Desarrollo y aplicación de estrategias (bio) analíticas para la evaluación de la toxicidad asociada a nanopartículas de plata en modelos in-vitro e in-vivo

Resumo

Nowadays, AgNPs, due to their strong antimicrobial activity, are used in a wide variety of consumer products as well as in biomedical applications, resulting in an increasing exposure of both humans and the environment to these nanoparticles. In particular, the size of AgNPs is one of the most important properties and a critical factor in terms of toxicity. For this reason, this Doctoral Thesis has evaluated the possible toxicity of these AgNPs depending on their size by developing and applying different analytical and proteomic strategies. For this purpose, an in vitro model (HepG2 cells) and an in vivo model (zebrafish larva) have been used. In the toxicological field, proteomics in combination with mass spectrometry plays a fundamental role as it allows the identification and quantification of a large number of proteins in complex biological samples. For this reason, one of the objectives of this PhD Thesis is to design a proteomic study to study the toxicity of AgNPs and to see how this differs according to the size of the nanoparticle used. First, several characterisation, viability and cell internalisation assays have been carried out. Then, two quantitative proteomics strategies, SILAC and super--SILAC, have been successfully implemented in HepG2 cells and zebrafish larvae, respectively, allowing us to elucidate the mechanisms of toxicity of these nanoparticles in a differential manner. The results obtained demonstrate that the size of AgNPs can induce the activation of unique biomolecular mechanisms and, therefore, specific effects that would not be activated by the same AgNPs of different particle size. It is also confirmed that AgNPs10, due to their small size, are able to internalise into the nucleus, produce nucleolar stress and DNA damage and, as a consequence, activate repair mechanisms of damaged DNA. With respect to the in vivo model, the altered proteins were mostly structural in both cases, which justifies the morphological and histological alterations observed during embryonic and cellular development of the larvae exposed to AgNPs. As in the in vitro model, the alteration observed was more pronounced when larvae were exposed to the smaller AgNPs, thus corroborating their greater toxicity. In this sense, while in larvae exposed to AgNP10 certain proteins related to apoptosis and cell cycle alteration were found, in larvae exposed to AgNP60 the alteration of proteins related to mitochondrial damage and induction of hypoxia was more pronounced, which in turn would be related to the generation of apoptosis and cell cycle alteration, while in larvae exposed to AgNP60 the alteration of proteins related to mitochondrial damage and induction of hypoxia was more pronounced. This, in turn, would be related to the generation of ROS and, ultimately, cell death. To complement the proteomic studies carried out in the in vivo