NANOBERRIES FOR TOPICAL DELIVERY OF ANTIOXIDANTS 477 These results could show that although the polar compounds in the extract are mainly dissolved in the inner aqueous space of the liposomes, some molecules interact with phos- phate groups of the phospholipids (57) in lamellar phase that are oriented to the interior of these unilamellar liposomes (58), as suggested earlier. This interaction did not affect the bilayer deformability. ANTIOXIDANT CAPACITY The radical scavenging of each extract, measured as IC50 is shown in Fig. 5. It was found that Blue Crisp IC50 O’Neal IC50 Millenia IC50 Millenia extract being the one with the highest antioxidant activity. The total antioxidant activity for all extracts in concen- trations of 0.75 mg of extract per milliliter was higher than 90% (data not shown). One month later, the Millenia and O’Neal extracts retained the 100% of their radical scavenging activity whereas the %AA for the Blue Crisp extract was a 25% lower (Fig. 6). It was because of its high antioxidant capacity and high content in polyphenols and antho- cyanins that the Millenia extract was selected for being loaded in UL. The antioxidant activ- ity of radical scavenging of the UL-B was 85% of that of the same amount of free extract. CYTOTOXICITY IN HaCaT CELLS The extracts were not cytotoxic on HaCaT cells in concentrations close to their IC50. Ten- fold higher concentrations were not cytotoxic either after a 24-h incubation. Only at Figure 5. IC50 of radical scavenging for ethanolic extracts of Millenia (ME), O’Neal (OE), and Blue Crisp (BE), expressed as milligrams of extract per milliliter. Figure 6. Retention of the antioxidant activity (AA) of the ethanolic extracts of Millenia (ME), O’Neal (OE), and Blue Crisp (BE) after 1-month storage at −18°C. Values have been obtained by measuring the AA on the 31st day on aliquots with the IC50 concentration of day 1.

JOURNAL OF COSMETIC SCIENCE 478 concentrations 100-fold higher than the IC50, the cell viability was lowered under the 50% when incubated with Millenia or O’Neal extracts, as shown in Fig. 7. Cell viability was around 80% when incubated with UL-B. Similar values were obtained with empty ultradeformable matrices in the same range of lipid concentration. Free ex- tracts in the same concentration than into UL-B did not affect cell viability. These results showed that the low cytotoxic effect of UL-B could be related to the lipid matrix more than to the loaded extracts. In total, the high cell viability at working concentrations of both extracts and liposomes on a corneocytic-originated cell line is a good advice with regard to the biocompatibility of this system intended for topical application. CONCLUSIONS Nanoberries were obtained by loading ethanolic extract of blueberry into UL. UV–Vis spectroscopy showed an interaction between the anthocyanins and the phosphate groups of lipids in the internal layer of the liposomes, and this was later confi rmed by DSC. This interaction could lead to the high retention of the antioxidant activity determined in UL- B. The antioxidant activity depended on the variety of blueberries. The cell viability after incubation with UL-B was over 80%. This nanosystem for topical delivery could be an important tool for providing high levels of antioxidant activity from a natural product beyond the barrier of the SC. ACKNOWLEDGMENTS Jorge Montanari, María Morilla, and Eder Romero are members of the Carrera del Inves- tigador del CONICET (National Council of Science and Technology Research in Argen- tina), which granted this work and gave approval to the “Native vegetal species extract-based nanocosmetics” research plan. Biotechnology students María Vera and Emanuel Mensi have been granted with the fellowship Beca Bicentenario of the Ministe- rio de Educación of the Argentine government. Jorge Montanari thanks Dr. Silvia Del Valle Alonso for her invaluable help in revising this work. The authors also thank Figure 7. Cytotoxicity in HaCaT cell line after exposure to ethanolic extracts of Millenia (light gray), O’Neal (medium gray), and Blue Crisp (dark gray) in concentrations of 0.4, 4.25, and 42.5 mg of extract per milliliter UL-B suspension in two concentrations expressed in terms of the loaded ethanolic extract of Mil- lenia empty UL (at 2 mM of phospholipids, intermediate between the concentration tested for UL-B) and the untreated control.