The field of nanoecotoxicology has been pioneered in recent years as concern grows in response to the potential environmental hazards of engineered nanoparticle release. Silver nanoparticle (AgNP) release through induction into commercial products as an antimicrobiont is of particular interest. Plausible routes of AgNPs to reach aquatic systems and their biological impacts have been investigated, but none have addressed the potential remediation of these waters using the heavy metal accumulating fern Azolla caroliniana. This study employed biological staining techniques and fluorescence microscopy to identify oxidative stress, wounding responses of cell wall and membrane, and cell death of A. caroliniana roots to assess the capability of this plant to withstand AgNP exposure. Two concentrations series were applied, 0-1.0ppm and 0-10.0ppm for 1, 3, 5 days after transfer (DAT), 0ppm being a control. Oxidative stress, measured in production of non-specific ROS, increased in a dose-dependent manner with increasing AgNP concentration. Callose (1,3-β-glucan) was deposited in response to potential cell wall damage and was also observed to be elevated in a dose-dependent manner. Cell vitality appeared from a general decline in fluorescence of nucleic content to visual nuclei lysis. Statistically significant and severe responses to AgNPs was observed at 1 DAT but recovery could be seen at 3~5 DAT. In sum, these data suggest a toxicity threshold of 1.0ppm at which A. caroliniana roots can mediate exposure.

Orientador: Oswaldo Luiz Alves / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-26T03:04:22Z (GMT). No. of bitstreams: 1 Simoes_MateusBatista_M.pdf: 4986679 bytes, checksum: 2b115e73f65ae444a8545b2ab4b05173 (MD5) Previous issue date: 2014 / Resumo: As diferenças nas propriedades observadas considerando um material no seu estado bulk e na sua escala nanométrica são, possivelmente, a característica mais marcante e fascinante da nanotecnologia. Os carbon dots são nanomateriais baseados em carbono que apresentam fluorescência quando menores do que 10 nm, mas que podem fluorescer após tratamento da sua superfície, quando em partículas da ordem de até 100 nm. É interessante notar que o comprimento de onda no qual ocorrerá a fluorescência é dependente do tamanho das partículas. Assim, é possível modular a fluorescência controlando o tamanho dos carbon dots, os quais apresentam grande potencial para aplicação em fotocatálise, bioimagem, sensores e optoeletrônica, sendo possível funcionalizar estes materiais, objetivando uma aplicação in vivo, a fim de aumentar sua biocompatibilidade. As sínteses hidrotérmicas vêm despertando interesse para a obtenção dos carbon dots, por ser uma técnica simples, econômico e eficiente. Além disto, é possível obter materiais com grande homogeneidade e com controle de morfologia e tamanho, fatores estes que irão influenciar a fluorescência. Desta forma, o presente trabalho teve como objetivo estudar a influência das condições de síntese hidrotérmica na fluorescência dos carbon dots, realizar a funcionalização deste material e avaliar a capacidade de utilização in vivo do material por meio de ensaios de hemólise. Carbon dots foram obtidos por meio da carbonização hidrotérmica de glicose e as condições de síntese foram otimizadas utilizando-se um planejamento fatorial. Observou-se que temperatura e tempos de síntese elevados e uma menor concentração inicial da fonte de carbono leva a nanopartículas com maior rendimento quântico (variando entre 3,3 e 5,8%). Os carbon dots foram caracterizados por espectroscopia na região do infravermelho, espectroscopia na região do ultravioleta-visível, microscopia eletrônica de transmissão, além de ter seu perfil de fluorescência estudado, sendo que o máximo de excitação ocorre na região do ultravioleta e o máximo de emissão na região do azul. Testes hemolíticos foram realizados com as nanopartículas que apresentaram maior rendimento quântico e mostraram que não há indução de hemólise, demonstrando que o material tem elevado potencial para aplicação in vivo. Por fim, utilizando-se as condições ótimas de síntese, carbon dots também foram obtidos por meio da carbonização hidrotérmica de pectina, demonstrando que o método de síntese é robusto e válido para fontes de carbono alternativas. Os carbon dots obtidos de pectina apresentam um rendimento quântico de 3,6% e foram caracterizados pelas mesmas técnicas utilizadas para os carbon dots