A Copaifera langsdorffii é uma das plantas de incentivo governamental para pesquisas científicas e possui diversas atividades biológicas. Em especial, as atividades antitumoral e tripanocida foram abordadas neste estudo tendo o ácido caurenoico (AC) como marcador. Devido à escassa literatura sobre a extração do AC e à sua baixa solubilidade, sua extração foi estudada com solvente orgânico e por fluido supercrítico. A extração por fluido supercrítico é vantajosa já que não fornece extratos com resíduos de solventes orgânicos e é ecologicamente correta. Visto as atividades antitumoral e tripanocida do AC e estas atividades serem objeto de estudo nacional e mundial, o AC foi inserido em lipossomas convencionais e furtivos com a finalidade de torná-lo mais biodisponível, permitir seu alcance ao sítio-alvo e diminuir seus efeitos adversos. Assim, os objetivos deste trabalho foram segmentados em três vertentes: a) realizar o estudo e otimização da extração do ácido caurenoico a partir das folhas de C. langsdorffii, b) obter lipossomas convencionais, secá-los pelos métodos de secagem liofilização (FD), spray drying (SD) e spray freeze drying (SFD), comparar suas características físico-químicas e avaliar suas atividade antitumoral e tripanocida, c) obter lipossomas furtivos e avaliar sua seletividade tumoral. De maneira geral, os resultados das extrações sólido-líquido e por fluido supercrítico apresentaram boa seletividade e eficiência visto que foram capazes de fornecer extratos com aproximadamente 20% de AC. A extração por fluido supercrítico foi mais eficaz extraindo 26,2% de AC. Já os lipossomas convencionais secos por SD e SFD apresentaram-se mais semelhantes entre si quanto à propriedade de fluxo e morfologia, à interação do AC com os componentes da bicamada lipídica e à dissolução/ liberação que quando secos por FD. Os três métodos de secagem foram capazes de prolongar em seis meses a estabilidade do AC nos lipossomas quando comparado com a dispersão aquosa. Os lipossomas convencionais secos apresentaram citotoxicidade maior frente a células tumorais e menor frente a células normais quando carregados com o AC e comparados com os lipossomas convencionais vazios (sem AC). Foi possível sintetizar com sucesso o lipídeo ligado ao polietilenoglicol e ao ácido fólico com o intuito de preparar os lipossomas furtivos. Entretanto, estes lipossomas não apresentaram ação antitumoral seletiva quando comparados às células normais. / Copaifera langsdorffii is one of the Brazilian plants which receives governmental support for scientific researches and also presents several biological activities. The kaurenoic acid (KA) is a diterpenic acid constituent of this specie that presents, in special, antitumoral and tripanocide activities which were taken into account in this study. Due to the lack of KA extraction from C. langsdorffii leaves in the literature and to KA low solubility, its extraction was studied using solid-liquid and supercritical fluid extraction methods. The supercritical fluid extraction is advantageous since provides solvent-free extracts and is an environment friendly method. Since KA is antitumoral and tripanocide and both activities are subject of national and internacional studies, in this study, KA was added in conventional and stealth liposomes in order to increase its biodiponibility, to allow it reaching the target tissue and decrease its adverse effects. So, this work was divided in three segments: a) study and optimize the KA extraction from C. langsdorffii leaves by solid-liquid (SLE) and supercritical fluid (SFE) extraction methods, b) obtain conventional liposomes, dry them by freeze drying (FD), spray drying (SD) and spray freeze drying (SFD), compare their physico-chemical features and evaluate their antitumor and tripanocide activities, c) obtain stealth liposomes and evaluate their selective tumor activity. In a general way, the extraction methods result in high KA selective extraction by both SLE and SFE since they were able to extract nearly 20% of KA. The SFE was a bit more efficacious than SLE providing 26,2% of KA. Conventional liposomes dried by SD and SFD were more similar in fluidity and morphology, KA and liposome lipid compounds interaction and dissolution/ release than liposomes dried by FD. The three drying methods provided more stable liposomes than the aqueous solution liposomes. Dried conventional liposomes presented higher cytotoxicity to tumor cells and lower one to normal cells when loaded with KA than unloaded conventional liposomes. The lipid linked to poliethyleneglycol and to folic acid to prepare stealth liposomes were successfully synthesized. However, the stealth liposomes did not presented selective antitumor action in relation to normal cells.

