Produção de lipossomas pelo metodo de injeção de etanol encapsulando agentes tuberculostaticos e avaliação do potencial de escalonamento do processo

Orientador : Angela Maria MoraesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia QuimicaResumo: Este trabalho teve por objetivo o escalonamento da produção de lipossomas projetados para a liberação controlada por inalação de fármacos utilizados no tratamento da tuberculose, visando a melhoria de sua eficácia terapêutica e a redução de sua toxicidade. O método de injeção de etanol foi selecionado para a produção das vesículas, por ser simples e de baixo custo. Os fármacos isoniazida, pirazinamida e rifampicina, comumente usados na terapia primária da tuberculose e o agente terapêutico kanamicina, empregado em casos de tuberculose resistente a múltiplos compostos terapêuticos, assim como o fármaco modelo doxorrubicina, foram incorporados nas vesículas unilamelares contendo basicamente lecitina natural purificada e colesterol, dentre outras formulações. Foram avaliados os efeitos de variáveis operacionais como temperatura, taxa de agitação, velocidade de injeção e concentração inicial de lipídios nas características finais dos lipossomas obtidos. As formulações preparadas foram caracterizadas quanto às concentrações finais de lipídios e de fármacos, ao tamanho dos lipossomas, a sua lamelaridade, à estabilidade de estocagem na forma de suspensão aquosa e de pó liofilizado reconstituído. Os principais resultados indicaram que as vesículas preparadas pelo método de injeção de etanol em escala de bancada permitiram encapsular ativamente os fármacos em estudo, embora o gradiente de pH estabelecido nestes lipossomas tenha se mostrado estável por menos de cinco dias. As características finais das vesículas preparadas por injeção de etanol em maior escala foram influenciadas pelo tipo e intensidade da agitação da fase aquosa, pela temperatura desta fase e pelo diâmetro da agulha de injeção. Foram obtidas altas eficiências de encapsulação ativa dos fármacos nestes lipossomas, atingindose até 100%, sendo tais eficiências influenciadas principalmente pela concentração inicial e pela composição lipídica das vesículas. As vesículas constituídas de fosfatidilcolina sintética mostraram-se mais apropriadas para a encapsulação dos fármacos, entretanto, seu custo é substancialmente maior. As populações mais freqüentes nos lipossomas apresentaram diâmetros médios variando de 132 a 156 nm. O processo de liofilização das vesículas provocou aumento em seu diâmetro médio e diminuição nas eficiências de encapsulação obtidas, e a adição dos criprotetores manitol e sacarose não preveniu a agregação e fusão dos lipossomas. A análise preliminar da viabilidade econômica da produção de lipossomas preparados por injeção de etanol em larga escala e a subseqüente incorporação do fármaco kanamicina indicou que o processo é economicamente viávelAbstract: The aim of this work was to the scale-up of the production of liposomes designed for the administration through inhalation of drugs used in tuberculosis therapy, seeking to improve drug therapeutic efficacy as well to reduce drug toxicity. The method based on ethanol injection was selected for vesicle production due to its simplicity and low cost. The drugs isoniazid, pyrazinamide and rifampin, indicated for tuberculosis primary treatment, as well as kanamycin, a drug used for multiresistant tuberculosis therapy and doxorubicin, a model drug, were incorporated in unilamellar vesicles basically prepared from purified natural lecithin and cholesterol, among other formulations. The effects of operational variables such as temperature, mixing rate, injection rate and initial lipid concentration on the characteristics of the vesicles were analyzed. The preparations were characterized concerning to lipid and drug concentration, vesicle diameter and lamelarity and also to liposomal storage stability both in aqueous solution and in the lyophilized form after rehydration. The most relevant results indicate that the vesicles prepared by ethanol injection in bench scale were able to actively encapsulate the studied drugs, however, the pH gradient stablished in liposomal membrane was stable for less than five days. The final characteristics of vesicles prepared by ethanol injection in larger scale were affected by aqueous phase mixing type and intensity, temperature as well as by the injection needle inner diameter. High active encapsulation efficiencies of the evaluated drugs in liposomes were achieved, reaching 100%, and those efficiency values were influenced by lipid concentration and composition. The vesicles containing synthetic phosphatidylcholine were more adequate for drug encapsulation, however, their cost is substantially higher. The most frequent populations in liposomes presented average diameters varying in the range from 132 to 156 nm. The lyophilization caused increases in vesicle mean diameters, reduced drug incorporation efficiency and the addition of the cryoprotectors mannitol and sucrose did not prevent vesicle aggregation and fusion. A preliminary analysis showed that the large scale production of liposomes through ethanol injection followed by kanamycin active loading is economically feasibleDoutoradoDesenvolvimento de Processos BiotecnologicosDoutor em Engenharia Químic

Evaluation Of In Vitro Anti-inflammatory Effects Of Crude Ginger And Rosemary Extracts Obtained Through Supercritical Co2 Extraction On Macrophage And Tumor Cell Line: The Influence Of Vehicle Type

