Nonaqueous sol-gel routes to doped metal oxide nanoparticles: synthesis, characterization, assembly and properties

The results presented in this thesis have been achieved under the Ph.D. project entitled “Nonaqueous Sol-Gel routes to doped metal oxide nanoparticles: Synthesis, characterization, assembly and properties”. The purpose of this study is the investigation of metal oxide nanostructures doped with metals of a diverse nature, leading to different type of applications. The easier control over the reaction kinetics in solvothermal routes, compared to aqueous methods, allows to better match the reactivity between metal oxide precursors, paving the way to a facile and low temperature production of doped oxides. In this manuscript diverse examples of the exploitation of the “Benzyl Alcohol Route” are discussed. Such a powerful pathway was utilized for the synthesis of transition metal doped zirconia, hafnia and various perovskites, and the study of their magnetic properties, as well as the synthesis of rare earth doped zirconium oxide. A further extension, proving the solidity of the synthetic method, is shown for the preparation of Li4Ti5O12 nanocrystals carrying excellent electrochemical properties for lithium-ion battery applications. Finally, the effect of doping and other reaction parameters on the assembly of the nanocrystals is discussed. These studies were carried out principally at the University of Aveiro, as well as at the University of Montpellier II and at the Seoul National University due to complementary available expertises and equipments.

