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Kato S.,Prefectural University of Hiroshima | Aoshima H.,Vitamin C60 BioResearch Corporation | Saitoh Y.,Prefectural University of Hiroshima | Miwa N.,Prefectural University of Hiroshima
Journal of Photochemistry and Photobiology B: Biology | Year: 2010

We previously reported biological safety of fullerene-C60 (C60) incorporated in liposome consisting of hydrogenated lecithin and glycine soja sterol, as Liposome-Fullerene (0.5% aqueous phase; a particle size, 76 nm; Lpsm-Flln), and its cytoprotective activity against UVA. In the present study, Lpsm-Flln was administered on the surface of three-dimensional human skin tissue model, rinsed out before each UVA-irradiation at 4 J/cm 2, and thereafter added again, followed by 19-cycle-repetition for 4 days (sum: 76 J/cm 2). UVA-caused corneum scaling and disruption of epidermis layer were detected by scanning electron microscopy. Breakdown of collagen type I/IV, DNA strand cleavage and pycnosis/karyorrhexis were observed in vertical cross-sections of UVA-irradiated skin models visualized with fluorescent immunostain or Hoechst 33342 stain. These skin damages were scarcely repressed by liposome alone, but appreciably repressed by Lpsm-Flln of 250 ppm, containing 0.75 ppm of C60-equivalent to a 1/3300-weight amount vs. the whole liposome. Upon administration with Lpsm-Flln [16.7 μM (12 ppm): C60-equivalent] on human abdomen skin biopsies mounted in Franz diffusion cells, C60 permeated after 24 h into the epidermis at 1.86 nmol/g tissue (1.34 ppm), corresponding to 0.3% of the applied amount and a 9.0-fold dilution rate, but C60 was not detected in the dermis by HPLC, suggesting no necessity for considering a toxicity of C60 due to systemic circulation via dermal veins. Thus Lpsm-Flln has a potential to be safely utilized as a cosmetic anti-oxidative ingredient for UVA-protection. © 2009 Elsevier B.V. All rights reserved.

Xiao L.,Prefectural University of Hiroshima | Aoshima H.,Vitamin C60 BioResearch Corporation | Saitoh Y.,Prefectural University of Hiroshima | Miwa N.,Prefectural University of Hiroshima
Journal of Cellular Biochemistry | Year: 2010

By Western blot and immunostaining we proved that polyvinylpyrrolidone (PVP)-wrapped fullerene molecules (PVP-fullerene) could combine the 8- and 53-kb proteins which localize in the membrane of human skin keratinocytes HaCaT. Only fullerene molecules are able to cross the lipid membrane and conjugate 53-kb proteins in the cytosol. There are no fullerene molecules detectable in the nucleus or cytoskeleton. Ultraviolet-A (UVA)-irradiation on HaCaT or normal human epidermal melanocytes (NHEM) caused nuclear fragmentations, lowering of intracellular DNA-contents below diploidy, concurrently with the repressed DNA synthesis and the increased DNA-3'OH cleavage terminals, all of which were repressed by PVP-fullerene, as shown by flow cytometry and PI- or TUNEL-stain fluorography. Translocation of the transcriptional factor NF-kappaB in the cytoplasm to the nucleus of the keratinocytes was caused with UVA and repressed by PVP-fullerene with cytoprotective effects. Thus, the PVP-fullerene may be developed as a UV-protective agent with DNA-preservative effects owing to its combinative ability to molecules in the cytoplasm and cytomembrane, and then represses cellular oxidative stress and blocks abnormal signal pathways. © 2010 Wiley-Liss, Inc.

Xiao L.,Prefectural University of Hiroshima | Aoshima H.,Vitamin C60 BioResearch Corporation | Saitoh Y.,Prefectural University of Hiroshima | Miwa N.,Prefectural University of Hiroshima
Biomaterials | Year: 2010

