Dispersione e caratterizzazione di microplastiche in matrici biologiche

In the recent decade increasing awareness about microplastic pollution has resulted in massive growth in the number of studies assessing the spread and impacts of microplastics. Research on this field focused on risk assessment studies but, despite all this effort, there is currently large debate on the issue of sources and pathways of microplastics pollution, as well as on methodologies to be used to detect and quantify microplastics, and on the assessment of the impacts of microplastics exposure on humans. To conduct risk assessments of microplastics, in vitro studies are needed, which are possible through the dispersion of microplastics in aqueous media, as cells are cultured mainly submersed in water-based media. In this thesis work, a protocol for the dispersion of hydrophobic microplastics in aqueous media and the characterization of the time evolution of the particles in two biological matrices i.e. cell culture media and simulated human digestion fluids has been successfully developed and validated for future applications. As case study has been selected a primary high-density polyethylene microplastics with a hydrophobic nature due to the material itself and the lack of surface modifications. Different amphiphilic substances as surfactants and proteins that potentially able to disperse hydrophobic molecules in a hydrophilic environment were considered. The standardized stock MPs dispersions were used to prepare the working dispersion in cell media. The “bioidentity” of micro plastic particles in cell culture media was assessed in term of size distribution (Sysmex FPIA-3000 particle shape and size analyser) and surface modification (Electrophoretic Light Scattering technique). Finally, the MPs dispersions were treated with a simulated human digestion system (SHDS) to get insight on the properties MPs when they come in contact with intestinal cells in vivo. Flotation kinetics studies showed that the concentration and the mean size distribution of particles decrease over time in a time scale of the hours. This makes essential to perform flotation kinetics when these suspensions are produced for typical in vitro tests. Furthermore, the results showed that microplastics undergoes biotransformation in cell culture media. Incubation in DMEM supplemented with FBS resulted in a modification of the protein corona by protein substitution on the surface of CD with serum proteins, and in a disaggregation of the micrometric CD particles. Concerning the simulated digestion, no changes in size distribution has been observed. However, ζ -potential results suggest a possible exchange of the protein BSA with the proteins of the simulated fluids. In perspective the characterization of microplastics treated or untreated with the SHDS in cell media are crucial to determine possible differences in bioidentity that, in turn, might modulate the biological response of cells to microplastics, and to gain insight into the biotransformation of microplastics that could occur in the human digestive apparatus.