Synthesis and functionalization of hydroxyapatite nanoparticles

Graças aos desenvolvimentos na área da síntese de nanomaterais e às potentes técnicas de caracterização à nanoescala conseguimos hoje visualizar uma nanopartícula (NP) como um dispositivo de elevado potencial terapêutico. A melhoria da sua efectividade terapêutica requer no entanto o aprofundamento e sistematização de conhecimentos, ainda muito incipientes, sobre toxicidade, selectividade, efeitos colaterais e sua dependência das próprias características físico-químicas da NP em análise. O presente trabalho, elegendo como alvo de estudo uma substância considerada biocompatível e não tóxica, a hidroxiapatite (Hap), pretende dar um contributo para esta área do conhecimento. Definiram-se como metas orientadoras deste trabalho (i) estudar a síntese de nanoparticulas de Hap (Hap NP), e a modificação das características físico-químicas e morfológicas das mesmas através da manipulação das condições de síntese; (ii) estudar a funcionalização das Hap NP com nanoestruturas de ouro e com ácido fólico, para lhes conferir capacidades acrescidas de imagiologia e terapêuticas, particularmente interessantes em aplicações como o tratamento do cancro (iii) estudar a resposta celular a materiais nanométricos, com propriedades físico-químicas diversificadas. No que se refere à síntese de Hap NP, comparam-se dois métodos de síntese química distintos, a precipitação química a temperatura fisiológica (WCS) e a síntese hidrotérmica (HS), em meios aditivados com ião citrato. A síntese WCS originou partículas de tamanho nanométrico, com uma morfologia de agulha, pouco cristalinas e elevada área superficial especifica. A síntese HS à temperatura de 180ºC permitiu obter partículas de dimensões também nanométricas mas com área específica inferior, com morfologia de bastonete prismático com secção recta hexagonal e elevada cristalinidade. Com o objectivo de aprofundar o papel de algumas variáveis experimentais na definição das características finais das partículas de hidroxiapatite, designadamente o papel do ião citrato (Cit), variou-se a razão molar [Cit/Ca] da solução reagente e o tempo de síntese. Demonstrou-se que o ião citrato e outras espécies químicas resultantes da sua decomposição nas condições térmicas (180ºC) de síntese tem um papel preponderante na velocidade de nucleação e de crescimento dessas mesmas partículas e por conseguinte nas características físico-químicas das mesmas. Elevadas razões [Cit/Ca] originam partículas de dimensão micrométrica cuja morfologia é discutida no contexto do crescimento com agregação. Com o objectivo de avaliar a citotoxicidade in vitro das nanopartículas sintetizadas procedeu-se à esterilização das mesmas. O método de esterilização escolhido foi a autoclavagem a 121º C. Avaliou-se o impacto do processo de esterilização nas características das partículas, verificando-se contrariamente às partículas WCS, que as partículas HS não sofrem alterações significativas de morfologia, o que se coaduna com as condições de síntese das mesmas, que são mais severas do que as de esterilização. As partículas WCS sofrem processos de dissolução e recristalização que se reflectem em alterações significativas de morfologia. Este estudo demonstrou que a etapa de esterilização de nanopartículas para aplicações biomédicas, por autoclavagem, pode alterar substancialmente as propriedades das mesmas, sendo pois criticamente importante caracterizar os materiais após esterilização. Os estudos citotoxicológicos para dois tipos de partículas esterilizadas (HSster e WCSster) revelaram que ambas apresentam baixa toxicidade e possuem potencial para a modelação do comportamento de células osteoblásticas. Tendo em vista a funcionalização da superfície das Hap NP para multifunções de diagnóstico e terapia exploraram-se condições experimentais que viabilizassem o acoplamento de nanopartículas de ouro à superfície das nanopartículas de Hidroxiapatite (Hap-AuNP). Tirando partido da presença de grupos carboxílicos adsorvidos na superfície das nanopartículas de Hap foi possível precipitar partículas nanométricas de ouro (1,5 a 2,5 nm) na superfície das mesmas adaptando o método descrito por Turkevich. No presente trabalho as nanopartículas de Hap funcionaram assim como um template redutor do ouro iónico de solução, propiciando localmente, na superfície das próprias nanopartículas de Hap, a sua redução a ouro metálico. A nucleação do ouro é assim contextualizada pelo papel redutor das espécies químicas adsorvidas, designadamente os grupos carboxílicos derivados de grupos citratos que presidiram à síntese das próprias nanopartículas de Hap. Estudou-se também a funcionalização das Hap NP com ácido fólico (FA), uma molécula biologicamente interessante por ser de fácil reconhecimento pelos receptores existentes em células cancerígenas. Os resultados confirmaram a ligação do ácido fólico à superfície das diferentes partículas produzidas HS e Hap-AuNPs. Graças às propriedades ópticas do ouro nanométrico (efeito plasmão) avaliadas por espectroscopia vis-UV e às potencialidades de hipertermia local por conversão fototérmica, as nanoestruturas Hap-AuNPs produzidas apresentam-se com elevado interesse enquanto nanodispositivos capazes de integrar funções de quimio e terapia térmica do cancro e imagiologia. O estudo da resposta celular aos diversos materiais sintetizados no presente trabalho foi alvo de análise na tentativa de se caracterizar a toxicidade dos mesmos bem como avaliar o seu desempenho em aplicações terapêuticas. Demonstrou-se que as Hap NP não afectam a proliferação das células para concentrações até 500 g/ml, observando-se um aumento na expressão genética da BMP-2 e da fosfatase alcalina. Verificou-se também que as Hap NP são susceptíveis de internalização por células osteoblásticas MG63, apresentando uma velocidade de dissolução intracelular relativamente reduzida. A resposta celular às Hap-AuNP confirmou a não citotoxicidade destas partículas e revelou que a presença do ouro na superfície das Hap NP aumenta a taxa proliferação celular, bem como a expressão de parâmetros osteogénicos. No seu conjunto os resultados sugerem que os vários tipos de partículas sintetizadas no presente estudo apresentam também comportamentos interessantes para aplicações em engenharia de tecido ósseo.

