Avaliação toxicológica de nanopartículas compostas de dióxido de titânio, superparamagnéticas de óxido de ferro e de poliuretano in vitro, in vivo, ex vivo e in silico

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2015.A nanotecnologia envolve a compreensão e manipulação da matéria em escala nanométrica, em que os materiais adquirem propriedades diferentes dos materiais na forma bruta, possibilitando aplicações nas mais diversas áreas e produtos de consumo. Porém, as propriedades dos materiais nessa escala de tamanho também podem determinar as interações e efeitos nos organismos vivos. Assim, os objetivos deste trabalho foram avaliar a toxicidade de nanopartículas de dióxido de titânio (TIO2-NPs), nanopartículas superparamagnéticas de óxido de ferro (SPIONS) e nanopartículas de poliuretano (PU-NPs), usando metodologias in vitro, in vivo, ex vivo e in silico. Os resultados obtidos foram divididos em capítulos, de acordo com o sistema nanoestruturado avaliado. O primeiro capítulo desse trabalho mostra os resultados da avaliação dos efeitos in vivo da administração intraperitoneal de TiO2-NPs em camundongos albinos. Observou-se que além da diminuição do ganho de peso, após o tratamento os animais apresentaram aumento significativo no acúmulo de gordura abdominal, inflamação do tecido adiposo e dano ao fígado e estômago. Por fim, identificou-se a presença de titânio no baço, fígado e rins dos camundongos. O segundo capítulo teve como objetivo a avaliação in vitro e in vivo de SPION-PEG350 e SPION-PEG2000, assim denominadas de acordo com o peso molecular da cadeia de PEG ligada as SPIONS. Em resumo, as SPIONS aqui avaliadas não induziram toxicidade in vitro, até a máxima concentração de 100 µg/ml, em três linhagens celulares. Ainda, ambas as formulações de nanopartículas foram captadas pelas células, com maior captação e acúmulo de SPION-PEG2000. A formulação de SPION-PEG2000 foi também avaliada in vivo, e de acordo com as concentrações e parâmetros avaliados, não induziu toxicidade, apesar do acúmulo em órgãos como baço, fígado, pulmão e coração. O terceiro capítulo trata da avaliação do efeito in vivo e ex vivo de PU-NPs. Para o estudo in vivo, a administração de três doses de PU-NPs foi feita pelas vias oral e intraperitoneal (i.p.), o que resultou em inflamação e prejuízo das funções do fígado nos animais tratados independentemente da via. Os estudos ex vivo, usando macrófagos derivados de monócitos primários humanos confirmaram o efeito pró-inflamatório de PU-NPs com o aumento significativo de citocinas pró-inflamatórias no meio de cultura celular após tratamento com as partículas. O quarto e último capítulo deste trabalho mostra os resultados obtidos no desenvolvimento de um modelo farmacocinético para avaliação do acúmulo de SPION-PEG2000 em camundongos e humanos. O modelo desenvolvido para camundongos foi validado com os resultados obtidos experimentalmente, com os camundongos tratados pela via endovenosa com três diferentes concentrações de SPION-PEG2000. Os resultados de distribuição e acúmulo obtidos com o modelo foram similares aos obtidos in vivo, descrevendo um maior acúmulo de SPION-PEG2000 no baço, fígado e pulmão. Com base nos resultados obtidos para camundongos, o mesmo modelo foi desenvolvido para humanos, considerando os parâmetros fisiológicos dessa espécie, sendo os resultados similares aos obtidos para animais, com maior acúmulo estimado para o pulmão, baço e fígado.Abstract : Nanotechnology involves the understanding and manipulation of matter at the nanoscale, where materials acquire distinct characteristics of the materials in the bulk form, which allows it?s applications in several areas and consumer products. However, the properties of materials in this size range also might determine their interaction and effects on living organisms. The objectives of this study was to assess the effects and potential toxicity of titanium dioxide nanoparticles (TiO2-NPs), superparamagnetic iron oxide nanoparticles (SPIONS) and polyurethane nanoparticles (PU-NPs) using in vitro, in vivo, ex vivo and in silico approaches. The results were presented in chapters according to the nanostructured system evaluated. The first chapter of this thesis shows the results of in vivo toxicological evaluation of intraperitoneal administration of TiO2-NPs in albino mice. It was observed that, in addition to significant decrease in body weight gain after treatment with TiO2-NPs, animals showed a significant increase in abdominal fat accumulation, adipose tissue inflammation, liver and stomach damage. Finally, it was identified the presence of titanium in the spleen, liver and kidneys of mice. The second chapter aimed to evaluate SPION-PEG350 and SPION-PEG2000, named according to the molecular weight of the PEG, using in vitro and in vivo methodologies. In summary, the SPIONS did not induce toxicity up to 100 µg/ml, in three cell lineages. Further, both formulations of nanoparticles were taken up by cells, with a higher uptake and accumulation of SPION-PEG2000. SPION-PEG2000 was also evaluated in vivo and according to the concentrations and parameters evaluated they did not induce toxicity, although the accumulation in organs such as the spleen, liver, lung and heart was observed. The third chapter is about the assessment of in vivo and ex vivo effect of PU-NPs. For the in vivo study, three doses of PU-NPs were administrated by oral or intraperitoneal (i.p.) routes, resulting in inflammation and impairment of liver function in both groups of treated mice. Ex vivo studies using human monocytes-derived macrophages have confirmed proinflammatory effects of PU-NPs with significant increase of proinflammatory cytokines after treatment of macrophages with nanoparticles. The fourth and last chapter of this study shows the results obtained in the development of a pharmacokinetic model to predict the pharmacokinetic SPION-PEG2000 in mice and humans. The model was validated toward in vivo results obtained with mice. The accumulation profile obtained with the model was similar to those obtained in vivo, describing higher accumulation of SPION-PEG2000 in the spleen, liver and lung. Based on the results obtained for mice, the same model was developed for humans, taking into account the physiological parameters of this species, and the results were similar to those obtained with animals, with higher accumulation in the lung, spleen and liver

