Eletroporação e estabilidade de membranas aniônicas com diferentes níveis de complexidade
Data
2022-07-19
Autores
Leomil, Fernanda dos Santos Costa 
Orientadores
Riske, Karin do Amaral
Tipo
Tese de doutorado
Título da Revista
ISSN da Revista
Título de Volume
Resumo
A estabilidade das membranas celulares é de importância vital para as células.
Após estímulos de alta intensidade, como os causados pela aplicação de pulsos
elétricos, poros são abertos na membrana (eletroporação) e se fecham em poucos
ms, permitindo que a integridade da membrana seja preservada, conforme observado
em vesículas unilamelares gigantes (GUVs) neutras de PC (fosfatidilcolina). No
entanto, a adição do lipídio negativo PG (fosfatidilglicerol) mostrou desempenhar um
papel importante na desestabilização das membranas após eletroporação: (i) alguns
poros se abrem indefinidamente, levando à completa perda da integridade da vesícula
(bursting) e (ii) GUVs que sobreviveram à poração exibem permeabilidade de longa
duração, revelando a permanência de poros submicroscópicos após o fechamento
dos macroporos.
Nesta tese, foi investigada a resposta mecânica de GUVs a campos elétricos,
com foco na estabilidade de membranas contendo frações crescentes de lipídios
aniônicos de conhecida relevância biológica, como o fosfatidilinositol (PI),
fosfatidilinositol 4,5-bisfosfato (PIP2), cardiolipina (CL), fosfatigilglicerol (PG) e extrato
lipídico de E. coli, bem como foi examinada a relevância da assimetria dos lipídios
negativos entre as monocamadas na estabilidade dessas membranas. Também foram
explorados sistemas mais complexos, como vesículas gigantes de membrana
plasmática (GPMVs), que representam um passo intermediário entre GUVs lipídicas
e células, apresentando as características lipídicas e proteicas das células que as
originaram. Métodos experimentais e computacionais foram desenvolvidos e
aperfeiçoados ao longo do trabalho para a realização e análise dos experimentos
executados.
Em GUVs com distribuição simétrica de lipídios aniônicos, medidas da
ocorrência de eventos desestabilizantes, como bursting e permeabilidade duradoura,
mostraram que as membranas contendo frações mais altas de lipídios negativos (50
mol%) são mais instáveis e propensas à desestabilização. Esses efeitos, que se
mostraram independentes do tipo de lipídio negativo utilizado, foram relacionados a
uma redução de cerca de 50% na tensão de linha do poro, propriedade que governa
o seu fechamento e que foi calculada através de um software desenvolvido durante o
projeto (PoET). GUVs com distribuição assimétrica de lipídio negativo entre as monocamadas da membrana foram obtidas por dois métodos diferentes: transferência
de fase e alterações do pH do meio externo das vesículas. A assimetria foi verificada
por meio de um ensaio de supressão de fluorescência aperfeiçoado ao longo do
estudo, em combinação com um algoritmo desenvolvido para medidas de
fluorescência em GUVs. Eventos desestabilizantes foram observados já para
pequenos graus de assimetria de carga (5 mol%), que potencializam a ocorrência de
eventos mais extremos, como o bursting, devido à geração de curvatura espontânea
acentuada. Quando consideramos modelos ainda mais relevantes, resultados
preliminares mostram que existem diferenças na forma com que GPMVs e GUVs
respondem a pulsos elétricos, provavelmente devido à maior complexidade exibida
pelas membranas das GPMVs.
Os resultados apresentados aqui evidenciam que os lipídios aniônicos
possuem um papel importante na desestabilização das membranas após
eletroporação, sendo os efeitos realçados no caso em que estes estão
assimetricamente distribuídos entre as monocamadas das membranas, como
observado nas membranas celulares. Acreditamos que os achados possam contribuir
para o desenvolvimento de sistemas de entrega intracelular de drogas baseados em
eletroporação mais eficientes. Ainda, julgamos que o aperfeiçoamento do ensaio de
supressão de fluorescência e o desenvolvimento do software para cálculo da tensão
de linha e do algoritmo para medidas de fluorescência serão de grande contribuição
para a comunidade científica, permitindo a realização de experimentos e obtenção de
dados de forma mais confiável e reprodutível.
