Cationic Liposomes Cause ROS Generation and Release of Neutrophil Extracellular Traps

摘要

Cationic liposomes are used as nanocarriers of drugs in chemotherapy, antibacterial therapy, and gene therapy. Positively charged stearylamine is commonly used in cationic liposome production. It has been shown that cationic liposomes, when applied intravenously, impact the functions and viability of blood cells, including neutrophils (innate immunity cells). In this work we studied the influence of phosphatidylcholine cationic liposomes containing stearylamine (SA liposomes) on the activity of human neutrophils–their ability of the formation of extracellular traps (NETs) and of the production of reactive oxygen species (ROS). The ratio of phosphatidylcholine to stearylamine in the liposomes was 9 : 1. Phosphatidylcholine liposomes (PC liposomes) were used as control. Liposomes were 140 ± 49 nm in diameter and differed in zeta-potential: PC liposomes had –1.74 ± 0.31 mV, while SA-liposomes had 11.40 ± 0.44 mV. NETs were visualized by light and fluorescent microscopy. It was found that the cells remained intact after a 90-min incubation with PC liposomes (control experiment), while incubation with SA liposomes caused an extrusion of thin DNA fibers into the extracellular space, i.e., the formation of NETs. ROS generation by neutrophils incubated with the liposomes and stimulated either with 0.5 mg/mL of zymozan, or with 40 nM of phorbol-12-miristate-13 acetate, or without stimulation was assessed by luminol-dependent chemiluminescence. It was shown that PC liposomes did not exert any significant effect on chemiluminescence of unstimulated and zymozan-stimulated neutrophils. PMA in the presence of liposomes did not exert any effect on the cells. SA liposomes had a complex impact on the activity of neutrophils: they inhibited the effects of zymozan and caused a prominent respiratory burst independently of the presence of the additional stimulation. The time before the maximal intensity of chemiluminescence peak after incubation with SA-liposomes ranged from 1 to 3 h and significantly differed from the time before the maximal intensity of chemoluminescence peak after incubation with zymozan or PMA (30–40 min). Apocynin, a NADPH-oxidase inhibitor, suppressed the respiratory burst induced by SA liposomes, indicating that NADPH oxidase has a role in ROS production caused by SA liposomes. To assess characteristics of the respiratory burst, lucigenin-dependent chemiluminescence sensitive to superoxide anion radical ( $${ext{O}}_{2}^{{ ullet -}}$$ ) was registered, and it was found that $${ext{O}}_{2}^{{ ullet -}}$$ is not produced after stimulation of neutrophils by SA liposomes. On the basis of these findings we conclude that SA liposomes are a polyfunctional factor that can affect the formation of NETs, suppress the action of zymozan, and stimulate ROS production by neutrophils.