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Authors
Abstract(s)
As vesículas extracelulares bacterianas -B-EVs- foram recentemente propostas como uma
potencial fonte valiosa de probióticos: preparações de microrganismos inanimados, e/ou seus
componentes, que conferem um benefício para a saúde do hospedeiro. No entanto, para que os
produtos à base de B-EVs possam ser utilizados em aplicações clínicas, são necessários
métodos adequados de formulação e administração que preservem a estrutura e a funcionalidade
dos B-EVs. As matrizes de microagulhas (MNAs) são sistemas promissores para a
administração transdérmica de fármacos, que estão a ser investigados para a administração
sistémica de uma série de compostos ativos, incluindo fármacos químicos e biológicos, vacinas
e células. Neste estudo, avaliámos a utilidade dos MNAs como sistema de administração de B-
EV. Os B-EVs da bactéria probiótica Gram-positiva Bacillus subtilis 168 foram isolados de
culturas bacterianas por ultracentrifugação seguida de cromatografia de exclusão de tamanho e
encerrados em MNAs de polivinilpirrolidona e acetato de polivinilo (PVP/PVA). Os B-EVs
foram então recuperados dos MNAs, dissolvendo-os em PBS estéril, e a sua estrutura e
propriedades ativas foram comparadas com as dos B-EVs não processados. A microscopia
eletrónica de transmissão confirmou que a estrutura dos B-EVs permaneceu inalterada após a
inclusão e subsequente libertação dos MNAs. Além disso, para avaliar o impacto do invólucro
e da libertação de microagulhas na sua atividade biológica, avaliámos a capacidade dos B-EVs
de B. subtilis de promover a cicatrização de feridas em culturas de queratinócitos humanos
imortalizados (células HaCaT). Os nossos dados preliminares mostram que tanto os B-EVs não
processados como os B-EVs derivados de microagulhas de B. subtilis 168 aceleram a
cicatrização de feridas in vitro, em comparação com o controlo PBS. Em conjunto, os nossos
resultados representam uma prova de conceito de que os MNAs podem ser recursos adequados
para formulações farmacêuticas baseadas em B-EVs e apoiam estudos futuros destinados a
avaliar a biodisponibilidade, a biodistribuição e a atividade de B-EVs administrados através
deste sistema in vivo.
Bacterial extracellular vesicles –B-EVs– have been recently proposed as a potential valuable source of probiotics: preparations of inanimate microorganisms, and/or their components, that confer a benefit for the host’s health. However, suitable formulation and delivery methods that preserve B-EV structure and functionality are required for B-EV-based products to be usable in clinical applications. Microneedle arrays (MNAs) are promising systems for transdermal drug delivery, which are being investigated for systemic administration of several active compounds, including chemical and biological drugs, vaccines and cells. In this study, we evaluated the usefulness of MNAs as a system for B-EV administration. B-EVs from the Gram- positive probiotic bacteria Bacillus subtilis 168 were isolated from bacterial cultures by ultracentrifugation followed by size-exclusion chromatography and enclosed in polyvinylpyrrolidone and polyvinyl acetate (PVP/PVA) MNAs. B-EVs were then recovered from MNAs by dissolving the latter in sterile PBS, and their structure and active properties were compared with those of unprocessed B-EVs. Transmission electron microscopy confirmed that the B-EV structure remained unaltered following inclusion in and subsequent release from MNAs. Moreover, as a proxy to evaluate the impact of microneedle enclosure and release on their biological activity, we assessed the ability of B. subtilis B-EVs to promote wound healing in cultured immortalized human keratinocytes (i.e., HaCaT cells). Our preliminary data show that both unprocessed and microneedle derived B-EVs from B. subtilis 168 accelerate wound healing in vitro, compared to the PBS control. Altogether, our results represent a proof of concept that MNAs may be suitable resources for B-EV-based pharmaceutical formulations and endorse future studies to evaluate the bioavailability, biodistribution, and activity of B-EVs administered through this system in vivo.
Bacterial extracellular vesicles –B-EVs– have been recently proposed as a potential valuable source of probiotics: preparations of inanimate microorganisms, and/or their components, that confer a benefit for the host’s health. However, suitable formulation and delivery methods that preserve B-EV structure and functionality are required for B-EV-based products to be usable in clinical applications. Microneedle arrays (MNAs) are promising systems for transdermal drug delivery, which are being investigated for systemic administration of several active compounds, including chemical and biological drugs, vaccines and cells. In this study, we evaluated the usefulness of MNAs as a system for B-EV administration. B-EVs from the Gram- positive probiotic bacteria Bacillus subtilis 168 were isolated from bacterial cultures by ultracentrifugation followed by size-exclusion chromatography and enclosed in polyvinylpyrrolidone and polyvinyl acetate (PVP/PVA) MNAs. B-EVs were then recovered from MNAs by dissolving the latter in sterile PBS, and their structure and active properties were compared with those of unprocessed B-EVs. Transmission electron microscopy confirmed that the B-EV structure remained unaltered following inclusion in and subsequent release from MNAs. Moreover, as a proxy to evaluate the impact of microneedle enclosure and release on their biological activity, we assessed the ability of B. subtilis B-EVs to promote wound healing in cultured immortalized human keratinocytes (i.e., HaCaT cells). Our preliminary data show that both unprocessed and microneedle derived B-EVs from B. subtilis 168 accelerate wound healing in vitro, compared to the PBS control. Altogether, our results represent a proof of concept that MNAs may be suitable resources for B-EV-based pharmaceutical formulations and endorse future studies to evaluate the bioavailability, biodistribution, and activity of B-EVs administered through this system in vivo.
Description
Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2023, Universidade de Lisboa, Faculdade de Farmácia.
Keywords
Bacterial extracellular vesicles (B-EVs)microneedles (MNAs); transdermal administration, probiotics, healing Microneedles (MNAs) Transdermal administration Probiotics Healing Mestrado Integrado - 2023