Empowering the therapeutic potential of clinically expired platelet concentrates: a new source of extracellular vesicles
Autor(a) principal: | |
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Data de Publicação: | 2022 |
Tipo de documento: | Dissertação |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/10362/141380 |
Resumo: | The limited shelf-life of platelet concentrates (PC) produced in blood centers leads to a significant portion of platelet donations being discarded before being purposed for transfusion. Consequently, PC are an enormous financial burden for the healthcare system, so, new applications for expired PC have been growing over the past years. Inspired by the high amounts of platelet-derived extracellular vesicles (pEV) that are released during PC storage and by the natural appetency of pEV to interact with cancer cells, we investigated the efficacy of pEV from expired PC as carriers of paclitaxel (PTX), an anti-angiogenic and anti-cancer drug. In this thesis, pEV were isolated from expired PC using different methods (density gradient ultracentrifugation (DGUC); size exclusion chromatography and the combination of both methods). The highest EV yield (2.03x1011/100 mL PC) was obtained using DGUC, so this was selected as the most suitable protocol. Furthermore, a low level of contaminating protein, together with the detection of specific EV markers (CD9, CD63 and FLOT2) were observed. In addition, a typical EV size distribution profile (100-300 nm) with a typical cup-shaped morphology was achieved. By direct incubation, pEV were loaded with PTX (PTX-pEV), and their functionality was assessed on endothelial and breast cancer cell lines (basal, MDA-MB-231 and luminal B, BT474). Our findings suggest that PTX-pEV showed higher anti-migratory and anti-angiogenic activity than the free drug. A comparison of the cytotoxic effect of PTX-pEV on breast cancer cells was carried out, which identified a decrease in cell viability (21.05%) and proliferation (41.04%) only in BT474. PTX delivery pathways for MDA-MB-231 and BT474 were also distinctly affected by EV uptake inhibitors, between cell lines, showing that dynamin-dependent endocytosis and heparin sulphate proteoglycan- dependent mechanism were only responsible by the PTX release on BT474. |
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Empowering the therapeutic potential of clinically expired platelet concentrates: a new source of extracellular vesiclesexpired platelet concentratesplatelet-derived extracellular vesiclesdrug delivery systempaclitaxeltherapeutic effectbreast cancerDomínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e TecnologiasThe limited shelf-life of platelet concentrates (PC) produced in blood centers leads to a significant portion of platelet donations being discarded before being purposed for transfusion. Consequently, PC are an enormous financial burden for the healthcare system, so, new applications for expired PC have been growing over the past years. Inspired by the high amounts of platelet-derived extracellular vesicles (pEV) that are released during PC storage and by the natural appetency of pEV to interact with cancer cells, we investigated the efficacy of pEV from expired PC as carriers of paclitaxel (PTX), an anti-angiogenic and anti-cancer drug. In this thesis, pEV were isolated from expired PC using different methods (density gradient ultracentrifugation (DGUC); size exclusion chromatography and the combination of both methods). The highest EV yield (2.03x1011/100 mL PC) was obtained using DGUC, so this was selected as the most suitable protocol. Furthermore, a low level of contaminating protein, together with the detection of specific EV markers (CD9, CD63 and FLOT2) were observed. In addition, a typical EV size distribution profile (100-300 nm) with a typical cup-shaped morphology was achieved. By direct incubation, pEV were loaded with PTX (PTX-pEV), and their functionality was assessed on endothelial and breast cancer cell lines (basal, MDA-MB-231 and luminal B, BT474). Our findings suggest that PTX-pEV showed higher anti-migratory and anti-angiogenic activity than the free drug. A comparison of the cytotoxic effect of PTX-pEV on breast cancer cells was carried out, which identified a decrease in cell viability (21.05%) and proliferation (41.04%) only in BT474. PTX delivery pathways for MDA-MB-231 and BT474 were also distinctly affected by EV uptake inhibitors, between cell lines, showing that dynamin-dependent endocytosis and heparin sulphate proteoglycan- dependent mechanism were only responsible by the PTX release on BT474.A data de validade limitada dos concentrados de plaquetas (PC) produzidos nos centros de transfusão de sangue, leva a que uma significante porção de doações de plaquetas seja descartada antes de serem transfundidas. Consequentemente, os PC são um enorme encargo financeiro para o sistema de saúde, e como tal, novas aplicações para PC expirados têm vindo a emergir nos últimos anos. Inspirados pelas elevadas quantidades de vesículas extracelulares derivadas de plaquetas (pEV) que são libertadas durante o período de armazenamento dos PC, e pela sua apetência natural para interagir com células cancerígenas, investigámos a eficácia das pEV de PC expirados como sistemas de entrega de paclitaxel (PTX), um fármaco anti angiogénico e anti cancerígeno. Neste trabalho, as pEV foram isoladas dos PC expirados através de diferentes métodos (ultracentrifugação em gradiente de densidade (DGUC); cromatografia de exclusão molecular e por combinação dos dois métodos). O maior rendimento em pEV (2.03x1011/100 mL PC) foi obtido por DGUC, e como tal, este foi selecionado o protocolo mais adequado. Adicionalmente, através