Immunotoxicity of zinc oxide nanoparticles with different size and electrostatic charge

Cheol-Su Kim,1,* Hai-Duong Nguyen,1,* Rosa Mistica Ignacio,2 Jae-Hyun Kim,1 Hyeon-Cheol Cho,1 Eun Ho Maeng,3 Yu-Ri Kim,4 Meyoung-Kon Kim,4 Bae-Keun Park,5 Soo-Ki Kim1,5 1Department of Microbiology, Wonju College of Medicine, Yonsei University, Wonju-si, Gangwon-do, Republic of Korea; 2Department of Environmental Medical Biology, Wonju College of Medicine, Yonsei University, Wonju-si, Gangwon-do, Republic of Korea; 3Healthcare Laboratory, Medical Device Evaluation Team, Korea Testing and Research Institute, Gimpo-si, Gyeonggi-do, Republic of Korea; 4Department of Biochemistry and Molecular Biology, Medical School and College, Korea University, Seoul, Republic of Korea; 5Institute of Lifestyle Medicine, Wonju College of Medicine, Yonsei University, Wonju-si, Gangwon-do, Republic of Korea *These authors contributed equally to this work Abstract: While zinc oxide (ZnO) nanoparticles (NPs) have been recognized to have promising applications in biomedicine, their immunotoxicity has been inconsistent and even contradictory. To address this issue, we investigated whether ZnO NPs with different size (20 or 100 nm) and electrostatic charge (positive or negative) would cause immunotoxicity in vitro and in vivo, and explored their underlying molecular mechanism. Using Raw 264.7 cell line, we examined the immunotoxicity mechanism of ZnO NPs as cell viability. We found that in a cell viability assay, ZnO NPs with different size and charge could induce differential cytotoxicity to Raw 264.7 cells. Specifically, the positively charged ZnO NPs exerted higher cytotoxicity than the negatively charged ones. Next, to gauge systemic immunotoxicity, we assessed immune responses of C57BL/6 mice after oral administration of 750 mg/kg/day dose of ZnO NPs for 2 weeks. In parallel, ZnO NPs did not alter the cell-mediated immune response in mice but suppressed innate immunity such as natural killer cell activity. The CD4+/CD8+ ratio, a marker for matured T-cells was slightly reduced, which implies the alteration of immune status induced by ZnO NPs. Accordingly, nitric oxide production from splenocyte culture supernatant in ZnO NP-fed mice was lower than control. Consistently, serum levels of pro/anti-inflammatory (interleukin [IL]-1Β, tumor necrosis factor-α, and IL-10) and T helper-1 cytokines (interferon-γ and IL-12p70) in ZnO NP-fed mice were significantly suppressed. Collectively, our results indicate that different sized and charged ZnO NPs would cause in vitro and in vivo immunotoxicity, of which nature is an immunosuppression. Keywords: immunosuppression, cytokine, ZnO, immune response, cytotoxicity, innate immunit