Assembly and Characterization of Biocompatible Coenzyme Q10 -Enriched Lipid Nanoparticles

Abstract

Coenzyme Q₁₀ (CoQ₁₀ ) is a strongly hydrophobic lipid that functions in the electron transport chain and as an antioxidant. CoQ₁₀ was conferred with aqueous solubility by incorporation into nanoparticles containing phosphatidylcholine (PtdCho) and apolipoprotein (apo) A-I. These particles, termed CoQ₁₀ nanodisks (ND), contain 1.0 mg CoQ₁₀ /5 mg PtdCho/2 mg apoA-I (97% CoQ₁₀ solubilization efficiency). UV/Vis absorbance spectroscopy of CoQ₁₀ ND revealed a characteristic absorbance peak centered at 275 nm. Incorporation of CoQ₁₀ into ND resulted in quenching of apoA-I tryptophan fluorescence emission. Gel filtration chromatography of CoQ₁₀ ND gave rise to a single major absorbance peak and HPLC of material extracted from this peak confirmed the presence of CoQ₁₀ . Incubation of cultured cells with CoQ₁₀ ND, but not empty ND, resulted in a significant increase in the CoQ₁₀ content of mitochondria as well as enhanced oxidative phosphorylation, as observed by a ~24% increase in maximal oxygen consumption rate. Collectively, a facile method to solubilize significant quantities of CoQ₁₀ in lipid nanoparticles has been developed. The availability of CoQ₁₀ ND provides a novel means to investigate biochemical aspects of CoQ₁₀ uptake by cells and/or administer it to subjects deficient in this key lipid as a result of inborn errors of metabolism, statin therapy, or otherwise.

Keywords: Coenzyme Q; HepG2; Mitochondria; Nanodisc; Reconstituted HDL.

Fig. 1

Model structure of CoQ₁₀ ND. In this depiction, PtdCho is organized as a disk-shaped bilayer whose perimeter is circumscribed, and stabilized, by apoA-I, which contacts PtdCho fatty acyl chains at the edge of the disk. CoQ₁₀, depicted as yellow dots, intercalates between phospholipid molecules in the ND structure

Fig. 2

UV/Vis absorbance spectrum of CoQ₁₀ ND. ND were formulated with 0, 0.25 and 0.5 mg CoQ₁₀, respectively. UV/Vis absorbance scans were then obtained on equivalent aliquots of each ND formulation (from 250 to 500 nm) on a Spectramax spectrophotometer. Curve (a) 75 μg CoQ₁₀ in ethanol solvent; curve (b) CoQ₁₀ ND (formulated with 0.5 mg CoQ₁₀) in PBS; curve (c) CoQ₁₀ ND (formulated with 0.25 mg CoQ₁₀) in PBS; curve (d) empty ND (formulated with 0 mg CoQ₁₀) in PBS. AU, absorbance units; PBS, phosphate buffered saline

Fig. 3

Effect of CoQ₁₀ incorporation on ND-associated apoA-I tryptophan fluorescence emission. Empty ND (curve a), CoQ₁₀ ND harboring 0.25 mg CoQ₁₀ (curve b), and CoQ₁₀ ND harboring 0.5 mg CoQ₁₀ (curve c), were excited at 280 nm and emission scanned from 300 to 425 nm. RFU, relative fluorescence units

Fig. 4

FPLC size exclusion chromatography profile of CoQ₁₀ ND. ND were formulated with 1 mg CoQ₁₀ or 0 mg CoQ₁₀ as described. Aliquot of each ND sample (corresponding to 0.2 mg CoQ₁₀ and an equivalent amount of empty ND) in PBS, was applied to a Superose 6 increase 10/300 GL column and elution monitored at 280 nm with collection of 1 mL fractions. PBS, phosphate buffered saline

Fig. 5

HPLC analysis of CoQ₁₀ extracted from mitochondria. HepG2 cells were incubated with empty ND (dashed line) or CoQ₁₀ ND (solid line) for 72 h. Following incubation, mitochondria were isolated and CoQ₉ internal standard added. The mitochondrial preparations were extracted with 1-propanol and aliquots applied to a C₁₈ revered phased column and eluted with methanol and 2-propanol (2:1 vol/vol). IS, internal standard

Fig. 6

Effect of incubation with CoQ₁₀ ND on mitochondrial CoQ₁₀ content. HepG2 cells were incubated with medium only, empty ND and CoQ₁₀ ND for 72 h as described in materials and methods. Following incubation, the cells were disrupted and mitochondria isolated by differential centrifugation. A protein assay was performed on the isolated mitochondria samples prior to addition of CoQ₉ internal standard and extraction with 1-propanol. The extracts were subjected to HPLC to determine the CoQ₁₀ content. Values reported are the mean ± standard error (n = 3) * p < 0.05 vs control and empty PtdCho ND

Fig. 7

Effect of CoQ₁₀ ND on oxygen consumption rates (OCR) of C2C12 myotubes. Differentiated C2C12 myotubes were incubated with DMEM supplemented with either 20 μM CoQ₁₀ ND (open bars) or an equivalent amount of empty ND (filled bars) for 72 h. Following incubation, cells were subjected to a mitochondrial stress test assay as described in the materials and methods. Upon completion of the mitochondrial stress test, OCR were determined for each well and normalized to nucleus count (as visualized following DAPI stain). The graph depicts (1) basal respiration rates and (2) maximal respiration rates for C2C12 cells incubated with control empty ND (filled bars) or with CoQ₁₀ ND (20 μM CoQ₁₀). Values reported are the mean ± standard error (n = 3) * p < 0.05 vs empty PtdCho ND. DMEM, Dulbecco’s minimal essential medium