Made available in DSpace on 2016-06-02T19:22:06Z (GMT). No. of bitstreams: 1 3661.pdf: 3457379 bytes, checksum: b8065e93e77882df9787154074c2cd19 (MD5) Previous issue date: 2010-12-17 / Financiadora de Estudos e Projetos / The fast development of the nanotechnology, and the use of this technology to develop new products to help in diagnosis diseases, drugs and treatments for many diseases is increasing. Some authors have been warning about the nanoparticles use, because the real affect, toxic or not, still unknown. Iron oxide nanoparticles have been used in contrast liquid for magnetic resonance imaging, cancer treatment, among others. The aim of this work was to analyze the possible effects of chronic treatment with these nanoparticles in adults rats. The animals were distributed in 3 groups: Control (saline 0.9% - 0.1 mL/100 of body weight), nFe 0.3 (magnetic iron oxide nanoparticles 0.3 mg/kg BW) and nFe 0.6 (magnetic iron oxide nanoparticles 0.6 mg/kg BW). The animals were treated by gastric gavage during 8 weeks, 5 days per week. The body weight of the animals from the nFe 0.6 group decreased when compared to animals from the control group, and the BW of the animals from nFe 0.3 group was not different from the control group. The animals from the nFe 0.6 group showed the higher blood levels of cholesterol, although the levels of glucose, urea, creatinine, AST, ALT and alkaline phosphatase, plus hematology were not different from the animals of control group. Moreover, the seminal gland and ventral prostate of the animals of nFe 0.6 were atrophied. All those parameters of animals from nFe 0.3 group were not different compared to control group. The bone (femur and vertebra) biometric, biomechanical and biophysical parameters were similar in the animals from the different experimental groups. These results showed that high concentrations of magnetic iron oxide nanoparticles could be toxic and dangerous to the rats when take daily, but in low concentration it could be safe. / Com o rápido desenvolvimento da nanotecnologia, o homem vem utilizando cada vez mais essa nova tecnologia para auxiliá-lo no desenvolvimento de novos produtos que auxiliem no diagnóstico de doenças, fármacos e tratamentos de inúmeras enfermidades. Alguns autores vêm chamando a atenção para o uso de nanopartículas em vários produtos comerciais, pois pouco se sabe sobre sua toxicidade. Especificamente as nanopartículas de óxido de ferro magnético vêm sendo utilizadas em líquidos de contraste de ressonância magnética, tratamento de câncer, entre outros. Com isso, o objetivo deste trabalho foi analisar os possíveis efeitos tóxicos dessas nanopartículas administradas cronicamente em ratos adultos jovens. Ratos Wistar machos foram distribuídos em 3 grupos experimentais: Controle (salina 0,9% - 0,1mL/100g MC), nFe 0,3 (nanopartículas de óxido de ferro - 0,3 mg/kg MC) e nFe 0,6 (nanopartículas de óxido de ferro - 0,6 mg/kg MC). O tratamento foi realizado por gavagem gástrica, 5 dias por semana, durante 8 semanas. As análises de massa corporal (MC) dos animais indicou que a concentração de 0,6 mg/kg MC de nanopartículas de óxido de ferro magnético levou à uma diminuição da MC dos animais em relação aos animais tratados com 0,3 mg/kg MC e aos animais tratados solução salina 0,9%. Os animais do grupo nFe 0,6 apresentaram aumento de colesterol sanguíneo, entretanto as concentrações de glicose, ferro, uréia, creatinina, AST, ALT e fosfatase alcalina, além da análise hematológica não foram diferentes dos animais do grupo controle. Além disso, as glândulas seminais e próstatas ventrais dos animais tratados com nFe 0,6 mg/kg MC atrofiaram, sendo que as dos animais do grupo nFe 0,3 não apresentaram diferença em relação as do grupo controle. Os parâmetros biométricos, biomecânicos e biofísicos dos ossos (fêmur e vértebra) foram semelhantes nos 3 grupos experimentais. Portanto, o tratamento crônico com nanopartículas na concentração de 0,6 mg/kg MC foi prejudicial à saúde dos animais, exceto no tecido ósseo, sendo que tal efeito não foi observado com a concentração mais baixa.

Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-09-15T17:49:26Z No. of bitstreams: 1 TeseTLLC.pdf: 3509437 bytes, checksum: 42003cf0915a332a24874db33a08def6 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-16T19:27:48Z (GMT) No. of bitstreams: 1 TeseTLLC.pdf: 3509437 bytes, checksum: 42003cf0915a332a24874db33a08def6 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-09-16T19:28:00Z (GMT) No. of bitstreams: 1 TeseTLLC.pdf: 3509437 bytes, checksum: 42003cf0915a332a24874db33a08def6 (MD5) / Made available in DSpace on 2016-09-16T19:28:10Z (GMT). No. of bitstreams: 1 TeseTLLC.pdf: 3509437 bytes, checksum: 42003cf0915a332a24874db33a08def6 (MD5) Previous issue date: 2015-03-06 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / This project aims to investigate the TiO2-NP toxicity after acute (48 h) and subchronic (14 d) exposure using hematology, biochemical and morphological biomarkers in P. lineatus. TiO2- NP only caused blood disorders after subchronic exposure and did not show geno- and neurotoxicity, but they affected the immunity of P. lineatus. In gills, acute exposure to TiO2- NP decreased reactive oxygen species (ROS) formation and increased glutathione (GSH) levels, while subchronic exposure increased ROS and GSH levels and inhibited superoxide dismutase (SOD) activity. In kidney, acute exposure to TiO2-NP did not cause ROS formation and changes in antioxidant enzymes activity, but increased GSH and lipid peroxidation (LPO) levels. Subchronic exposure inhibited catalase activity in kidney tissue, but redox balance was not impaired. In the liver, acute exposure to TiO2-NP did not alter the antioxidant enzymes activity and metallothionein levels, but ROS formation decreased and GSH levels increased. After subchronic exposure, SOD activity did not change, but GPx and GST activity and GSH levels increased in the liver. Additionally, morphological damage was found in gills, liver and kidneys. Gills showed slight changes after acute exposure and slight to moderate changes after subchronic exposure. In kidney and liver tissue, degenerative changes and necrosis occurred after TiO2-NP exposure. TiO2-NP also inhibited enzymes of osmoregulation activity in gills after acute exposure, but had no effect after subchronic exposure and in renal tissue. Mitochondria-rich cells (MRC) density increased in gill filaments and renal tissue after subchronic exposure, while plasma osmolality decreased and calcium ions (Ca2+) concentration increased after acute exposure. Osmolality, however, was restored after subchronic exposure. These results suggest that GSH played an important role in preventing ROS formation. In liver and kidneys, TiO2-NP can cause moderate to severe tissue damage the long term, may lead to organ dysfunction, as considerable portion of the organ was reduced due to necrosis and degenerative damage. Osmoregulatory system impairment was caused after acute exposure to TiO2-NP, but no after subchronic exposure, probably due to proliferation of new MRC and morphological changes in gills. However, these morphological changes may hinder gases exchange and osmotic and ionic balance in the long term. / O presente projeto teve como objetivo investigar a toxicidade das NP-TiO2 após exposição aguda (48 h) e subcrônica (14 d) utilizando biomarcadores de hematologia, bioquímicos e morfológicos em P. lineatus. As NP-TiO2 causaram distúrbios hematológicos apenas após exposição subcrônica e não mostraram genotoxicidade e neurotoxicidade, porém afetaram a imunidade dos animais. Nas brânquias, a exposição aguda a NP-TiO2 diminuiu a produção de espécies reativas de oxigênio (ERO) e aumentou os níveis de glutationa (GSH), enquanto a exposição subcrônica aumentou as ERO e os níveis de GSH e diminuiu a atividade da superóxido dismutase (SOD). Nos rins, a exposição aguda a NP-TiO2 não causou formação de ERO e alteração na atividade das enzimas antioxidantes, porém causou um aumento nos níveis de GSH e lipoperoxidação (LPO). Após exposição subcrônica a atividade da catalase diminuiu sem causar desbalanço oxidativo. No fígado, a exposição aguda a NP-TiO2 não alterou as enzimas antioxidantes e metalotioneína, mas causou diminuição de ERO e aumento de GSH. Após exposição subcrônica ocorreu uma diminuição na atividade da SOD e aumento da atividade das enzimas GPx e GST e do conteúdo de GSH no fígado. Danos morfológicos também foram encontrados em brânquias, fígado e rins. As brânquias mostraram alterações leves após exposição aguda e alterações leves e moderadas após exposição subcrônica. No tecido renal e hepático, mudanças degenerativas e necrose ocorreram após exposição a NPTiO2. As NP-TiO2 inibiram a atividade das enzimas com função de osmorregulação branquiais após exposição aguda sem nenhuma alteração após exposição subcrônica e no tecido renal. A densidade de células ricas em mitocôndrias (CRM) aumentou nos filamentos branquiais e tecido renal após exposição subcrônica, enquanto a exposição aguda diminuiu a osmolalidade plasmática e aumentou a concentração de íons cálcio (Ca2+). No entanto, a osmolalidade foi restabelecida após exposição subcrônica. Esses resultados sugerem que a GSH pode ser uma das principais vias no combate as ERO. No fígado e nos rins, as NP-TiO2 podem causar danos moderados a severos no tecido que podem levar à perda da função do órgão em longo prazo, uma vez que a porção do tecido funcional reduziu devido aos danos degenerativos e necrose. Desequilíbrio osmorregulatório foi causado pela exposição aguda a NP-TiO2, mas não após exposição subcrônica devido à proliferação de novas CRM e mudanças morfológicas nas brânquias. No entanto, essas mudanças morfológicas podem dificultar a troca de gases e o equilíbrio osmótico e iônico em longo prazo.