Effects of squalane-dissolved fullerene-C60 (Sql-fullerene) on macrophage activation and adipose conversion with oxidative stress were studied using an inflammatory adipose-tissue equivalent (ATE) and OP9 mouse stromal preadipocyte-U937 lymphoma cell co-culture systems. Differentiation of OP9 cells was initiated by insulin-rich serum replacement (SR) as an adipogenic stimulant, and then followed by accumulation of intracellular lipid droplets and reactive oxygen species (ROS), both of which were significantly inhibited by Sql-fullerene. In the OP9-U937 cell co-culture system, U937 cells rapidly differentiated to macrophage-like cells during SR-induced adipogenesis in OP9 cells. The ROS accumulation was in the co-culture more marked than in OP9 cells alone, suggesting that the interaction between adipocytes and monocytes/macrophages promotes inflammatory responses. Sql-fullerene significantly inhibited macrophage activation and low-grade adipogenesis in the OP9-U937 co-culture system. We developed a three-dimensional inflammatory adipose-tissue model " ATE" consisting of, characteristically, U937 cells in the culture-wells, and, in addition, mounted a culture insert containing OP9 cells-populated collagen gel. ATE is enabled with suitable stimulation to represent the pathology of inflammatory disorders, such as macrophage infiltration in adipose tissue. Five-day culturing of ATE in SR medium occurred U937 macrophage migration and intracellular oil-droplet accumulation that were significantly inhibited by Sql-fullerene. Our results suggest that Sql-fullerene might be explored as a potential medicine for the treatment of metabolic syndrome or other obesity-related disorders. © 2010 Elsevier Ltd.

Xiao L.,Prefectural University of Hiroshima | Xiao L.,The Nippon Dental University | Aoshima H.,Vitamin C60 BioResearch Corporation | Saitoh Y.,Prefectural University of Hiroshima | Miwa N.,Prefectural University of Hiroshima
Free Radical Biology and Medicine | Year: 2011

Adipose tissue is a crucial site for pathologic changes in obesity/metabolic syndrome-related diseases. Interaction between adipogenesis and reactive oxygen species (ROS) in adipose tissue involving chronic low-grade inflammation is postulated to be causal in the development of insulin resistance and other metabolic consequences. We used different culture systems to investigate the relationship between ROS and adipogenesis at three levels: within adipocytes, during adipocyte-monocyte interactions, and in a subcutaneous adipose tissue model. The effects of highly hydroxylated fullerene (HHF; C 60(OH) 36) on adipogenesis-accompanying oxidative stress and inflammatory changes were examined using these three systems. We demonstrated that H 2O 2 stimulates lipid accumulation in 3T3-L1 preadipocytes, and lipid uptake causes ROS generation in OP9 preadipocytes, both of which were then markedly suppressed with HHF treatment. HHF significantly inhibited the adipogenic stimulant insulin-rich serum replacement (SR)-induced triacylglycerol accumulation, ROS production, and macrophage activation in cultured OP9 cells and an OP9-U937 monocyte-like cell coculture system. H 2O 2-induced intracellular ROS production in OP9 adipocytes was also notably inhibited by HHF. We developed a three-dimensional subcutaneous adipose-tissue equivalent (SATE) consisting of air-exposed cultures of HaCaT keratinocytes on an OP9 adipocyte-populated collagen gel in a culture insert. With SR stimulation and under suitable conditions, fat accumulation, ROS generation, and macrophage infiltration were observed in the SATE and significantly inhibited by HHF. By western blotting, we demonstrated that HHF localized at the cytoskeleton, which controls the transport of lipids. In conclusion, HHF is able to inhibit oxidative stress in adipocytes and adipogenesis-related macrophage activation in adipose tissues through its antioxidation. © 2011 Elsevier Inc. All rights reserved.

Kato S.,Prefectural University of Hiroshima | Aoshima H.,Vitamin C60 BioResearch Corporation | Saitoh Y.,Prefectural University of Hiroshima | Miwa N.,Prefectural University of Hiroshima
Journal of Nanoscience and Nanotechnology | Year: 2011

The aim of this study is to examine antioxidant activity of fullerene-C 60 (C 60) 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 C 60 of 0.3%. Hydroxyl radicals (·OH), generated from UVA-or UVB-irradiated H 2O 2, were scavenged by LpsmFlln but not by C 60-lacking Lpsm as assessed by ESR, showing that the active principle is C 60 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 (C 60-eq.:16.7 μM) to 34.1% or 78.3% upon irradiation with UVA (12 J/cm 2) or UVB (500 mJ/cm 2), 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/cm 2, but retained to 60.6% with 0.025% LpsmFlln (C 60-eq.: 0.84 μM) together with prevention of cell-morphological degeneration, in contrast to scarce effects of C 60-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 μM L-ascorbic acid (Asc) or Trolox, respectively, whereas 0.5% Lpsm-Flln (C 60-eq:16.7 μM, 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 β-carotene discoloration (% of control) for 60 min was in the order, based on the same molar or weight concentration: 1.3%:3.34 μM Asc < 25.0%:0.1% Lpsm < 36.3%:0.1% LpsmFlln (C 60-eq.:3.34 μM) < 57.2%:3.34 μM Trolox, indicating the preventive effect of LpsmFlln against β-carotene oxidation. Thus, LpsmFlln was demonstrated for an antioxidant ability characteristic of long-term persistence, and is attributed to fullerene-C 60 but scarcely to Lpsm in all the tests examined, and is expected as the skin-protecting agent against oxidative stress. Copyright © 2011 American Scientific Publishers All rights reserved.