de glicose / Abstract: The differences in observed properties considering a material in its bulk state and its nanoscale are possibly the most striking and fascinating feature of nanotechnology. Carbon dots are carbon-based nanomaterials that present fluorescence when smaller than 10 nm, but may fluoresce after treatment of its surface considering particles of the order of until 100 nm. Interestingly, the wavelength at which the fluorescence occurs is dependent on the particle size. Thus, it is possible to modulate the fluorescence controlling the size of the carbon dots, which have great potential for application in photocatalysis, bioimage, optoelectronics and sensors, being possible to functionalize these materials, aiming an application in vivo, in order to increase its biocompatibility. The hydrothermal syntheses have attracted interest for obtaining the carbon dots, being a simple, cheap and efficient technique. Moreover, it is possible to obtain materials with high homogeneity and controlled morphology and size, factors that will influence the fluorescence. Thus, the present work aimed to study the influence of the conditions of hydrothermal synthesis in the fluorescence of carbon dots, perform the functionalization of this material and evaluate the ability to in vivo use of the material by hemolytic trials. Carbon dots were obtained by hydrothermal carbonization of glucose and the synthesis parameters were optimized by a factorial design of experiments. It was observed that higher temperature and time of synthesis and a lower initial concentration of the carbon source leads to nanoparticles with a higher quantum yield (varying between 3.3 and 5.8%). The carbon dots were characterized by infrared spectroscopy, ultraviolet-visible spectroscopy, transmission electron microscopy, beyond to have its fluorescence profile studied, and it was observed that the maximum excitation occurs at the ultraviolet range and the maximum emission at the blue range of the spectrum. Hemolytic trials were performed with the nanoparticles of highest quantum yield, and the results showed that no hemolysis was provoked, demonstrating that he material have a raised potential to in vivo applications. Lastly, with the optimized synthesis parameters, carbon dots were also obtained by hydrothermal carbonization of pectin, evidencing that the synthesis protocol is robust and effectual to alternatives carbon sources. The carbon dots of pectin presented a quantum yield of 3.6% and were characterized by the same techniques utilized to the carbon dots of glucose / Mestrado / Quimica Inorganica / Mestre em Química

Orientador: Oswaldo Luiz Alves / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-19T14:26:47Z (GMT). No. of bitstreams: 1 Martinez_DiegoStefaniTeodoro_D.pdf: 3429324 bytes, checksum: cc064a83a4c293e8f0dbeb800c8212cb (MD5) Previous issue date: 2011 / Resumo: Neste trabalho foram estudados aspectos de purificação e caracterização de nanotubos de carbono de parede múltiplas, bem como suas interações com diferentes níveis de organização dos biossistemas. Os nanotubos foram purificados e caracterizados após tratamentos químicos com HNO3, HCl e NaOH em sistema de refluxo convencional, sendo obtido nanotubos com pureza > 98,5 % (livres de ferro) e livres de resíduos carbonáceos. Para expressas os reflexos da interação dos nanotubos purificados com os biossistemas selecionados foi adotada uma abordagem sistêmica (Nível Ecológico > Nível Imunológico > Nível Celular-molecular). Os principais resultados desta interação foram: i) ausência de ecotoxicidade aguda para o organismo aquático Daphnia similis, quando dispersos em biossurfactantes produzidos pelo microorganismo Bacillus subtilis; ii) ausência de efeito mutagênico sobre linhagens de Salmonella typhimurium (Teste de Ames); iii) desenvolvimento de um eficiente processo para tratamento de efluente contendo resíduos carbonáceos provenientes da etapa de purificação dos nanotubos; iv) efeito imunoestimulatório (adjuvante) em camundongos geneticamente selecionados e, v) capacidade de internalização celular e efeito citotóxico em astrócitos primários in vitro, via indução de morte celular programada (apoptose) / Abstract: In this work, aspects of the purification and characterization of multiwalled carbon nanotubes were studied as well as their interactions with different organization levels of biological systems. The carbon nanotubes were purified and characterized