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

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.