功能自噬通量调节 AgNP 的摄取，而被内化的纳米颗粒通过时间调节通量来决定肿瘤细胞的命运。

BACKGROUND: Silver nanoparticles (AgNPs) are known to induce the conserved, cellular, homeostatic process- autophagy in tumor cells. Previous studies primarily focus on the pro-survival role of autophagy post AgNP exposure in tumor cells, but seldom on its role in AgNP uptake, or on the functional significance of autophagy temporal dynamics. Our study sheds more light on the extensive crosstalk that exists between AgNP and autophagy, which can be critical to the improvement of AgNP-induced therapeutic effects. METHODS: β-cyclodextrin (β-CD) coated AgNPs of two different sizes were synthesized by nucleation method and characterized by transmission electron microscopy. Fluorescence microscopy and flow cytometry were used to probe intracellular uptake of AgNPs. Endocytic mechanism of AgNPs was classically analyzed through use of various endocytosis inhibitors. Autophagy was evaluated by immunoblot and fluorescence microscopy. Additionally, immunoblot was performed to monitor Janus Kinase (JNK) signalling, ubiquitination of proteins, expression of endo-lysosomal and apoptotic markers in correlation to AgNP-induced autophagy. RESULTS: The intra-cellular route of entry for the small NPs (~~9 nm; ss-AgNPs) was different than the large NPs (~~19 nm; ls-AgNPs) studied. However, irrespective of their unique route of entry an inhibition of autophagic flux by chloroquine (CQ) reduced uptake of both the AgNPs. In contrary, rapamycin (Rapa), an autophagy inducer enhanced it. Importantly, JNK activation was required for autophagy induction and AgNP uptake. Furthermore, effect of AgNPs on autophagy showed temporal dependency. An enhanced autophagic flux was noted at early time points; however, prolonged exposure resulted in inhibition of flux marked by increase in Rab7, LC3B-II and p62 proteins. Inhibition of flux was associated with lysosomal dysfunction, decreased LAMP1 expression and an increased accumulation of ubiquitinated (Ub) proteins. This resulted in heightened reactive oxygen species (ROS) and consequent cytotoxicity. CONCLUSION: In this study, we observed that a functional autophagic flux aids AgNP uptake, but AgNPs in turn, overtime, inhibits flux and endo-lysosomal function. We provide critical, novel insights into crosstalk between AgNP and autophagy which can be vital to future AgNP-based therapy development.

背景：已知银纳米粒子（AgNPs）可诱导肿瘤细胞中保守的、细胞内的、动态平衡的自噬过程。先前的研究主要集中在 AgNP 暴露后自噬在肿瘤细胞中的生存作用，但很少关注其在 AgNP 摄取中的作用，或自噬时间动态的功能意义。我们的研究更深入地探讨了 AgNP 与自噬之间存在的广泛相互作用，这对于提高 AgNP 诱导的治疗效果可能至关重要。

方法：通过成核法合成了两种不同大小的β-环糊精（β-CD）包覆的 AgNPs，并通过透射电子显微镜进行了表征。荧光显微镜和流式细胞术用于探测 AgNPs 的细胞内摄取。通过使用各种内吞作用抑制剂，经典地分析了 AgNPs 的内吞作用机制。通过免疫印迹和荧光显微镜评估自噬。此外，还进行了免疫印迹以监测 Janus 激酶（JNK）信号、蛋白质的泛素化、内体-溶酶体和凋亡标志物的表达与 AgNP 诱导的自噬相关联。

结果：小 NPs（~~9nm；ss-AgNPs）的细胞内进入途径与研究的大 NPs（~~19nm；ls-AgNPs）不同。然而，无论其独特的进入途径如何，氯喹（CQ）抑制自噬流都会减少两种 AgNPs 的摄取。相反，自噬诱导剂雷帕霉素（Rapa）增强了它。重要的是，JNK 激活是自噬诱导和 AgNP 摄取所必需的。此外，AgNPs 对自噬的作用表现出时间依赖性。在早期时间点观察到增强的自噬流；然而，延长暴露会导致通量抑制，表现为 Rab7、LC3B-II 和 p62 蛋白增加。通量抑制与溶酶体功能障碍、LAMP1 表达减少和泛素化（Ub）蛋白积累增加有关。这导致活性氧（ROS）增加和随之而来的细胞毒性。

结论：在这项研究中，我们观察到功能正常的自噬流有助于 AgNP 的摄取，但 AgNP 反过来又会随着时间的推移抑制通量和内体-溶酶体功能。我们为 AgNP 与自噬之间的相互作用提供了重要的、新颖的见解，这对于未来基于 AgNP 的治疗发展可能至关重要。