Scientific research using nanotechnology is a relatively recent development with a variety of potential applications in many fields of science. Within this field of research, many new products, with improved performances, have been developed. Despite increased research on its toxicity to ecosystem, the knowledge about this area is still limited. To evaluate the toxicity and genotoxicity of different sizes silica nanoparticles (SiNP)to the environment, different species, on different trophic levels (Vibrio fisheri, Raphidocelissubcapitata, Daniorerio and Allium cepa) were exposed to TM40 (22 nm), HS30 (12 nm), SM30 (7 nm) with concentrations ranging from0.19 to 163.8 g/L (TM40) and 0.29 to 122.85 g/L (HS30 and SM30), and the following parameters were monitored during exposure: production of bioluminescence (V. fischeri), growth rate (R. subcapitata), embryonic development and DNA damage (D. rerio) and germination rate, growth and DNA damage (A. cepa). Within each test SiNPpresent a size dependent chronic toxicity. The bioluminescence test present a EC50 of 29.11, 32.34 and 4.58 g/L for TM40, HS30 and SM30, respectively. For the growth rate assay the EC50 was 9.32, 9.07 and 7.93 g/L for TM40, HS30 and SM30, respectively. And for the zebra fish embryonic development test for TM40, HS30 and SM30, the EC50 was 5.85, 1.13 and 2.68 g/L respectively. All particles also induce phytotoxicity in A.cepa, growth and germination reduce significatively when expose to SiNP. Futhermoregenotoxic effects were also induced by the particles for both A.cepaand D. rerio. Therefore, SiNP can cause toxicity to the environment and size can strongly influence this toxicity / Com uma variedade de aplicações potenciais, em diversos campos da ciência, as pesquisas científicas utilizando nanotecnologia são de desenvolvimento relativamente recente. Dentro deste campo de pesquisa, vários novos produtos, com desempenhos melhorados têm sido desenvolvidos. Apesar do aumento de pesquisas sobre a toxicidade dessas tecnologias à biota, o conhecimento sobre esta área ainda é limitado. Visando avaliar a toxicidade e genotoxicidade denanopartículasde sílica (SiNP) no meio ambiente diferentes espécies pertencentes a diversos níveis tróficos (Vibriofisheri, Raphidocelissubcapitata, DaniorerioandAllium cepa) foram expostos a Ludox TM40 (22 nm), Ludox HS30 (12 nm) e Ludox SM30 (7 nm). As espécies de teste foram expostas a concentrações de nanopartículas (NP) variando de 0.29 a 163.8 g/L (TM40) e 0.19 a 122.85 g/L (HS30 e SM30) e os seguintes parâmetros monitorizados durante a exposição: a produção de bioluminescência (V. fischeri), o crescimento taxa (R. subcapitata), inibição de alimentação (D. magna), desenvolvimento embrionário e dano ao DNA (D. rerio) e taxa de germinação, crescimento e danos ao DNA (A. cepa). Nos testes feitos com as SiNPfoi observado que a toxicidade é dependente do tamanho da partícula. O ensaio de bioluminescência apresentou um EC50 de 29.11, 32.34 e 4.58 g/L para TM40, HS30 e SM30, respectivamente. Para o ensaio de taxa de crescimento o EC50 foi 9.32, 9.07 e 7.93 g/Lpara TM40, HS30 e SM30, respectivamente. E para o teste de desenvolvimento embrionário com peixe zebra, para o TM40, HS30 e SM30 o EC50 foi de 5.85, 1.13 e 2.68g/L, respectivamente. Todas as partículas também induziram fitotoxicidade em A. cepa, crescimento e germinação reduziram significativamente quando o organismo foi exposto a SiNP. Efeitos genotóxicos também foram induzir pelas partículas, tanto para A. cepa quanto paraD. rerio. Portanto, as SiNP podem causar toxicidade ao ambiente e o tamanho pode influenciar fortemente a essa toxicidade

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The association of haloperidol (HP) with polymeric nanocapsules causes a significant improve in therapeutic efficacy prolongs the drug time of action and reduces motor side effects in rats. However, in view of the HP toxicity on organs such as liver and kidney and besides due the lack of knowledge about the toxicity of polymeric nanocapsules, the objective of this study was to evaluate the effects of formulation containing haloperidol-loaded lipid-core nanocapsules on biochemical parameters and oxidative stress (OS) markers in the same tissues, besides DNA damage in blood. For this study, 28 rats were divided in four groups (n = 7) and treated with aqueous solution containing 5% polysorbate 80 (v/v) (C group), free haloperidol suspension (FH group), blank nanocapsules suspension (B-Nc group) and haloperidol-loaded lipid-core nanocapsules suspension (H-Nc group). All suspensions were administered in the animals (0,5 mg/kg-ip) once a day, for 28 days. The results showed that a subchronic treatment with FH increased oxidative damage evidenced by the elevation in lipid peroxidation levels and diminution in antioxidant defenses like vitamin C and superoxide dismutase enzyme, decreased cell integrity and increased plasma levels of AST and ALT enzymes. FH also caused damage to kidney, but to a lesser extent, and caused damage to blood DNA. On the other hand, rats treated with H-Nc did not present these alterations. Through this comparative study was possible evidence that H-Nc did not show subchronic toxicity in liver and kidney of animals, preserving these tissues from oxidative damage and loss of cell integrity, which were observed in animals treated with free drug. / A associação de haloperidol (HP) a nanocápsulas poliméricas proporciona uma significante melhora na eficácia terapêutica, prolonga o tempo de ação e reduz efeitos adversos motores em ratos. No entanto, tendo em vista a toxicidade do HP sobre órgãos como fígado e rim e o pouco conhecimento sobre a toxicidade das nanocápsulas poliméricas, o objetivo deste estudo foi avaliar os efeitos da formulação contendo haloperidol nanoencapsulado sobre parâmetros bioquímicos e marcadores de estresse oxidativo (EO) nestes mesmos tecidos, além do dano no DNA no sangue. Para este estudo, 28 ratos foram separados em quatro grupos experimentais (n=7) e tratados com solução aquosa contendo 5% polissorbato 80 (v/v) (grupo C), suspensão de haloperidol livre (grupo FH), suspensão de nanocápsulas branca (grupo B-Nc) e suspensão de haloperidol nanoencapsulado (grupo H-Nc). Todas as suspensões foram administradas aos animais (0,5 mg/kg-ip) uma vez por dia, durante 28 dias. Os resultados mostraram que o tratamento subcrônico com FH causou danos no fígado, evidenciado pelo aumento nos níveis de peroxidação lipídica e diminuição das defesas antioxidantes como vitamina C e superóxido dismutase, diminuição na integridade celular e aumento nos níveis plasmáticos das enzimas AST e ALT. O FH também causou danos no rim, mas em menor extensão, e causou danos ao DNA sanguíneo. Por outro lado, ratos tratados com H-Nc não apresentaram estas alterações. A partir deste estudo comparativo foi possível evidenciar que o haloperidol nanoencapsulado (H-Nc) não causou toxicidade subcrônica hepática e renal aos animais, preservando estes tecidos dos danos oxidativos e da perda da integridade celular, os quais foram observados nos animais tratados com o fármaco livre.

