Mechanism of engineered nanoparticle-induced lysosomal membrane permeability

摘要 纳米技术已成为世界上最有前途的新技术之一。由于 纳米技术领域的快速发展，对工人和 消费者是有保证的。工程纳米材料（ENM）广泛应用于电子产品， 医疗保健和消费品。由于ENM种类繁多， 很难评估其毒性。生物活性ENM已被证明会导致 肺泡巨噬细胞释放促炎细胞因子白细胞介素-1 β（IL-1β）。一些 ENM与溶酶体膜渗透性（LMP）有关，这一点通过释放 来自溶酶体的组织蛋白酶B。胞质组织蛋白酶B启动NLRP 3炎性体组装， 这反过来又导致半胱天冬酶-1活化，随后将IL-1β原切割成其 活动形式。还已知的是，ENM被吞噬体中的巨噬细胞内化。 然而，导致ENM启动LMP的机制仍不清楚。有人建议 溶酶体膜中流动性增加或脂质紊乱增加LMP的机会。这 该提案将调查ENM启动LMP的机制。一组特征良好的ENM 将用于确定影响LMP的不同物理和化学差异。模型 膜系统、纳米盘和脂质体以及小鼠肺泡巨噬细胞也将被 利用。为了评估膜流动性的变化，磷脂的有序/无序， 溶剂化显色荧光探针和时间分辨荧光光谱/显微镜将 用于测量荧光寿命、荧光各向异性和荧光相关性。 还将使用毛地黄皂苷提取方法测量LMP的量，以测量LMP的浓度。 细胞溶质溶酶体酶的活性，而不渗透溶酶体。该专用 这项研究的问题非常重要，将在本建议中得到回答。

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.