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As doenças lisossômicas de depósito (DLDs) compreendem cerca de 70 desordens genéticas ocasionadas por mutações em proteínas críticas para o funcionamento dos lisossomos. Apesar de serem consideradas raras quando isoladas, as DLDs afetam coletivamente 1 a cada 5.000 nascidos vivos, tornando-as significativas doenças metabólicas da infância. Dentre as DLDs, encontra-se a doença de Niemann-Pick C (NPC), a qual é caracterizada por um engarrafamento lipídico intracelular ocasionado pela mutação nos genes NPC1 ou NPC2 e consequente acúmulo de colesterol e glicoesfingolipídios dentro do compartimento endossomal/lisossomal das células. A NPC apresenta, assim como a maioria das DLDs, envolvimento neurológico. Ainda não existe cura para NPC, mas inúmeros estudos vêm demonstrando a utilização benéfica da - -CD) devido à sua capacidade de mobilizar o colesterol acumulado dos lisossomos para a corrente sanguínea. Entretanto, a principal -CD é a sua incapacidade em atravessar a barreira hemato-encefálica (BHE). A procura por novos sistemas de liberação de fármacos tem sido muito relevante no sentido de se estabelecerem alternativas terapêuticas mais eficientes, que possibilitem administrar os fármacos com maior segurança, com efeitos colaterais minimizados e nos locais de ação específicos. Nesse sentido, a estrutura tridimensional flexível torna as nanopartículas lipídicas (fases esponja e microemulsões) formadas por monoleína excelentes candidatas ao carreamento de fármacos e enzimas. No desenvolvimento de carreadores nanométricos direcionados ao sistema nervoso central (SNC), o polissorbato 80 vem demonstrando ser o tensoativo mais indicado. Considerando o exposto, o objetivo deste trabalho foi o desenvolvimento de uma nanopartícula-plataforma, capaz de atravessar a BHE e liberar enzimas/fármacos dentro dos lisossomos. Além disso, utilizando a plataforma como ponto de partida, objetivou- -CD e assim o desenvolvimento de uma nanopartícula para o tratamento da NPC. Por fim, buscou-se avaliar o potencial citotóxico das nanopartículas previamente desenvolvidas. As nanopartículas foram caracterizadas por espalhamento dinâmico de luz (DLS), espalhamento de raios-X a baixos ângulos (SAXS) e 11 por criomicroscopia eletrônica de transmissão (cryo-MET). As nanopartículas consideradas como plataformas (NS) apresentaram diâmetro médio de cerca -CD apresentaram (NS -CD) diâmetro médio de cerca de 120 nm. Os perfis de espalhamento obtidos por SAXS para ambas nanopartículas desenvolvidas ajustaram-se ao modelo matemático de Teubner-Strey, aplicado com sucesso para descrever microemulsões e fases esponja. Os resultados de cryo-TEM corroboraram os resultados de SAXS e DLS, demonstrando a presença de estruturas com fase interna bicontínua desordenada com tamanho médio próximo a 100 nm. Através da utilização de cumarina-6 foi possível determinar que as NS e NS -CD atingiram os lisossomos após 1 hora de incubação na cultura celular. As nanopartículas foram rastreadas in vivo e se mostraram capazes de atingir o tecido cerebral, conferindo grande vantagem em relação às terapias comumente utilizadas. As nanopartículas foram capazes, também, de atingir o tecido hepático, que está amplamente comprometido em doenças como a NPC. Além disso, a NS -CD mostrou atividade em reduzir os níveis de colesterol acumulado em fibroblastos dérmicos derivados de pacientes NPC. Tanto NS, quanto NS -CD não causaram hemólise, um pré-requisito para administração parenteral, não apresentaram citotoxicidade e genotoxiciddade. Além disso, não causaram perturbações oxidativas/nitrativas quando incubadas com fibroblastos de pacientes NPC. As nanopartículas desenvolvidas neste projeto apresentaram resultados bastante promissores quanto à sua utilização para o tratamento de DLDs e, em particular, de NPC. Lysosomal storage diseases (LSDs) comprise about 70 genetic disorders caused by mutations in critical proteins to lysosomes functioning. Although they are considered rare when isolated, LSDs collectively affect 1 in 5,000 live births, making them significant childhood metabolic diseases. Among the LSDs is Niemann-Pick type C (NPC) disease, which is characterized by intracellular lipid impairment caused by mutation in NPC1 or NPC2 genes and consequent accumulation of cholesterol and glycosphingolipids inside the endosomal/lysosomal cell compartment. NPC presents neurological impairment, as most LSDs. There is still no cure for NPC, but several studies have demonstrated the beneficial use of -cyclodextrin ( -CD) because of its ability to mobilize accumulated cholesterol from