The present PhD thesis, entitled: "Essential oil components encapsulated in mesoporous silica supports: a bioactive properties evaluation and a toxicological approach" focuses on the study of protection and controlled release of natural bioactive agents, derived from essential oil components (EOCs), encapsulated in mesoporous silica particles (MSPs). In addition, this thesis evaluatesthe silica-based supports to reduceundesirable sensorial propertiesandfor ensuring a low-health risk. The first section of the thesis shows the effect of encapsulation of EOCs in mesoporous silica supports. This study evaluates the efficiency of free and encapsulated EOCs to reduce the viability of cancer colon cell lines. This sectionalso shows the selectivity of encapsulated EOCs against cancer linesandtheir effect onnormal (non-cancer) colon cells. Results indicate that EOCs effect can be enhanced and sustained in time when EOCs are encapsulated. Moreover, EOCs' encapsulation shows promising specificity indices, reaching to double effect on colon cancer cells above normal cells. On the other hand,the encapsulation supports and their surface functionalization allows the odour masking of high volatility EOCs. Therefore, the delivery system based on MSPs represents an excellent alternative to promote controlled EOCs release, taking advance of their bioactive properties and solving the technical disadvantages related to volatility and unpleasant odours. Finally, samples used for garlic components encapsulation were immobilised in nanofibers to provide homogeneous and easy-to-handle hybrid system for controlling delivery.The developed 'composite' has potential applications on food, pharmacology, medical or engineering fields. The second sectionof the thesis evaluates the toxicity of the mesoporous silica supports through in vitro and in vivo assessments. Cell viability allows to identify the cytotoxic impact based on the kind of silica-based support, and their features (doses range, size and surface structure changes). Furthermore, the use of Caednorhabditis elegansmodel,shows the in vivo effects afterMSPs ingestion. The toxicological study confirms that size and surface structure, are decisiveMSPs' featuresfor reducing the toxicity risks for health. In summary, the present thesis evaluates the mesoporous silica-based particles as supports for EOCs encapsulation and identifies the main MSPs' features forreducingthe health-toxicity impact. Results of this thesis show that MSPs improve the EOCs activity and help to solve technical problemsof EOCs' volatility.Moreover, these results open up a suitable and safety option for oral delivery devices. / La presente tesis titulada: "Componentes de aceites esenciales encapsulados en soportes mesoporosos de sílice: una evaluación de sus propiedades bioactivas y un enfoque toxicológico" se centra enla evaluación de las propiedades funcionales y organolépticas de agentes naturales bioactivos, derivados de componentes de aceites esenciales, encapsulados en materiales mesoporosos de sílice; a la vez que evalua la toxicidad de los soportes utilizados,con el fin de proponer nuevos sistemas de liberación controlada por vía oral. La primera sección de esta tesis muestra el efecto de la encapsulación de los compuestos de aceites esenciales (EOCs, por sus siglas en inglés) en soportes mesoporosos de sílice. Por un lado, seevalúa la eficiencia de los EOCs libres y encapsulados para reducir la viabilidad en líneas celulares de cáncer de colon. Además, se evalúa la selectividad de los EOCs frente a células de colon normales (líneas no tumorales). Por otro lado, seestudia la capacidad de enmascaramiento de olor de los soportes. Los resultados obtenidos, evidencian en primer lugar, que los EOCs encapsulados mejoran su actividad frente a células de cáncer,en comparacióncon la respuesta de los compuestos sin encapsular. La encapsulación hace que el efecto de los EOCs sea sostenido en el tiempo, y muestra índices de especificidad prometedores, cuandose evalua el efecto toxico de los EOCsfrente a células de cáncer de colon y células normales. Los resultados de esta primera sección, indican que los soportes basados en partículas de sílice mesoporosa (MSPs, por sus siglas en inglés) protegen y liberan eficientemente los compuestos, sino que, a la vez que la funcionalización de la superficie de las MSPs permite enmascarar el olor de los compuestos de mayor volatilidad, y con mayores inconvenientes a nivel sensorial (p.e. compuestos derivados del ajo). Por lo tanto, el sistema de encapsulación se plantea como una excelente alternativa para (i) promover la liberación controlada de EOCs, (ii) aprovechar y mejorar el efecto de sus propiedades bioactivas en células de cáncer de colón y (iii) controlar las desventajas técnicas relacionadas con la volatilidad y limitaciones organolepticas. Por último, se ha comprobado que los soportes empleados en la encapsulación de los compuestos derivados de ajo, mantienen su funcionalidad luego der ser inmovilizados en nanofribras de nylon.Con esto, se busca desarrollar un nuevo sistema de 'composite';un material híbrido y homogéneo, fácil de manejar, que libera controladamente los compuestos encapsulados desde soportes tipo fibras (composites).Estoexpande el abanico de aplicaciones de los EOCs en laindustria alimentaria y farmacológica. La segunda sección de esta tesis, evalúa la toxicidad de los soportes de sílice mesoporosa (MSPs) mediante ensayos in vitro e in vivo. En primer lugar, la viabilidad celular permite identificar el impacto citotóxico de los MSPs sobre líneas celulares de colón. En particular, se evalúa los soportes mesoporosos de sílice, tipo MCM41, en función de (i) las dosis empleadas, (ii) la diferencia de tamaño (micro y nanopartículas) y (iii) el efecto que la funcionalización de la superficie genera en la viabilidad celular. Por otro lado, empleando el modelo Caednorhabditis elegans, yadministrando por vía oral las MSPs,se evalua la influencia de las características de laspartículas (MSPs) en función de la