are a promising alternative for the production of CL and the complexation with pDNA in continuous mode, because of the technological and biological potentialities, which contributes to the development of feasible processes, for the production of new pharmaceutical products for gene and vaccine therapies / Mestrado / Desenvolvimento de Processos Biotecnologicos / Mestre em Engenharia Química

Wound healing, as a normal biological process in the human body, is achieved through four precise and highly programmed phases: hemostasis, inflammation, proliferation, and remodeling. For a wound to heal successfully, all four phases must occur in the proper sequence and time frame. However, many factors can interfere with one or more of these phases, thus causing improper or impaired wound healing. Maintaining a moist wound environment is crucial in facilitating the wound-healing process. The beneficial effects of a moist versus a dry wound environment include re-epithelization, tissue granulation, and repair. The use of hydrocolloid occlusive dressings in maintaining a moist wound environment has proven to be a useful adjunct in facilitating wound healing. Although hydrocolloid dressings have been widely used clinically in wound management, bacterial resistance, poor solubility and sustained drug release remain to be a problem for many of the drugs used in wound therapy. In chronic wound management, where patients normally undergo long treatments and frequent dressing changes, a system that delivers drugs into a wound site in a controlled fashion can improve patient compliance and therapeutic outcomes. Liposomes are small phospholipid vesicles that have been widely investigated as drug carriers for the delivery of therapeutic agents. They are spherical lipid vesicles consisting of phospholipid bilayers that improve the efficacy of the drugs by fusing with biological membranes, and eventually releasing their entrapped content into the cells or bacteria. The aim of this study therefore, is to develop a new bacitracin-based controlled release hydrocolloid dressing, with good absorptive properties for improving the efficacy of antibiotics in wound healing. HPLC (high-pressure liquid chromatography) assay of bacitracin was performed for quantification of the drug. Liposomes were prepared using thin film hydration and extrusion methods. Liposomes were also characterized based on their ideal particle size and encapsulation efficiency, and then incorporated into the different ratios of chitosan/gelatin hydrocolloid films. The films were prepared with increase in gelatin concentration and were evaluated for folding endurance, tensile strength, water absorption capacity, morphology, drug release kinetics, antimicrobial activity and stability. The morphology of these films was found to be very smooth and homogeneous proving a good compatibility between the two polymers. With increase in gelatin concentration, folding endurance, water absorption capacity, tensile strength, drug release kinetics and antimicrobial activity were increased. The antibacterial activity against various bacterial species was improved in the bacitracin loaded hydrocolloid films as compared to the blank films. Based on the findings above, it can be concluded that chitosan/gelatin films at 1:3 proportion is a successful wound dressing for wound management with improved wound healing properties than other formulations. This formulation is a potential candidate for the development of alternative pharmaceutical dosage forms, for the treatment of bacterial infected wounds, based on the activity of the eco-friendly chitosan matrix added to the bacitracin activity. In this work, chitosan also demonstrated a great potential as a dressing for advanced wound therapy and confirmed its good biocompatibility and potential to provide, in combination with liposomes, sustained drug release which is highly beneficial for wound treatment. The addition of gelatin improved the water affinity of the films and facilitated water mediated cross-linking process.

Master of Science (Nanoscience) / Neuroblastomas (NBs) are the most common solid extra-cranial tumours diagnosed in childhood and characterized by a high risk of tumour relapse. Like in other tumour types, there are major concerns about the specificity and safety of available drugs used for the treatment of NBs, especially because of potential damage to the developing brain. Many plant-derived bioactive compounds have proved effective for cancer treatment but are not delivered to tumour sites in sufficient amounts due to compromised tumour vasculature characterized by leaky capillary walls. Betulinic acid (BetA) is one such naturally-occurring anti-tumour compound with minimum to no cytotoxic effects in healthy cells and rodents. BetA is however insoluble in water and most aqueous solutions, thereby limiting its therapeutic potential as a pharmaceutical product. Liposomes are self-assembling closed colloidal structures composed of one or more concentric lipid bilayers surrounding a central aqueous core. The unique ability of liposomes to entrap hydrophilic molecules into the core and hydrophobic molecules into the bilayers renders them attractive for drug delivery systems. Cyclodextrins (CDs) are non-reducing cyclic oligosaccharides which proximate a truncated core, with features of a hydrophophilic outer surface and hydrophobic inner cavity for forming host-guest inclusion complexes with poorly water