Lutein nanodisks protect human retinal pigment epithelial cells from UV light-induced damage

Abstract

The hydrophobic carotenoid, lutein, was conferred with aqueous solubility upon formulation into reconstituted discoidal high density lipoprotein particles, termed lutein nanodisks (ND). When formulated with phosphatidylcholine (PC), apolipoprotein (apo) A-I and lutein (formulation ratio = 5 mg PC/2 mg apoA-I/1 mg lutein), lutein solubilization efficiency in phosphate buffered saline (PBS) was ~90%. The UV/Vis absorbance maxima for lutein ND in PBS were red shifted by 6-13 nm versus the corresponding lutein absorbance maxima in ethanol. FPLC gel filtration chromatography gave rise to a single major absorbance peak in the size range of ND. Incubation of cultured ARPE-19 cells with lutein ND resulted in lutein uptake, as determined by HPLC analysis of cell extracts. Compared to control incubations, ARPE-19 cells incubated with lutein ND were protected from UV light-induced loss of cell viability. Consistent with this, reactive oxygen species generation, induced by exposure to UV irradiation, was lower in lutein-enriched cells than in control cells. Thus, uptake of ND-associated lutein protects ARPE-19 cells from UV light-induced damage. Taken together, the data indicate ND provide an aqueous lutein delivery vehicle for biotechnological or therapeutic applications.

Keywords: UV irradiation; lutein; macular degeneration; nanodisc; retinal pigment epithelial cells.

FIGURE 1

UV/Vis absorbance spectroscopy of lutein. Spectra were collected for (A) control EYPC rHDL (no lutein) in PBS; (B) lutein (10 μg) in ethanol; (C) lutein ND (10 μg as lutein) in PBS. Samples were scanned from 350 to 570 nm on a Spectramax M5 spectrophotometer.

FIGURE 2

FPLC gel filtration chromatography of lutein ND. A 400 μl aliquot of freshly prepared lutein ND (corresponding to 0.8 mg apoA-I and 0.4 mg lutein) was analyzed by FPLC gel filtration chromatography, with absorbance continuously monitored at 280 nm (A). Fractions corresponding to Peak 1 from Panel a were pooled, concentrated and subjected to FPLC gel filtration analysis under the same conditions (B).

FIGURE 3

Lutein uptake by cultured ARPE-19 cells. ARPE-19 cells were incubated for 72 h in media only or media supplemented with lutein ND (8.8 μM as lutein). Following incubation, cells were washed to remove unincorporated lutein and suspended in methanol containing fucoxanthin as internal standard. The samples were homogenized, filtered and lutein content analyzed by reversed phase HPLC. Values reported are the mean ± standard error (n = 3); p = p < 0.05 vs. control.

FIGURE 4

Effect of lutein ND on ARPE-19 cell viability. ARPE-19 cells were incubated for 72 h in media only, media supplemented with EYPC rHDL (no lutein), or media supplemented with indicated concentrations of lutein ND. Following incubation, cell viability assays were performed. Sample fluorescence emission was measured at 590 nm (excitation 530 nm). Values reported are the mean ± standard error (n = 6); pp = p < 0.01, pppp = p < 0.0001 vs. control.

FIGURE 5

Effect of UV irradiation on ARPE-19 cell viability. Cells were incubated for 72 h in media only, media + 0.4% DMSO, media + EYPC rHDL, media + lutein (8.8 μM) in DMSO, or media + lutein ND (8.8 μM as lutein). Following incubation, cells in indicated treatment groups were exposed to UV irradiation for 2 h. Following irradiation, cell viability was assayed and sample fluorescence emission monitored at 590 nm (excitation 530 nm). Values reported are the mean ± standard error (n = 10); **** = p < 0.0001 vs. control, ns = not significant.

FIGURE 6

Effect of lutein ND on UV irradiation-induced ROS generation in cultured ARPE-19 cells. Cells were incubated for 72 h in media only, media plus 0.4% DMSO, EYPC rHDL, lutein (8.8 μM in DMSO), and lutein ND (8.8 μM as lutein). Following incubation, cells in indicated treatment groups were exposed to UV irradiation for 2 h. Following irradiation, cellular ROS levels were determined. For assay readout, sample fluorescence emission was measured at 535 nm (excitation 485 nm). Statistical values reported are the mean ± standard error (n = 10); ** = p < 0.01 vs. control, *** = p < 0.001 vs. control, **** = p < 0.0001 vs. control. Ns = not significant.

FIGURE 7

Structure and organization of lutein ND. A lutein ND particle is depicted showing a disk-shaped phospholipid bilayer with integrated lutein molecules (yellow dots). The disk is circumscribed by apolipoprotein scaffold molecules which contact otherwise solvent exposed phospholipid fatty acyl chains at the edge of the bilayer. In the expanded view, a plausible spatial orientation of lutein, with respect to the phospholipid bilayer, is presented. In this depiction lutein aligns parallel to phospholipid fatty acyl chains, such that its two hydroxyl moieties remain solvated, each localizing near phospholipid polar head groups at the aqueous interface.