esperanza de vida (lifespan) y la calidad con la que viven y envejecen (healthspan) los nematodos. Los resultados de este estudio,muestran que el tamaño y la estructura de la superficie de las partículas, son parámetros determinantesal momento de diseñar soportes de bajoriesgo toxicológico. En resumen, la presente tesis ha evaluado las características de la sílice mesoporosa, micro y nanoparticulada, como soporte de encapsulación para mejorar la actividad y las aplicaciones de los compuestos de aceites esenciales, al mismo tiempo / La present tesi titulada: "Components d'olis essencials encapsulats en suports mesoporosos de sílica: una avaluació de les seves propietats bioactives i un enfocament toxicològic" se centra en estudis de protecció i alliberament controlat d'agents naturals bioactius, derivats de components d'olis essencials, encapsulats en materials mesoporosos de sílica. Els components d'olis essencials encapsulats milloren les seves propietats funcionals i redueixen els problemes sensorials per aplicacions futures, garantint, al mateix temps, la baixa toxicitat dels suports desenvolupats. La primera secció de la tesi mostra l'efecte d'encapsulació dels components d'olis essencials (EOCs, per les seves sigles en anglès) en suports mesoporosos de sílica sobre la millora de les seues propietats bioactives i el camuflament de problemes sensorials. Este estudi avalua l'eficiència dels EOCs lliures i encapsulats per a reduir la viabilitat en línies cel¿lulars de càncer còlon. A més, la selectivitat dels EOCs es va provar enfront de cèl¿lules de còlon normals (no canceroses). Els resultats han demostrat que l'efecte dels EOCs pot ser millorat i sostingut en el temps quan els EOCs estan encapsulats. Encara més, l'encapsulació dels EOCs mostra índexs d'especificitat prometedors, arribant a duplicar la toxicitat en l'efecte en les cèl¿lules de càncer de còlon amb comparacio en les cèl¿lules normals. Els resultats també mostren que els suports basats en partícules de sílice mesoporoses (MSPs, per les seves sigles en anglès) no sols protegixen i alliberen EOCs eficientment, sinó que, a més, la funcionlització en superfície de les MSPs permet emmascarar l'olor dels EOCs d'alta volatilitat, que té una aplicació limitada a causa dels seus problemes sensorials(p.e. compostos derivats de l'all). Per tant, el sistema de subministrament proposat resulta una excel¿lent alternativa per a (i) promoure l'alliberament controlat de EOCs, (ii) avançant en les seues propietats bioactives en cel¿lulas de càncer còlon i (iii) controlant els desavantatges tècnics relacionats amb la volatilitat i la disseminació desagradable de les olors. Finalmet, les mostres utilitzades per encapsulació de compostos d'all es van immobilitzar en nanofibres per a proporcionar un sistema híbrid homogeni i fàcil de manejar amb administració controlada i característiques bioactives, per aplicacions potencials en l'àrea d'alimentació, farmacologia, medicina o enginyeria. La segona secció avalua la toxicitat del suports de sílice mesoporosa per mitjà d'avaluacions in vitro e in vivo. La viabilitat cel¿lular permet identificar l'impacte citotòxic basat en el tipus de suport base de sílice i les seues característiques (rang de dosi, grandària i canvis en l'estructura superficial).A més, utilitzant el model in vivo Caednorhabditis elegants, s'ha estudiat la influència de les característiques de la sílice mesoporosa, administrant micro i nanopartícules de base sílice, no sols en l'esperança de vida, sinó també en el comportament dels nematodes durant el seu envelliment. Aquest estudi ha demostrat que la grandària i l'estructura superficial, són decisius per a reduir el risc de toxicitat dels suports de sílice mesoporosa i obrir la possibilitat d'utilitzar estos materials en aplicacions d'ingesta oral. En resum, la present tesi ha avaluat les característiques de les partícules de sílice mesoporosa, com a suports d'encapsulació per a millorar l'activitat i les aplicacions dels EOCs, alhora que es va avaluar el seu principal risc tòxicologic. En conseqüència, els resultats obrin una opció adequada i de seguretat per als dispositius d'administració oral. / Acosta Romero, C. (2017). Essential oil components encapsulated in mesoporous silica supports: a bioactive properties evaluation and toxicological approach [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90653 / TESIS

Mechanism underlying nanotoxicity has remained elusive. Hence, efforts to understand whether nanoparticle properties might explain its toxicity are ongoing. Considering three different types of organo-coated silver nanoparticles (AgNPs): citrate-coated AgNP, polyvinylpyrrolidone-coated AgNP, and branched polyethyleneimine-coated AgNP, with different surface charge scenarios and core particle sizes, herein we systematically evaluate the potential role of particle size and surface charge on the toxicity of the three types of AgNPs against two model organisms, Escherichia coli and Daphnia magna. We find particle size, surface charge, and concentration dependent toxicity of all the three types of AgNPs against both the test organisms. Notably, Ag+ (as added AgNO3) toxicity is greater than each type of AgNPs tested and the toxicity follows the trend: AgNO3>BPEI-AgNP>Citrate-AgNP>PVP-AgNP. Modeling particle properties using the general linear model (GLM), a significant interaction effect of primary particle size and surface charge emerges that can explain empirically-derived acute toxicity with great precision. The model explains 99.9% variation of toxicity in E. coli and 99.8% variation of toxicity in D. magna, revealing satisfactory predictability of the regression models developed to predict the toxicity of the three organo-coated AgNPs. We anticipate that the use of GLM to satisfactorily predict the toxicity based on nanoparticle physico-chemical characteristics could contribute to our understanding of nanotoxicology and underscores the need to consider potential interactions among nanoparticle properties to explaining nanotoxicity.