Lanthanide oxide and phosphate nanoparticles for thermometry and bimodal imaging

Nesta tese relatam-se estudos de fotoluminescência de nanopartículas de óxidos e fosfatos dopados com iões trivalentes de lantanídeos, respectivamente, nanobastonetes de (Gd,Eu)2O3 e (Gd,Yb,Er)2O3 e nanocristais de (Gd,Yb,Tb)PO4, demonstrando-se também aplicações destes materiais em revestimentos inteligentes, sensores de temperatura e bioimagem. Estuda-se a transferência de energia entre os sítios de Eu3+ C2 e S6 dos nanobastonetes Gd2O3. A contribuição dos mecanismos de transferência de energia entre sítios para o tempo de subida 5D0(C2) é descartada a favor da relaxação directa 5D1(C2) 5D0(C2) (i.e., transferência de energia entre níveis). O maior tempo de decaimento do nível 5D0(C2) nos nanobastonetes, relativamente ao valor medido para o mesmo material na forma de microcristais, é atribuído, quer à existência de espaços livres entre nanobastonetes próximos (factor de enchimento ou fracção volúmica), quer à variação do índice de refracção efectivo do meio em torno dos iões Eu3+. A dispersão de nanobastonetes de (Gd,Eu)2O3 em três resinas epoxi comerciais através da cura por UV permite obter nanocompósitos epoxi- (Gd,Eu)2O3. Relatam-se estudos cinéticos e das propriedades térmicas e de fotoluminescência destes nanocompósitos. Estes, preservam as típicas propriedades de emissão do Eu3+, mostrando o potencial do método de cura por UV para obter revistimentos inteligentes e fotoactivos. Considera-se um avanço significativo a realização de uma nanoplataforma óptica, incorporando aquecedor e termómetro e capaz de medir uma ampla gama de temperaturas (300-2000 K) à escala nano, baseada em nanobastonetes de (Gd,Yb,Er)2O3 (termómetros) cuja superfície se encontra revestida com nanopartículas de ouro. A temperature local é calculada usando, quer a distribuição de Boltzmann (300-1050 K) do rácio de intensidades da conversão ascendente 2H11=2!4I15=2/4S3=2!4I15=2, quer a lei de Planck (1200-2000 K) para uma emissão de luz branca atribuída à radiação do corpo negro. Finalmente, estudam-se as propriedades de fotoluminescência correspondentes às conversões ascendente e descendente de energia em nanocristais de (Gd,Yb,Tb)PO4 sintetizados por via hidrotérmica. A relaxividade (ressonância magnética) do 1H destes materiais são investigadas, tendo em vista possíveis aplicações em imagem bimodal (luminescência e ressonância magnética nuclear).

Silver nanoparticles flow in an aquatic trophic chain

Silver nanoparticles (AgNP) have been produced and applied in a variety of products ranging from personal care products to food package containers, clothing and medicine utilities. The antimicrobial function of AgNP makes it very useful to be applied for such purposes. Silver (Ag) is a non-essential metal for organisms, and it has been historically present in the environment at low concentrations. Those concentrations of silver increased in the last century due to the use of Ag in the photographic industry and lately are expected to increase due to the use of AgNPs in consumer products. The presence of AgNP in the aquatic environment may pose a risk for aquatic species, and the effects can vary from lethal to sublethal effects. Moreover, the contact of aquatic organisms with AgNP may not cause immediately the death of individuals but it can be accumulated inside the animals and consequently transferred within the food chain. Considering this, the objective of this work was to study the transfer of silver nanoparticles in comparison to silver ions, which was used as silver nitrate, within an aquatic food chain model. To achieve this goal, this study was divided into four steps: the toxicity assessment of AgNP and AgNO3 to aquatic test-species, the bioaccumulation assessment of AgNP and AgNO3 by Pseudokirchneriella subcapitata and Daphnia magna under different exposure scenarios, and finally the evaluation of the trophic transfer of Ag through an experimental design that included the goldfish Carassius auratus in a model trophic chain in which all the species were exposed to the worse-case scenario. We observed that the bioconcentration of Ag by P. subcapitata is mainly driven by ionic silver, and that algae cannot internalize these AgNPs, but it does internalizes dissolved Ag. Daphnia magna was exposed to AgNP and AgNO3 through different exposure routes: water, food and both water and food. The worse-case scenario for Daphnia Ag bioaccumulation was by the joint exposure of contaminated water and food, showing that Ag body burdens were higher for AgNPs than for AgNO3. Finally, by exposing C. auratus for 10 days through contaminated water and food (supplied as D. magna), with another 7 days of depuration phase, it was concluded that the 10 days of exposure were not enough for fish to reach a plateau on Ag internal concentration, and neither the 7 days of elimination were sufficient to cause total depuration of the accumulated Ag. Moreover, a higher concentration of Ag was found in the intestine of fish when compared with other organs, and the elimination rate constant of AgNP in the intestine was very low. Although a potential for trophic transfer of AgNP cannot be suggested based in the data acquired in this study, there is still a potential environmental risk for aquatic species.

