Superparamagnetic Nanoparticles for the Assessment of Intracellular Nanoparticle-Cell Interactions

Since their discovery, nanomaterials have been employed in an increasing number of products and applications. Thanks to their unique properties they enabled the advancement of many technologies and improved our lives under several aspects. Because of their nanometric dimensions, nanomaterials interact with the biological matter in a completely different way compared to their smaller (molecular scale) or larger (macro scale) counterparts. Despite the efforts of the scientific community in unravelling the network of machineries involved in the interaction of nanomaterials with the cells and human body, our understanding of the bio-nano interactions is still limited. The lack of knowledge around the dynamics that regulate the nanoparticles’ (NPs) trafficking in the human body is limiting the development of nanomedicine and, at the same time, is rising concerns in the regulatory bodies for the safe commercialisation of nanomaterial-based products.
One of the main issues related to this critical gap in the knowledge is the lack of methodologies and tools that prevents unravelling the complexity of the bio-nano interactions. Techniques commonly used for the study of the intracellular dynamics result limiting for the study of the NPs intracellular trafficking for which the combination of different analysis and methodologies is required to obtain reliable and robust results. However, the correlation of data from different techniques is difficult when different NPs systems are used for the experiments.
In this thesis, we develop new tools and methodologies for the study of the bio-nano interactions, exploiting one of the unique properties of iron oxide NPs, the superparamagnetism. In this context, superparamagnetic NPs are extremely useful tools because allow to label the machineries involved in the intracellular trafficking of NPs and enable their isolation from the biological matrix, which in order allows a more in-depth downstream analysis. To label the machineries involved in the intracellular trafficking with superparamagnetic NPs, two strategies are possible: exploiting the natural uptaking mechanisms of the cells or targeting specific compartments with NPs designed ad hoc.
To exploit the first strategy, we designed multifunctional core-shell NPs with iron oxide multicores that provided the superparamagnetic properties and a silica shell doped with an organic fluorophore. Combining the magnetic and fluorescent properties in one nanoconstruct enabled to resolve the NPs intracellular trafficking by optical microscopy and to isolate the vesicles loaded with the NPs by magnetic separation, at different stages of their voyage inside the cell. The vesicles were then analysed with a set of technique to evaluate their integrity and functionality.
To exploit the second strategy, we designed antibody grafted iron oxide NPs for the targeting of specific biological species. For this purpose, we adopted a thoughtful strategy of NPs surface modification that enabled the grafting of the antibody through bio-orthogonal chemistries, stabilise the particles in biological conditions, and limit the adsorption of undesired biomolecules typically responsible for NPs off-targeting. Although this second nanoconstruct is still under development, the preliminary results showed excellent targeting ability and specificity.
Overall, the work presented in this thesis provides a solid base for the isolation, by magnetic separation, of biological species involved in the NPs intracellular trafficking and for the development of methodologies for the investigation of the machineries involved in the process.