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Otimização do direcionamento para células tumorais de nanosistemas lipídicos para uso terapêutico
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Resumo(s)
Lipossomas de longo tempo de circulação têm surgido como opções muito promissoras de sistemas de entrega de fármacos a células cancerígenas. Estas partículas são revestidas por polietilenoglicol (PEG), cuja função pode ser alargada para além dos horizontes de melhoria da sua biodistribuição. A possibilidade de modificação funcional por ligação covalente nas suas extremidades levou ao aparecimento de estratégias de direccionamento activo, nomeadamente com pequenas moléculas como ácido fólico (AF), explorando a sobreexpressão dos receptores de folato em inúmeros tipos de tumores. Neste trabalho, a ligação do AF aos lipossomas previamente formados foi explorada como um método único e alternativo aos actualmente existentes na preparação deste tipo de partículas. Com este objectivo, AF foi activado com um grupo sulfidrilo e ligado covalentemente aos lipossomas contendo DSPE-PEG2000-maleimida. Este processo não alterou o tamanho nem a carga dos lipossomas e permitiu obter quantidades de AF (detectado após a ligação) até 34 % (mol AF/maleimida). Adicionalmente foram preparados lipossomas modificados com o péptido antagonista G para direccionar os lipossomas para as células do carcinoma de pulmão, aplicando o mesmo método de ligação e por um método alternativo em que o péptido ligado ao DSPE-PEG-maleimida é inserido em lipossomas previamente formados (método de pós-inserção), ambos com elevados rendimentos de ligação, em comparação ao ácido fólico (71 % e 43 % mol péptido/maleimida, respectivamente). Os lipossomas foram testados in vitro em diferentes linhas celulares tumorais. O direccionamento com AF foi testado em células HeLa e MCF7 (elevada expressão e sem expressão de receptores de folato, respectivamente). Por outro lado, o direccionamento com antagonista G foi testado em células H69 que sobreexpressam receptores de neuropéptidos. A internalização de lipossomas direccionados, marcados com fosfatidiletanolamina de ovo marcada com rodamina, foi observada por microscopia de fluorescência.
Drug delivery systems targeted to tumor cells are an ever-growing field in cancer research. One of the most successful strategies in this field consists of employing long circulating liposomes as drug carriers. The presence of poly-(ethylene glycol) (PEG) on their surface allows membrane stabilization, decreases protein-liposome interactions and allows surface modification with molecules that recognize cell-specific surface markers. One of the most commonly and successfully employed targeting molecules is folic acid, which binds with high affinity to folate receptors, overexpressed in a wide variety of tumors. The present study addressed the development of a simple, yet unique method of linking covalently a targeting molecule to the surface of pre-formed PEGylated liposomes. Herein, the protocol of grafting liposomes with folic acid after their preparation was studied and optimized. For this purpose, folic acid was activated with a sulfhydryl group and linked to liposomes, containing DSPE-PEG2000-maleimide. This process did not interfere with liposome size or ζ potential and it was possible to detect this vitamin up to 34 % (mol FA/maleimide) in liposomes after linkage. Additionally, liposomes targeted to the lung carcinoma cells with the peptide antagonist G were prepared by the same method and by the post-insertion method (where DSPE-PEG-peptide was inserted in the liposomal bilayer), both with high linkage efficiency (71 % and 43 % mol peptide/maleimide respectively). In order to assess the targeting ability of prepared liposomes, in vitro internalization studies were conducted with different cell lines. Folate receptor overexpressing and non-expressing cell lines HeLa and MCF7 were cultured in presence of FA targeted liposomes and neuropeptide receptor overexpressing cells, H69 (lung carcinoma) were cultured in presence of antagonist G targeted liposomes. The cellular uptake of fluorescently labeled liposomes (egg-phospatidylethanolamine-rhodamine) was assessed by fluorescence microscopy. However, no qualitative differences were observed in the internalization patterns for all cell lines.
Drug delivery systems targeted to tumor cells are an ever-growing field in cancer research. One of the most successful strategies in this field consists of employing long circulating liposomes as drug carriers. The presence of poly-(ethylene glycol) (PEG) on their surface allows membrane stabilization, decreases protein-liposome interactions and allows surface modification with molecules that recognize cell-specific surface markers. One of the most commonly and successfully employed targeting molecules is folic acid, which binds with high affinity to folate receptors, overexpressed in a wide variety of tumors. The present study addressed the development of a simple, yet unique method of linking covalently a targeting molecule to the surface of pre-formed PEGylated liposomes. Herein, the protocol of grafting liposomes with folic acid after their preparation was studied and optimized. For this purpose, folic acid was activated with a sulfhydryl group and linked to liposomes, containing DSPE-PEG2000-maleimide. This process did not interfere with liposome size or ζ potential and it was possible to detect this vitamin up to 34 % (mol FA/maleimide) in liposomes after linkage. Additionally, liposomes targeted to the lung carcinoma cells with the peptide antagonist G were prepared by the same method and by the post-insertion method (where DSPE-PEG-peptide was inserted in the liposomal bilayer), both with high linkage efficiency (71 % and 43 % mol peptide/maleimide respectively). In order to assess the targeting ability of prepared liposomes, in vitro internalization studies were conducted with different cell lines. Folate receptor overexpressing and non-expressing cell lines HeLa and MCF7 were cultured in presence of FA targeted liposomes and neuropeptide receptor overexpressing cells, H69 (lung carcinoma) were cultured in presence of antagonist G targeted liposomes. The cellular uptake of fluorescently labeled liposomes (egg-phospatidylethanolamine-rhodamine) was assessed by fluorescence microscopy. However, no qualitative differences were observed in the internalization patterns for all cell lines.
Descrição
Tese de mestrado em Bioquímica (Bioquímica Médica), Universidade de Lisboa, Faculdade de Ciências, 2014
Palavras-chave
Lipossomas Polietilenoglicol Ácido fólico Antagonista G Direccionamento activo Teses de mestrado - 2014