Effect assessment of nanoparticles toxicity in the terrestrial compartment

Over 11 million tons of nanomaterials (NMs) have been produced in 2012 and predictions point the increase in production. Despite predictions and extended usage via consumer products and industry, the understanding of the potential impact of these materials on the environment is virtually absent. The main aim of this thesis is to understand how a selected group of nanomaterials (metal based particles) may impact soil invertebrates, with special focus on the mechanisms of response. Since a case-by-case Environmental Risk Assessment (ERA) of all the emerging contaminants (particularly NMs) is impossible, among others due to time and cost reasons, to gain understanding on the mechanism of action and response is very important to reach a common paradigm. Understanding the modes of action provides predictive characters in cross particle extrapolation. Besides, it also provides insight for the production of new and sustainable materials. Overall, the effects of the selected NMs (Copper and Silver, Titanium and Zirconium oxides) and the respective salt forms, were investigated at the gene expression (using high-throughput tools, microarray and qPCR technology), biochemical (using enzymatic assays for analysis of oxidative stress markers) and organism (survival and reproduction as in OECD test guidelines) levels, this using standard soil species (Enchytraeus albidus, Enchytraeus crypticus, Eisenia fetida). Gene expression analysis provided valuable information on the mechanisms affected by each of the NMs. The gene expression profile highlighted a (nano)material signature and the effect of the duration of exposure. The functional analyses integrated with the biochemical and organism data, revealed a good understanding power. The biochemical parameters (oxidative stress related) were distinct across the materials and also influenced by duration of exposure and concentration. The standardized organismal responses differed the least between the various materials. The overall outcome is that, in this context of NMs effect assessment, gene expression and enzymatic assays introduced a very important knowledge gap, which could not had been achieved by the standard organismal effects alone. A reoccurring issue with some metal based NMs is the possible dissolution and subsequent release of ions that then causes toxicity e.g. Cu-NPs or Ag-NPs release Cu2+ or Ag+. The oxidation state of the particles was investigated, although this was not the focus of the thesis. The study of fate, e.g. dissolution of NPs, is also only in its beginning and the appropriate techniques are currently being developed. The results showed a specific nanoparticle effect. The UV exposure with titanium dioxide nanoparticles increased its effect.

Preparation and in vivo evaluation of insulin-loaded biodegradable nanoparticles prepared from diblock copolymers of PLGA and PEG

The aim of this study was to design a controlled release vehicle for insulin to preserve its stability and biological activity during fabrication and release. A modified, double emulsion, solvent evaporation, technique using homogenisation force optimised entrapment efficiency of insulin into biodegradable nanoparticles (NP) prepared from poly (dl-lactic-co-glycolic acid) (PLGA) and its PEGylated diblock copolymers. Formulation parameters (type of polymer and its concentration, stabiliser concentration and volume of internal aqueous phase) and physicochemical characteristics (size, zeta potential, encapsulation efficiency, in vitro release profiles and in vitro stability) were investigated. In vivo insulin sensitivity was tested by dietinduced type II diabetic mice. Bioactivity of insulin was studied using Swiss TO mice with streptozotocin-induced type I diabetic profile. Insulin-loaded NP were spherical and negatively charged with an average diameter of 200–400 nm. Insulin encapsulation efficiency increased significantly with increasing ratio of co-polymeric PEG. The internal aqueous phase volume had a significant impact on encapsulation efficiency, initial burst release and NP size. Optimised insulin NP formulated from 10% PEG-PLGA retained insulin integrity in vitro, insulin sensitivity in vivo and induced a sustained hypoglycaemic effect from 3 hours to 6 days in type I diabetic mice.

