Preparation of reactive oxygen species scavenging Ce6-Luminol encapsulated liposomes and their protective effect against radiation pneumonitis in mice

Objective‍ ‍To prepare reactive oxygen species (ROS) scavenging Ce6-Luminol encapsulated liposomes (CLL) and to explore the protective effect of CLL against radiation pneumonitis in mice. Methods‍ ‍ROS scavenging material Ce6-Luminol was chemically synthesized, and then was encapsulated using liposome technology to prepare ROS scavenging CLL liposomes. A laser particle size analyzer was used to characterize particle size, polydispersity index, and zeta potential of prepared CLL liposomes. Flow cytometry was employed to detect the uptake of CLL by A549 cells, HUVEC and Raw 264.7 cells, respectively, and CCK-8 assay was utilized to evaluate the cell viability after CLL uptake. The distribution of CLL in the lung tissue of mouse model of radiation lung injury was investigated using In Vivo Imaging System (IVIS). A total of 75 male C57BL/6J mice (6~8 weeks old, 17~19 g) were randomly divided into a normal control group (Normal) and CLL treatment groups (0, 2, 10, and 50 mg/kg), with 15 animal in each group. After the mice in the CLL treatment groups received 15 Gy X-ray irradiation on their chests, they received CLL injection at the corresponding through tail vein on days 0.25, 3, and 5 post-irradiation. Then on day 7 post-irradiation, the contents of H2O2, inflammatory cytokines TNF-α and IL-1β, and chemokines monocyte chemoattractant protein (MCP-1) and keratinocyte chemoattractant (KC) in lung tissue, and the proportion of neutrophils in bronchoalveolar lavage fluid (BALF) were measured to evaluate the efficacy of CLL in treatment of radiation pneumonitis in mice. Results‍ The ROS scavenging material Ce6-Luminol was successfully synthesized, and CLL liposomes were prepared, with an average particle size of 127.8±0.5 nm, a polydispersity index of 0.164±0.005, and a Zeta potential of -23.6±1.4 mV. The prepared CLL liposomes were taken up by A549 cells, HUVEC and Raw264.7 cells in a time- and dose-dependent manner, without a sharply decrease in cell viability. IVIS revealed that there were significantly more CLL liposomes distributed in the lung tissues of radiation pneumonitis mice than in normal mice (F=351.741, P<0.05). CLL intervention significantly reduced the lung contents of H2O2 (F=15.183, P<0.05), TNF-α (F=5.150, P<0.05), IL-1β (F=10.036, P<0.05), MCP-1 (F=3.777, P<0.05) and KC (F=3.755, P<0.05), and the proportion of neutrophils in BALF (F=17.751, P<0.05) from the radiation pneumonitis mice. Conclusion‍ ‍ROS scavenging CLL liposomes can alleviate radiation pneumonitis in mice, which may be due to the protective role against radiation pneumonitis.