LipoFullerene has been developed by Vitamin C60 BioResearch Corporation, a pioneer in the active ingredients business, with a focus on fullerenes. LipoFullerene markedly reduced superoxide anion radical production in vitro in the mouse stromal preadipocyte and lymphoma cell co-culture system.
It also prevented cell injury and apoptosis-like cell death induced by 2,4-nonadienal. In addition, we enrolled 23 women in an eightweek- long randomised, matched-pair double-blind study on LipoFullerene-blended cream. LipoFullerene (1%)-blended cream significantly reduced the percentage of the wrinkle area as compared to the placebo cream, without any severe side effects. These results suggest that LipoFullerene can remove excess free radicals and thus prevent wrinkle formation on skin.
Exposure to ultraviolet (UV) light and ageing decreases the antioxidant level in the skin. This in turn causes excess generation of free radicals and reactive oxygen species in the skin, because of which lipid peroxides and reactive carbonyl compounds are produced.1 Many reports suggest that these reactive molecules cause not only cosmetic problems such as pigmentation, wrinkles, and erythema but also dermatological diseases such as malignant skin tumours, cutaneous vasculitis, and acne. Table 1 shows the cytological or histological changes in skin cells caused by free radicals and consequent cosmetic problems such as wrinkle formation and pigmentation. Currently, the free radical theory of ageing is among the most widely accepted theories explaining the causes of ageing. Topical administration of antioxidants is believed to be useful for the removal of the excess free radicals and reactive oxygen species from the skin tissues. The following two challenges should be considered while developing such skin formulations. First, product stability is crucial. Antioxidants are highly unstable and may be oxidised and thus inactivated before reaching the skin. Second, the antioxidants must be properly absorbed into the skin, reach the target tissue in the active form, and remain there for long enough to produce the desired effects. Although cosmetic companies have been developing novel formulations to overcome these challenges for a long time, there are only a few studies on controlled clinical trials in humans that examine the role of antioxidants in preventing or decelerating skin ageing.2
What is fullerene?
Fullerene, like diamond, is a carbon allotrope (a molecule composed of a single element), and it is also the generic chemical term for a polyhedron composed of 60 carbon atoms (C60). A C60 molecule is about 0.7 nm in diameter (1 nm = 1/1,000,000,000 m) and is structurally a truncated icosahedron resembling a soccer ball. Fullerene was accidently discovered in 1985 by Dr Kroto who was then studying interstellar molecules (trace material in space) at the University of Sussex, Great Britain, and Drs. Curl and Smalley of Rice University, US, who were studying the spectroscopy of clusters.3 All three researchers were awarded the Nobel Prize in Chemistry in 1996 for their contribution to the discovery of fullerene (C60) and the prediction of its structure. In 1991, a paper published in Science4 shocked researchers in the field of life sciences: it reported that fullerene (C60) absorbs and eliminates free radicals and detoxifies these entities. Vitamin C60 BioResearch Corporation, established in 2003, was the first company that aimed to manufacture products that effectively utilise the biological properties of fullerene. In one of our studies, “Radical Sponge”, polyvinylpyrrolidone-solubilised fullerenes (PVP/fullerenes), did not exhibit pro-oxidant activity in presence of Fe3+ or Cu2+.5 In addition, it has been reported that ascorbic acid continues to produce ascorbyl radicals even 48 hours after UV-B irradiation, and this sutained production of ascorbyl radicals can be prevented by using PVP/fullerenes. These data show that fullerenes exhibit antioxidant activity even 48 hours after application.6 Despite the low solubility of fullerenes in hydrocarbon oils such as squalane, we were successful in stably solubilising fullerenes in vegetable squalane and, thus, developed our product LipoFullerene. We conducted in vitro experiments to evaluate the skin-protective effect that LipoFullerene exerts by inhibiting free radicals and a clinical trial to determine whether LipoFullerene (now referred to as ‘the anti-ageing active’) has an anti-wrinkle effect.