Effects of nanoparticles exposure in the mussel Mytilus Galloprovincialis

Tese de dout., Ciências do Mar, Terra e Ambiente (Ecotoxicologia), Faculdade de Ciências e Tecnologia, Univ. do Algarve, 2012

Synthesis and evaluation of polymer nanoparticles for delivery in RPE cells

Ocular pathologies are among the most debilitating medical conditions affecting all segments of the population. Traditional treatment options are often ineffective, and gene therapy has the potential to become an alternative approach for the treatment of several pathologies. Methacrylate polymers have been described as highly biocompatible and are successfully used in medical applications. Due to their cationic nature, these polymers can be used to form polyplexes with DNA for its delivery. This work aims to study the potential of PDMAEMA (poly(2-(N,N’-dimethylamino)ethyl methacrylate)) as a non viral gene delivery system to the retina. The first part of this work aimed to study the potential for gene delivery of a previously synthesized PDMAEMA polymer of high molecular weight (354kDa). In the second part, we synthesized by RAFT a PDMAEMA with a lower molecular weight (103.3kDa) and similarly, evaluated its ability to act as a gene delivery vehicle. PDMAEMA/DNA polyplexes were prepared at 5, 7.5, 10, 12.5 and 20 nitrogen/phosphorous (N/P) ratio for the 354kDa PDMAEMA and at 5 and 7.5 for the 103.3kDa PDMAEMA. Dynamic light scattering and zeta potential measurements confirmed the nanosize and positive charge of polyplexes for all ratios and for both polymers. Both high and low Mw PDMAEMA were able to efficiently complex and protect DNA from DNase I degradation. Their cytotoxicity was evaluated using a non-retinal cell line (HEK293) and a retinal pigment epithelium (RPE) cell line (D407). We have found that cytotoxicity of the free polymer is concentration and time dependent, as expected, and negligible for all the concentrations of the PDMAEMA-DNA polyplexes. Furthermore, for the concentrations to be used in vivo, the 354kDa PDMAEMA showed no signs of inflammation upon injection in the intravitreal space of C57BL/6 mice. The transfection efficiency, as evaluated by fluorescence microscopy and flow cytometry, showed that the D407 retinal cells were transfected by polyplexes of both high and low Mw PDMAEMA, but with varied efficiency, which was dependent on the N/P ratio. Althogether, these results suggest that PDMAEMA is a feasible candidate for non-viral gene delivery to the retina, and this work constitutes the basis of further studies to elucidate the bottleneck in transfection and further optimization of the material.

Effects of mixture of nanoparticles under different salinity in the clams Ruditapes philippinarum and Ruditapes decussatus

Dissertação de mestrado, Biologia Marinha, Faculdade de Ciências e Tecnologia, Universidadde do Algarve, 2015

Development of a melanoma therapeutic vaccine candidate by the coencapsulation of melanoma antigen peptides and toll-like receptor ligands as adjuvants into polymeric nanoparticles

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2014

Control of endosomal Toll-like receptor (TLR) signaling by nanoparticles and applications in cancer therapy

Thesis (Ph.D.)--University of Washington, 2014

Application of green zero-valent iron nanoparticles to the remediation of soils contaminated with ibuprofen

Zero-valent iron nanoparticles (nZVIs) are often used in environmental remediation. Their high surface area that is associated with their high reactivity makes them an excellent agent capable of transforming/degrading contaminants in soils and waters. Due to the recent development of green methods for the production of nZVIs, the use of this material became even more attractive. However, the knowledge of its capacity to degrade distinct types of contaminants is still scarce. The present work describes the study of the application of green nZVIs to the remediation of soils contaminated with a common anti-inflammatory drug, ibuprofen. The main objectives of this work were to produce nZVIs using extracts of grape marc, black tea and vine leaves, to verify the degradation of ibuprofen in aqueous solutions by the nZVIs, to study the remediation process of a sandy soil contaminated with ibuprofen using the nZVIs, and to compare the experiments with other common chemical oxidants. The produced nZVIs had nanometric sizes and were able to degrade ibuprofen (54 to 66% of the initial amount) in aqueous solutions. Similar remediation efficiencies were obtained in sandy soils. In this case the remediation could be enhanced (achieving degradation efficiencies above 95%) through the complementation of the process with a catalyzed nZVI Fenton-like reaction. These results indicate that this remediation technology represents a good alternative to traditional and more aggressive technologies.

