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Development of lipid-polymer hybrid nanoparticles as a strategy to treat Alzheimer’s Disease
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A doença de Alzheimer (AD) é uma doença neurodegenerativa crónica responsável pela maioria dos casos de demência no mundo. Apesar da grande incidência da AD na população mundial, ainda não existe uma abordagem de tratamento eficaz para a patologia. A terapia farmacológica que existe para a doença de AD apenas trata os sintomas, não sendo capaz de induzir ou prevenir regressão da doença.
Devido aos muitos fatores que estão envolvidos na patologia da AD, uma terapia de múltiplos alvos seria a ideal para esta patologia. Isto, aliado ao facto de a AD ser uma doença do sistema nervoso central (CNS) e de o CNS estar protegido por uma barreira altamente seletiva, a barreira hematoencefálica (BBB), explica a ausência de um tratamento eficaz da doença.
Outra limitação para tratar a perturbação é a falta de um diagnóstico preciso. No passado, um diagnóstico preciso desta patologia era mais difícil. No entanto, hoje em dia o uso de técnicas de neuroimagem como a ressonância magnética (MRI) podem dar importantes informações sobre biomarcadores.
Devido a todas as limitações existentes para criar um tratamento eficaz e local da AD, esta tese de mestrado propõe o desenvolvimento de um nanossistema composto por nanopartículas híbridas de tamanho suficientemente pequeno para passar pelo BBB, que encapsulam, por sua vez, nanopartículas superparamagnéticas de óxidos de ferro (SPIONs) e um fármaco (curcumina). O nanossistema incluirá também a funcionalização destas nanopartículas com um péptido penetrante de células (CPP) que permite a translocação de membranas celulares.
O nanossistema proposto pode ser considerado um agente de teranóstico, uma vez que é capaz de ter tanto um efeito terapêutico como de diagnóstico na AD. O efeito terapêutico é dado pela curcumina presente nas nanopartículas, e o efeito de diagnóstico é dado pelas SPIONs encapsuladas uma vez que podem atuar como agentes de contraste para ressonância magnética, permitindo o diagnóstico da AD.
Todas as nanopartículas foram caracterizadas durante as diferentes fases do trabalho. As SPIONs produzidas foram analisadas por microscopia eletrónica de transmissão e o tamanho médio obtido foi de 12,3 ± 2,3 nm. Em seguida, todas as nanopartículas produzidas durante a otimização das nanopartículas híbridas foram caracterizadas utilizando a técnica DLS. Os diâmetros hidrodinâmicos obtidos para as nanopartículas não funcionalizadas foram de 91,6 ± 1,6 nm e para o nanossistema final (funcionalizado) foram de 120,1± 0,1 nm.
Para avaliar a eficiência de internalização das nanopartículas nas células, foi utilizada uma linha celular de células endoteliais humanas do cérebro. A mesma linha celular foi também utilizada para realizar ensaios de citotoxicidade. Os ensaios celulares mostraram que as nanopartículas conseguem internalizar nas células, sendo que o nanossistema mostrou uma capacidade de internalização mais elevada do que as nanopartículas híbridas + SPIONs. Os dois tipos de nanopartículas testados também mostraram uma tendência para se tornarem citotóxicos nas concentrações testadas mais elevadas.
As características do nanossistema produzido nesta tese de mestrado mostram-se promissoras para atravessar a BBB devido à sua capacidade de internalização numa linha celular de células endoteliais do cérebro humano in vitro.
