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Mitochondrial dysfunction represents a hallmark of both brain aging and age-related neurodegenerative disorders including Alzheimer disease (AD). AD-related mitochondrial dysfunction is characterized by an impaired electron transport chain (ETC), subsequent decreased adenosine triphoshpate (ATP) levels, and elevated generation of reactive oxygen species (ROS). The bioactive citrus flavanone hesperetin (Hst) is known to modulate inflammatory response, to function as an antioxidant, and to provide neuroprotective properties. The efficacy in improving mitochondrial dysfunction of Hst nanocrystals (HstN) with increased bioavailability has not yet been investigated. Human SH-SY5Y cells harboring neuronal amyloid precursor protein (APP695) acted as a model for the initial phase of AD. MOCK-transfected cells served as controls. The energetic metabolite ATP was determined using a luciferase-catalyzed bioluminescence assay. The activity of mitochondrial respiration chain complexes was assessed by high-resolution respirometry using a Clarke electrode. Expression levels of mitochondrial respiratory chain complex genes were determined using quantitative real-time polymerase chain reaction (qRT-PCR). The levels of amyloid β-protein (Aβ1-40) were measured using homogeneous time-resolved fluorescence (HTRF). ROS levels, peroxidase activity, and cytochrome c activity were determined using a fluorescence assay. Compared to pure Hst dissolved in ethanol (HstP), SH-SY5Y-APP695 cells incubated with HstN resulted in significantly reduced mitochondrial dysfunction: ATP levels and respiratory chain complex activity significantly increased. Gene expression levels of RCC I, IV, and V were significantly upregulated. In comparison, the effects of HstN on SY5Y-MOCK control cells were relatively small. Pure Hst dissolved in ethanol (HstP) had almost no effect on both cell lines. Neither HstN nor HstP led to significant changes in Aβ1-40 levels. HstN and HstP were both shown to lower peroxidase activity significantly. Furthermore, HstN significantly reduced cytochrome c activity, whereas HstP had a significant effect on reducing ROS in SH-SY5Y-APP695 cells. Thus, it seems that the mechanisms involved may not be linked to altered Aβ production. Nanoflavonoids such as HstN have the potential to prevent mitochondria against dysfunction. Compared to its pure form, HstN showed a greater effect in combatting mitochondrial dysfunction. Further studies should evaluate whether HstN protects against age-related mitochondrial dysfunction and thus may contribute to late-onset AD.

Poor aqueous solubility of drug substances is associated with poor bioavailability and thus hampers the effective use of many potent active pharmaceutical ingredients. Various strategies to overcome poor solubility are available, whereby drug nanocrystals represent one of the most powerful formulation strategies to enhance the kinetic solubility and dissolution rate of poorly soluble drugs. Nanocrystals are simply obtained by milling large-sized drug powders to sizes < 1 µm. The so obtained nanocrystals possess an increased dissolution rate and kinetic solubility when compared with larger-sized bulk material. The aim of this study was to produce differently sized hesperetin nanocrystals and to investigate the influence of nanocrystal size on the bioefficacy of the natural antioxidant hesperetin in two cell culture models for the prevention and treatment of Alzheimer’s disease. Results showed that the testing of poorly soluble compounds is challenging and requires incredibly careful characterization. Reasons for this are possible changes of the formulations in cell culture media which can occur due to various reasons. If the changes are not considered, results obtained can be misleading and even lead to a false interpretation of the results obtained. Besides, results demonstrate the increase in dissolution rate with decreasing particle size that is especially pronounced with particle sizes < 200 nm. Data also provide clear evidence that smaller nanocrystals with higher kinetic solubility possess higher antioxidant capacity. This results in lower amounts of free radicals in the cell culture models, suggesting that hesperetin nanocrystals, that improve the poor aqueous solubility of hesperetin, are promising for the prevention and treatment of Alzheimer’s disease. Graphical abstract "Image missing"

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