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Background: Numerous plants from have been investigated due to their anti-inflammatory activity and, among then, extracts or components of ginger (Zingiber officinale Roscoe) and rosemary (Rosmarinus officinalis L.), sources of polyphenolic compounds. 6-gingerol from ginger rhizome and carnosic acid and carnosol from rosemary leaves present anti-tumor, anti-inflammatory and antioxidant activities. However, the evaluation of the mechanisms of action of these and other plant extracts is limited due to their high hydrophobicity. Dimethylsulfoxide (DMSO) is commonly used as a vehicle of liposoluble materials to mammalian cells in vitro, presenting enhanced cell penetration. Liposomes are also able to efficiently deliver agents to mammalian cells, being capable to incorporate in their structure not only hydrophobic molecules, but also hydrophilic and amphiphilic compounds. Another strategy is based on the use of Pluronic F-68, a biocompatible low-foaming, non-ionic surfactant, to disperse hydrophobic components. Here, these three delivery approaches were compared to analyze their influence on the in vitro anti-inflammatory effects of ginger and rosemary extracts, at different concentrations, on primary mammalian cells and on a tumor cell line. Methods: Ginger and rosemary extracts free of organic solvents were obtained by supercritical fluid extraction and dispersed in DMSO, Pluronic F-68 or liposomes, in variable concentrations. Cell viability, production of inflammatory mediators and nitric oxide (NO) release were measured in vitro on J774 cell line and murine macrophages primary culture stimulated with bacterial lipopolysaccharide and interferon-gamma after being exposed or not to these extracts. Results: Ginger and rosemary extracts obtained by supercritical CO2 extraction inhibited the production of pro-inflammatory cytokines and the release of NO by peritoneal macrophages and J774 cells. The delivery vehicles influenced the anti-inflammatory effects. Comparatively, the ginger extract showed the highest anti-inflammatory activity on the tumor cell line. Controversially, rosemary extract dispersed on DMSO induced a more significant IL-1 and TNF-alpha reduction than ginger extract in primary macrophages. Conclusions: Amongst the tested delivery vehicles, DMSO was the most suitable, presenting reduced cytotoxicity, followed by Pluronic F-68 and liposomes, provably due to differences in their form of absorption, distribution and cellular metabolism. Co-administration of liposomes and plant extracts may cause death of macrophages cells and induction of NO production. It can be concluded that some of the beneficial effects attributed to extracts of ginger and rosemary may be associated with the inhibition of inflammatory mediators due to their high antioxidant activity. However, these effects were influenced by the type of delivery vehicle.15Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2005/04496-4, 2015/21365-2, 2005/03507-2]CNPq [484547/2006-2

Efeito da adição de precursores na produção de alcaloide anticancerígeno usando a técnica de planejamento experimental

The effect of precursors on the anticancer alkaloid production by submerged fermentation using M. anisopliae 3935 was studied, according to complete experimental design 2² with three central points. The results showed that lysine was the most important variable, however, when both lysine and glucose were added to the fermentation medium, the alkaloid production reached, approximately, 17 mg L-1 after 120 hours of fermentation. Then, the scale-up of the process was carried out and these results were confirmed. Finally, 35 mg L-1 of alkaloid at 192 h were attained after increment of added aminoacid lysine

Evaluation Of In Vitro Anti-inflammatory Effects Of Crude Ginger And Rosemary Extracts Obtained Through Supercritical Co2 Extraction On Macrophage And Tumor Cell Line: The Influence Of Vehicle Type.

Numerous plants from have been investigated due to their anti-inflammatory activity and, among then, extracts or components of ginger (Zingiber officinale Roscoe) and rosemary (Rosmarinus officinalis L.), sources of polyphenolic compounds. 6-gingerol from ginger rhizome and carnosic acid and carnosol from rosemary leaves present anti-tumor, anti-inflammatory and antioxidant activities. However, the evaluation of the mechanisms of action of these and other plant extracts is limited due to their high hydrophobicity. Dimethylsulfoxide (DMSO) is commonly used as a vehicle of liposoluble materials to mammalian cells in vitro, presenting enhanced cell penetration. Liposomes are also able to efficiently deliver agents to mammalian cells, being capable to incorporate in their structure not only hydrophobic molecules, but also hydrophilic and amphiphilic compounds. Another strategy is based on the use of Pluronic F-68, a biocompatible low-foaming, non-ionic surfactant, to disperse hydrophobic components. Here, these three delivery approaches were compared to analyze their influence on the in vitro anti-inflammatory effects of ginger and rosemary extracts, at different concentrations, on primary mammalian cells and on a tumor cell line. Ginger and rosemary extracts free of organic solvents were obtained by supercritical fluid extraction and dispersed in DMSO, Pluronic F-68 or liposomes, in variable concentrations. Cell viability, production of inflammatory mediators and nitric oxide (NO) release were measured in vitro on J774 cell line and murine macrophages primary culture stimulated with bacterial lipopolysaccharide and interferon-γ after being exposed or not to these extracts. Ginger and rosemary extracts obtained by supercritical CO2 extraction inhibited the production of pro-inflammatory cytokines and the release of NO by peritoneal macrophages and J774 cells. The delivery vehicles influenced the anti-inflammatory effects. Comparatively, the ginger extract showed the highest anti-inflammatory activity on the tumor cell line. Controversially, rosemary extract dispersed on DMSO induced a more significant IL-1 and TNF-α reduction than ginger extract in primary macrophages. Amongst the tested delivery vehicles, DMSO was the most suitable, presenting reduced cytotoxicity, followed by Pluronic F-68 and liposomes, provably due to differences in their form of absorption, distribution and cellular metabolism. Co-administration of liposomes and plant extracts may cause death of macrophages cells and induction of NO production. It can be concluded that some of the beneficial effects attributed to extracts of ginger and rosemary may be associated with the inhibition of inflammatory mediators due to their high antioxidant activity. However, these effects were influenced by the type of delivery vehicle.1539

