Potential of Fairy Chemicals as Functional Cosmetic Ingredients: Effect of 2-Aza-8-Oxohypoxanthine on Skin Lightness.

Among the "fairy chemicals" involved in forming the natural phenomenon of "fairy rings," we focused on 2-aza-8-oxohypoxanthine (AOH) as a candidate functional cosmetic ingredient. In previous studies, AOH was confirmed to be safe for use on human skin, and no adverse reactions were observed in any of the safety studies. In this study, we report the results of a clinical trial using a lotion containing AOH. Our analysis using the L* value for indices of skin lightness indicated that the AOH application significantly increased the L* value after 8 weeks. Since a previous DNA microarray study using normal human epidermal cells showed that AOH suppressed the expression of a group of genes that induce inflammatory cytokines (prostaglandin E synthase, prostaglandin-endoperoxide synthase 2 [cyclooxygenase-2], and interleukin-18), our results suggest that the AOH-induced suppression of inflammatory factors results in skin lightening.

Potential Suppressive Effects of Two C60 Fullerene Derivatives on Acquired Immunity.

The therapeutic effects of fullerene derivatives on many models of inflammatory disease have been demonstrated. The anti-inflammatory mechanisms of these nanoparticles remain to be elucidated, though their beneficial roles in allergy and autoimmune diseases suggest their suppressive potential in acquired immunity. Here, we evaluated the effects of C60 pyrrolidine tris-acid (C60-P) and polyhydroxylated fullerene (C60(OH)36) on the acquired immune response in vitro and in vivo. In vitro, both C60 derivatives had dose-dependent suppressive effects on T cell receptor-mediated activation of T cells and antibody production by B cells under anti-CD40/IL-4 stimulation, similar to the actions of the antioxidant N-acetylcysteine. In addition, C60-P suppressed ovalbumin-specific antibody production and ovalbumin-specific T cell responses in vivo, although T cell-independent antibodies responses were not affected by C60-P. Together, our data suggest that fullerene derivatives can suppress acquired immune responses that require T cells.

Hydroxylated fullerene: a potential antiinflammatory and antioxidant agent for preventing mouse preterm birth.

OBJECTIVE: Intrauterine infection such as by Escherichia coli and Ureaplasma spp induce placental inflammation and are one of the leading causes of preterm birth. Here we evaluated hydroxylated fullerene (C60[OH]44) for its in vitro antiinflammatory and antioxidant effects against host cellular responses to the ureaplasma toll-like receptor 2 (TLR2) ligand, UPM-1. In addition, we investigated the preventative effects of C60(OH)44 in vivo in a mouse preterm birth model that used UPM-1. STUDY DESIGN: TLR2-overexpressing cell lines and the primary cultures of mouse peritoneal macrophages were pretreated with C60(OH)44. After UPM-1 addition to the cell lines, the activation of the nuclear factor kappa-light chain-enhancer of activated B cells (NF-kappaB) signaling cascade and the production of reactive oxygen species were monitored. The levels of expression of inflammatory cytokines of interleukin (IL)-6, IL-1ß, tumor necrosis factor (TNF)-α, and the production of reactive oxygen species were quantified after stimulation with UPM-1. The in vivo preventative effects of C60(OH)44 on mice preterm birth were evaluated by analyzing the preterm birth rates and fetal survival rates in the preterm birth mouse model with placental histological analyses. RESULTS: Pretreatment with C60(OH)44 significantly suppressed UPM-1-induced NF-kappaB activation and reactive oxygen species production in TLR2-overexpressing cell lines. In the primary culture of mouse peritoneal macrophages, UPM-1-induced production of reactive oxygen species and the expression of inflammatory cytokines such as IL-6, IL-1ß, and TNF-α were significantly reduced by pretreatment with C60(OH)44. In the UPM-1-induced preterm birth mouse model, the preterm birth rate decreased from 72.7% to 18.2% after an injection of C60(OH)44. Placental examinations of the group injected with C60(OH)44 reduced the damage of the spongiotrophoblast layer and reduced infiltration of neutrophils. CONCLUSION: C60(OH)44 was effective as a preventative agent of preterm birth in mice.