Aoshima H.,Vitamin C60 BioResearch Corporation | Yamana S.,Vitamin C60 BioResearch Corporation | Nakamura S.,Keio University | Mashino T.,Keio University
Journal of Toxicological Sciences | Year: 2010

We evaluated the safety of water-soluble polymer-enwrapped fullerenes (PVP/fullerenes) as antioxidants in cosmetic and pharmaceutical preparations by studying the genotoxicity, phototoxicity, and pro-oxidant effects of these fullerenes. These materials were not mutagenic to any of the tested bacterial strains and did not induce chromosomal aberrations in cultured mammalian cells. The PVP/fullerenes did not exhibit cytotoxicity under ultraviolet or sham irradiation in the alternative phototoxicity test. Moreover, they did not show any pro-oxidant effect in the presence of Fe2+ or Cu2+. Thus, we concluded that PVP/fullerenes are safe for use in cosmetic and pharmaceutical applications. This is the first study in which toxicity tests were performed on PVP/fullerenes.

Kokubo K.,Osaka University | Shirakawa S.,Osaka University | Kobayashi N.,Osaka University | Aoshima H.,Vitamin C60 BioResearch Corporation | Oshima T.,Osaka University
Nano Research | Year: 2011

A water-soluble polyhydroxylated fullerene, i. e. a fullerenol, with 44 hydroxyl groups and 8 secondary bound water molecules, C60(OH)44·8H2O (estimated average structure), has been synthesized in a facile one step reaction from pristine C60 by hydroxylation with hydrogen peroxide in the presence of a phase-transfer catalyst, tetra-n-butylammonium hydroxide (TBAH), under organic/aqueous bilayer conditions. The fullerenol exhibited high water solubility, up to 64.9 mg/mL, under neutral (pH = 7) conditions. Dynamic light-scattering (DLS) analysis showed a narrow particle size distribution, of 1-2 nm, indicating that the fullerenol had high dispersion properties in water. The results of particle size analyses, which both focused on a single nanoregion and were conducted using a novel induced grating (IG) method and a scanning probe microscope (SPM), were consistent with the DLS results. A plausible reaction mechanism, which includes fullerene oxide intermediates detected by liquid chromatography-mass spectrometry (LC-MS), has been proposed. © 2010 Tsinghua University Press and Springer-Verlag Berlin Heidelberg.

Inui S.,Osaka University | Aoshima H.,Vitamin C60 BioResearch Corporation | Nishiyama A.,Osaka University | Itami S.,Osaka University
Nanomedicine: Nanotechnology, Biology, and Medicine | Year: 2011

Oxidative stress plays a major role in acne formation, suggesting that oxygen radical scavengers are potential therapeutic agents. Fullerene is a spherical carbon molecule with strong radical sponge activity; therefore, we studied the effectiveness of fullerene gel in treating acne vulgaris. We performed an open trial using a fullerene gel twice a day; at 4 and 8 weeks, the mean number of inflammatory lesions (erythematous papules and pustules) significantly (P < 0.05) decreased from 16.09 ± 9.08 to 12.36 ± 7.03 (reduction rate 23.2%) and 10.0 ± 5.62 (reduction rate 37.8%), respectively. The number of pustules, consisting of accumulation of neutrophils, was significantly (P < 0.05) decreased from 1.45 ± 1.13 to 0.18 ± 0.60 (reduction rate 87.6%), and further in vitro assays of sebum production in hamster sebocytes revealed that 75 μM polyvinylpyrrolidone-fullerene inhibits sebum production, suggesting that fullerene suppresses acne through decreasing neutrophil infiltration and sebum production. After treatment for 8 weeks, the water content of the skin significantly (P < 0.05) increased from 51.7 ± 7.9 to 60.4 ± 10.3 instrumental units. Therefore, the fullerene gel may help in controlling acne vulgaris with skin care benefit. From the Clinical Editor: Fullerenes, spherical carbon cages with strong oxygen radical scavenging, with formulated into a gel and used to successfully treat acne vulgaris, an inflammatory disease associated oxidative stress. © 2011 Elsevier Inc.

Vitamin C60 BioResearch Corporation | Date: 2016-02-12

Chemicals for use in the manufacture of skin care, facial care and make-up products.

Vitamin C60 BioResearch Corporation | Date: 2016-07-05

Chemicals for use in the manufacture of skin, facial, and body care products and make-up products.

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