after chemical treatments with HNO3, HCl and NaOH under conventional reflux system. It was obtained nanotubes with purity > 98.5% (iron free) and without carbonaceous byproducts. A systemic approach (Ecological level > Immunological level > Cellular-molecular level) was used to represent the carbon nanotube interactions with biological systems. The main results regarding the interactions were: i) absence of acute ecotoxicity to Daphnia similis aquatic organism when the nanotubes were dispersed with biosurfactant produced by Bacillus subtilis microorganism; ii) absence of mutagenic effect to Salmonella typhimurium strains (Ames Test); iii) development of an effective process for effluent treatment containing carbonaceous byproducts originated from carbon nanotube purification process; iv) immunostimulant effect (adjuvant) in genetically selected mice; v) cellular internalization capacity and cytotocity effect on in vitro primary astrocytes by inducting programmed cell death (apoptosis) / Doutorado / Quimica Inorganica / Doutor em Ciências

The rapid development of nanotechnology has caused concerns about nanoproducts on human health throughout their lifecycle. As part of the consortium NEPHH (nanomaterial related environmental pollution on human health through their life cycle, funded by EU-FP7), this project aimed to assess the potential effect of novel polymer-silicon composites on human health from a lifecycle perspective, focusing on in vitro toxicity of raw silica nanoparticles (SiNP) and dust nanoparticles (NP) released from silicon-based polymer composites. The main objectives were to characterise a group of amorphous SiNP and dust NP in water and cell culture medium; assess NP toxicity potential in in vitro models; and establish mode of SiNP action. The selection of SiNP of size 7-14 nm was based on their wide use in developing polymer nanocomposites. Dust NP were generated from mechanical processing of polymer composites made of polyamide-6 (PA6), polyurethane (PU) and polypropylene (PP), each incorporated with SiNP or 3 other different silicon reinforcement materials. The dispersion and size of NP in water and in cell culture medium were characterized using dynamic light scattering, scanning electron microscopy and transmission electron microscopy. The chemical composition of NP was assessed by infra-red spectroscopy. NP were assessed in vitro for induction of membrane damage, intracellular reactive oxygen species (ROS), loss of cell viability, and cellular uptake by flow cytometry and confocal microscopy. In order to identify potential biomarkers for toxicity prediction, miRNA array and extracellular metabonomic assays were performed. The size of SiNP (10-100 µg/ml) ranged from ~200-500 nm in water and ~20- 500 nm in culture medium, indicating the presence of aggregates. The infra-red spectrum of SiNP dried from culture medium showed a slight difference as compared with that dried from water, indicating protein adsorption. SiNP induced acute ROS increase, cell membrane damage, and reduction in cell viability after 48 h in human lung carcinoma epithelial A549 cells, lung fibroblast MRC-5 cells and skin HaCaT keratinocytes. SiNP were up taken by all 3 cell types, and located in the cytosol. Six early ( < 48h) SiNP responsive miRNAs were identified in A549 cells. SiNP also induced early changes in metabolites including glucose, lactate, ethanol, phenylalanine, histidine and tyrosine. Dust NP generated from PA6 group materials were more toxic than those from other polymer composites when assessed at 25-100 µg/ml at 72 h in A549 cells. The results obtained from this study suggest that 1) both small and larger SiNP aggregates are taken up into the target cells; 2) conventional cytotoxicity assays combined with miRNA and metabonomic assays provide insight into the molecular mechanisms of the nanotoxicity; 3) metabonomics and miRNA assays can serve as robust tools for recognising sub-toxic dose-effect relationships; 4) the toxicity of dust NP from polymer composites depends on polymer type but not reinforcement materials. This study demonstrated the importance of lifecycle analysis as opposed to single stage analysis of novel materials. Further studies need to improve study design to enable interpretation of cytotoxicity in relation to NP size, physiochemical property and intracellular dose, and to simulate the health effect of polymer-silicon composites under more realistic scenarios.