The diploma thesis is focused on so called new risks, which currently include the risks associated with various nanomaterials - their production, introduction into practice and use. The paper summarizes the up-to-date knowledge about the characteristics of nanoparticles used and it proposes a procedure to estimate potential consequences of their exposure. The experimental part of the paper is represented by a case study, which introduces the practical application of the proposed procedure aiming at estimated exposure to nanoparticles on the workplace where metal working is executed and resulting occupational risks. The aim of this part of the study was to measure selected parameters of aerosols using a special measuring system working on the principle of laser photometry combined with diffusion charger and detector using Faraday cage. The object of the measurements was to determine the numerical concentration of nanoparticles present in working environment and the representation of size groups of submicron particles. The measurement procedure was designed in accordance with ČSN EN 689, ČSN EN ISO 16000-1 and ČSN ISO 8756 standards and data gained were subsequently interpreted. Afterwards the results were used to design the measures to reduce the risk of health damage when facing chronic inhalation...

Les nanoparticules (NP) figurent aux premiers rangs des contaminants émergents prioritaires dans le champ de surveillance des grands organismes de santé et sécurité du travail. Parmi les NP les plus utilisées, on peut citer les NP d’argent (Ag). L’exposition humaine aux NP d’Ag augmente alors inévitablement avec l’accroissement de leur production et leur utilisation généralisée ce qui suscite des préoccupations sur les risques à la santé. L'objectif du projet est de mieux documenter le devenir des NP d’Ag dans l’organisme, à partir d’études expérimentales chez l’animal exposé sous différentes conditions par inhalation, la principale voie d’exposition des travailleurs. Ces informations sont nécessaires pour le développement sécuritaire de ces nouvelles technologies. Dans un premier temps, le profil toxicocinétique des NP d’Ag inhalées a été documenté. Les rats ont été exposés « nez seulement » à des NP de 20 nm pendant 6 h à une concentration cible de 15 mg/m3. L'évolution temporelle de l'élément Ag dans les poumons, le sang, les tissus et les excrétas a été déterminée pendant 14 jours après le début de l'inhalation. La cinétique des NP d’Ag inhalées a été ensuite comparée avec la cinétique d’une forme soluble de l’élément Ag suite à l’exposition au nitrate d’argent (AgNO3) dans de mêmes conditions expérimentales pour mieux comprendre leur comportement, principalement, en raison de leur dissolution et leur capacité à libérer progressivement des ions Ag+ en milieu biologique. Ainsi, dans un dernier temps, dans le but de déterminer la meilleure métrique à utiliser pour mieux évaluer les risques associés à ces NP d’Ag, nous avons étudié l’impact de la cinétique entre un nombre plus faible et un nombre plus élevé de particules. Les profils cinétiques des NP d’Ag inhalées ont montré que la fraction de la dose inhalée qui a atteint les poumons est rapidement éliminée au cours des 72 premières heures suivant l'inhalation, puis la fraction restante de la dose est lentement éliminée par la suite. La dose inhalée éliminée des poumons semble être transférée dans la circulation systémique et atteint un maximum entre 48 et 72 h après l'inhalation. Cependant, les niveaux d'Ag dans le sang étaient faibles, ce qui suggère une biodistribution rapide dans les tissus tels que le foie, l’organe cible des NP d’Ag chez le rat après inhalation. Une translocation vers le bulbe olfactif et les ganglions lymphatiques était évidente durant l'exposition par inhalation de 6 h jusqu'à 6 h après la fin de l'exposition, démontrant l’occurrence d’un transport direct des NP d’Ag via le nerf nasal par le transport axonal et via la circulation lymphatique après la clairance pulmonaire, respectivement. Les profils d'excrétion ont également révélé que l'excrétion fécale est la voie d'excrétion dominante pour les NP d’Ag. Les résultats obtenus après l'inhalation d'AgNO3 ont montré des différences dans la cinétique de l’Ag sous la forme soluble par rapport à la forme insoluble (nanoparticulaire) avec des niveaux plus élevés dans le sang, le tractus GI et les tissus extrapulmonaires, mais des niveaux plus faibles dans les poumons. En plus de ces observations, l'évolution temporelle de l’Ag dans le tube digestif et les fèces après l'exposition à la forme soluble était associée à une réabsorption intestinale de l'Ag. Une fraction plus élevée de la dose a été également récupérée dans les reins et l'urine pour les formes solubles d’Ag; en effet, la filtration glomérulaire des agrégats de NP d’Ag peut être limitée alors que le cation monovalent dissous peut plus facilement passer dans le filtrat du sang. Notre étude a également révélé des différences significatives dans les profils temporels de l'Ag dans les poumons, le sang, les ganglions lymphatiques et le tractus gastro-intestinal entre les rats exposés à des aérosols de NP d'Ag avec un nombre faible et un nombre élevé de particules, mais dont la concentration massique est identique. Certaines similitudes entre les deux conditions ont également été notées, telles que la distribution tissulaire relative, le temps jusqu'aux niveaux de pointe (Tmax) et les profils d'excrétion. Cependant, pour confirmer si le modèle de biodistribution des NPs d'Ag est conditionné par le nombre de particules, des investigations supplémentaires sont nécessaires. / Nanoparticles (NPs) are among the top priority emerging contaminants in the monitoring field of the major occupational health and safety organizations. Among the most widely used nanoparticles, we can cite silver nanoparticles (Ag). Human exposure to Ag NPs inevitably increases with the increase in their production and their widespread use which raises concerns about the health risks. The objective of the project is to better document the fate of Ag nanoparticles in the body, based on experimental studies in animals exposed under different conditions by inhalation, the main route of exposure for workers. This information is necessary for the safe development of these new technologies. First, the toxicokinetic profile of inhaled Ag NPs was documented. Rats were exposed "nose only" to 20 nm NPs for 6 h at a target concentration of 15 mg/m3. The temporal evolution of the Ag element in the lungs, blood, tissues and