deste obtiveram-se baixos níveis de contaminação proteíca, bem como se detetaram marcadores específicos de EV (CD9, CD63 e FLOT2). Para além disto, obtiveram-se pEV com um perfil de distribuição de tamanho típico (100-300 nm) e com uma morfologia típica. Por incubação direta, as pEV foram encapsuladas com o PTX (PTX-pEV), e a sua funcionalidade foi avaliada em células endoteliais e em duas linhas celulares de cancro da mama (basal, MDA-MB-231 e luminal B, BT474). Os resultados sugerem que as PTX-pEV apresentaram uma maior atividade anti migratória e anti angiogénica que o fármaco livre. Quando comparado o efeito citotóxico das PTX-pEV nas células de cancro da mama, identificou-se um decréscimo na viabilidade (21.05%) e proliferação (41.04%) celulares apenas para as BT474. Após incubação com inibidores de internalização de EV, as via de internalização celular das pEV foram também diferencialmente afetadas entre MDA-MB-231 e BT474, mostrando-se desta forma que as vias de endocitose dependente de dinamina e dependente de proteoglicanos heparina sulfato estavam apenas envolvidas na entrega do PTX nas BT474.Serra, MargaridaSalvador, DanielaRUNMeliciano, Ana Eduarda Lopes2022-07-05T14:04:28Z2022-012022-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/141380enginfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T05:18:46Zoai:run.unl.pt:10362/141380Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:49:59.114887Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Empowering the therapeutic potential of clinically expired platelet concentrates: a new source of extracellular vesicles |
title |
Empowering the therapeutic potential of clinically expired platelet concentrates: a new source of extracellular vesicles |
spellingShingle |
Empowering the therapeutic potential of clinically expired platelet concentrates: a new source of extracellular vesicles Meliciano, Ana Eduarda Lopes expired platelet concentrates platelet-derived extracellular vesicles drug delivery system paclitaxel therapeutic effect breast cancer Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
title_short |
Empowering the therapeutic potential of clinically expired platelet concentrates: a new source of extracellular vesicles |
title_full |
Empowering the therapeutic potential of clinically expired platelet concentrates: a new source of extracellular vesicles |
title_fullStr |
Empowering the therapeutic potential of clinically expired platelet concentrates: a new source of extracellular vesicles |
title_full_unstemmed |
Empowering the therapeutic potential of clinically expired platelet concentrates: a new source of extracellular vesicles |
title_sort |
Empowering the therapeutic potential of clinically expired platelet concentrates: a new source of extracellular vesicles |
author |
Meliciano, Ana Eduarda Lopes |
author_facet |
Meliciano, Ana Eduarda Lopes |
author_role |
author |
dc.contributor.none.fl_str_mv |
Serra, Margarida Salvador, Daniela RUN |
dc.contributor.author.fl_str_mv |
Meliciano, Ana Eduarda Lopes |
dc.subject.por.fl_str_mv |
expired platelet concentrates platelet-derived extracellular vesicles drug delivery system paclitaxel therapeutic effect breast cancer Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
topic |
expired platelet concentrates platelet-derived extracellular vesicles drug delivery system paclitaxel therapeutic effect breast cancer Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
description |
The limited shelf-life of platelet concentrates (PC) produced in blood centers leads to a significant portion of platelet donations being discarded before being purposed for transfusion. Consequently, PC are an enormous financial burden for the healthcare system, so, new applications for expired PC have been growing over the past years. Inspired by the high amounts of platelet-derived extracellular vesicles (pEV) that are released during PC storage and by the natural appetency of pEV to interact with cancer cells, we investigated the efficacy of pEV from expired PC as carriers of paclitaxel (PTX), an anti-angiogenic and anti-cancer drug. In this thesis, pEV were isolated from expired PC using different methods (density gradient ultracentrifugation (DGUC); size exclusion chromatography and the combination of both methods). The highest EV yield (2.03x1011/100 mL PC) was obtained using DGUC, so this was selected as the most suitable protocol. Furthermore, a low level of contaminating protein, together with the detection of specific EV markers (CD9, CD63 and FLOT2) were observed. In addition, a typical EV size distribution profile (100-300 nm) with a typical cup-shaped morphology was achieved. By direct incubation, pEV were loaded with PTX (PTX-pEV), and their functionality was assessed on endothelial and breast cancer cell lines (basal, MDA-MB-231 and luminal B, BT474). Our findings suggest that PTX-pEV showed higher anti-migratory and anti-angiogenic activity than the free drug. A comparison of the cytotoxic effect of PTX-pEV on breast cancer cells was carried out, which identified a decrease in cell viability (21.05%) and proliferation (41.04%) only in BT474. PTX delivery pathways for MDA-MB-231 and BT474 were also distinctly affected by EV uptake inhibitors, between cell lines, showing that dynamin-dependent endocytosis and heparin sulphate proteoglycan- dependent mechanism were only responsible by the PTX release on BT474. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-07-05T14:04:28Z 2022-01 2022-01-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10362/141380 |
url |
http://hdl.handle.net/10362/141380 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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1799138097174151168 |