soluble molecules. CDs and liposomes have recently gained interest as novel drug delivery vehicles by allowing lipophilic/non-polar molecules into the aqueous core of liposomes, hence improving the therapeutic load, bioavailability and efficacy of many poorly water-soluble drugs. The aim of the study was to develop nano-drug delivery systems for BetA in order to treat human neuroblastoma (NB) cancer cell lines. This was achieved through the preparation of BetA liposomes (BetAL) and improving the percent entrapment efficiency (% EE) of BetA in liposomes through double entrapment of BetA and gamma cyclodextrin BetA inclusion complex (γ-CD-BetA) into liposomes (γ-CD-BetAL). We hypothesized that the γ-CD-BetAL would produce an increased % EE compared to BetAL, hence higher cytotoxic effects. Empty liposomes (EL), BetAL and γ-CD-BetAL were synthesized using the thin film hydration method followed by manual extrusion. Spectroscopic and electron microscopic characterization of these liposome formulations showed size distributions of 1-4 μm (before extrusion) and less than 200 nm (after extrusion). As the liposome size decreased, the zeta-potential (measurement of liposome stability) decreased contributing to a less stable liposomal formulation. Low starting BetA concentrations were found to be more effective in entrapping higher amounts of BetA in liposomes while the incorporation of γ-CD-BetA into liposomes enhanced the % EE when compared to BetAL, although this was not statistically significant. Cell viability studies using the WST-1 assay showed a time-and concentration-dependent decrease in SK-N-BE(2) and Kelly NB cell lines exposed to free BetA, BetAL and γ-CD-BetAL at concentrations of 5-20 ug/ml for 24, 48 and 72 hours treatment durations. The observed cytotoxicity of liposomes was dependant on the % EE of BetA. The γ-CD-BetAL was more effective in reducing cell viability in SK-N-BE(2) cells than BetAL whereas BetAL was more effective in KELLY cells at 48-72 hours. Exposure of all cells to EL showed no toxicity while free BetA was more effective overall than the respective liposomal formulations. The estimated IC₅₀ values following exposure to free BetA and BetAL were similar and both showed remarkable statistically significant decrease in NB cell viability, thus providing a basis for new hope in the effective treatment of NBs.

Magister Pharmaceuticae - MPharm / Sutherlandia frutescens is a traditional plant medicine widely used in South Africa. Traditionally, the leaves of S. frutescens are mainly used as a tea, but these traditional dosage forms have several disadvantages, including that they are not particularly convenient to prepare and store, encourage dosage inaccuracy and are highly susceptible to microbial contamination. To solve these problems, dried aqueous extract forms, e.g. freeze dried aqueous extract (FDAE) of S. frutescens were prepared, but they, in turn, may still suffer from instability and contain mainly hydrophilic phytoconstituents that are poorly absorbed and delivered for in vivo activity. Modified forms of the FDAE, i.e. the active phytopharmaceutical ingredient (API), may be a better solution. Therefore this study sought to prepare liposomes and phytosomes of the freeze dried aqueous extract of Sutherlandia frutescens, as a means of increasing the total the surface area of the API, thus improving its release and dissolution in gastrointestinal fluids. Liposomes and phytosomes of the FDAE of Sutherlandia frutescens obtained were prepared using a thin film hydration method at ratios of lecithin: S. frutescens (3:1) and phosphatidylcholine: S. frutescens (2:1) respectively. The physical characteristics (i.e. particle size, size distribution, zeta potential, and morphology), of flavonoid glycosides (i.e. sutherlandins A to D; API) as well as content and release profiles of each dosage form (i.e. FDAE liposome or phytosomes) at pH 1.2 and pH 6.8 was determined. A validated HPLC assay was used to determine and compare the flavonoid glycoside content and release profiles of the liposomes and phytosomes. Both liposomes and phytosomes were successfully prepared, in moderate yields (± 30 %, and ± 50 %, respectively), using the thin film hydration method. The liposomes had a significantly smaller size, lower size distribution, higher zeta potential and better stability than the phytosomes (p < 0.05). The phytosomes, however, had significantly higher flavonoid glycoside encapsulation efficiency than the liposomes (±50 % vs ±26 %; p < 0.01). In addition, the release at 120 minutes, of flavonoid glycosides from the liposomes (63%, 58%, 76% and 46% % at pH 1.2, and 78%, 76%, 87% and 89 % at pH 6.8 for sutherlandins A, B, C and D, respectively) was significantly higher and faster than that of the phytosomes (52%, 41%, 51% and 39 % at pH 1.2, and 31% 31%, 33%and 45% % at pH 6.8, for sutherlandins A, B, C and D, respectively). The differences in release were likely due to differences in particle size and size distribution of the two modified API forms. Overall, liposomes and phytosomes can be considered promising vehicles for delayed delivery of herbal crude extracts. Based on its characteristics (i.e. narrower size distribution, and better stability), the liposomes were preferred compared to the phytosomes offering a better kinetic release profile. The phytosomes had higher encapsulation than the liposomes that may be due to complex formation between the API and the lipid.

