Fluorescent head-labelled phospholipid coumarin bioconjugate as a chemical reporter for antioxidant protection in liposomes

The oxygen radical absorbance capacity (ORAC) assay is considered a valuable analytical tool for measuring the protection afforded by an antioxidant compound to a target probe. Fluorescein is the classical probe used in the ORAC assays. In addition to not being a biological target for oxidation, fluorescein acts as a free molecule within the reaction media. Moreover, the probe specific location is the aqueous medium where its oxidation occurs. Hence, fluorescein is not ideal for inferring about the interactions of radicals with biomimetic membrane models within the liquid-lipid interface or deeper in the bilayer system. The aim of the present work was to explore the use of POPE-COUM probe, a 2-oleoyl-1-palmitoyl-sn-glycero-3phosphoethanolamine labelled with 3-carboxycoumarin, to report the protection of antioxidants interacting with biomembranes using liposomes as a biomimetic model. The probe was obtained applying an improved bioconjugation by using continuous flow synthesis to afford the desired compound in just 5 min in 80% yield. The lipid peroxidation induced by 2,2 & PRIME;-azobis(2-methylpropionamidine) dihydrochloride (AAPH) derived radicals was monitored directly by the fluorescence intensity decay of the POPE-COUM, without resorting to any other reporter, in large unilamellar vesicles (LUVs). The results obtained showed a delay on the oxidation of the probe compared to aqueous phase fluorescein which might indicate that POPE-COUM is not accessible to the attack by the AAPH-derived radicals and is protected towards the inside of the vesicle. Hence, the access to the embedded probe only happens when alteration of the LUV structure occurs. Fluorescence lifetime imaging microscopy studies support the fact that the fluorescent POPE-COUM probe is restricted to the lipidic layers, and no background fluorescence was observed. Ultimately, these outcomes represent the proof-of-concept for the use of POPE-COUM in ORAC assays and its suitability as biomarker of the oxidation state and integrity of cellular membrane biomimetic systems such as liposomes.