Nanoparticles (particles less than 100 nm in at least one dimension) exhibit chemical properties that differ from their bulk counterparts. Furthermore, they exhibit increased potential for systemic toxicities due to their deposition deep within pulmonary tissue upon inhalation. Thus, standard regulatory assays alone may not always be appropriate for evaluation of their full spectrum of toxicity. Systems biology (e.g., the study of molecular processes to describe a system as a whole) has emerged as a powerful platform proposed to provide insight in potential hazard, mode of action and human disease relevance. This work makes use of systems biology to characterize carbon black nanoparticle-induced toxicities in pulmonary and extra-pulmonary tissues (i.e., liver and heart) in mice over dose and time. This includes investigations of gene expression profiles, microRNA expression profiles, tissue-specific phenotypes and plasma proteins. The data are discussed in the context of potential use in human health risk assessment. In general, the work provides an example of how toxicogenomics can be used to support human health risk assessment.

>Magister Scientiae - MSc / Nanotechnology is a rapidly growing field of research. Due to major innovations brought about by developments in nanotech, several consumer products are currently available containing nanomaterials. The increase of nanomaterial production and use is accompanied by the increased potential of human, plant and animal exposure to these nanomaterials. As a relatively new nanomaterial, carbon quantum dots (CQDs) are being extensively used and researched due to its unique properties. Although many studies have assessed the toxic potential of CQDs, and found them to exhibit low toxicity, there is lack of work assessing the effects on the immune system. In the present study, RAW 264.7 murine macrophages were used as model to assess the immunomodulatory potential of CQDs. RAW cells exposed to varying concentrations of CQDs (0-500μg/ml), showed that CQDs caused a reduction at cell viability. In the absence of a mitogen CQDs, induced an inflammatory response by stimulating the release of various cytokines and chemokines such as, TNFα, MIP-1α, MIP-1β, MIP-2, IP-10, G-CSF, GM-CSF, and JE.

Zinkoxid-Nanopartikel (ZnO-NP) finden in vielen Produkten des täglichen Verbrauchs Verwendung. Daten über die toxikologischen Eigenschaften von ZnO-NP werden kontrovers diskutiert. Die menschliche Haut ist in Bezug auf die ZnO-NP Exposition das wichtigste Kontakt-Organ. Intakte Haut stellt eine suffiziente Barriere gegenüber NP dar. Bei defekter Haut ist ein Kontakt zu den proliferierenden Stammzellen möglich, sodass diese als wichtiges toxikologische Ziel für NP darstellen. Das Ziel dieser Dissertation war die Bewertung der genotoxischen und zytotoxischen Effekte an humanen mesenchymalen Stammzellen (hMSC) durch niedrig dosierte ZnO-NP nach 24 stündiger Exposition, repetitiven Expositionen und im Langzeitversuch bis zu 6 Wochen. Zytotoxische Wirkungen von ZnO-NP wurden mit 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid-Test (MTT) gemessen. Darüber hinaus wurde die Genotoxizität durch den Comet-Assay bewertet. Zur Langzeitbeobachtung bis zu 6 Wochen wurde die Transmissionselektronenmikroskopie (TEM) verwendet. Zytotoxizität nach 24-stündiger ZnO-NP-Exposition war ab einer Konzentration von 50 µg/ml nachweisbar. Genotoxizität konnten bereits bei Konzentrationen von 1 und 10 µg/ml ZnO-NP beschrieben werden. Wiederholte Exposition verstärkte die Zyto-, aber nicht die Genotoxizität. Eine intrazelluläre NP-Akkumulation mit Penetration der Zellorganelle wurde bei einer Exposition bis zu 6 Wochen beobachtet. Die Ergebnisse deuten auf zytotoxische und genotoxisches Effekte von ZnO-NP hin. Bereits geringe Dosen von ZnO-NP können bei wiederholter Exposition toxische Wirkungen hervorrufen sowie eine langfristige Zellakkumulation. Diese Daten sollten bei der Verwendung von ZnO-NP an geschädigter Haut berücksichtigt werden. / Zinc oxide nanoparticles (ZnO-NP) are widely used in many products of daily consumption. Data on the toxicological properties of the ZnO-NP used are discussed controversially. Human skin is the most important organ in terms of ZnO-NP exposure. Intact skin has been shown to provide an adequate barrier against NPs, while defective