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Doctoral Thesis
DOI
10.11606/T.5.2017.tde-15122017-092710
Document
Author
Full name
Arthur Cassio de Lima Luna
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2017
Supervisor
Committee
Maria, Durvanei Augusto (President)
Afeche, Solange Castro
Capelozzi, Vera Luiza
Kubrusly, Marcia Saldanha
Title in Portuguese
Potencial antitumoral da formulação lipossomal DODAC/fosfoetanolamina sintética no modelo de hepatocarcinoma
Keywords in Portuguese
Carcinoma hepatocelular
Fosfoetanolamina sintética
Fosfolipídios antitumorais
Lipossomas
Nanocarreadores
Abstract in Portuguese
A fosfoetanolamina sintética (FO-S), um fosfomonoéster, apresenta relevante atividade antitumoral. Contudo, a utilização de um carreador para encapsular a FO-S em lipossomas poderia favorecer a sua disponibilidade no microambiente tumoral, possibilitando o aumento da sua eficácia. Desta forma, o presente estudo avaliou a eficiência de encapsulamento da FO-S em lipossomas de DODAC e o seu potencial antitumoral. Os lipossomas foram preparados por ultrasonicação e caracterizados físicoquimicamente. A citotoxidade foi avaliada nas linhagens tumorais B16F10 (melanoma murino), Hepa1c1c7 (hepatocarcinoma murino) e Skmel-28 (melanoma humano) e nas células normais HUVEC, após o tratamento com diferentes concentrações dos lipossomas DODAC/FO-S, no tempo de 24 horas. A internalização dos lipossomas e o potencial elétrico mitocondrial foram analisados por microscopia confocal a laser. Adicionalmente, a expressão das proteínas caspases 3 e 8 ativas, receptor DR4, citocromo c, p53, p21, Bax, p27, CD44, CD90, Bcl-2 e ciclina D1 foi quantificada por citometria de fluxo. Para os estudos in vivo, os camundongos C57BL/6J portadores de hepatocarcinoma foram tratados com FO-S, DODAC/FO-S e DODAC, pelas vias intraperitoneal (IP) e intrahepática (IH), durante 20 dias. Os resultados demonstraram que os lipossomas apresentaram aspecto esférico e alta eficiência de encapsulação da FO-S, como também promoveram maior citotoxicidade nas linhagens tumorais estudadas, em comparação com FO-S. Além disto, nas células B16F10 e Hepa1c1c7, ocasionou parada nas fases S e G2/M do ciclo celular. A linhagem Hepa1c1c7 foi a mais sensível ao tratamento com os lipossomas DODAC/FO-S, os quais foram internalizados em até 6 horas e promoveram a diminuição de CD90, CD44, ciclina D1 e Bcl-2, o aumento de p53, p21, p27, Bax e caspases 8 e 3 ativas e a liberação do citocromo c. O aumento significativo das caspases 8 e 3 ativas, expressão do receptor DR4 e a liberação do citocromo c também ocorreu nas linhagens B16F10 e Skmel-28. Os resultados in vivo mostraram que os lipossomas DODAC/FO-S e a FO-S não induziram hepatotoxicidade, nefrotoxicidade e caquexia. Os lipossomas DODAC/FO-S não ocasionaram mielossupressão e hemólise, apresentando menor toxicidade em relação a FO-S, administrada pelas vias IP e IH. Além disto, os tratamentos com DODAC/FO-S (IH) e FO-S (IH e IP) foram efetivos em diminuir o número de células na fase S. Contudo, apenas os lipossomas DODAC/FO-S (IH) reduziram significamente os focos tumorais, aumentando as áreas de necrose, promovendo também o aumento da expressão gênica da p53, ciclina B1 e caspases 8 e 3. O conjunto dos resultados in vivo e in vitro demonstraram que a formulação lipossomal DODAC/FO-S foi capaz de maximizar os efeitos antitumorais da FO-S, ativando as vias intrínsecas e extrínsecas da apoptose
Title in English
Potential antitumor of the DODAC/PHO-S liposomal formulation in the model of hepatocellular carcinoma
Keywords in English
Antitumor phospholipids
Hepatocellular carcinoma
Liposomes
Nanocarriers
Synthetic phosphoethanolamine
Abstract in English
Synthetic phosphoethanolamine (PHO-S) - a phosphomonoester - has shown relevant anticancer effects. However, the utilization of a carrier to encapsulate the PHOS in liposomes can maximize its availability in the tumor microenvironment, allowing an increase in its effectiveness. Thus, the present study has evaluated efficiency of PHO-S encapsulation in DODAC liposomes and its antitumor potential. The liposomes were prepared by ultrasonication and physico-chemically characterized. The cytotoxic effects were evaluated on B16F10 cells (murine melanoma), Hepa1c1c7 cells (murine hepatocellular carcinoma), Skmel-28 (human melanoma) and in endothelial cells HUVEC, after treatment with DODAC/PHO-S liposomes at different concentrations for 24 hours. The internalization of the liposomes and mitochondrial electrical potential were analyzed by confocal laser microscopy. Additionally, the expression of active caspases 3 and 8, receptor DR4, cytochrome c, p53 p53, p21, Bax, p27, CD44, CD90, Bcl-2 and cyclin D1 proteins was quantified by flow cytometry. For in vivo studies, C57BL/6J mice with hepatocellular carcinoma were treated with PHO-S, DODAC/PHO-S and DODAC, by intraperitoneal (IP) and intratumoral (IT) routes for 20 days. The results demonstrated that liposomes presented spherical aspect and high PHO-S encapsulation efficiency, as also promoted high cytotoxic effect - compared with PHO-S. Furthermore, in B16F10 and Hepa1c1c7 cells, the liposomes induced S and G2/M cell cycle arrest. Hepa1c1c7 cells showed greater sensitivity to the DODAC/PHO-S formulation, which were internalized until 6 hours and promoted a decrease in the expression of CD90, CD44, cyclin D1 and Bcl-2, an increase of de p53, p21, p27, Bax and active caspases 8 and 3 and the liberation of cytochrome c. The significant increase in the expression of active caspases 3 and 8, DR4 receptor and liberation of cytochrome c also occurred in B16F10 and Skmel-28 cells. In vivo results showed that DODAC/PHO-S liposomes and PHO-S did not induce nephrotoxicity, hepatotoxicity and cachexia. DODAC/PHO-S liposomes did not cause myelosuppression and hemolysis, presenting lower toxicity in relation to PHO-S - when administered by IP and IT routes. Moreover, treatment with DODAC/PHO-S (IT) and PHO-S (IT and IP) effectively decreased the number of cells in S phase. However, only DODAC/PHO-S liposomes significantly reduced the number of tumor foci, increasing area of necrosis, and also promoting an increase in gene expression of p53, cyclin B1 and caspases 8 and 3. The set of in vitro and in vivo results demonstrated that DODAC/PHO-S liposomal formulation was capable of maximizing the PHO-S antitumor effects, activating the intrinsic and extrinsic pathways of the apoptosis
 
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Publishing Date
2017-12-19
 
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