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Master's Dissertation
DOI
https://doi.org/10.11606/D.60.2019.tde-20092021-192623
Document
Author
Full name
Gabriela Favero Galvão
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
Ribeirão Preto, 2019
Supervisor
Committee
Lopez, Renata Fonseca Vianna (President)
Annichini, Maria Sol Brassesco
Favorin, Leila Aparecida Chiavacci
Title in Portuguese
Avaliação da iontoforese no transporte celular de lipossoma e imunolipossoma
Keywords in Portuguese
Iontoforese
Nanoparticulas
Tráfego intracelular
Abstract in Portuguese
A iontoforese é um método físico que utiliza uma corrente elétrica constante de baixa intensidade para aumentar a permeação de fármacos através da pele. Sua aplicação associada a lipossomas tem mostrado resultados positivos no tratamento tópico do carcinoma de células escamosas (SCC), os quais superexpressam o receptor de fator de crescimento epidermal (EGFR). Assim, a funcionalização dos lipossomas com o anti-EGFR cetuximabe (imunolipossomas) aumenta a eficiência do tratamento iontoforético. A translocação das nanopartículas lipossomais através da membrana plasmática das células tumorais pode ser afetada pela iontoforese e influenciar no tráfego intracelular das nanopartículas e do fármaco que elas carreiam impactando na resposta biológica. Desta forma, o objetivo desse trabalho foi investigar a influência da iontoforese nas vias de internalização e tráfego intracelular de lipossomas e imunolipossomas de cetuximabe, contendo ou não o quimioterápico 5-fluouracil (5-FU). As nanopartículas foram obtidas e caracterizadas na presença do marcador fluorescente 3,3'-dioctadeciloxacarbocianina perclorato (DiO) para facilitar a visualização do uptake, vias de endocitose e tráfego intracelular dos lipossomas e imunolipossomas brancos (sem fármaco) e contendo 5-FU pelas células de SCC da pele humana A431. A ionotoforese aumentou o uptake das nanopartículas em pelo menos 6 vezes, sendo que a internalização dos imunolipossomas foi aproximadamente 2,5 vezes maior do que a dos lipossomas independente do tempo de tratamento. A expressão do EGFR nas células A431 foi confirmada por ensaios de degradação do receptor por immunoblotting, assim como a interação dos imunolipossomas com o EGFR. As vias de endocitose das nanopartículas foram significativamente modificadas pela iontoforese. Passivamente (sem iontoforese) os lipossomas foram internalizados por vias não endocíticas; já os imulolipossomas foram auxiliados por vias dependentes de clatrina, caveolina e macropinocitose. Quando a iontoforese foi aplicada, a internalização dos lipossomas foi mediada por macropinocitose e caveolina, enquanto a dos lipossomas foi mediada por macropinocitose e clatrina. O tráfego intracelular das nanopartículas, mas principalmente dos receptores de membrana EGFR, também foi alterado na presença da iontoforese: a membrana plasmática se rearranjou e passou a englobar um grupo de células, a marcação do EGFR diminuiu sensivelmente, a colocalização das nanopartículas com os endossomos primários foi alterada e a expressão dos lisossomos foi significativamente reduzida, principalmente após iontoforese dos imunolipossomas. A presença do quimioterápico, 5-FU, não interferiu nos processo de internalização e tráfego intracelular. Conclui-se, desta forma, que a iontoforese, além de aumentar a permeação cutânea de fármacos, altera as vias de internalização celular das nanopartículas e o padrão de expressão do EGFR e dos lisossomos, podendo ser vantajosa na diminuição da resistência aos tratamentos com anticorpos e na maior eficiência na biodisponibilidade de quimioterápicos.
Title in English
Evaluation of the iontophoresis in liposome and immunoliposome cell transport
Keywords in English
Intracellular traffic
Iontophoresis
Nanoparticles
Abstract in English
Iontophoresis is a physical method that uses a constant low intensity electric current to increase the permeation of drugs through the skin. Its application associated with liposomes has shown positive results in the topical treatment of squamous cell carcinoma (SCC), which overexpress the epidermal growth factor receptor (EGFR). Thus, liposome functionalization with anti-EGFR cetuximab (immunoliposomes) increases the efficiency of iontophoretic treatment. The translocation of liposomal nanoparticles across the tumor cell membrane may be affected by iontophoresis and influence the intracellular traffic of the nanoparticles and of the drugs they carry, impacting the biological response. Therefore, the objective of this study was to investigate the influence of iontophoresis on the internalization pathways and intracellular traffic of liposomes and immunoliposomes, whether or not containing 5- fluouracil (5-FU) chemotherapy. Nanoparticles were obtained and characterized in the presence of the fluorescent marker 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) to facilitate visualization of uptake, endocytosis pathways and intracellular traffic of blank nanoparticles (without drug) and containing 5-FU by A431 human skin SCC cells. Ionotophoresis increased nanoparticle uptake by at least 6-fold, and the internalization of immunoliposomes was approximately 2.5-fold greater than that of liposomes regardless of treatment time. EGFR expression in A431 cells was confirmed by immunoblotting receptor degradation assays as well as the interaction of immunoliposomes with EGFR. The endocytosis pathways of nanoparticles were significantly modified by iontophoresis. Passively (without iontophoresis) the liposomes were internalized by non-endocytic pathways; imuloliposomes were aided by clathrin, caveolin and macropinocytosis dependent pathways. When iontophoresis was applied, liposome internalization was mediated by macropinocytosis and caveolin, while liposome internalization was mediated by macropinocytosis and clathrin. Intracellular traffic of nanoparticles, but mainly EGFR membrane receptors, was also altered in the presence of iontophoresis: the cell membrane rearranged to encompass a group of cells, EGFR labeling decreased significantly, colocalization of nanoparticles with early endosomes was altered, and lysosome expression was significantly reduced, especially after immunoliposome iontophoresis. The presence of 5-FU chemotherapy doesn't interfere in the process of internalization and intracellular traffic.Thus, in addition to increasing the skin permeation of drugs, iontophoresis alters nanoparticle cell internalization pathways and the expression pattern of EGFR and lysosomes. Such modifications may be advantageous in reducing cell resistance to antibody treatments and in the greater bioavailability of chemotherapeutic drugs.
 
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Release Date
2021-09-20
Publishing Date
2021-09-24
 
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