excreta was determined for 14 days after the start of inhalation. Thus, to better understand their behavior, mainly because of their dissolution and their capacity to progressively release Ag+ ions in the biological medium, the kinetics of inhaled Ag NPs were compared with the kinetics of a soluble form of the element Ag following exposure to silver nitrate (AgNO3) under the same experimental conditions. Thus, as a last step, in order to determine the best metric to use to better assess the risks associated with these Ag NPs, we studied their kinetic from inhalation studies by comparing the effect of a lower -number with a higher- number of particles. The kinetic profiles of inhaled Ag nanoparticles showed that the fraction of the inhaled dose that reached the lungs is rapidly eliminated during the first 72 hours after inhalation, and the remaining fraction of the dose is slowly eliminated thereafter. The inhaled dose cleared from the lungs appears to be transferred to the systemic circulation and reaches a maximum between 48 and 72 hours after inhalation. However, Ag levels in the blood were low, suggesting rapid biodistribution to tissues such as the liver, the target organ of Ag nanoparticles in rats after inhalation. A translocation of Ag NPs in olfactory bulbs and lymph nodes was apparent, demonstrating the occurrence of direct transport of Ag NPs through nasal nerve by axonal transport and via lymphatic circulation after lung clearance, respectively. The excretion profiles also revealed that fecal excretion is the dominant excretion route for Ag nanoparticles. The results obtained after inhalation of AgNO3 showed differences in the kinetics of soluble AgNO3 compared to insoluble Ag NPs, with higher levels in blood, GI tract and extrapulmonary tissues, but lower levels in lungs. In addition to these observations, the time courses of Ag elements in the GI tract and feces following ionic form exposure were compatible with an intestinal reabsorption of Ag. A higher fraction of the dose was further recovered in kidneys and urine after AgNO3 inhalation compared to Ag NP inhalation. Indeed, filtration of Ag NP aggregates may be restricted while the dissolved Ag+ monovalent ion can more easily pass into the filtrate from blood. Our study also revealed significant differences in the time profiles of Ag element in lungs, blood, lymphatic nodes and GI tract between rats exposed to Ag NPs aerosols of lower- and higher-total particle number counts, but with the same mass concentration. Some similarities between the two conditions were also noted, such as the relative tissue distribution, time-to-peak levels (Tmax) and excretion profiles. However, to confirm if the biodistribution pattern of Ag NPs is conditioned by the particle number, further investigations are needed.

Engineered nanomaterials (ENMs) have become ubiquitous in consumer products and industrial applications, and consequently the environment. Much of the environmentally released ENMs are expected to enter terrestrial ecosystems via land application of nano-enriched biosolids to agricultural fields. Among the organisms most likely to encounter nano-enriched biosolids are the key soil bacteria known as plant growth promoting rhizobacteria (PGPR). I reviewed what is known concerning the toxicological effects of ENMs to PGPR and observed the need for high-throughput methods to evaluate lethal and sublethal toxic responses of aerobic microbes. I addressed this issue by developing high-throughput microplate assays which allowed me to normalize oxygen consumption responses to viable cell estimates. Oxygen consumption is a crucial step in cellular respiration which may be examined relatively easily along with viability and may provide insight into the metabolic/physiological response of bacteria to toxic substances. Because many of the most toxic nanomaterials (i.e. metal containing materials) exhibit some level of ionic dissolution, I first developed my methods by examining metal ion responses in the PGPR, Bacillus amyloliquefaciens GB03. I found this bacterium exhibits differential oxygen consumption responses to Ag+, Zn2+, and Ni2+. Exposure to Ag+ elicited pronounced increases in O2 consumption, particularly when few viable cells were observed. Also, while Ni2+ and Zn2+ are generally thought to induce similar toxic responses, I found O2 consumption per viable cell was much more variable during Ni2+ exposure and that Zn2+ induced increased O2 utilization to a lesser extent than Ag+. Additionally, I showed my method is useful for probing toxicity of traditional antibiotics by observing large increases in O2 utilization in response to streptomycin, which was used as a positive control due to its known effects on bacterial respiration. After showing the utility of my method for examining metal ion responses in a single species of PGPR, I investigated the toxicity of silver ENMs (AgENMs) and ions to three PGPR, B. amyloliquefaciens GB03, Sinorhizobium meliloti 2011, and Pseudomonas putida UW4. The ENM exposures consisted of untransformed, polyvinylpyrrolidone coated silver ENMs (PVP-AgENMs) and 100% sulfidized silver ENMs (sAgENMs), which are representative of environmentally transformed AgENMs. I observed species specific O2 consumption responses to silver ions and PVP-AgENMs. Specifically, P. putida exhibited increased O2 consumption across the observed range of viable cells, while B. amyloliquefaciens exhibited responses similar to those found in my first study. Additionally, S. meliloti exhibited more complex responses to Ag+ and PVP-AgENMs, with decreased O2 consumption when cell viability was ~50-75% of no metal controls and increased O2 consumption when cell viability was <50%. I also found the abiotically dissolved fraction of the PVP-AgENMs was likely responsible for most of the toxic response, while abiotic dissolution did not explain the toxicity of sAgENMs. My work has yielded a straightforward, cost-effective, and high-throughput method of evaluating viability and oxygen consumption in aerobic bacteria. I have used this method to test a broad range of toxic substances, including, metal ions, antibiotics, and untransformed and transformed ENMs. I observed species specific toxic responses to Ag+, PVP-AgENMs, and sAgENMs in PGPR. These results not only show the clear utility of the methodology, but also that it will be crucial to continue examining the responses of specific bacterial strains even as nanotoxicology, as a field, must move toward more complex and environmentally relevant systems.

