Cubosomal lipid formulation of nitroalkene fatty acids: Preparation, stability and biological effects

Abstract

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.

Keywords

• Nitrooleate
• Monoacyl glycerol
• Nitric oxide
• Lipidic mesophase
• Cubosome
• Keratinocytes