skin allows NP contact with proliferating cells. Among proliferating cells, stem cells are the main toxicological target for NPs. Therefore, the aim of this dissertation was to evaluate the genotoxic and cytotoxic effects of human mesenchymal stem cells (hMSC) by low-dose ZnO-NP after 24 hours of exposure, repetitive exposures and in long-term experiments up to 6 weeks. Cytotoxic effects of ZnO-NP were measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT). In addition, genotoxicity was assessed by the comet assay. Transmission electron microscopy (TEM) was used for long-term observation after 6 exposure periods. The results of the study show that ZnO-NP has a cytotoxic effect starting at high concentrations of 50 µg/mL and could demonstrate genotoxic effects in hMSC exposed to 1 and 10 µg/ml ZnO-NP. Repeated exposure enhanced cytotoxicity but not genotoxicity. Intracellular NP accumulation with penetration of the cell organelles was observed at exposure up to 6 weeks. The results indicate the cytotoxic and genotoxic potential of ZnO-NP. Even small doses of ZnO-NP can cause toxic effects with repeated exposure and long-term cell accumulation. These data should be considered when using ZnO-NP on damaged skin.

No description available.

Carbon nanotubes (CNT) are a new form of industrially relevant nano-scale particle and are seen as the cutting edge of the burgeoning nanotechnology revolution which promises to impact on all our lives. Due to high length to diameter ratio, CNT are perhaps the most well known of a growing collection of high aspect ratio nanoparticles (HARN). However the production and use of carbon nanotubes has presented an interesting toxicological question based on its structure and raised the question ‘are carbon nanotubes like asbestos?’. Few people are unaware of the devastating global pandemic of diseases caused by asbestos and similarities in needle-like shape between asbestos and nanotubes have raised fears that nanotubes may mimic asbestos-type disease. The purpose of this study was to investigate this link, based on the wealth of information known about the toxic effects of certain forms of fibre on the respiratory system. From this we hope to identify those carbon nanotubes which are hazardous whilst not prejudicing the use of those industrially relevant materials which can be used safely. Within fibre toxicology there exists a central paradigm which outlines the main properties a fibrous particle must possess if it is to exert pathogenic effects in the body. This paradigm outlines the importance of length, thinness and biopersistence to a fibre and an absence of one or more of these attributes results in a loss of pathogenicity. We took this paradigm and, using suitable asbestos and non-asbestos controls, applied it various morphological forms of carbon nanotubes using an in vivo model. The resultant data demonstrates for the first time that asbestos-like pathogenic behaviour associated with carbon nanotubes is closely linked to the morphology of the nanotubes and their aggregates. Specifically our results showed that CNT which possessed a long, straight length were highly inflammogenic and fibrogenic within the peritoneal cavity of mice; a model sensitive to the pathogenic effects of fibres. As well as length, the importance of biopersistence in the pathogenesis of fibrous particles has been known for many years and is a central attribute affecting the pathogenicity of fibres. Amphibole asbestos is known to be durable, a commercially exploited attribute and as such is biopersistent in the lung which is a key feature of its pathogenicity. Glass fibre on the other hand is bio-soluble, and whilst long and inhalable, does not cause significant disease due to its lack of biopersistence. Based on the grapheme structure of CNT which impart exceptional strength and rigidity and the chemical inertness of carbon we would hypothesis that CNT would be biopersistent and therefore fulfil another of the criteria of the fibre pathogenicity paradigm (FPP). Our aim therefore has been to establish the durability of CNT against fibrous particles of known durability using a synthetic solution maintained at a pH to simulate the lung environment. Using a range of 4 CNT and using both durable and non-durable fibres such as amphibole asbestos and glass fibre to bench mark