A Copaifera langsdorffii é uma das plantas de incentivo governamental para pesquisas científicas e possui diversas atividades biológicas. Em especial, as atividades antitumoral e tripanocida foram abordadas neste estudo tendo o ácido caurenoico (AC) como marcador. Devido à escassa literatura sobre a extração do AC e à sua baixa solubilidade, sua extração foi estudada com solvente orgânico e por fluido supercrítico. A extração por fluido supercrítico é vantajosa já que não fornece extratos com resíduos de solventes orgânicos e é ecologicamente correta. Visto as atividades antitumoral e tripanocida do AC e estas atividades serem objeto de estudo nacional e mundial, o AC foi inserido em lipossomas convencionais e furtivos com a finalidade de torná-lo mais biodisponível, permitir seu alcance ao sítio-alvo e diminuir seus efeitos adversos. Assim, os objetivos deste trabalho foram segmentados em três vertentes: a) realizar o estudo e otimização da extração do ácido caurenoico a partir das folhas de C. langsdorffii, b) obter lipossomas convencionais, secá-los pelos métodos de secagem liofilização (FD), spray drying (SD) e spray freeze drying (SFD), comparar suas características físico-químicas e avaliar suas atividade antitumoral e tripanocida, c) obter lipossomas furtivos e avaliar sua seletividade tumoral. De maneira geral, os resultados das extrações sólido-líquido e por fluido supercrítico apresentaram boa seletividade e eficiência visto que foram capazes de fornecer extratos com aproximadamente 20% de AC. A extração por fluido supercrítico foi mais eficaz extraindo 26,2% de AC. Já os lipossomas convencionais secos por SD e SFD apresentaram-se mais semelhantes entre si quanto à propriedade de fluxo e morfologia, à interação do AC com os componentes da bicamada lipídica e à dissolução/ liberação que quando secos por FD. Os três métodos de secagem foram capazes de prolongar em seis meses a estabilidade do AC nos lipossomas quando comparado com a dispersão aquosa. Os lipossomas convencionais secos apresentaram citotoxicidade maior frente a células tumorais e menor frente a células normais quando carregados com o AC e comparados com os lipossomas convencionais vazios (sem AC). Foi possível sintetizar com sucesso o lipídeo ligado ao polietilenoglicol e ao ácido fólico com o intuito de preparar os lipossomas furtivos. Entretanto, estes lipossomas não apresentaram ação antitumoral seletiva quando comparados às células normais. / Copaifera langsdorffii is one of the Brazilian plants which receives governmental support for scientific researches and also presents several biological activities. The kaurenoic acid (KA) is a diterpenic acid constituent of this specie that presents, in special, antitumoral and tripanocide activities which were taken into account in this study. Due to the lack of KA extraction from C. langsdorffii leaves in the literature and to KA low solubility, its extraction was studied using solid-liquid and supercritical fluid extraction methods. The supercritical fluid extraction is advantageous since provides solvent-free extracts and is an environment friendly method. Since KA is antitumoral and tripanocide and both activities are subject of national and internacional studies, in this study, KA was added in conventional and stealth liposomes in order to increase its biodiponibility, to allow it reaching the target tissue and decrease its adverse effects. So, this work was divided in three segments: a) study and optimize the KA extraction from C. langsdorffii leaves by solid-liquid (SLE) and supercritical fluid (SFE) extraction methods, b) obtain conventional liposomes, dry them by freeze drying (FD), spray drying (SD) and spray freeze drying (SFD), compare their physico-chemical features and evaluate their antitumor and tripanocide activities, c) obtain stealth liposomes and evaluate their selective tumor activity. In a general way, the extraction methods result in high KA selective extraction by both SLE and SFE since they were able to extract nearly 20% of KA. The SFE was a bit more efficacious than SLE providing 26,2% of KA. Conventional liposomes dried by SD and SFD were more similar in fluidity and morphology, KA and liposome lipid compounds interaction and dissolution/ release than liposomes dried by FD. The three drying methods provided more stable liposomes than the aqueous solution liposomes. Dried conventional liposomes presented higher cytotoxicity to tumor cells and lower one to normal cells when loaded with KA than unloaded conventional liposomes. The lipid linked to poliethyleneglycol and to folic acid to prepare stealth liposomes were successfully synthesized. However, the stealth liposomes did not presented selective antitumor action in relation to normal cells.

