细胞培养基中纳米银形态的转化及其细胞毒性效应研究

Studying the cytotoxicity of silver nanoparticles (AgNPs) is essential to evaluate its environmental and biological safety. Currently, the mechanisms of AgNP toxicity remain controversial, mainly due to the neglected transformation of AgNPs in cell culture medium, which could lead to an alteration of their cytotoxicity before or during cell exposure tests. Therefore, the project aims to study physicochemical transformation of AgNPs in cell culture medium and its impacts on the cytotoxicity. Using MEM, DMEM and RPMI-1640 as the model of cell culture medium, the physical transformation of AgNPs will be studied by the coupling of size-exclusion chromatogram with inductively coupled plasma mass spectrometry (SEC-ICP-MS), high resolution transmission electron miscroscope (HRTEM) and dynamic light scattering (DLS). Then, the released Ag+ will be detected with SEC-ICP-MS and ultrafiltration. In addition, the generation of silver-containing nanoparticles will be monitored by SEC-ICP-MS, and the mechanisms of which will be further studied by the coupling of HRTEM with energy dispersive spectrometer (EDS) and Raman spectra. Finally, based on the transformation of AgNPs in cell culture medium of HepG2 exposure experiment, the effects of these transformation on the cytotoxicity will be investigated. The implement of this project is helpful for understanding the transformation of AgNPs in cell culture medium and its impacts on cytotoxicity of AgNPs, which is of great importance to elucidate cytotoxicity mechanisms and evaluate environmental and biological safety of AgNPs.

纳米银的细胞毒性研究是评估其环境和生物安全性的重要基础，但目前其细胞毒性机制尚存在争议，这是由于现有研究大多忽视了细胞培养基中纳米银物理、化学形态转化对细胞毒性的影响。本项目旨在研究细胞培养基中纳米银形态的转化机制及其细胞毒性效应。拟以MEM、DMEM和RPMI-1640为模型培养基，利用尺寸排阻色谱与电感耦合等离子体质谱联用（SEC-ICP-MS）、高分辨透射电镜、动态光散射等技术研究细胞培养基中纳米银的物理形态转化；基于SEC-ICP-MS和超滤技术研究纳米银在细胞培养基中银离子的释放及监控含银纳米颗粒的生成；并利用高分辨透射电镜与能谱联用、拉曼光谱阐明含银纳米颗粒的生成机制；以HepG2为模式细胞进行暴露实验，明确纳米银形态转化对其细胞毒性效应的影响。项目的实施，有望阐明细胞培养基中纳米银形态的转化及相应的细胞毒性效应，对揭示纳米银的细胞毒性机制及评估其环境和生物安全性具有重要意义。

细胞培养基中纳米银的物理和化学形态转化是决定其细胞毒性的关键因素，而相关研究的缺乏严重制约了其细胞致毒机制的研究。本项目围绕复杂基质中纳米银形态分析的新方法、转化机制及效应研究等方面的若干科学问题，开展了较为系统的研究，主要取得了一下代表性成果：（1）发展了复杂基质中不同化学形态纳米银的测定的新方法。将浊点萃取、尺寸排阻色谱与电感耦合等离子体质谱等检测技术联用，发展了实际水样和细胞培养基中痕量硫化银纳米颗粒和纳米银的定量测定新方法。（2）揭示了细胞培养基中纳米银的物理和化学形态的转化机制。发现了细胞培养基质中蛋白质诱导纳米银释放银离子，无机盐类介导释放的银离子转化为含银纳米颗粒，揭示了表面性质（如包裹剂）、无机盐、蛋白质等对纳米银的银离子释放、团聚、再分散和再合成等行为的影响机制。上述研究成果已发表标注本项目资助的SCI论文7篇，包括环境分析化学领域权威期刊Anal. Chem. (2篇)，Environ. Sci. Technol. (1篇）。申请国家发明专利4件；在国内学术会议作了3个学术报告。项目可为科学评估纳米银的生物安全性提供关键技术和基础数据。