In vitro experiments
Antioxidant activity of the anti-ageing active
To evaluate the potential of the anti-ageing active in reducing the levels of superoxide anion (a type of free radical), we performed a modified nitroblue tetrazolium (NBT) colorimetric assay. Superoxide anion radicals reduce NBT to dark blue, insoluble formazan. The level of the radicals can be determined by assessing the production of formazan, which is determined by measuring its absorbance at 610 nm. Co-culture of OP9 cells (mouse stromal preadipocytes) with U937 monocytes was found to induce superoxide anion production in the former. The anti-ageing active markedly decreased anion production in the OP9 cells at 1%–8% concentrations, while the vehicle (squalane) had no effect (Fig. 1).7
Prevention of cell injury by the anti-ageing active
2,4-nonadienal is one of the most toxic unsaturated aliphatic aldehydes among the secondary products of hydroperoxide autoxidation, and it induces cell injury. We measured the cell viability using the WST assay, which is based on mitochondrial function. 2,4-nonadienal was found to injure human skin keratinocyte membranes and reduce cell viability. Anti-ageing active (1%–4%) administration at five hours before 2,4-nonadienal administration preserved cell membrane integrity and cell viability, while the vehicle (squalane) had no effect (Fig. 2).8 The terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) technique was used to detect DNA fragmentation, a characteristic of apoptotic cells. 2,4-nonadienal also induced DNA fragmentation, as shown in Fig. 3b; further, anti-ageing active administration at five hours before 2,4-nonadienal administration appreciably suppressed and prevented apoptosis-like cell death (Fig. 3d).8 The results show that the anti-ageing active acts as a free-radical scavenger in skin cells and prevents skin cell damage such as mitochondrial injury and DNA fragmentation.
Controlled clinical test to evaluate the anti-wrinkle effect of the anti-ageing active
On the basis of the promising results of the in vitro tests, we evaluated whether the anti-ageing active has an anti-wrinkle effect. As shown in Fig. 4, 23 women with initial wrinkle grades of 2–3 were enrolled in an 8-week randomised matched-pair double-blind trial of the anti-ageing active (1%)-blended cream. Anti-ageing active (1%)-blended cream was applied twice daily on any one half of the face, and antiageing active-free cream was applied as the placebo on the other half. The wrinkles in the area of the lateral angle of the eyes were evaluated by skin replica analysis. For this analysis, the percentage of the wrinkle area and overall average wrinkle depth were calculated by two-dimensional analysis of the target areas on replicas of the skin surface, according to previously described procedures.9
Results
Image analysis of the replicas revealed that the anti-ageing active-blended cream improved wrinkles. The percentage of the wrinkle area was lower at four and eight weeks of anti-ageing active treatment as compared to that of 0 weeks of treatment, but it increased over time with the placebo. The anti-ageing active significantly reduced the percentage of the wrinkle area between 0 and eight weeks of treatment as compared to the placebo (P=0.021; Fig. 5). The overall average wrinkle depth was lower at four and eight weeks of the antiageing active treatment than at 0 weeks of treatment, but it increased over time with the placebo. The wrinkle depth values between 0 and eight weeks of treatment were lower trend in the halves treated with the anti-ageing active than in those treated with the placebo (P=0.078; Fig. 6). All subjects were asked to fill out questionnaires to check for problems with the use of the creams and to ascertain usage and efficacy. No serious undesirable effects were noted during the period of the trial. Any adverse events that occurred during the trial were subjective symptoms, and the doctors supervising the trial pronounced these symptoms as “mild”. For five adverse events that occurred during the trial, including two events of skin roughness, one event of oily skin, and two events of pimples, the possibility of a causal relationship between their occurrence and the treatment was considered as “not likely” or “might”. These events were mild and the nature of their expression was transient. For all other events, the possibility of a causal relationship between their occurrence and the treatment was ruled out. As such, it was confirmed that application of the anti-ageing active-blended cream did not cause any safety-related problems under this condition. The subjects were blinded to which half of their faces was treated with the antiageing active-blended cream. However, the answers to the questionnaires indicated that they perceived the effects of the antiageing active -blended cream to be six times better than those of the placebo cream. The subjects felt the following on the anti-ageing active-treated side: reduced dryness (4), decreased wrinkle formation (2), skin whitening (1), improved skin texture (1), and reduced makeup deterioration (1) (Fig. 7). They did not feel any of this on the placebo cream-treated side.
