Evaluating Anti-inflammatory Nanoparticles in Whole Blood

Freshly withdrawn whole blood in Li-heparin Monovettes (Sarstedt) from healthy adult donors that had not received any anti-inflammatory treatment the last 10 days was provided by the Institute of Transfusion Medicine, Jena University Hospital. The blood was incubated for different periods with either BRP-201 or NPs (Ace-DEX, Ace-DEX[BRP-201], PLGA, PLGA[BRP-201]) and stimulated with pathogenic E. coli (O6:K2:H1; 1 × 10⁹ cells per mL blood) for 180 min. The reaction was stopped with ice-cold methanol containing the deuterium-labeled internal standards d8-5S-HETE, d4-LTB₄, d5-LXA₄, d5-RvD2, and d4-PGE₂ (500 pg, each). Samples were kept at − 20 °C for 1 day to allow protein precipitation. After centrifugation (2000×g, 4 °C, 10 min) 8 mL acidified water was added (final pH = 3.5). The samples were subjected to solid phase extraction and analyzed by UPLC–MS–MS as described previously [18 (link)], see above (Sect. Determination of lipid mediator signature profiles in human monocyte-derived macrophages).