Lipid nitroalkenes - nitro-fatty acids (NO2-FAs) are formed in vivo via the interaction of reactive nitrogen species with unsaturated fatty acids. The resulting electrophilic NO2-FAs play an important role in redox homeostasis and cellular stress response. This study investigated the physicochemical properties and reactivity of two NO2-FAs: 9/10-nitrooleic acid (1) and its newly prepared 1-monoacyl ester, (E)-2,3-hydroxypropyl 9/10-nitrooctadec-9-enoate (2), both synthesized by a direct radical nitration approach. Compounds 1 and 2 were investigated in an aqueous medium and after incorporation into lipid nanoparticles prepared from 1-monoolein, cubosomes 1@CUB and 2@CUB. Using an electrochemical analysis and LC-MS, free 1 and 2 were found to be unstable under acidic conditions, and their degradation occurred in an aqueous environment within a few minutes or hours. This degradation was associated with the production of the NO radical, as confirmed by fluorescence assay. In contrast, preparations 1@CUB and 2@CUB exhibited a significant increase in the stability of the loaded 1 and 2 up to several days to weeks. In addition to experimental data, density functional theory-based calculation results on the electronic structure and structural variability (open and closed configuration) of 1 and 2 were obtained. Finally, experiments with a human HaCaT keratinocyte cell line demonstrated the ability of 1@CUB and 2@CUB to penetrate through the cytoplasmic membrane and modulate cellular pathways, which was exemplified by the Keap1 protein level monitoring. Free 1 and 2 and the cubosomes prepared from them showed cytotoxic effect on HaCaT cells with IC50 values ranging from 1 to 8 μM after 24 h. The further development of cubosomal preparations with embedded electrophilic NO2-FAs may not only contribute to the field of fundamental research, but also to their application using an optimized lipid delivery vehicle.

Department of Analytical Chemistry Faculty of Science Palacký University 17 listopadu 12 77146 Olomouc Czech Republic

Department of Chemistry Faculty of Science University of South Bohemia Branišovská 31 České Budějovice 370 05 Czech Republic

Department of Medical Chemistry and Biochemistry Faculty of Medicine and Dentistry Palacký University Hněvotínská 3 775 15 Olomouc Czech Republic

Department of Medical Chemistry and Biochemistry Faculty of Medicine and Dentistry Palacký University Hněvotínská 3 775 15 Olomouc Czech Republic; The Czech Academy of Sciences Institute of Biophysics Kralovopolská 135 Brno 612 65 Czech Republic

Department of Organic Chemistry Faculty of Science Palacký University 17 listopadu 12 771 46 Olomouc Czech Republic

Faculty of Chemistry University of Warsaw Pasteura 1 02 093 Warsaw Poland

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Lipid-based liquid crystalline materials in electrochemical sensing and nanocarrier technology