Determination of airborne nanoparticles in elderly care centres

According to numerous studies, airborne nanoparticles have a potential to produce serious adverse human health effects when deposited into the respiratory tract. The most important parts of the lung are the alveolar regions with their enormous surface areas and potential to transfer nanoparticles into the blood stream. These effects may be potentiated in case of the elderly, since this population is more susceptible to air pollutants in general and more to nanoparticles than larger particles. The main goal of this investigation was to determine the exposure of institutionalized elders to nanoparticles using Nanoparticle Surface Area Monitor (NSAM) equipment to calculate the deposited surface area (DSA) of nanoparticles into elderly lungs. In total, 193 institutionalized individuals over 65 yr of age were examined in four elderly care centers (ECC). The occupancy daily pattern was achieved by applying a questionnaire, and it was concluded that these subjects spent most of their time indoors, including the bedroom and living room, the indoor microenvironments with higher prevalence of elderly occupancy. The deposited surface area ranged from 10 to 46 mu m(2)/cm(3). The living rooms presented significantly higher levels compared with bedrooms. Comparing PM10 concentrations with nanoparticles deposited surface area in elderly lungs, it is conceivable that living rooms presented the highest concentration of PM10 and were similar to the highest average DSA. The temporal distribution of DSA was also assessed. While data showed a quantitative fluctuation in values in bedrooms, high peaks were detected in living rooms.

Nanoparticles for the remediation of agrogenic contaminated soil and groundwater – a review

The wide spread use and strong reliance on both fertilizers and pesticides made of agrigenic pollution one of the major contemporary threats to environment and human health. Impacts on the environment vary from local effects, such as eutrophycation1, 2, loss of biodiversity and diminished ecosystem health3, to global effects, such as the aggravation of global warming2, 4 and ozone layer depletion5. The novelty of nanoremediation and its early successes, reported for various contexts, present the prospect for the development of relevant applications for agrigenic contaminants.

Nanoparticles based Permeable Reactive Barriers as an Eco-efficient Technology for nitrate remediation in soil and groundwater

The need to increase agricultural yield led, among others, to an increase in the consumption of nitrogen based fertilizers. As a consequence, there are excessive concentrations of nitrates, the most abundant of the reactive nitrogen (Nr) species, in several areas of the world. The demographic changes and projected population growth for the next decades, and the economic shifts which are already shaping the near future are powerful drivers for a further intensification in the use of fertilizers, with a predicted increase of the nitrogen loads in soils. Nitrate easily diffuses in the subsurface environments, portraying high mobility in soils. Moreover, the presence of high nitrate loads in water has the potential to cause an array of health dysfunctions, such as methemoglobinemia and several cancers. Permeable Reactive Barriers (PRB) placed strategically relatively to the nitrate source constitute an effective technology to tackle nitrate pollution. Ergo, PRB avoid various adverse impacts resulting from the displacement of reactive nitrogen downstream along water bodies. A four stages literature review was carried out in 34 databases. Initially, a set of pertinent key words were identified to perform the initial databases searches. Then, the synonyms of those initial key words were used to carry out a second set of databases searches. The third stage comprised the identification of other additional relevant terms from the research papers identified in the previous two stages. Again, databases searches were performed with this third set of key words. The final step consisted of the identification of relevant papers from the bibliography of the relevant papers identified in the previous three stages of the literature review process. The set of papers identified as relevant for in-depth analysis were assessed considering a set of relevant characterization variables.