This diploma thesis deals with the preparation and characterization of stealth liposomes and their combination with trimethylchitosan (TMC). This complex could find application in the field of inhalation administration. Stealth liposomes were prepared from neutral phophatidylcholine, negatively charged fosfatidic acid and polyethyleneglycol bounded to phosphatidylethanolamine. We have managed to prepare stealth liposomes with suitable properties that should guarantee passive targeting without evocation an immune response, despite the content of the negative component. We also found a suitable method of preparation for stealth liposome–TMC complex, where the change of size and zeta potential confirmed the non–covalent bound between two components despite the content of the polyethyleneglycol.

Targeting the anticancer agents selectively to cancer cells is desirable to improve the efficacy and to reduce the side effects of anticancer therapy. Previously reported passive tumor targeting by PEGylated liposomes (stealth liposomes) have resulted in their higher tumor accumulation. However their interaction with cancer cells has been minimal due to the steric hindrance of the PEG coating. This dissertation reports two approaches to enhance the interaction of stealth liposomes with cancer cells. First, we designed a lipid-hydrazone-PEG conjugate that removes the PEG coating at acidic pH as in the tumor interstitium. However, such a conjugate was highly unstable on shelf. Targeting the anticancer agents selectively to cancer cells is desirable to improve the efficacy and to reduce the side effects of anticancer therapy. Previously reported passive tumor targeting by PEGylated liposomes (stealth liposomes) have resulted in their higher tumor accumulation. However their interaction with cancer cells has been minimal due to the steric hindrance of the PEG coating. This dissertation reports two approaches to enhance the interaction of stealth liposomes with cancer cells. First, we designed a lipid-hydrazone-PEG conjugate that removes the PEG coating at acidic pH as in the tumor interstitium. However, such a conjugate was highly unstable on shelf. Second we developed lipids with imidazole headgroups. Such lipids can protonate to provide positive charges on liposome surface at lowered pH. Additionally, negatively charged PEGylated phospholipids can cluster with the protonated imidazole lipids to display excess positive charges on the surface of the liposomes, thus enhancing their interaction with negatively charged cancer cells. We prepared convertible liposome formulations I, II and III consisting of one of the three imidazole-based lipids DHI, DHMI and DHDMI with estimated pKa values of 5.53, 6.2 and 6.75, respectively. Zeta potential measurement confirmed the increase of positive surface charge of such liposomes at lowered pHs. DSC studies showed that at pH 6.0 formulation I formed two lipid phases, whereas the control liposome IV remained a one-phase system at pHs 7.4 and 6.0. The interaction of such convertible liposomes with negatively charged model liposomes mimicking biomembranes at lowered pH was substantiated by 3-4 times increase in average sizes of the mixture of the convertible liposomes and the model liposomes at pH 6.0 compared to pH 7.4. The doxorubicin-loaded convertible liposomes show increased cytotoxicity in B16F10 (murine melanoma) and Hela cells at pH 6.0 as compared to pH 7.4. Liposome III shows the highest cell kill at pH 6.0 for both the cells. The control formulation IV showed no difference in cytotoxicity at pH 7.4 and 6.0. Uptake of convertible liposome II by B16F10 cells increased by 57 % as the pH was lowered from 7.4 to 6.0.