Fullerene-C60 incorporated in liposome exerts persistent hydroxyl radical-scavenging activity and cytoprotection in UVA/B-irradiated keratinocytes

The aim of this study is to examine antioxidant activity of fullerene-C60 (C60) incorporated in liposome (LpsmFlln, a diameter of 75.6 nm). LpsmFlln is water-soluble, and composed of hydrogenated lecithin of 89.7%, glycine soja sterol of 10% and C60 of 0.3%. Hydroxyl radicals (*OH), generated from UVA- or UVB-irradiated H2O2, were scavenged by LpsmFlln but not by C60-lacking Lpsm as assessed by ESR, showing that the active principle is C60 as scanty as 1/415 weight versus LpsmFlln; the *OH amount (% of non-additive control) was decreased, LpsmFlln-dose-dependently, and for 0.5% LpsmFlln (C60-eq.:16.7 microM) to 34.1% or 78.3% upon irradiation with UVA (12 J/cm2) or UVB (500 mJ/cm2), respectively, showing the superiority for UVA to UVB in terms of the *OH scavenging of LpsmFlln. Cell viability of human skin keratinocytes HaCaT decreased to 41.1% upon UVA-irradiation at 10 J/cm2, but retained to 60.6% with 0.025% LpsmFlln (C60-eq.: 0.84 microM) together with prevention of cell-morphological degeneration, in contrast to scarce effects of C60-lacking Lpsm. The scavenging activity for Fenton reaction-generated *OH, detected by DMPO/ESR, was 96.2% or 72.2% (% of no-additive control) at 1 min and decreased time-dependently to 24.8% or 28.3% at 12 min with 16.7 microM L-ascorbic acid (Asc) or Trolox, respectively, whereas 0.5% LpsmFlln (C60-eq:16.7 microM, the same concentration as for Asc) diminished *OH by 90.9% at 1 min and 91.5% even at 12 min, demonstrating the superiority of LpsmFlln to Asc or Trolox in terms of persistence of *OH-scavenging ability. Repressive efficacy on beta-carotene discoloration (% of control) for 60 min was in the order, based on the same molar or weight concentration: 1.3%:3.34 microM Asc < 25.0%:0.1% Lpsm < 36.3%:0.1% LpsmFlln (C60-eq.:3.34 microM) < 57.2%:3.34 microM Trolox, indicating the preventive effect of LpsmFlln against beta-carotene oxidation. Thus, LpsmFlln was demonstrated for an antioxidant ability characteristic of long-term persistence, and is attributed to fullerene-C60 but scarcely to Lpsm in all the tests examined, and is expected as the skin-protecting agent against oxidative stress.