Mechanism of engineered nanoparticle-induced lysosomal membrane permeability

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Abstract Nanotechnology has emerged as one of the world’s most promising new technologies. Due to the rapid growth of the nanotechnology field, heath risk assessment for both workers and consumers is warranted. Engineered nanomaterials (ENM) are widely used in electronics, healthcare and consumer goods. Due to the wide variety of ENM, making sweeping assessments of their toxicity is difficult. Bioactive ENM have been demonstrated to cause alveolar macrophages to release the pro-inflammatory cytokine interleukin-1beta (IL-1β). Some ENM have been linked to lysosome membrane permeability (LMP) as evident by the release of cathepsin B from lysosomes. Cytosolic cathepsin B initiates NLRP3 inflammasome assembly, which in turn results in the activation caspase-1, followed by the cleavage of pro- IL-1β to its active form. What is also known is that ENM are internalized by macrophages in phagosomes. However, the mechanism causing ENM initiated LMP is still unclear. It has been suggested that increased fluidity or lipid disorder in the lysosome membrane increases the chance of LMP. This proposal will investigate the mechanism of ENM initiated LMP. A well-characterized set of ENM will be used to determine different physical and chemical differences that affect LMP. Model membrane systems, nanodiscs and liposomes, and mouse alveolar macrophages will also be utilized. To assess changes in membrane fluidity, the order/disorder of the phospholipids, solvatochromic fluorescence probes and time-resolved fluorescent spectroscopy/microscopy will be used to measure fluorescence lifetime, fluorescence anisotropy and fluorescence correlation. The amount of LMP will also be measured using a digitonin extraction method to measure the activity of cytosolic lysosome enzymes without permeabilizing lysosomes. The purposed questions of this study are of great importance and will be answered in this proposal.