Safety of the anti-ageing active
We previously evaluated the safety of Biofullerene, the major ingredient of the anti-ageing active. We found that Biofullerene was safe for use as a cosmetic ingredient in all the tests. Then, we found that Biofullerene was safe for use as a cosmetic ingredient in all the tests, i.e. single-dose oral toxicity, primary skin irritation, cumulative skin irritation, eye irritation, skin sensitisation, skin photosensitisation, contact phototoxicity, bacterial reverse mutation, chromosomal aberration, and human patch tests, which we conducted for application as an additive in quasi-drug in Japan.10,11 In all these tests, only very mild physical effects (transient conjunctival redness and corneal epithelium defects) were observed upon the application of insoluble Biofullerene powder to rabbit eyes. Therefore, Biofullerene was considered as being “minimally irritating” in eye irritation test. We also reported that the anti-ageing active did not exhibit any significant biotoxic effects, such as photocytotoxicity and bacterial reverse mutagenesis. In addition, the anti-ageing active could permeate into the epidermis via the corneum but not the dermis as noted in the human skin biopsy specimens obtained 24 hours after administration; this suggests that fullerenes toxicity in the body would not matter when the anti-ageing active is applied to the skin’s surface.12 According to our records till October 2010, a cosmetic ingredient developed by us that contains fullerenes has been used in more than 500 cosmetic items, and all cosmetic product manufacturers are satisfied with our product. Therefore, we are confident that our products are safe.
Conclusion
LipoFullerene can prevent skin cell injury caused from free radicals and therefore may be used as an active ingredient of anti-wrinkle cosmetics. We believe that LipoFullerene diffused from the test cream into the epidermis; protected keratinocytes from UV light, lipid hydroperoxides, and reactive carbonyl compounds; and contributed to the normal differentiation and turnover of these cells, thereby reducing the existing wrinkles and preventing the appearance of new wrinkles. LipoFullerene is suitable for use in a wide range of anti-wrinkle formulations, such as creams, gels, and liquids. Although fullerenes do not exhibit pro-oxidant effects,5 it does have long-lasting antioxidant effect.6 Fullerenes can strongly protect skin from ageing, thanks to its characteristic ability, different from that of other antioxidants.
Acknowledgement
• The authors are grateful to Prof. Dr Nobuhiko Miwa (Prefectural University of Hiroshima, Laboratory of Cell-Death Control Bio Technology, Faculty of Life and Environmental Sciences) for providing professional consultation, data and analysis.
References
1 Bickers D.R., Athar M. Oxidative stress in the pathogenesis of skin disease. J Invest Dermatol 2006; 126: 2565-2575. 2 Allemann I.B., Baumann L. Antioxidants used in skin care formulations. Skin Therapy Lett 2008; 13: 5-9. 3 Kroto H.W., Heath J.R., O’Brien S.C., Curl R.F., Smalley R.E. C60: Buckminsterfullerene. Nature 1985; 318: 162-163. 4 Krusic P.J, Wasserman E., Keizer P.N., Morton PPCC J.R., Preston K.F. Radical reactions of C60. Science 1991; 254: 1183-1185. 5 Aoshima H., Yamana S., Nakamura S., Mashino T. Biological safety of water-soluble fullerenes evaluated by genotoxicity, phototoxicity studies, and pro-oxidant activity. J Toxicol Sci 2010; 35: 401-409. 6 Ito S., Itoga K., Yamato M., Akamatsu H., Okano T. The co-application effects of fullerene and ascorbic acid on UV-B irradiated mouse skin. Toxicology 2010; 267: 27-38. 7 Xiao L., Aoshima H., Saitoh Y., Miwa N. The effect of squalane-dissolved fullerene-C60 on adipogenesis-accompanied oxidative stress and macrophage activation in a preadipocytemonocyte co-culture system. Biomaterials 2010; 31: 5976-5985. 8 Kato S., Aoshima H., Saitoh Y., Miwa N. 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. J Biomed Nanotechnol 2010; 6: 52-58. 9 Task force committee for evaluation of anti-aging function, Guidelines for evaluation of anti-wrinkle products, Journal of Japanese Cosmetic Science Society 2007; 31 (Suppl): 411-431. 10 Mori T., Takada H., Ito S., Matsubayashi K., Miwa N., Sawaguchi T. Preclinical studies on safety of fullerene upon acute oral administration and evaluation for no mutagenesis. Toxicology 2006; 225: 48-54. 11 Aoshima H., Saitoh Y., Ito S., Yamana S., Miwa N. Safety evaluation of highly purified fullerenes (HPFs): based on screening of eye and skin damages. J Toxicol Sci 2009; 34 (5): 555-562. 12 Kato S., Aoshima H., Saitoh Y., Miwa N. Biological safety of LipoFullerene composed of squalane and fullerene-C60 upon mutagenesis, photocytotoxicity, and permeability into the human skin tissue. Basic Clin Pharmacol Toxicol 2009; 104: 483-487.
