Kenneth A. DawsonAMIRAH SAAD ALAHMARI2022-06-052019-08-212022-06-0578569https://drepo.sdl.edu.sa/handle/20.500.14154/67127Nanoparticles hold great promise for applications in a diversity of medical scenarios; however, a thorough investigation of their toxicological profile prior to clinical applications is currently hampered by a lack of suitable in vitro models for studying the long-term intracellular behaviour of nanoparticles. Consequently, despite various studies reporting on the destiny of internalised nanoparticles in the short term, very little is known about the fate of commonly used nanomaterials over longer periods of time. Acquiring this information is critical to fully understanding the effects of their use as medicine, as some particle types can persist in intracellular compartments for long periods of time. The work presented in this thesis details the development of a novel long-lived threedimensional (3D) cell culture system that allows the in vitro study of the intracellular behaviour of nanoparticles over extended time periods. Human alveolar basal epithelial (A549) cells were used as a model cell type, and the newly established 3D cultures resembled phenotypes closer to that of an in vivo profile, indicating they may serve as a more appropriate model to study biological processes in an in vitro environment. Additionally, the clusters proved to be promising for application in nanoparticle interaction studies over longer time periods (up to 1 month). The different physicochemical properties of nanoparticles will largely dictate their behaviour in a biological system; however, for many commonly used particle types (such as silica) there is contradictory information with respect to their fate in vitro and in vivo. In this work, various silica and cerium oxide nanoparticles were applied to the newly established 3D cell model to determine their intracellular behaviour during many weeks in culture. The various types of silica nanoparticles tested accumulated in the lysosomes and persisted for several weeks; however, they displayed different degrees of degradation depending on the synthesis method. Additionally, for the first time, remarkable spike-like features were observed on the structure of the cerium oxide nanoparticles after incubation with the cell clusters. These unique features are thought to be a result of a form of biotransformation taking place with the cerium oxide particles during their intracellular residence in cells. This work confirmed that the majority of nanoparticles will end up and persist for many weeks in the lysosomes; therefore, the effect of the various nanoparticles biotransformation/ degradation on lysosomal function was also evaluated. It was found that although there were some indications of the lysosomes working harder in the presence of a nanoparticle load, there was no significant detrimental effect on lysosomal function or cell viability. The work presented in thesis highlights the use of a more relevant 3D cell culture model that permits the analysis of the long-term effects of nanoparticle interactions in vitro. Having been successfully applied to study the fate of various silica and nanoceria particles, new information was revealed about their intracellular behaviour over many weeks. This represents a significant step towards understanding their biological fate without the need for animal models or human testing, which will in turn help improve the design and application of nanoparticles for medical applications.263enThesis