Estratégias para avaliação da toxicidade de sistemas nanoestruturados

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde, Programa de Pós-Graduação em Farmácia, Florianópolis, 2011Os termos nanociência e nanotecnologia correspondem à área do conhecimento que manipula a matéria em escala nanométrica e referem-se ao estudo e as aplicações tecnológicas de objetos e dispositivos que tenham ao menos uma das suas dimensões físicas na ordem de alguns nanômetros. O objetivo deste trabalho foi avaliar a toxicidade de nanopartículas lipídicas (F1, F2, F3, F4, F5, F7 e F8), poliméricas (F9) e metálicas (dióxido de titânio), além do ácido fosfotúngstico (HPW), in vitro, em duas linhagens celulares, de fibroblasto de rim de macaco (Vero) e fibroblasto de rim de cachorro (MDCK), e in vivo, em camundongos. Através dos ensaios in vitro foi possível dizer que as nanopartículas lipídicas e poliméricas estudadas mostraram-se biocompatíveis, uma vez que apenas algumas delas mostraram uma baixa toxicidade, com alto valor de IC50, quando comparadas com a IC50 de outras nanopartículas comumente usadas. Para a nanopartícula de TiO2, a mesma parece interferir nos métodos de avaliação da toxicidade in vitro, sendo que a toxicidade desta nanopartícla pôde ser comprovada através da análise do ciclo celular. Da mesma forma, o HPW demonstrou citotoxicidade nos ensaios de viabilidade in vitro, porém ambos, TiO2 e HPW não demonstraram toxicidade em concentrações abaixo de 100 µg/mL. A avaliação da toxicidade das nanopartículas in vivo foi realizada com as nanopartículas F3, F5, F9 e de TiO2. Foi avaliado o peso dos animais de cada grupo no primeiro e último dia de tratamento, o peso dos órgãos de cada animal após os mesmos serem sacrificados, além de parâmetros hematológicos e bioquímicos, usados comumente como indicativos de toxicidade. Através dos ensaios de toxicidade in vivo, as nanopartículas F3 e F9 demonstraram toxicidade renal e hepática, respectivamente, através de parâmetros bioquímicos que são usados para identificar tais alterações. As nanopartículas de TiO2 causaram alterações nos parâmetros hematológicos e bioquímicos, que caracterizam dano hepático, além de esplenomegalia

A physiologically based pharmacokinetic model to predict the superparamagnetic iron oxide nanoparticles (SPIONs) accumulation in vivo

AbstractSuperparamagnetic iron oxide nanoparticles (SPIONs) have been identified as a promising material for biomedical applications. These include as contrast agents for medical imaging, drug delivery and/or cancer cell treatment. The nanotoxicological profile of SPIONs has been investigated in different studies and the distribution of SPIONs in the human body has not been fully characterized. The aim of this study was to develop a physiologically-based pharmacokinetic (PBPK) model to predict the pharmacokinetics of SPIONs. The distribution and accumulation of SPIONs in organs were simulated taking into consideration their penetration through capillary walls and their active uptake by specialized macrophages in the liver, spleen and lungs. To estimate the kinetics of SPION uptake, a novel experimental approach using primary macrophages was developed. The murine PBPK model was validated against in vivo pharmacokinetic data, and accurately described accumulation in liver, spleen and lungs. After validation of the murine model, a similar PBPK approach was developed to simulate the distribution of SPIONs in humans. These data demonstrate the utility of PBPK modeling for estimating biodistribution of inorganic nanoparticles and represents an initial platform to provide computational prediction of nanoparticle pharmacokinetics.</jats:p