Fullerene-C60 derivatives prevent UV-irradiation/ TiO2-induced cytotoxicity on keratinocytes and 3D-skin tissues through antioxidant actions.

Microcorpuscular titanium dioxide (TiO2), a useful sunscreen agent, photocatalyzes generation of reactive oxygen species (ROS). We assessed protective effects of fullerene-C60 derivatives or microcolloidal platinum (Pt) against ultraviolet ray (UV)-irradiation in the presence of TiO2 in vitro. UV-irradiation (8 J/cm2, mixed UVA and UVB) in the presence of 15 ppm TiO2 on HaCaT keratinocytes decreased cell viability as quantified by WST-1 assay, and increased both intracellular ROS and cell-membrane-lipid peroxidation, as quantified by nitroblue-tetrazolium (NBT) assay and diphenyl-1-pyrenylphosphine (DPPP) assay, respectively, whereas all of three phototoxicity-related symptoms were appreciably repressed almost to UV-unirradiational levels by pretreatment with polyvinylpyrrolidone-entrapped fullerene-C60 (C60/PVP) or fullerene-C60 dissolved in squalane (C60/Sqn) in a dose-dependent manner of C60, but scarcely by PVP alone or Sqn alone. In contrast, Pt repressed intracellular ROS generation, but did not prevent either peroxidation of cell-membrane-lipid or cell mortality. Then in the epidermis of 3-dimensional human skin tissue model, UV-irradiation in the presence of TiO2 extensively induced two symptoms such as ROS-generation around perinuclear regions and membrane-lipid peroxidation, both of which were repressed by C60/PVP or C60/Sqn, whereas Pt did not prevent membrane-lipid peroxidation adequately. Thus the advantageous application of the lipophilic antioxidant fullerene-C60 which effectively protects cell membrane against peroxidation. In conclusion, fullerene-C60 can be expected to serve as an antioxidant for scavenging of TiO2-photocatalyzed ROS in the skin surface, and therefore provide a functional improvement of TiO2-containing sunscreens.

Inhibition of sebum production and Propionibacterium acnes lipase activity by fullerenol, a novel polyhydroxylated fullerene: potential as a therapeutic reagent for acne.

Oxidative stress plays a major role in acne formation; this suggests that oxygen-radical scavengers could be potential therapeutic agents. Fullerenol C60(OH)44, a recently developed polyhydroxylated fullerene, is a spherical carbon molecule that has many hydroxyl groups capable of potent radical-scavenging activity. We have investigated its inhibitory effects in vitro on sebum production in hamster sebocytes and in Propionibacterium acnes lipase activity. Sebum production was significantly reduced by 1.5 microM of fullerenol in cells that had been irradiated with 10 mJ/cm2 UVB, although it was not altered in the non-irradiated cells, indicating that fullerene is a sebum suppressor for sebocytes under oxidative stress, such as that induced by UVB. It was also found that fullerenol has inhibitory activity against P. acnes lipase. These results suggest that fullerenol could be a beneficial skin care reagent for controlling acne vulgaris by suppressing sebum in the inflammatory response and by reducing P. acnes lipase activity.

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.

Highly hydroxylated fullerene localizes at the cytoskeleton and inhibits oxidative stress in adipocytes and a subcutaneous adipose-tissue equivalent.

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(2)O(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(2)O(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.

Silica and titanium dioxide nanoparticles cause pregnancy complications in mice.

The increasing use of nanomaterials has raised concerns about their potential risks to human health. Recent studies have shown that nanoparticles can cross the placenta barrier in pregnant mice and cause neurotoxicity in their offspring, but a more detailed understanding of the effects of nanoparticles on pregnant animals remains elusive. Here, we show that silica and titanium dioxide nanoparticles with diameters of 70 nm and 35 nm, respectively, can cause pregnancy complications when injected intravenously into pregnant mice. The silica and titanium dioxide nanoparticles were found in the placenta, fetal liver and fetal brain. Mice treated with these nanoparticles had smaller uteri and smaller fetuses than untreated controls. Fullerene molecules and larger (300 and 1,000 nm) silica particles did not induce these complications. These detrimental effects are linked to structural and functional abnormalities in the placenta on the maternal side, and are abolished when the surfaces of the silica nanoparticles are modified with carboxyl and amine groups.