lysosomes into the bloodstream. However, the main disadvantage presented by free -CD is the inability to cross the blood-brain barrier (BBB). The search for new drug delivery systems has been very relevant in order to establish more efficient therapeutic alternatives, that allow to administer drugs with greater security, minimized side effects and in the specific sites of action. In this sense, the flexible threedimensional structure makes the lipidic nanoparticles (sponge phases and microemulsions) formed by monoolein excellent candidates for the transport of drugs and enzymes. In the development of nanometric carriers directed to the central nervous system (CNS), polysorbate 80 has been shown to be the most indicated surfactant. Considering the above, the objective of this work was the development of a nanoparticle-platform, able to cross the BBB and release drugs/enzymes into the lysosomes. In addition, using the platform as a starting point, the objective was to incorporate -CD in a nanoparticle for NPC treatment. Finally, we aimed to evaluate the cytotoxic potential of previously developed nanoparticles. The nanoparticles were characterized by dynamic light scattering (DLS), small angle X ray scattering (SAXS) and by cryogenic transmission electron microscopy (cryo-TEM). The nanoparticles considered as platforms (NS) had an average diameter of about 115 nm, whereas nanoparticles containing -CD (NS -CD) had a mean diameter of about 120 nm. The scattering profiles obtained by SAXS for both nanoparticles were adjusted 13 to the Teubner-Strey mathematical model, successfully applied to describe microemulsions and sponge phases. The cryo-TEM results corroborated the results of SAXS and DLS, demonstrating the presence of structures with a disordered bicontinuous inner phase and with an average size close to 100 nm. By using coumarin-6 it was possible to track NS and NS -CD into cells, and results showed that the NS reached the lysosomes after 1 hour of incubation with healthy fibroblasts, and NS -CD remain in NPC fibroblasts for at least 3 hours. The nanoparticles were tracked in vivo and reached the brain tissue, which confers great advantage over the commonly used therapies. The nanoparticles were also able to target hepatic tissue, which is extensively involved in diseases such as NPC. In addition, NS -CD induced a decrease in cholesterol accumulation when incubated with dermal NPC fibroblasts patientsderived. Both NS and NS -CD did not cause hemolysis, a pre-requisite for parenteral administration, did not show cytotoxicity and genotoxicity. In addition, they did not cause oxidative/nitrative disturbances when incubated with fibroblasts from NPC patients. The nanoparticles developed in this project presented very promising results regarding their use for the treatment of LSDs, in particular NPC.

Introduction: Homocysteine (Hcy) tissue accumulation occurs in a metabolic disease characterized biochemically by cystathionine β-synthase (CBS) deficiency and clinically by mental retardation, vascular problems, and skeletal abnormalities. Previous studies indicate the occurrence of DNA damage secondary to hyperhomocysteinemia and it was observed that DNA damage occurs in leukocytes from CBS-deficient patients. This study aimed to investigate whether an oxidative mechanism could be involved in DNA damage previously found and investigated the in vitro effect of N-acety-L-cysteine (NAC) on DNA damage caused by high Hcy levels. Methods: We evaluated a biomarker of oxidative DNA damage in the urine of CBS‑deficient patients, as well as the in vitro effect of NAC on DNA damage caused by high levels of Hcy. Moreover, a biomarker of lipid oxidative damage was also measured in urine of CBS deficient patients. Results: There was an increase in parameters of DNA (8-oxo-7,8-dihydro-2’- deoxyguanosine) and lipid (15-F2t-isoprostanes levels) oxidative damage in CBS-deficient patients when compared to controls. In addition, a significant positive correlation was found between 15-F2t-isoprostanes levels and total Hcy concentrations. Besides, an in vitro protective effect of NAC at concentrations of 1 and 5 mM was observed on DNA damage caused by Hcy 50 μM and 200 μM. Additionally, we showed a decrease in sulfhydryl content in plasma from CBS-deficient patients when compared to controls. Discussion: These results demonstrated that DNA damage occurs by an oxidative mechanism in CBS deficiency together with lipid oxidative damage, highlighting the NAC beneficial action upon DNA oxidative process, contributing with a new treatment perspective of the patients affected by classic homocystinuria.