Effect of the precursor addition on the anticancer alkaloid production using experimental design methodology

The effect of precursors on the anticancer alkaloid production by submerged fermentation using M. anisopliae 3935 was studied, according to complete experimental design 2² with three central points. The results showed that lysine was the most important variable, however, when both lysine and glucose were added to the fermentation medium, the alkaloid production reached, approximately, 17 mg L-1 after 120 hours of fermentation. Then, the scale-up of the process was carried out and these results were confirmed. Finally, 35 mg L-1 of alkaloid at 192 h were attained after increment of added aminoacid lysine.13941398Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Preparação e caracterização de lipossomas para a administração por inalação de compostos terapeuticos utilizados na terapia da tuberculose

Orientador: Angela Maria MoraesDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia QuimicaResumo: A tuberculose, atualmente, é um dos mais sérios problemas de saúde pública mundiais, especialmente em países em desenvolvimento, estando associada à morte de pelo menos três milhões de pessoas por ano. Dentro deste contexto, o presente trabalho visou a preparação e caracterização de lipossomas (vesículas lipídicas) destinados ao aumento da eficácia de drogas freqüentemente utilizadas nas terapias primária e secundária da Tuberculose, para a liberação controlada por inalação das mesmas. Lipossomas unilamelares foram preparadas a partir de 60 mol % de Distearoilfosfatidilcolina e 40 mol % de Colesterol. As drogas Isoniazida, Pirazinamida, Rifampicina e Etionamida, foram incorporadas nos lipossomas por encapsulamento passivo no cerne aquoso ou na bicamada lipídica das vesículas. As preparações, foram caracterizadas quanto às concentrações finais de lipídios e de drogas, ao diâmetro médio dos lipossomas, e à estabilidade de estocagem na forma de suspensão a '5 GRAUS¿C. Para as amostras com melhores desempenhos quanto ao encapsulamento e à estabilidade de estocagem, foram realizados ensaios complementares de avaliação da estabilidade das vesículas quando em presença do tensoativo não-iônico 'C IND. 12¿¿E IND. 5¿. Ensaios similares foram também realizados para as mesmas drogas, além da Estreptomicina, empregando-se a técnica de incorporação ativa estabelecendo-se um gradiente de pH na bicamada lipídica de 3,4 unidades... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digitalAbstract: Nowadays, Tuberculosis, is one of the most serious problems of public helth worldwide, specially in development countries, being associated to the death of at least three million people yearly. In this context, the present work aimed the preparation nd characterization of liposomes (lipid vesicles) designed to increase the effectiveness of drugs frequently used in Tuberculosis primary and secondary therapy, through the controlled release of these drugs by inhalation. Unilamellar liposomes were prepared with 60% mol of distearoylpHosHatidylcoline and 40% mol of cholesterol. The drugs Isoniazid, Pyrazinamide, Rifampin and Ethionamide were incorporated in the liposomes by passive loading in vesicle aqueous core or in its lipid bilayer. The formulations obtained were characterized in terms of their final lipid and drug concentrations, vesicle mean diameter, and stability during storage at '5 DEGREES¿C as an aqueous suspension. The sample presenting the best encapsulation efficiencies and storage stability were also characterized concerning to their stability when in contact with the non-ionic surfactant 'C IND. 12¿¿E IND. 5¿. Similar encapsulation studies were also accomplished for the same drugs, besides Streptomycin, using the active loading technique based on imposing a transmembrane pH gradient of 3.4 units. The results indicated a strong association among the experimental protocols, the trapping efficiency and the stability of the obtained systems... Note: The complete abstract is available with the full electronic digital thesis or dissertationsMestradoDesenvolvimento de Processos BiotecnologicosMestre em Engenharia Químic