*2.8. Determination of FLAP-Dependent 5-LO Product Formation in PMNL*

The evaluation of the effects on FLAP was performed according to our established protocol [21]. We assessed FLAP-dependent 5-LO product formation in human PMNL,

cells (5 <sup>×</sup> <sup>10</sup><sup>6</sup> mL−<sup>1</sup> ) were pre-incubated with BRP-187 or NPs for indicated timepoints at 37 ◦C. The cells were stimulated with 2.5 µM Ca2 + -ionophore A23187 (Cayman, Ann Arbor, USA) for 10 min, and the incubation was stopped with 1 mL ice-cold methanol containing 200 ng mL−<sup>1</sup> PGB<sup>1</sup> as an internal standard. Samples were subjected to solid phase extraction, and the formed lipid mediators (leukotriene B<sup>4</sup> (LTB4), trans-isomers of LTB4, 5-hydroxyeicosatetraenoic acid (5-HETE)) were separated and analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC) as previously described [24]. Statistical analysis was performed with log-transformed values to obtain Gaussian-distributed data sets. Experiments were analyzed via one-way ANOVA and Tukey's multicomparison test with GraphPad Prism 9.1.2 (GraphPad, La Jolla, CA, USA).
