Synthesis, Characterization and Toxicological Assessment of a new model of Nanoplastics

Synthetic plastics were first introduced into the consumer markets in the 1950’s. Over the next 70 years, annual global production increased reaching to 367 million metric tons in 2020 1. While plastic is a unique material in terms of its versatility, cost-effectiveness and resistance, these same properties, combined with the intensive consumption and rapid disposal of plastic products, have caused its accumulation in the environment. Nowadays, it has been estimated that 0.1% of the total global production (>8.3 billion metric tons) has reached the ocean as plastic marine debris2–7 and can degrade into small micro and nano sized fragments. Microplastics (MPs) have been detected in numerous aquatic organisms and also in human samples, making significantly important to adopt a risk assessment of this pollutant for the human health. The lack of appropriate methodologies to collect the nanoplastics (NPs) from water systems imposes the formation of engineered models to explore their interactions with biological systems. Frequently, these models are colloidal and chemically synthesized, proving low correlated with the real case conditions. This thesis explores the possibility to fabricate diverse types of plastic particles that can be used as alternative models to the chemical model for a more realistic investigation. In particular, the adopted top-down fabrication approach, based on the laser ablation of polymer films in water, allows obtaining NPs and submicron sized plastic particles starting from bulk materials, mimicking, thus, a naturally occurred degradation pathway. Different typologies of NPs have been developed in this study; starting from the previously reported polyethylene terephthalate (PET) NPs, the methodology was further modified in order to fabricate polycarbonate (PC) NPs. It was revealed that the variation of the energy fluence (F), during the ablation process results in the modification of the size of the formed plastic particles. PC NPs were carefully characterized in terms of chemical/physical properties and stability in different media. Moreover, during the PC NPs formation, various byproducts are also released, also detectable in the PC photo-degradation reactions. Therefore with the herein proposed approach it is offered the possibility to obtain a fully integrated product composed by all the components that are expected to be released in the environment during the photo-degradation of the PC plastic litter. As the oral route has been defined as the main route for human exposure to NPs with the liver a secondary organ of the exposure, the impact of the PC NPs is evaluated using upcyte® hepatocytes as a human liver cell model, an advanced system for assessing in vitro nanomaterial hepatotoxicity. Additionally, considering the different types of NPs expected to be present in the environment and the limited outcomes of the conventional cytotoxicity evaluation methods for nanoparticulate matter, different in vitro technologies have been explored for the evaluation of different types of NPs. In particular, the toxicological assessment of PC and PET NPs (PET1) synthetized by laser ablation and of PET NPs (PET2) made by nanoprecipitation was explored using combinations of conventional and advanced cytotoxicity evaluation methods. In comparison to the particles made by nanoprecipitation, the laser-ablated NPs are generally smaller, with broader size distributions and a higher degree of oxidation on their surface. Two different cell lines were used: single and a monolayer of HePG2 cells, a standard cell model for hepatic organ, and undifferentiated and differentiated Caco-2 cells, a standard model for the intestinal epithelial barrier. Using conventional colorimetric tests no cytotoxic effects of the exposure to NPs were observed, in terms of both cell viability and membrane integrity. However, iii using the High Content Screening (HCS), a multiparametric approach that allows for the monitoring of different cellular characteristics through a high-throughput fluorescence imaging it was revealed that PC NPs are more toxic than PET1 NPs, whereas the PET2 NPs showed no toxicity. Interestingly, on a model of the cellular monolayer and intestinal barrier, none of the NPs induced any measurable toxicity. Therefore, the laser ablation approach allows the formation of NPs of different nature, expanding its application to the different types of polymers. This approach allows obtaining a fully integrated product with all the expected components in the environment, with byproducts by the PC photo-degradation in water added to the NPs population. The results of the toxicological assessment do not show toxicity in cell viability on more complex cellular models, such as gastrointestinal barrier and hepatic monolayer. Still, a potential effect on the hepatic functionality has been revealed on cells closer to the human hepatocyte. The fabrication method has a significant impact on the evaluation of the toxicity of the model. Moreover, it might overcome the possible limitations of the technique used for the toxicological assessment by comparing different technologies.