Improvement of acne vulgaris by topical fullerene application: unique impact on skin care.

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.

Super-highly hydroxylated fullerene derivative protects human keratinocytes from UV-induced cell injuries together with the decreases in intracellular ROS generation and DNA damages.

Polyhydroxylated fullerenes (fullerenols: C(60)(OH)(n)) are known as the major water-soluble fullerene derivatives which possess particular significance as free radical scavengers or antioxidants in biological systems. Recently, the novel polyhydroxylated fullerene (C(60) (OH)(44)·8H(2)O: SHH-F) was successfully synthesized. In the present study, we investigated the radical-scavenging effects and cytoprotective effects of three types of fullerenols (C(60)(OH)(6-12): LH-F, C(60) (OH)(32-34)·7H(2)O: HH-F, and C(60) (OH)(44)·8H(2)O: SHH-F) on UV-irradiation-induced cell injuries. HH-F and SHH-F exerted hydroxyl-radical scavenging activities as shown by DMPO-spin trap/ESR method, more markedly than LH-F. UVA or UVB irradiation-induced injuries in human skin keratinocytes HaCaT were significantly suppressed by HH-F and SHH-F, but scarcely by LF-H. The cytoprotective effects of SHH-F had a tendency to be superior to that of HH-F. And the cytoprotective effects of SHH-F against UVB-induced injuries were more effective than those of UVA. Irradiation with UVB to HaCaT cells was shown to cause rapid increases in cell-injury-associated symptoms such as intracellular oxidative stress levels, the formation of cyclobutane pyrimidine dimers and chromatin condensation, all of which were repressed by SHH-F. Thus, UVB-induced diverse harmful effects could be prevented by SHH-F, which was suggested to exert the cytoprotective effects through intracellular reactive oxygen species-scavenging in the keratinocytes.

Clinical evaluation of fullerene-C60 dissolved in squalane for anti-wrinkle cosmetics.

Highly purified and organic solvent-free fullerene-C60 was dissolved, at nearly saturated concentration of 278 ppm, in squalane prepared from olive oil, which is designated as LipoFullerene (LF-SQ) and was examined for usage as a cosmetic ingredient with antioxidant ability. The aim of this study was to assess the anti-wrinkle formation efficacy of LF-SQ in subjects. A total of 23 Japanese women (group I: age 38.9 +/- 3.8, n = 11, group II; age 39.4 +/- 4.3, n = 12) were enrolled in an 8-week trial of LF-SQ blended cream in a randomized, matched pair double-blind study. The LF-SQ cream was applied twice daily on the right or left half of the face, and squalane blended cream (without fullerene-C60) was applied as the placebo on another half of the face. As clinical evaluations of wrinkle grades, visual observation and photographs, and silicone replicas of both crow's feet areas were taken at baseline (0 week) and at 4th and 8th weeks. Skin replicas were analyzed using an optical profilometry technique. The wrinkle and skin-surface roughness features were calculated and statistically analyzed. Subsequently, trans-epidermal water loss (TEWL), moisture levels of the stratum corneum, and visco-elasticity (suppleness: RO and elasticity: R7) were measured on cheeks by instrumental analysis. LF-SQ cream enhanced the skin moisture and the anti-wrinkle formation. LF-SQ cream that was applied on a face twice daily was not effective at 4th week, but significantly more effective than the placebo at 8th week (p < 0.05) without severe side effects. The roughness-area ratio showed significant improvement (p < 0.05) at 8th week with LF-SQ cream as compared to 0 week with LF-SQ cream, but no significant difference was detected between LF-SQ cream and the placebo. We suggest that LF-SQ could be used as an active ingredient for wrinkle-care cosmetics.

Novel polyhydroxylated fullerene suppresses intracellular oxidative stress together with repression of intracellular lipid accumulation during the differentiation of OP9 preadipocytes into adipocytes.