Cromoblastomicose é uma micose crônica que acomete a pele e o tecido subcutâneo. Vários tratamentos têm sido utilizados, mas a eficácia é extremamente baixa, não permitindo eleger uma terapia de escolha. No presente trabalho foram realizados: I – ensaios de suscetibilidade in vitro de agentes da cromoblastomicose contra antifúngicos comerciais; II - caracterização molecular de amostras oriundas de casos clínicos, bem como a descrição destes casos; III - a produção e caracterização de duas nanoemulsões, uma de anfotericina B e uma de itraconazol produzidas pela técnica de homogeneização à alta pressão; IV - a avaliação da atividade antifúngica destas nanoemulsões in vitro e in vivo em agentes da cromoblastomicose; V - a verificação do nível de comprometimento renal e hepático causados pelas nanoemulsões; VI - a avaliação da toxicidade das formulações produzidas. De modo geral, os agentes da cromoblastomicose, apresentaram maior suscetibilidade à terbinafina e ao itraconazol, respectivamente. A combinação de anfotericina B e terbinafina foi sinérgica para quatro dos cinco grupos avaliados. Quanto aos casos clínicos, no primeiro foi identificada uma infecção por E. spinifera e no segundo uma por Fonsecaea monophora. As nanoemulsões foram elaboradas com composição passível de administração parenteral, uma de anfotericina B e uma de itraconazol, pelo método de homogeneização à alta pressão. Não foi possível determinar as CIMs da nanoemulsão de anfotericina B e Abelcet® in vitro, enquanto que a nanoemulsão de itraconazol apresentou CIMs muito semelhantes às do fármaco livre. Em modelo animal de cromoblastomicose, a nanoemulsão de anfotericina B foi mais ativa que o fármaco livre, Fungizone® e Abelcet®. A nanoemulsão de itraconazol também apresentou melhor atividade quando comparada com o fármaco livre. Os níveis de uréia foram mais elevados nos animais que receberam anfotericina B livre e Fungizone®. A enzima alanina aminotransferase foi encontrada em níveis menores nos animais tratados com a nanoemulsão de itraconazol do que naqueles que receberam itraconazol livre. A anfotericina B livre e Fungizone® causaram graves danos aos rins. Nos animais tratados com Abelcet® e com a nanoemulsão de anfotericina B foi possível verificar apenas necrose focal. Da mesma forma, a nanoemulsão de itraconazol protegeu os animais contra danos hepáticos quando comparada com o fármaco livre. Em relação aos ensaios de toxicidade, a anfotericina B foi citotóxica em concentrações a partir de 4μg/mL, sendo que com a nanoemulsão esta toxicidade não foi observada em concentrações mais elevadas. O itraconazol foi citotóxico, sendo que este efeito não foi visto com a nanoemulsão. É de extrema importância a avaliação da suscetibilidade dos agentes da cromoblastomicose a fim de orientar a clínica. A identificação molecular de agentes isolados de casos clínicos pode contribuir para delinear o perfil epidemiológico da doença. As nanoemulsões de anfotericina B e itraconazol apresentaram atividades superiores in vivo quando comparadas aos demais tratamentos e foram capazes de reduzir os efeitos adversos causados por estes antifúngicos. Através de ensaios in vitro foi confirmada a redução da citotoxidade do fármaco quando veiculado na nanoemulsão. Assim, as nanoemulsões produzidas poderiam ser alternativas terapêuticas para o tratamento da cromoblatomicose. / Chromoblastomycosis is a chronic mycosis that affects the skin and subcutaneous tissue. Various treatments have been used, but the efficacy is extremely low and does not allow choosing a therapy of choice. In the present work was performed: I - in vitro susceptibility testing for chromoblastomycosis agents against commercial antifungal; II - molecular characterization of samples from clinical cases as well as the description of these cases III - production and characterization of two nanoemulsions , one of amphotericin B and one of itraconazole, produced by high pressure homogenization technique; IV - assessing the in vitro and in vivo antifungal activity of these nanoemulsions against chromoblastomycosis agents; V - checking the level of impairment caused in the kidney and liver by nanoemulsions; VI - evaluation of toxicity of the formulations produced. In general, the chromoblastomycosis agents showed greater susceptibility to terbinafine and to itraconazole, respectively. The combination of amphotericin B and terbinafine was synergistic to four of the five groups. As for clinical cases, in the first was identified an infection by E. spinifera and in the second one by Fonsecaea monophora. The nanoemulsions were prepared with composition amenable of parenteral administration, one of amphotericin B and one of itraconazole, by the at high pressure homogenization method. Could not be determined the MIC of amphotericin B nanoemulsion and Abelcet® in vitro, while the itraconazole nanoemulsion showed MICs very similar to free drug. In a chromoblastomycosis animal model, the amphotericin B nanoemulsion was more active than free drug, Abelcet® and Fungizone®. The nanoemulsion of itraconazole also showed better activity compared to the free drug. Urea levels were higher in the animals receiving amphotericin B free and Fungizone®. The enzyme alanine aminotransferase was found in lower levels in animals treated with itraconazole nanoemulsion than in those who received itraconazole free. Amphotericin B free and Fungizone® caused severe damage to the kidneys. Already in animals treated with Abelcet® and the amphotericin B nanoemulsion was verified only focal necrosis. Likewise, the itraconazole nanoemulsion protected against liver damage when compared with the free drug. Regarding toxicity assays, amphotericin B was cytotoxic at concentrations from 4 μg/mL, while with the nanoemulsion this toxicity was not observed at higher concentrations. Itraconazole was cytotoxic, and this effect was not observed with the nanoemulsion. It is extremely important to evaluate the susceptibility of chromoblastomycosis agents to guide the clinic. Molecular identification of agents isolated from clinical cases can contribute to outline an epidemiological profile of the disease. The amphotericin B and itraconazole nanoemulsions showed higher activities in vivo when compared to other treatments and were able to reduce the adverse effects caused by these antifungals. Through in vitro assays were confirmed the reduction of the cytotoxicity of the drug when vehiculated in the nanoemulsion. Therefore, the nanoemulsions may be produced therapeutic alternatives for the chromoblastomycosis treatment.