MDA-lipid oxidation assay (TBARS assay)

The TBARS assay was performed according to the Lipid Peroxidation (MDA) Assay Kit (Sigma-Aldrich).³⁷ Copper oxidized LDL was reconstituted in 20 μL PBS buffer and mixed with 500 μL of 42 mM sulfuric acid in a 1.5 mL Eppendorf tube. 125 μL of phosphotungstic acid was added and vortexed for 2 minutes. The sample and standards were incubated at room temperature for 15 minutes and centrifuged at 13 000g for 5 minutes. LDL was oxidized with 5 μM Cu²⁺ for 4 h at 37 °C and Cu²⁺ was removed by dialysis (with and without treated SBTG1 nitroxide NPs). The top layer was collected and mixed with 2 μL of BHT in 100 μL of deionized water and the final volume was adjusted to 200 μL. Finally, 600 μL of TBA solution was mixed together with the 200 μL analyte solution and incubated at 95 °C for 60 minutes and the UV absorbance of the solution measured at 532 nm.