Differential Autophagic Responses to Nano-Sized Materials
Popp, Lauren Bailey
Doctor of Philosophy
Recent progress in the field of nanotechnology has led to a dramatic increase in the number of engineered nanomaterials currently being produced and introduced in the marketplace, raising concerns regarding the risk of human exposure to these specially designed, highly reactive materials. Engineered nanomaterials exert a diverse range of effects on biological systems, reflecting the heterogeneity and complexity of their properties. The effects of nanomaterials on biological systems, which range from inflammatory response to carcinogenicity and neurodegeneration, originate from the interaction between nanomaterials and cellular components, which also operate at the nanoscale. Cellular clearance mechanisms are typically activated in response to internalization of nano-sized materials. In particular, the cellular response to nanomaterials is mediated by the lysosome-autophagy system, which is the main catabolic pathway in mammalian cells that mediates degradation of a variety of nano-sized materials that are encountered by the cell through the lysosome. The lysosome-autophagy system is thus at the forefront of the cellular response to the uptake of nanomaterials: depending on the nanomaterial’s physicochemical properties, this response can vary dramatically and may culminate in enhancement of cellular clearance or blockage of the autophagic flux and, eventually, cell death. Due to its important role in maintaining cellular homeostasis and survival, defects in the lysosome-autophagy system may have deleterious effects on cells, possibly leading to pathologic conditions. The objective of this study was to characterize the response of the lysosome-autophagy system to different types of nanomaterials, namely genetically encoded adeno-associated virus (AAV) particles, which are of particular interest due to their potential as gene delivery vectors, and engineered titanium dioxide and zinc oxide nanoparticles, which are used in a variety of consumer products. I investigated a comprehensive set of makers of the lysosome-autophagy system with the ultimate goal of elucidating the specific nature of the autophagic response to nanomaterial uptake and whether it leads to biocompatible or bioadverse effects on cell physiology. Analyses of the molecular mechanisms underlying the autophagic response to AAV nanoparticles revealed that uptake of AAV induces activation of autophagy, which, in turn, results in reduction in transgene delivery. Upregulation of autophagy induced by AAV also causes enhanced degradation of potentially toxic autophagic substrates. These results provide important insights for the design of AAV-based gene delivery systems as well as nanotherapeutics for the treatment of diseases characterized by insufficient autophagic clearance. Titanium dioxide nanoparticles of different primary particle diameters were found to induce transcriptional upregulation of the lysosome-autophagy system. Prolonged exposure to titanium dioxide nanoparticles, however, induced lysosomal membrane permeabilization, leading to blockage of autophagic flux and accumulation of autophagic substrates. These results point to the complexity of the autophagic response to nanomaterials, which may involve activation of the lysosome-autophagy system, but also impairment of some of its components, leading to accumulation of autophagic vesicles that cannot be cleared by lysosomes. Exposure to zinc oxide nanoparticles was also found to cause transcriptional upregulation of the lysosome-autophagy system and formation of autophagic vesicles. Cell treatment with bare zinc oxide nanoparticles (~85 nm) or coated with a highly stable silicone derivative (triethoxycaprylylsilane) enhanced the formation and turnover of autophagosomes, while exposure to larger zinc oxide particles (~200-1000 nm) caused blockage of autophagic flux, resulting in accumulation of autophagosomes. Results from this study provide important insights into the effect of nanomaterials on the lysosome-autophagy system and will inform the design of the next generation of nanomaterials with predictable autophagy-modulating properties for a variety of industrial and medical applications.