Along with differentiation of mouse stromal preadipocytes OP9 into adipocytes, intracellular ROS, especially superoxide anion radicals detected by NBT reduction assay, were found to appreciably increase, mainly in cytoplasmic area, parallelling with increases in intracellular lipid-droplet accumulation, whereas undifferentiated OP9 cells kept lower levels of ROS and lipid-droplets. beta-Carotene bleaching assay showed that super-highly hydroxylated fullerene (SHH-F; C(60) (OH)(44)) exerted higher antioxidant ability than highly hydroxylated fullerene (HH-F; C(60) (OH)(32-34)) or lowly hydroxylated fullerene (LH-F; C(60) (OH)(6-12)). Differentiation-dependent lipid-droplet accumulation was suppressed by SHH-F or HH-F more efficiently than LH-F. Furthermore, SHH-F significantly repressed intracellular ROS generation accompanied by adipocyte differentiation. Thus, lipid-droplet accumulation was shown to positively correlate with ROS upon the differentiation of OP9 preadipocytes into adipocytes and SHH-F significantly suppressed intracellular ROS together with repression of intracellular lipid accumulation.

Fullerene-polyvinylpyrrolidone clathrate localizes in the cytoplasm to prevent Ultraviolet-A ray-induced DNA-fragmentation and activation of the transcriptional factor NF-kappaB.

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.

Biological safety of water-soluble fullerenes evaluated using tests for genotoxicity, phototoxicity, and pro-oxidant activity.

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 Fe(2+) or Cu(2+). 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.

Defensive effects of fullerene-C60 dissolved in squalane against the 2,4-nonadienal-induced cell injury in human skin keratinocytes HaCaT and wrinkle formation in 3D-human skin tissue model.

We dissolved fullerene-C60 in squalane (LipoFullerene; LF-SQ, C60-eq.: 500 ppm) and examined its defensive effects against 2,4-nonadienal (NDA)-induced cell injury in HaCaT keratinocytes and wrinkle formation in three dimensional (3D)-human skin tissue model. NDA is an analog of 4-hydroxynonenal, one of major causes for human body odor indicative of aging and a lipophilic cell injury factor. Cell viability (% of the control) decreased to 31.6% on treatment with NDA (40 microM), but it increased to 66.0-97.5% when LF-SQ of 1-4% (C60-eq.: 5-20 ppm) was administered for 5 hr before NDA addition. The defensive effect by LF-SQ was superior to that of "squalane" alone at the same doses. NDA-induced DNA-fragmentation in HaCaT cells was suppressed by LF-SQ administered for 5 hr before NDA treatment, and LF-SQ protected HaCaT cells against apoptosis-like cell death. LF-SQ did not appreciably defend against hydrogen peroxide, though LF-SQ effectively defended against tert-butylhydroperoxide, a type of the intermediate hydrophilicity-lipophilicity degree out of other reactive oxygen species. The scanning electron microscopy demonstrated that NDA caused wrinkles and abnormal scales on keratinocytes of 3D-human skin tissue model, and structural homogeneity of the interstratum was broken, any of which were, however, markedly suppressed with LF-SQ. Squalane alone exhibited defensive effect against the skin tissue injury to some extent, but which was inferior to LF-SQ. LF-SQ might effectively capture and scavenge lipid radicals generated inside the cell membrane, because squalane acts as a lipophilic carrier of C60. C60 dissolved in squalane can be expected to serve as a cosmeceutical ingredient for anti-wrinkle formation.

The effect of squalane-dissolved fullerene-C60 on adipogenesis-accompanied oxidative stress and macrophage activation in a preadipocyte-monocyte co-culture system.

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.

Defensive effects of fullerene-C60/liposome complex against UVA-induced intracellular reactive oxygen species generation and cell death in human skin keratinocytes HaCaT, associated with intracellular uptake and extracellular excretion of fullerene-C60.

The UVA-irradiation of 10 J/cm(2) on HaCaT keratinocytes increased 59.1% of the intracellular reactive oxygen species (ROS) by NBT assay and the cell viability decreased to 31.5% by WST-1 assay, comparing to the non-irradiated control. In the presence of fullerene-C60 (C60) incorporated in phospholipid membrane vehicle (LiposomeFullerene: Lpsm-Flln) of 250-500 ppm, they were restored to -9.1% to +2.3% of the ROS and 83.0-84.8% of the cell viability, but scarcely restored by the liposome without C60 (Lpsm). In HaCaT cells administered with Lpsm-Flln (150 ppm), C60 was ingested at the intracellular concentrations of 1.4-21.9 ppm for 4-24 h, and, intracellular C60 was excreted by 80% at 4h after rinsing-out, and decreased to 2-10% after 24-48 h. C60 was predominantly distributed around the outside of nuclear membrane without deterioration of intact cell morphology according to fluorescent immunostain. Thus Lpsm-Flln is found to be an effective antioxidant that could preserve HaCaT keratinocytes against UVA-induced cellular injury. Lpsm-Flln has a potential to serve as a cosmetic material for skin protection against UVA.