Alzheimer’s disease (AD) is a chronic neurodegenerative disorder responsible for most cases of dementia in the world. Despite the large incidence of AD in the world population, there is still no effective treatment approach to the pathology. The pharmacologic therapy that exists for AD only treat the symptoms, it is not able to induce or prevent disease regression. Due to the many factors that are involved in AD pathology, a multiple-target therapy would be the preferred treatment for this pathology. This, allied to the fact that AD is a central nervous system (CNS) disorder and that the CNS is protected by a highly selective barrier, the blood-brain barrier (BBB), explains the absence of effective treatment for the disorder. Another limitation in treating the disorder is the lack of an accurate diagnosis. In the past, an accurate diagnosis of this pathology was more difficult. Nowadays the use of neuroimaging techniques such as magnetic resonance imaging (MRI) can give important biomarker information. Due to all the existing limitations to creating an effective and local treatment for AD, this master thesis proposes the development of a nanosystem composed of hybrid nanoparticles with a small enough size to pass through the BBB, that encapsulates superparamagnetic iron oxide nanoparticles (SPIONs) and a drug (curcumin). The nanosystem will also include the functionalization of these nanoparticles with a cell-penetrating peptide (CPP) that allows the translocation of cell membranes. The proposed nanosystem can be considered a theranostics agent, since it is able to both have a therapeutic and diagnostic effect on AD. The therapeutic effect is given by the curcumin present in the nanoparticles, and the diagnostic effect is given by the SPIONs encapsulated since they can act as contrast agents in MRI, allowing AD diagnosis. All nanoparticles were characterized during the different stages of the work. The produced SPIONs were analyzed by transmission electron microscopy and the obtained average size was 12.3 ± 2.3 nm. Then, all the nanoparticles produced during the optimization of the hybrid nanoparticles were characterized using DLS technique. The hydrodynamic diameters obtained for the nanoparticles not functionalized was 91.6 ± 1.6 nm and for the final nanosystem (functionalized) was 120.1± 0.1 nm. To assess the efficiency of the nanoparticles in internalizing in cells, a cell line of human brain endothelial cells was used. The same cell line was also used to perform cytotoxicity assays. Cellular assays showed that the nanoparticles can internalize in the cells. However, the nanosystem showed a higher internalization capacity when compared to the hybrid nanoparticles + SPIONs. Both types of nanoparticles tested also showed a tendency to become cytotoxic at the higher concentrations tested. The characteristics of the produced nanosystem in this master thesis are promising to overcome the BBB due to their ability to internalize in a cell line of human brain endothelial cells in vitro.
Alzheimer’s disease (AD) is a chronic neurodegenerative disorder responsible for most cases of dementia in the world. Despite the large incidence of AD in the world population, there is still no effective treatment approach to the pathology. The pharmacologic therapy that exists for AD only treat the symptoms, it is not able to induce or prevent disease regression. Due to the many factors that are involved in AD pathology, a multiple-target therapy would be the preferred treatment for this pathology. This, allied to the fact that AD is a central nervous system (CNS) disorder and that the CNS is protected by a highly selective barrier, the blood-brain barrier (BBB), explains the absence of effective treatment for the disorder. Another limitation in treating the disorder is the lack of an accurate diagnosis. In the past, an accurate diagnosis of this pathology was more difficult. Nowadays the use of neuroimaging techniques such as magnetic resonance imaging (MRI) can give important biomarker information. Due to all the existing limitations to creating an effective and local treatment for AD, this master thesis proposes the development of a nanosystem composed of hybrid nanoparticles with a small enough size to pass through the BBB, that encapsulates superparamagnetic iron oxide nanoparticles (SPIONs) and a drug (curcumin). The nanosystem will also include the functionalization of these nanoparticles with a cell-penetrating peptide (CPP) that allows the translocation of cell membranes. The proposed nanosystem can be considered a theranostics agent, since it is able to both have a therapeutic and diagnostic effect on AD. The therapeutic effect is given by the curcumin present in the nanoparticles, and the diagnostic effect is given by the SPIONs encapsulated since they can act as contrast agents in MRI, allowing AD diagnosis. All nanoparticles were characterized during the different stages of the work. The produced SPIONs were analyzed by transmission electron microscopy and the obtained average size was 12.3 ± 2.3 nm. Then, all the nanoparticles produced during the optimization of the hybrid nanoparticles were characterized using DLS technique. The hydrodynamic diameters obtained for the nanoparticles not functionalized was 91.6 ± 1.6 nm and for the final nanosystem (functionalized) was 120.1± 0.1 nm. To assess the efficiency of the nanoparticles in internalizing in cells, a cell line of human brain endothelial cells was used. The same cell line was also used to perform cytotoxicity assays. Cellular assays showed that the nanoparticles can internalize in the cells. However, the nanosystem showed a higher internalization capacity when compared to the hybrid nanoparticles + SPIONs. Both types of nanoparticles tested also showed a tendency to become cytotoxic at the higher concentrations tested. The characteristics of the produced nanosystem in this master thesis are promising to overcome the BBB due to their ability to internalize in a cell line of human brain endothelial cells in vitro.
Descrição
Tese de mestrado, Ciências Biofarmacêuticas, 2022, Universidade de Lisboa, Faculdade de Farmácia.
Palavras-chave
Alzheimer’s Disease BBB CPP SPIONs Hybrid nanoparticles Teses de mestrado - 2022