纳米CuO离子化水平及其对细胞毒性影响观察

赵康峰; 宋予晴; 顾雯; 董力; 张兴; 戴洪兴; 白雪涛

doi:10.13421/j.cnki.hjwsxzz.2018.03.001

纳米CuO离子化水平及其对细胞毒性影响观察

Assessment of Copper Oxide Nanoparticles(CuO-NPs) Ionization Level in in vitro Culture System and its Contribution to Cytotoxicity

摘要

摘要:
目的试验测评纳米CuO在体外培养条件下离子化水平及其对细胞毒性贡献率。
方法粒径为40和200 nm的近球型CuO纳米颗粒（CuO-NPs）用于测试。以超滤离心滤过液中铜元素的含量代表CuO-NPs的离子化水平。ICP-MS方法用于铜元素含量测定。细胞毒性试验设CuO-NPs（40和200 nm粒径）组、CuSO₄组、HPMC组和无处理组。在添加和不添加D-青霉胺（D-PA）情况下给A549细胞染毒，分别于染毒后6和24 h采用刃天青法测算细胞抑制率（IR）并计算离解铜离子对细胞毒性贡献率。
结果在CuO-NPs-DMEM/F12体系中，40和200 nm CuO-NPs在孵育6 h时滤过液中铜离子测定浓度分别为（3.6±0.1）和（2.8±0.2）mg/L，在孵育24 h滤过液中铜离子测定浓度分别为（3.9±0.1）和（1.8±0.1）mg/L，上述两个时间点下40 nm CuO-NPs滤过液中铜离子测定浓度均显著高于200 nm CuO-NPs（P < 0.05）。细胞毒性试验结果显示，在添加D-青霉胺情况下，40和200 nm CuO-NPs对A549细胞作用6 h的细胞抑制率分别是64.7%和47.2%，作用24 h的细胞抑制率分别是87.5%和70.9%。在不添加D-青霉胺情况下，40和200 nm CuO-NPs对A549细胞作用6 h的细胞抑制率分别是90.3%和69.4%，作用24 h的细胞抑制率分别是95.2%和86.0%。求得40和200 nm CuO-NPs对A549细胞作用6 h的离子化毒性贡献率分别为28.3%和32.0%，作用24 h的离子化毒性贡献率分别为8.1%和17.6%。
结论 CuO-NPs在体外培养体系中发生离子化现象，离子化水平与CuO-NPs粒径和时间有关。离子化对CuO-NPs细胞毒性具有一定贡献。

Abstract:
Objectives To assess the ionization level of copper oxide nanoparticles(CuO-NPs) in in vitro culture system and its contribution to cytotoxicity.
Methods Both 40 and 200 nm sized CuO-NPs with a globoid shape were synthesized to used in the study. Ionization level of CuO-NPs was represented by the content of copper element in the ultrafiltration fluid. Inductively coupled plasma mass spectrometry(ICP-MS) method was used to determine copper content. Cytotoxicity test was divided into 5 groups including 40 nm CuO-NPs group, 200 nm CuO-NPs group, CuSO₄ group, HPMC group and no-treatment group. When test, A549 cells were exposed to 40 and 200 nm CuO-NPs, CuSO₄ and HPMC with the dosages ofIC50(6 h)+IC50(24 h)/2 respectively in with or without addition of D-penicillamine(D-PA) situation. At the 6^th and 24^th hour respectively after the initial exposure, the cell inhibition rate(IR) was determined by resazurin method, and the contribution(C) of the dissociated copper ions to cytotoxicity was calculated.
Results In 40 nm and 200 nm CuONPs-DMEM/F12 suspension system, copper contents in the ultrafiltration fluid were 3.6±0.1(40 nm) and 2.8±0.2(200 nm) mg/L respectively when incubation for 6 hours, and were 3.9±0.1(40 nm) and 1.8±0.1(200 nm) mg/L respectively when incubation for 24 hours The values of 40 nm CuO-NPs were both significantly higher than 200 nm CuO-NPs'(P < 0.05) in two different hours. Cytotoxicity test result showed the IR of 40 and 200 nm CuO-NPs on A549 cells were 64.7% and 47.2%(with D-PA) and 90.3% and 69.4%(without D-PA) respectively when treated for 6 hours, were 87.5% and 70.9%(with D-PA) and 95.2% and 86.0% (without D-PA) respectively when treated for 24 hours. The cytotoxicity contribution rate of 40 and 200 nm CuO-NPs were calculated 28.3% and 32.0%(treated for 6 hours) and 28.3% and 32.0%(treatment for 24 hours) respectively.
Conclusions CuO-NPs can ionize in in vitro culture system. The ionization level of CuO-NPs is related to their size and storage time. Furthermore, CuO-NPs do some contribution in cytotoxicity.

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