L'huile essentielle de clou de girofle (HECG) et son constituant majeur l'eugénol (Eug) sont reconnus pour leurs propriétés biologiques. Ces principes actifs naturels peuvent constituer des alternatifs aux agents antimicrobiens, antioxydants et anti-inflammatoires de synthèse dans les formulations alimentaires et pharmaceutiques. Cependant, leur utilisation est limitée en raison de leur faible solubilité aqueuse, volatilité et sensibilité à la lumière. Notre travail de thèse porte sur la préparation et la caractérisation des vésicules lipidiques encapsulant l'HECG et l'Eug ainsi que les complexes d'inclusion cyclodextrine/Eug. Dans une première étape, la méthode d'injection éthanolique est utilisée à l'échelle du laboratoire où les paramètres de préparation ont été optimisés. Des phospholipides naturels de soja saturés (Phospholipon 80H et Phospholipon 90H) et insaturés (Lipoid S100) ont été utilisés pour étudier l'effet de l'hydrogénation et de la composition des phospholipides sur les caractéristiques des liposomes. Les conditions optimales ont été par la suite appliquées pour préparer les liposomes à grande échelle par contacteur à membrane et à l'échelle pilote. Des résultats similaires en termes de taille, indice de polydispersité, potentiel zêta, morphologie et taux d'incorporation de phospholipides sont obtenus à petite et grande échelle. Ceci indique la reproductibilité de ces procédés de préparation. Par ailleurs, des complexes d'inclusion d'HP-β-CD/Eug et d'HP-β-CD/HECG sont préparés dans une solution aqueuse et ensuite incorporés dans les liposomes formant un système combiné « drug in cyclodextrin in liposomes, DCL ». Un système en double encapsulation (DCL2) a été également préparé où l'Eug ou l'HECG sont ajoutés dans la phase organique et leurs complexes d'inclusion dans la phase aqueuse. En comparant à une simple incorporation dans les liposomes, DCL et DCL2 améliorent le rendement d'encapsulation de l'Eug et possèdent des tailles plus petites. Les résultats ont montré que les liposomes et les DCLs sont stables et maintiennent l'activité anti-oxydante de l'Eug. De plus, les liposomes protègent l'Eug contre la dégradation induite par les rayons UVC. Les DCLs, dont la particularité est de maintenir une huile essentielle volatile dans un lyophilisat en dépit des pressions très basses appliquées, peuvent être considérés comme un système de vectorisation prometteur de l'HECG et de l'Eug permettant leur utilisation en tant qu'ingrédients dans les préparations cosmétiques, pharmaceutiques, et agroalimentaires / Clove essential oil (CEO) and its major constituent eugenol (Eug) are recognized for their biological properties. These molecules may constitute natural alternatives to synthetic antimicrobial, antioxidant, and anti-inflammatory agents in food and pharmaceutical formulations. However, CEO constituents are volatile, sensitive to light and possess low aqueous solubility, which may limit their wide applications. Our thesis focuses on the preparation and characterization of lipid vesicles encapsulating CEO, Eug and the inclusion complexes cyclodextrin/Eug. In a first step, the ethanol injection method is applied at laboratory scale where the preparation parameters have been optimized. Natural hydrogenated (Phospholipon 80H, Phospholipon 90H) and non-hydrogenated (Lipoid S100) soybean phospholipids were used to study the effect of hydrogenation and phospholipid composition on the characteristics of liposomes. Optimal conditions were then applied to prepare liposomes at large scale by membrane contactor and at pilot scale. Similar results in terms of size, polydispersity index, zeta potential, morphology and phospholipid loading rate were obtained at laboratory and large scale. This indicates the reproducibility of the preparation methods. In addition, HP-β-CD/Eug and HP- β-CD/CEO inclusion complexes were prepared in aqueous solution and were then incorporated into liposomes forming a combined system « drug in cyclodextrin in liposomes, DCL ». Double loaded liposomes (DCL2) were also prepared where CEO or Eug were added in the organic phase and their inclusion complexes in the aqueous phase. Compared to CEO and Eug loaded liposomes, DCL and DCL2 improved the loading rate of Eug and possessed smaller vesicles size. Results showed that both liposomes and DCLs are stable and maintain the antioxidant activity of Eug. In addition, liposomes protect Eug from degradation induced by UVC irradiation. DCLs, whose characteristic is to keep a volatile essential oil in a lyophilized form despite the very low applied pressures, could be considered as a promising carrier system of CEO and Eug permitting their use as ingredients in cosmetic, pharmaceutical and food industries