J. Cosmet. Sci., 64, 469–481 (November/December 2013) 469 Nanoberries for topical delivery of antioxidants JORGE MONTANARI, MARÍA VERA, EMANUEL MENSI, MARÍA MORILLA, and EDER ROMERO, Nanomedicine Research Program, Universidad Nacional de Quilmes, B1876BXD Bernal, Pcia. de Buenos Aires, Argentina. Accepted for publication May 20, 2013. Synopsis With the aim of improving the antioxidant activity of polyphenols from blueberries (Vaccinium myrtillus) on skin targets after topical application, ethanolic extracts from three blueberry varieties (named Millenia, O’Neal, and Blue Crisp) were loaded into ultradeformable liposomes. These nanocarriers are known to be capable of penetrating through the stratum corneum reaching its deeper layers and the viable epidermis. On the other hand, blueberries contain large amounts of polyphenols, whose antioxidant properties as tissue protectors against processes mediated by reactive oxygen species have been extensively proved. Blueberries are usually consumed as edible products, but their antioxidant compounds are poorly absorbed. The antioxi- dant properties of the extracts were screened before and after being loaded into ultradeformable liposomes made of soy phosphatidylcholine and sodium cholate, of nearly 100 nm in size at 0.223 extract/lipid w/w. The ethanolic extract-loaded ultradeformable liposomes (nanoberries) from Millenia variety retained an 85% of the antioxidant capacity of the free extract and showed low cytotoxicity on HaCaT cells (less than 20%) at active concentration against free radicals. INTRODUCTION Polyphenols include a great diversity of compounds, with more than one hydroxyl group on an aromatic ring among which fl avonoids and several classes of nonfl avonoids are usu- ally distinguished (1). Polyphenols are highly reactive and also act as substrates for en- zymes such as polyphenoloxidases, peroxidases, glycosidases and esterases. They can be found in fruits (2) and plant foods (3) to which they provide organoleptic properties such as color and fl avor. In particular, the added value related to blueberry (Vaccinium myrtillus) ingestion results from its high content of fl avonoids, mainly positive-charged derivatives from anthocyani- dines named anthocyanins (4). Anthocyanins are highly soluble in polar solvents such as water, and possess antioxidant (5–7), anti-infl ammatory (8), antitumor (9), and cardio- protective (10,11) properties. Blueberries are rich in anthocyanins, with a mean total content in organic extracts of up to 6 g anthocyanins/kg of fresh fruit (12). Address all correspondence to Jorge Montanari at jmontanari@unq.edu.ar.

JOURNAL OF COSMETIC SCIENCE 470 In view of the potential carcinogenicity of synthetic antioxidants (13,14), the search for new sources of natural antioxidants has gained growing interest. Blueberries are usually con- sumed as fresh fruit or fruit juice and polyphenols undergo absorption and metabolization along the gastrointestinal tract, the liver, and the skin, to be fi nally eliminated via urine and bile (15). Compounds such as dietary fl avonoids, with relative molecular mass over 500 which can form hydrogen bond interactions are generally unable to cross biological mem- branes by passive diffusion (16) thus, having practically no chance to be passively absorbed at the gastrointestinal level. This was confi rmed by bioavailability studies showing levels lower to 1% of the administered dose (17). In addition, cell plasma membranes generally block the fl avonoid diffusion into peripheral tissues. Anthocyanins—the main fl avonoid compounds in blueberries—are rapidly absorbed in the stomach but also rapidly elimi- nated, showing poor effi ciency in general (17). To improve the availability of natural anti- oxidants at their intended sites of action appears as a crucial issue. Environmentally generated free radicals act on the skin at two levels: direct oxidation (damag- ing cell membranes, nucleic acids, and proteins) and indirectly by activating transcription factors for matrix metalloproteases including collagenases (18,19). An alternative strategy to increase the antioxidant effect of anthocyanins on the skin could be based on their topical ap- plication in suitable vehicles, as a key factor on the accurate reaction of these compounds with the damaging reactive oxygen species relies on their appropriate location (4,20). With that aim, we prepared and characterized V. myrtillus extracts in ultradeformable liposomes (UL) (nanoberries) made of soy phosphatidylcholine and sodium cholate (NaChol). The elastic modulus of UL is nearly 20-fold lower than that of conventional liposomes (21). This enables UL to penetrate the stratum corneum (SC) presumably driven by the transepithelial humidity gradient (22), to shuttle their inner aqueous con- tent within the viable epidermis (23) (several tens of micrometers of depth) instead of aggregate or coalesce on the skin surface as conventional liposomes do (24). Conventional liposomes for topical applications are mainly used as depots for sustained release on the skin surface. When they have been studied for the delivery of cosmetic actives into the skin, no evidence of vesicle penetration was found (25) even when actives could be effec- tively loaded into vesicles of different lamellarity and size (26). Nonocclusive application of UL could be a powerful tool for controlled/targeted delivery of cosmetic and/or skin- therapeutic actives (27) and it has been found that their depth of penetration beyond the SC depends on their applied amount per surface area (28). Although conventional lipo- somes fail to penetrate through the SC into the viable epidermis (29) where the antioxi- dant compounds should be delivered, UL provide a higher penetration/accumulation of hydrosoluble actives into the epidermis and potentially into the dermis, in absence of conventional permeation enhancers such as low molecular weight alcohols (30), dimethyl sulfoxide (31), or oleic acid in synergy with propylene glycol (32). Thus, although con- ventional liposomes cannot penetrate below the fi rst layers of the SC after application on the skin (23), ultradeformable vesicles have been reportedly found in channel-like pene- tration pathways across the SC (33). Several studies of UL as delivery systems for small drugs have been reported in the last years (34–36). UL matrix composition only differs from conventional liposomes in the presence of a minor fraction of a common surfactant (e.g., NaChol, Tween 20) (29) added to the main phospholipid, whereas the process for obtaining them practically does not differ from the conventional technique for obtaining large unilamellar vesicles (23). UL have been reviewed between potentially important delivery systems for both cosmetic and cosmeceuticals (37).