Fullerene-C60/liposome complex: Defensive effects against UVA-induced damages in skin structure, nucleus and collagen type I/IV fibrils, and the permeability into human skin tissue.

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, 76nm; 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 microM (12 ppm): C60-equivalent] on human abdomen skin biopsies mounted in Franz diffusion cells, C60 permeated after 24h 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.

Safety evaluation of highly purified fullerenes (HPFs): based on screening of eye and skin damage.

The safety of highly purified fullerenes (HPFs) for utilization as antioxidants in the cosmetic industry was evaluated by studying the toxicity and effects on laboratory animals, human epidermal keratinocytes, and human fibroblasts. The HPFs did not induce primary or cumulative skin irritation, skin sensitization, skin photosensitization or contact phototoxicity. No skin reaction was observed in the patch test on human skin. In the primary eye-irritation test on rabbits, conjunctival redness and corneal epithelial defects were observed in all animals of the eye-unwashed group at 1 and 24 hr after application, but disappeared by 48 hr after application. The irritation may have been caused by administration of insoluble fullerene powder. Therefore, the HPFs were assessed as "minimally irritating" in the eye-irritation test. By comparing these results with previously published data, we concluded that HPFs can be safely used in cosmetic ingredients for human skin application. This is the first study performing all the toxicity tests on the same fullerene material for approval as an additive in quasi-drugs.

Highly hydroxylated or gamma-cyclodextrin-bicapped water-soluble derivative of fullerene: the antioxidant ability assessed by electron spin resonance method and beta-carotene bleaching assay.

Antioxidant ability of the water-soluble derivative of fullerene (C60), prepared by high-degree hydroxylation [C60-(OH)(32) x 8H(2)O] or C60/gamma-cyclodextrin (1:2 mol/mol) clathrate formation [C60/(gamma-CD)(2)], was assessed by electron spin resonance method and beta-carotene bleaching assay. These C60 derivatives have an ability to diminish a 1:2:2:1 quartet ESR spectrum attributed to hydroxyl radicals ((.)OH) as shown by DMPO-spin trap/ESR method. Meanwhile, a singlet radical-signal different from ()OH-attributed signals increased in a manner dependent on concentrations of C60-(OH)(32) x 8H(2)O. This might suggest that C60-(OH)(32) x 8H(2)O scavenges (.)OH owing to dehydrogenation of C60-(OH)(32) x 8H(2)O, and is simultaneously oxidized to a stable radical species, which may be a dehydrogenated fullerenol radical (C60-O(.)). Furthermore, these water-soluble derivatives of C60 suppressed fading of yellowish color characteristic of intact beta-carotene in beta-carotene bleaching assay. Antioxidant abilities of these derivatives were assessed as retention of yellowish color (viz absorbance at 470 nm) for 180 min. Namely, beta-carotene-attributed chromaticity (% relative absorbance at 470 nm compared with the control) after 180 min was 69% for C60-(OH)(32) x 8H(2)O (400 microM: C60-eq.), and 32% for C60/(gamma-CD)(2) (400 microM: C60-eq.), whereas it was 6% for l(+)-ascorbic acid (400 microM) which is hydrophilic, and 85% for (+/-)-alpha-tocopherol (400 microM) which is lipophilic, respectively. Thus C60-(OH)(32) x 8H(2)O and C60/(gamma-CD)(2) can scavenge (.)OH, and have a distinct antioxidative activity in the aqueous system containing linoleic acid which is abundantly contained in the cell membrane together with other unsaturated lipids. These C60 derivatives have a potential to protect the cell membrane from oxidative stress due to (.)OH.