A Mucopolissacaridose tipo IVA (MPS IVA), é uma doença lisossômica de depósito ocasionada pela degradação deficiente dos glicosaminoglicanos (GAG) queratan sulfato e condroitin-6-sulfato devido à deficiência da enzima N-acetilgalactosamina 6-sulfatase. Como a fisiopatologia desta doença ainda não está totalmente elucidada e muitos estudos vêm demonstrando o envolvimento do estresse oxidativo e inflamação na patogênese de outros tipos de mucopolissacaridoses, o objetivo principal deste trabalho foi investigar os parâmetros de estresse oxidativo e mediadores inflamatórios em pacientes MPS IVA sob terapia de reposição enzimática (TRE). Foram analisadas amostras de urina e sangue de pacientes MPS IVA sob TRE (n=17) e controles saudáveis pareados por idade (n=10-14). Os pacientes apresentaram diminuição significativa nos níveis de defesas antioxidantes, medida através dos níveis de glutationa reduzida (GSH), e aumento da enzima superóxido dismutase (SOD) em eritrócitos. Em relação ao dano a biomoléculas, foi observado nos pacientes um aumento de lipoperoxidação (aumento dos níveis de isoprostanos urinários) e de dano oxidativo a proteínas (aumento dos níveis urinários de di-tirosina e diminuição dos grupamentos sulfidrila no plasma) comparativamente aos controles. Nossos resultados também mostraram que os pacientes MPS IVA sob TRE apresentaram maior dano ao DNA, sendo este dano de origem oxidativa e atingindo bases purínicas e pirimidínicas. Além disso, os pacientes apresentaram níveis significativamente aumentados de interleucina-6 e esta, por sua vez, apresentou correlação negativa com GSH, mostrando uma possível relação entre inflamação e estresse oxidativo nesta doença. Considerando que os níveis de GAG urinários ainda se encontravam elevados nos pacientes em comparação com o grupo controle, pode-se supor que os mesmos estejam, pelo menos em parte, correlacionados com os danos oxidativos encontrados nestes pacientes. Os dados encontrados no presente trabalho sugerem que pacientes MPS IVA sob TRE apresentam uma condição pró-inflamatória e oxidativa e que a suplementação com antioxidantes em combinação com a TRE deve ser investigada com o intuito de melhorar a qualidade de vida dos pacientes. Cabe ressaltar que este é o primeiro estudo em pacientes que relaciona MPS IVA com estresse oxidativo e inflamação. The Mucopolysaccharidosis type IVA (MPS IVA) is a lysosomal storage disease caused by impaired degradation of keratan sulfate and chondroitin-6-sulfate glycosaminoglycans (GAG), due to a deficiency on the enzyme N-acetylgalactosamine-6-sulfatase. Since the pathophysiology of this disease is still not totally elucidated and many studies demonstrated the involvement of oxidative stress and inflammation in the pathogenesis of other mucopolysaccharidoses types, the principal objective of this study was to investigate oxidative stress parameters and inflammatory mediators in MPS IVA patients under enzyme replacement therapy (ERT). Urine anda blood samples of MPS IVA patients under ERT (n= 17) and healthy age-matched controls (n= 10-14) were evaluated. Patients presented a significant decrease in antioxidant defenses levels, assessed by reduced glutathione (GSH), and increased superoxide dismutase (SOD) activity in erythrocytes. With regard to the biomolecules damage, was observed that patients presented lipid peroxidation (increase of isoprostanes urinary levels) and protein damage (increase of di-tyrosine urinary levels and decrease of sulfhydryl groups in plasma), when compared to controls. Our results showed higher DNA damage levels in MPS IVA patients compared to control group, in both pyrimidines and purines bases. The pro-inflammatory cytokine interleukin 6 (IL-6) was significantly increased in patients and showed an inverse correlation with GSH levels, showing a possible link between inflammation and oxidative stress in MPS IVA disease. Considering that GAG urinary level were still high in ERT patients compared to the control group, we propose that GAG are, at least in part, related with oxidative damage found in MPS IVA patients. The data presented suggest that pro-inflammatory and pro-oxidant states occur in MPS IVA patients under ERT, and the supplementation of antioxidants in combination with ERT can be investigated with the purpose of improving the patient’s life quality. To the best of our knowledge, this is the first study in patients relating MPS IVA with oxidative stress and inflammation.