单壁碳纳米管的生物可利用性、生物可降解性及毒性效应研究

The wide use of single-walled carbon nanotubes (SWCNT) in environmental and medical fields has attracted a great deal of concerns on their potential adverse effects on environment and human health. The toxicity of SWCNT is related to the bioavailability and biodegradation of SWCNT, which determines the internal exposure concentration of SWCNT. However, very limited data are available on how physicochemical properties of SWCNT influence the accumulation and degradation of SWCNT within biological system. Generally, pollutants including nanomaterials have to be accessible to cells and taken up by cells before the occurrence of toxic effects. Studies have showed a non-linear relationship between the length of SWCNT and its toxicity. CNTs in appropriate length tend to be readily taken up by cells resulting in higher internal concentration, while very long CNTs are resistant to cellular internalization, causing frustrated phagocytosis and eliciting inflammation response. Recently, biodegradation of SWCNT within biological system has been demonstrated by incubating SWCNT with phagocytes and found that once engulfed, only carboxylated SWCNT can be effectively digested by cells via the catalysis of myeloperoxidase, while pristine SWCNT was kept intact. Therefore, it is of significance to understand the relationship between physicochemical properties and bioavailability and biodegradation for the safe development of SWCNT in medical and environmental applications. In this proposal, we proposed to study the relationship between length and surface functionalization of SWCNT and their bioavailability, biodegradation as well as toxicity. The accumulation of SWCNTs in various forms within cells can be firstly determined by SDS-PAGE gel electrophoresis. The internalization pathway and toxicity of these SWCNTs can be determined by using endocytosis inhibitors and flow cytometry analysis, respectively. After that, the biodegradation and integrity of various SWCNTs in cells can be monitored through a series of spectroscopic methods including FT-IR, Raman, and UV-vis-NIR etc. Simultaneously, SWCNT biodegradation can be further verified by using isolated phagolysosomes and phagolysosomal simulating fluid to detect the direct degradation of CNT. Furthermore, we proposed to test the degradation of SWCNTs by purified haloperoxidases and NADPH oxidase to identify the enzymes responsible for SWCNT degradation. At last, molecular simulation method will be adopted to understand the interactions between SWCNT and enzymes and shed light on the degradation mechanisms.A better understanding of the bioavailability and biodegradation of nanomaterials is important for hazard assessment and could allow their safety by design and production of new medical and environmental applications.

单壁碳纳米管在环境与医学等领域的广泛应用已使人们开始极为关注其潜在的环境和健康危害。SWCNT的毒性与其生物可利用性和可降解性密切相关，而目前这方面的研究报道很少。本课题拟对具有不同表面修饰和长度分布的SWCNT的生物可利用性和可降解性及其毒性进行研究。通过考察不同SWCNT在细胞内的累积，检测SWCNT进入细胞的能力和方式及其产生的毒性变化；通过一系列光谱和电镜方法考察暴露于细胞前后SWCNT的结构变化来研究细胞对SWCNT的降解能力；同时在无细胞体系，研究吞噬溶酶体、溶酶体模拟液以及过氧化物酶溶液分别对不同SWCNT的降解能力来进一步研究SWCNT生物可降解性的原因。在此基础上，采用分子模拟方法研究SWCNT与酶蛋白的作用位点和方式，以阐释SWCNT可降解性的机制。阐明SWCNT特性与其生物可利用性和可降解性的关系将极大地有助于指导我们设计生产和使用更加安全、环保的纳米材料。

单壁碳纳米管在环境与医学等领域的广泛应用已使人们开始极为关注其潜在的环境和健康危害。SWCNT的毒性与其生物可利用性和可降解性密切相关，而目前这方面的研究报道很少。本课题对具有不同表面修饰和长度分布的SWCNT的生物可利用性和可降解性及其毒性进行了研究。通过考察不同长度SWCNT在细胞内的累积和排出，检测SWCNT进入细胞的能力和方式及其机制；研究发现SWCNT不同长度对细胞的摄入影响较小，这主要是因为中等长度的SWCNT既能快速地被细胞摄入，又能快速地被细胞所排出，因此可作为一类良好的穿梭载体。研究表明，细胞摄入SWCNT的主要机制为巨胞饮，同时其他的机制，如小窝蛋白、网格蛋白介导的内吞作用也不同程度地起作用。我们首次发现细胞通过P2X7受体依赖方式将SWCNT排出，这也是一类潜在的CNT与环境污染物联合毒性作用的机制。通过一系列光谱和电镜方法考察了暴露于细胞前后不同表面基团修饰的SWCNT的结构变化来研究细胞对SWCNT的降解能力；同时在无细胞体系，研究了体外呼吸爆发酶模拟液对这些不同SWCNT的降解来进一步分析SWCNT生物可降解性的结构原因。发现只有初始的CNT中含有一定量的缺陷位点，才能成为生物降解的靶点和底物。同时，我们的研究还发现碳纳米材料具有一定的引起细胞自噬的作用，同时能对细胞自噬流产生抑制作用。阐明SWCNT特性与其生物可利用性和可降解性的关系将极大地有助于指导我们设计生产和使用更加安全、环保的纳米材料。本研究所揭示的细胞内吞、外排CNT的分子机制，有助于对CNT用于生物医药载体的效率进行调控，也为纳米材料的毒性控制提供了一种有效的手段。