our result; we demonstrated that 3 of the 4 CNT tested displayed exceptional durability whilst the fourth lost approximately 30% of its mass during the experiment with concomitant reduction in pathogenicity. As well as length and biopersistence, the surface of a particle has been shown to contribute to the overall toxicity of a particle and in certain circumstances, such as that of quartz, the surface of the particle can be the biologically active component. In the case of carbon nanotubes, surface functionalisation is commonly used for various endpoints including the addition of various tags and labels for tracking. As such our further aim was to investigate the relationship between the length-dependent pathogenicity of a fibre sample and the surface of the fibre. By using different forms of functional groups attached to the surface of a pathogenic carbon nanotube we aim to critically test if the level of inflammation and fibrosis triggered in vivo can be altered by simple alteration of the surface. Our results showed that surface modification of CNT could alter the inflammogenic and fibrogenic effects of CNT which may have important implications when considering the hazard assessment of functionalised HARN. As CNT are not the only form of fibrous nanomaterial and within this project we also attempted to determine the applicability of the FPP to further high aspect ratio nanomaterials. In order to do this we set out to determine the generality of this hypothesis by asking whether nickel nanowires, a radically different form of HARN to CNT, show length-dependent pathogenicity. Nickel oxide nanowires synthesised to be predominantly long (>20 μm) act similarly to amphibole asbestos in showing the ability to elicit strong inflammation in the mouse peritoneal model in a dose dependent manner; inflammation was not seen with the short (<5 μm) nanowires. In summation, the results from this study are the first to show that long HARN can indeed behave like asbestos when in contact with the sensitive mesothelium. This study suggests a potential link between inhalation exposure to long nanotubes and asbestos-related disease, especially mesothelioma and as such this may have immediate implications across many disciplines if care is to be taken to avoid a long term legacy of harm.

Electrospun materials have potential use in many biomedical applications such as soft tissue replacements or as scaffolds to target drug delivery to local sites. Electrospinning is a polymer processing technique that can be used to create materials composed of fibers with diameters ranging from the micron to the nanoscale. We investigated the effects of microfibrous and nanofibrous electrospun polycaprolactone (EPCL) on innate, cell-mediated, and humoral components of the immune system. Results demonstrated that in both young (12 week) and old (6 month) mice, EPCL had no effect on various immune parameters. With its lack of immunotoxicity, EPCL presents an excellent polymer scaffold for use in delivering drugs to local sites. Drug delivery studies focused on using EPCL nanofiber scaffolds with the known immunosuppressive compound dexamethasone (DEX) incorporated within the matrix. The ability of the EPCL-DEX scaffold to suppress cell-mediated immunity (CMI) was evaluated using the delayed-type hypersensitivity (DTH) response to Candida albicans. Preliminary studies were conducted following subcutaneous implantation of a single disk (6-mm or 3-mm diameter) with 3, 10, 30, or 100 % w/w DEX in EPCL in the thigh region. Based on footpad swelling, dose -responsive suppression of the DTH was observed based on DEX equivalent units (DEU) at all but the lowest dose. The animals that received the high dose (100% in 6-mm) had decreased spleen weights, however no change in spleen weight was observed at the lower doses. Thymus weights were only affected at the four highest doses. These preliminary results suggest that implantation of a drug-containing electrospun scaffold may achieve local immunosuppression without systemic toxicity. Finally, we evaluated the EPCL-DEX scaffold in an acute inflammatory model (keyhole limpet hemocyanin) and a mouse model of rheumatoid arthritis (collagen induced arthritis). While similar trends were observed in the other models, the EPCL-DEX system achieved greatest success in the DTH model.