Lipid membrane behavior of nitro-fatty acids and their loading into liposomes to activate Nrf2 pathway in RAW264.7 cells with impact on intracellular NO production

Nitro-fatty acids (NO₂-FAs) are endogenous electrophilic signalling modulators, and some of them have been proposed as drug candidates. The main ones include nitro-oleic acid (NO₂-OA) and other derivatives of unsaturated fatty acids such as nitro-linoleic acid (NO₂-LA). In this study, we describe the behavior of 9/10-NO₂-OA, 10-NO₂-LA and the conjugated nitro-linoleic acid (9/12-NO₂-cLA) in a model POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) membrane using molecular dynamics and selected experimental approaches. We showed that when loaded in liposomes, NO₂-FAs undergo degradation (a decay reaction) to a very limited extent, in contrast to the free molecular form in an aqueous environment. This was confirmed by the electron paramagnetic resonance spectroscopic analysis of NO radical release. In general, NO₂-FAs suppress membrane hydration, especially in the segment where the ester groups are located. Further, in the presence of NO₂-FAs, there is increased membrane fluidity and a decrease in the degree of lipid order. These effects are greater for NO₂-FAs than for their non-nitrated versions. The presence of a nitro group in close contact with the polar head groups was confirmed. This drives the tilt of the lipid chain which in turn induces membrane disorder. Protonated NO₂-FAs penetrated more easily/deeper into the membrane structure than the dissociated forms and this makes the membrane bilayer surface more negatively charged based on zeta potential measurement. We also found that NO₂-FAs incorporated into POPC liposomes retained their ability to activate the Nrf2 pathway. This was documented by an increased expression of heme oxygenase-1 at the level of mRNA, with a parallel decrease in protein levels of Keap1, in murine macrophage RAW264.7 cells. The NO₂-FAs treatment resulted in an increase in intracellular NO level in vitro as determined by a genetically encoded G-geNOp sensor. This was confirmed at statistically significant level only for NO₂-OA, not for NO₂-LA or NO₂-cLA. The results indicate that biologically relevant NO release may be strictly dependent on which NO₂-FA is investigated. This study supports the hypothesis that NO₂-FAs are distributed (co-localized) in cells and tissues in the lipid or aqueous phase, which affects whether they are mobile, stable, and thus biologically active.