Membrane stability is of vital importance to cells. After high-intensity stimuli, such as those caused by the application of electric pulses, pores are opened in the membrane (electroporation) and close within a few ms, allowing the integrity of the membrane to be preserved, as observed in neutral PC (phosphatidylcholine) giant unilamellar vesicles (GUVs). However, the addition of the negative lipid PG (phosphatidylglycerol) has shown to play an important role in the destabilization of membranes after electroporation: (i) some pores open indefinitely, leading to complete loss of vesicle integrity (bursting) and (ii) GUVs that survived poration exhibit longlasting permeability, revealing the persistence of submicroscopic pores after macropores have closed. In this thesis, the mechanical response of GUVs to electric fields was investigated, focusing on the stability of membranes containing increasing fractions of anionic lipids of known biological relevance, such as phosphatidylinositol (PI), phosphatidylinositol 4,5-bisphosphate (PIP2), cardiolipin (CL), phosphatidylglycerol (PG) and E. coli lipid extract, as well as the relevance of the asymmetry of negative lipids between the membrane monolayers in the stability of these membranes. More complex systems were also explored, such as Giant Plasma Membrane Vesicles (GPMVs), which represent an intermediate step between lipid GUVs and cells, exhibiting the lipid and protein characteristics of the cells that originated them. Experimental and computational methods were developed and improved throughout the work to execute and analyze the experiments performed. In GUVs with symmetric distribution of anionic lipids, measurements of the occurrence frequency of destabilizing events, such as bursting and long-lasting permeability, showed that membranes containing higher fractions of negative lipids (50 mol%) are more unstable and prone to destabilization. These effects, which proved to be independent of the type of negative lipid used, were related to a reduction of about 50% in the pore edge tension, a property that governs its closure and that was calculated using a software developed during the project (PoET). GUVs with asymmetric negative lipid distribution between the membrane monolayers were obtained by two different methods: phase transfer and changes in the pH of the external solution of the vesicles. Asymmetry was verified using a fluorescence quenching assay improved throughout the study, in combination with an algorithm developed for fluorescence measurements in GUVs. Destabilizing events were already observed for small amounts of charge asymmetry (5 mol%), which enhances the occurrence of more extreme events, such as bursting, due to the generation of accentuated spontaneous curvature. When we consider even more relevant models, preliminary results show that there are differences in the way GPMVs and GUVs respond to electric pulses, probably due to the greater complexity exhibited by GPMV membranes. The results presented here show that anionic lipids play an important role in the destabilization of membranes after electroporation, the effects being enhanced in the case where they are asymmetrically distributed between the membrane monolayers, as observed in cell membranes. We believe that the findings may contribute to the development of more efficient electroporation-based intracellular drug delivery systems. Furthermore, we believe that the improvement of the fluorescence quenching assay and the development of the software for calculating the edge tension and the algorithm for fluorescence measurements will be of great contribution to the scientific community, allowing the performance of experiments and data collection in a more reliable and reproducible way.
Membrane stability is of vital importance to cells. After high-intensity stimuli, such as those caused by the application of electric pulses, pores are opened in the membrane (electroporation) and close within a few ms, allowing the integrity of the membrane to be preserved, as observed in neutral PC (phosphatidylcholine) giant unilamellar vesicles (GUVs). However, the addition of the negative lipid PG (phosphatidylglycerol) has shown to play an important role in the destabilization of membranes after electroporation: (i) some pores open indefinitely, leading to complete loss of vesicle integrity (bursting) and (ii) GUVs that survived poration exhibit longlasting permeability, revealing the persistence of submicroscopic pores after macropores have closed. In this thesis, the mechanical response of GUVs to electric fields was investigated, focusing on the stability of membranes containing increasing fractions of anionic lipids of known biological relevance, such as phosphatidylinositol (PI), phosphatidylinositol 4,5-bisphosphate (PIP2), cardiolipin (CL), phosphatidylglycerol (PG) and E. coli lipid extract, as well as the relevance of the asymmetry of negative lipids between the membrane monolayers in the stability of these membranes. More complex systems were also explored, such as Giant Plasma Membrane Vesicles (GPMVs), which represent an intermediate step between lipid GUVs and cells, exhibiting the lipid and protein characteristics of the cells that originated them. Experimental and computational methods were developed and improved throughout the work to execute and analyze the experiments performed. In GUVs with symmetric distribution of anionic lipids, measurements of the occurrence frequency of destabilizing events, such as bursting and long-lasting permeability, showed that membranes containing higher fractions of negative lipids (50 mol%) are more unstable and prone to destabilization. These effects, which proved to be independent of the type of negative lipid used, were related to a reduction of about 50% in the pore edge tension, a property that governs its closure and that was calculated using a software developed during the project (PoET). GUVs with asymmetric negative lipid distribution between the membrane monolayers were obtained by two different methods: phase transfer and changes in the pH of the external solution of the vesicles. Asymmetry was verified using a fluorescence quenching assay improved throughout the study, in combination with an algorithm developed for fluorescence measurements in GUVs. Destabilizing events were already observed for small amounts of charge asymmetry (5 mol%), which enhances the occurrence of more extreme events, such as bursting, due to the generation of accentuated spontaneous curvature. When we consider even more relevant models, preliminary results show that there are differences in the way GPMVs and GUVs respond to electric pulses, probably due to the greater complexity exhibited by GPMV membranes. The results presented here show that anionic lipids play an important role in the destabilization of membranes after electroporation, the effects being enhanced in the case where they are asymmetrically distributed between the membrane monolayers, as observed in cell membranes. We believe that the findings may contribute to the development of more efficient electroporation-based intracellular drug delivery systems. Furthermore, we believe that the improvement of the fluorescence quenching assay and the development of the software for calculating the edge tension and the algorithm for fluorescence measurements will be of great contribution to the scientific community, allowing the performance of experiments and data collection in a more reliable and reproducible way.
Descrição
Citação
LEOMIL, F.S.C. Eletroporação e estabilidade de membranas aniônicas com diferentes níveis de complexidade. São Paulo, 2022. 163 f. Tese (Doutorado em Biologia Molecular) - Escola Paulista de Medicina (EPM), Universidade Federal de São Paulo (UNIFESP). São Paulo, 2022.