State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China.
University of Chinese Academy of Sciences, Beijing, 100049, China.
Sci Rep. 2017 May 10;7(1):1518. doi: 10.1038/s41598-017-01746-9.
Cellular uptake and exocytosis of SWCNTs are fundamental processes determining their intracellular concentration and effects. Despite the great potential of acid-oxidized SWCNTs in biomedical field, understanding of the influencing factors on these processes needs to be deepened. Here, we quantitatively investigated uptake and exocytosis of SWCNTs in three lengths-630 (±171) nm (L-SWCNTs), 390 (±50) nm (M-SWCNTs), and 195 (±63) nm (S-MWCNTs) in macrophages. The results showed that the cellular accumulation of SWCNTs was a length-independent process and non-monotonic in time, with the most SWCNTs (3950 fg/cell) accumulated at 8 h and then intracellular SWCNTs dropped obviously with time. The uptake rate of SWCNTs decreased with increasing concentration, suggesting that intracellular SWCNTs accumulation is a saturable process. After refreshing culture medium, we found increasing SWCNTs in supernatant and decreasing intracellular SWCNTs over time, confirming the exocytosis occurred. Selective inhibition of endocytosis pathways showed that the internalization of SWCNTs involves several pathways, in the order of macropinocytosis> caveolae-mediated endocytosis> clathrin-dependent endocytosis. Intriguingly, clathrin-mediated endocytosis is relatively important for internalizing shorter SWCNTs. The dynamic processes of SWCNTs uptake and exocytosis and the mechanisms revealed by this study may render a better understanding on SWCNT toxicity and facilitate the design of CNT products with mitigated toxicity and desired functions.
细胞摄取和 SWCNTs 的胞吐作用是决定其细胞内浓度和作用的基本过程。尽管酸氧化 SWCNTs 在生物医学领域具有巨大的潜力，但需要深入了解这些过程的影响因素。在这里，我们定量研究了三种长度的 SWCNTs（L-SWCNTs：630（±171）nm、M-SWCNTs：390（±50）nm 和 S-MWCNTs：195（±63）nm）在巨噬细胞中的摄取和胞吐作用。结果表明，SWCNTs 的细胞积累是一个与长度无关的过程，并且时间上是非单调的，在 8 h 时积累了最多的 SWCNTs（3950 fg/细胞），然后随着时间的推移，细胞内 SWCNTs 明显减少。SWCNTs 的摄取速率随着浓度的增加而降低，表明细胞内 SWCNTs 的积累是一个饱和过程。更换培养液后，我们发现随着时间的推移，上清液中 SWCNTs 增加，细胞内 SWCNTs 减少，证实了胞吐作用的发生。对细胞内吞途径的选择性抑制表明，SWCNTs 的内化涉及几种途径，顺序为巨胞饮>小窝介导的内吞>网格蛋白依赖的内吞。有趣的是，网格蛋白依赖的内吞对于较短 SWCNTs 的内化相对重要。本研究揭示的 SWCNTs 摄取和胞吐作用的动态过程和机制，可能使我们更好地理解 SWCNTs 的毒性，并有助于设计具有减轻毒性和所需功能的 CNT 产品。
