Direct Assessment of Nitrative Stress in Lipid Environments: Applications of a Designer Lipid-Based Biosensor for Peroxynitrite

ABSTRACT Lipid environments can be chemically impacted by peroxynitrite (ONOO − ), a reactive species generated under nitrative stress. Molecular tools used for investigating ONOO − reactivity in biological membranes remain underdeveloped, available probes lack the ability of subcellular localization, and the standard methods for detecting ONOO − in vivo are indirect. Here we investigated ONOO − in diverse lipid environments (biomimetic giant vesicles, live mammalian cells, and within the lung lining) using a biocompatible and membrane-localized phospholipid named DPPC-TC-ONOO − . This designer lipid and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine self-assemble to giant vesicles that respond to ONOO − by generating fluorescence. These vesicles remain intact after sensing ONOO − and exhibit excellent selectivity against other redox species. We delivered DPPC-TC-ONOO − into live HeLa and RAW cells via lipid nanoparticles (LNPs). Cytokine-induced nitrative stress led to enhanced fluorescence of the lipid clusters, primarily in the endoplasmic reticulum. These LNPs allowed the detection of ONOO − reactivity and nitrative stress around bronchioles within precision cut lung slices in response to acute lung injury (ALI). Furthermore, the use of the LNPs allowed for the detection of pulmonary macrophages from bronchoalveolar lavage following ALI in C57BL6/J but not in Nos2 −/– mice. These investigations revealed significant advantages of DPPC-TC-ONOO − over its non-amphiphilic analog. Our work presents (i) an unprecedented function for biomimetic membranes, (ii) the potential of LNPs for delivering designer lipids into cells and tissues, (iii) real-time imaging of endogenous ONOO − at the organelle level in mammalian cells, and (iv) a direct method of studying nitrative stress due to ALI ex vivo and in vivo .