Biosafety Evaluation Of ZnO Nanoparticles By Utilizing RTCA And Other Experimental Methods

With the wide application of ZnO nanoparticles (ZnO NPs) in many areas, their biological safety has become a great concern. ZnO NPs in surrounding environment could enter human bodies through respiration and other pathways, and subsequently cause biological/toxic effects in respiratory and other systems. Understanding the biological effects and biosafety of ZnO NPs has important scientific and public health significances.In this thesis, we used human alveolar type Ⅱ epithelial cell line A549, human lung epithelial cell line BEAS-2B, non-small cell lung cancer cell lines H1975 and HCC827 as experimental models to evaluate the biological effects and biosafety of ZnO NPs in vitro. Observation of cell morphology, RTCA real-time label-free cell analysis technology, WST-1 and other experimental methods were carried out for this purpose in the above normal and cancer cell lines. Meanwhile, RTCA Cardio real-time analysis technique was used to determine the toxicity of ZnO NPs on neonatal rat primary cardiomyocytes. In addition, by detecting ROS levels, LDH levels, the cell cycle changes and induction of apoptotic cell death, this thesis explored the mechanism involved in the biological effects of ZnO NPs in vitro.Our data indicate that ZnO NPs, at the concentration range between 12.5 μg/ml and 100 μg/ml, showed dose-dependent toxicity to the above respiratory normal and cancer cell lines. Meanwhile, we demonstrated that at the concentration range between 12.5 μg/ml and 100 μg/ml, ZnO NPs produced toxic effects on the cell viability, beating rate and amplitude of primary cardiomyocytes. The reversibility of these toxic effects on cardiomyocytes depends on the concentration and treatment duration of ZnO NPs.Preliminary mechanism study indicated that ZnO NPs at concentration of 12.5 μg/ml and higher led to a significant increase in ROS level in the human lung epithelial cell line BEAS-2B and the rat myocardial cell line H9C2. Similar increase of ROS level was observed in A549 cells treated with 50 μg/ml and higher concentrations of ZnO NPs. It is known that once the cell membrane is damaged and broken, LDH will be released from cytoplasm to extracellular medium. By administration of 25 μg/ml and higher concentrations of ZnO NPs, a high level of LDH was detected in the extracellular medium of cultured A549, BEAS-2B and H9C2 cells. These findings indicated that oxidative stress is closely related with the toxicity of ZnO NPs.Further PI staining and Annexin V/PI double staining and subsequent flow cytometric analysis showed that after exposure to ZnO NPs for 24 hr, the cell cycle distribution of A549, BEAS-2B and H9C2 cells were all changed, with the induction of apoptotic cell death. With the increase of ZnO NPs concentration, the cell proportion in GO/G1 phase was significantly reduced and the cell proportion of G2/M phase was significantly increased, with the increase of apoptotic cell death.These data has provided valuable information for evaluating the bioligical effects of ZnO NPs. On the other hand, ZnO NPs had extremely slight impact on the RTCA electrode, while RTCA analysis system showed multiple advantages in determining the biological effects of ZnO NPs, e.g. good sensitivity, predictive and real-time data acquisition, adjustable test cycle length, suggesting that RTCA technology is an ideal and convenient method for biological studies of nanomaterials, which has further expanded the application scope of RTCA technology.