Abstract: OBJECTIVES:Use of two methods to compare the cytotoxicity of CuO nanoparticles on brain microvascular endothelial cells (BMECs) in neonatal rats.METHODS:Growth curves of BMECs were determined by Real-Time Cell Analysis (RTCA) which was used to determine the optimal number of cells to be inoculated and the optimal exposure time for the toxicity experiment.Cells were treated with CuO-NPs at 1.5,0.75,0.38,0.19,0.09 mg/mL,and the culture medium without cell was used as control (0 mg/mL).The cytotoxicity of CuO-NPs was analyzed by two methods:RTCA and Resazurin.Correlations of the two methods were determined by comparing the IC₅₀ values obtained.RESULTS:The growth curves of BMECs show that 2.5×10³ cells/well was the optimal inoculation concentration,and 40 h after inoculation was the best exposure time.RTCA results show that BMECs were transiently unstable at 2-3 h after exposure to 1.5,0.75,and 0.38 mg/mL CuO-NPs,followed by progressive death.The cell viability of BMECs at the 1.5 and 0.75 mg/mL groups began to decrease at about 2 h after exposure,and all cells died at 4 h.The cell viability was measured by the Resazurin method at 3,12,24,36,and 48 h after exposure from the same doses.The Resazurin method yielded similar results to RTCA,ie,the 1.5 and 0.75 mg/mL groups had the greatest cytotoxicity,and the cytotoxicity of CuO-NPs on BMECs showed a dose-dependent trend.Results from correlation analyses show that IC₅₀ obtained by the two methods has a good correlation,and correlation coefficient was 0.999,P=0.000.Paired t test results show the difference was not statistically significant (t=1.125,P=0.323).From the calculation of R² value of IC₅₀,R² value of RTCA fitting curve is higher than R² value of the Resazurin method Logistic regression coefficient.CONCLUSION:CuO-NPs demonstrated dose-dependent cytotoxic effects on BMECs in vitro.The results of cytotoxicity of BMECs as determined by the RTCA and Resazurin assays were related,but results obtained by the RTCA method appear to be more accurate.Therefore,RTCA may be more suitable for the research of cytotoxicity of CuO-NPs.

Key words: copper oxide nanoparticles, brain microvascular endothelial cells, real time cell analysis, resazurin method, cytotoxicity