Serum from morbidly obese patients affects melanoma cell behavior in vitro

Here we examined whether serum from obese patients could create a growth-enhancing microenvironment that alters gene expression in&nbsp;BRAF- and&nbsp;NRAS-mutated melanoma cell lines. SK-Mel-28 (BRAF-mutated) and Sk-Mel-147 (NRAS-mutated) cells were treated with pooled serum from 10 severely obese patients (BMI &gt; 40 kg/m2), pooled serum from 6 healthy lean individuals (BMI = 18.5-24.9 kg/m2), or recombinant TNF-α. We found that obese patient serum enhanced migration capacity and increased&nbsp;NRAS&nbsp;expression levels in both&nbsp;BRAF- and&nbsp;NRAS-mutated melanoma cells. Although TNF-α is the major obesity-related cytokine and TNF-α levels were found to be increased in the serum of obese individuals, this cytokine made only a modest contribution to the migration capacity of melanoma cells. These results indicate that other components present in the serum of severely obese patients may be responsible for enhancing the migration capacity of melanoma cells. As TNF-α alone did not seem to significantly affect tumor cell behavior, anti-tumor strategies aimed at blocking TNF-α should be considered with caution in future studies, particularly when&nbsp;in vitro&nbsp;models are used as screening platforms for antitumor activity

Influence of Surfactant and Lipid Type on the Physicochemical Properties and Biocompatibility of Solid Lipid Nanoparticles

Nine types of solid lipid nanoparticle (SLN) formulations were produced using tripalmitin (TPM), glyceryl monostearate (GM) or stearic acid (SA), stabilized with lecithin S75 and polysorbate 80. Formulations were prepared presenting PI values within 0.25 to 0.30, and the physicochemical properties, stability upon storage and biocompatibility were evaluated. The average particle size ranged from 116 to 306 nm, with a negative surface charge around −11 mV. SLN presented good stability up to 60 days. The SLN manufactured using SA could not be measured by DLS due to the reflective feature of this formulation. However, TEM images revealed that SA nanoparticles presented square/rod shapes with an approximate size of 100 nm. Regarding biocompatibility aspects, SA nanoparticles showed toxicity in fibroblasts, causing cell death, and produced high hemolytic rates, indicating toxicity to red blood cells. This finding might be related to lipid type, as well as, the shape of the nanoparticles. No morphological alterations and hemolytic effects were observed in cells incubated with SLN containing TPM and GM. The SLN containing TPM and GM showed long-term stability, suggesting good shelf-life. The results indicate high toxicity of SLN prepared with SA, and strongly suggest that the components of the formulation should be analyzed in combination rather than separately to avoid misinterpretation of the results

Influence of Surfactant and Lipid Type on the Physicochemical Properties and Biocompatibility of Solid Lipid Nanoparticles

Nine types of solid lipid nanoparticle (SLN) formulations were produced using tripalmitin (TPM), glyceryl monostearate (GM) or stearic acid (SA), stabilized with lecithin S75 and polysorbate 80. Formulations were prepared presenting PI values within 0.25 to 0.30, and the physicochemical properties, stability upon storage and biocompatibility were evaluated. The average particle size ranged from 116 to 306 nm, with a negative surface charge around −11 mV. SLN presented good stability up to 60 days. The SLN manufactured using SA could not be measured by DLS due to the reflective feature of this formulation. However, TEM images revealed that SA nanoparticles presented square/rod shapes with an approximate size of 100 nm. Regarding biocompatibility aspects, SA nanoparticles showed toxicity in fibroblasts, causing cell death, and produced high hemolytic rates, indicating toxicity to red blood cells. This finding might be related to lipid type, as well as, the shape of the nanoparticles. No morphological alterations and hemolytic effects were observed in cells incubated with SLN containing TPM and GM. The SLN containing TPM and GM showed long-term stability, suggesting good shelf-life. The results indicate high toxicity of SLN prepared with SA, and strongly suggest that the components of the formulation should be analyzed in combination rather than separately to avoid misinterpretation of the results