Nanoscale Res Lett (2010) 5:224–230 solubilize lipophilic drugs, nanoemulsions, which were composed of nanoscale droplets of one immiscible liquid dispersed within another, had been widely designed to deliver drugs by various routes of administration (e.g., intravenous, oral or ocular delivery) for therapeutic needs [13–17]. Few studies reported nanoemulsions were used as a topical carrier with following signiﬁcant advantages including powerful penetrability and a high drug-loading capacity [18–21]. Most of nanoemulsions consisted of synthetic surfactant, and nanoemulsions without any syn- thetic surfactant were rarely reported. In the present study, we report a LNE system without any synthetic surfactant composed of snake oil [22], soy- bean lecithin, glycerol and water. The effects of the glyc- erol to water ratio and soybean lecithin concentration on the emulsion’s physical properties (droplet size, viscosity, morphology and stability) were studied. Furthermore, the skin penetration ability and skin hydration levels of this LNE system were determined to evaluate its topical delivery potential. Materials and Methods Materials Soybean lecithin was purchased from Cargill Texturizing Solutions Deutschland GmbH & Co. KG., USA. Reﬁned snake oil and o/w cream were obtained from Jiangsu Longliqi Bioscience Co., Ltd., China. Nile red (NR) was obtained from Sigma–Aldrich, USA. 2-Propanol and opti- mal cutting temperature compound (OCT) were purchased from Leica microsystem, German. Glycerol, ethane and hexane were reagent grade. Preparation of LNE The LNE was composed of 15% (w/w) snake oil, soybean lecithin at varying concentrations of 2.5, 5 and 7.5% (w/w) and an aqueous glycerol solution with glycerol concentra- tions of 0, 25, 50, 75 and 100% (w/w) as shown in Table 1. Soybean lecithin was dissolved into the snake oil at 50 C. The oil phase was then stirred in the glycerol aqueous solution at 350 rpm and 50 C for about 10 min. The mixture was emulsiﬁed by stirrer at 1,200 rpm for 2 min. Subsequently, the pre-emulsion obtained was passed through a high pressure homogenizer (NS1001L, Niro So- avi, Italy) at a pressure of 1,000 bar for 4 cycles. Afterward, the hot LNE was cooled to room temperature. Samples were stored at 25 C for 180 days. To study their stability, samples were stored in a sealed glass bottle at 25 C and were evaluated in terms of droplet size at ﬁxed times during the ﬁrst day, 90 and 180 days after the production day. Table 1 Composition of LNE formulation Formulation Ingredients concentration (%,w/w) Snake oil Soybean lecithin Glycerol Water F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 15 2.5 5 7.5 2.5 5 7.5 2.5 5 7.5 2.5 5 7.5 2.5 5 7.5 0 0 0 20.6 20 19.4 41.25 40 38.75 61.9 60 58.1 82.5 80 77.5 225 82.5 80 77.5 61.9 60 58.1 41.25 40 38.75 20.6 20 19.4 0 0 0 Droplet Size and Polydispersity Index (PI) Droplet size and the PI of LNE were measured by photon correlation spectroscopy (PCS; Zetasize 2000, Malvern Instruments, UK). The LNE was diluted with deionized water by 50-fold. Droplet size and PI value were obtained as the average of three measurements at 25 C. Viscosity Measurement The viscosity of the LNE was measured using the small sample adapter of a Brookﬁeld rheometer (Model DV-III, Brookﬁeld Engineering Labs., Inc., Stoughton, MA, USA) at 25 C. An average of three data points was obtained to determine the viscosity at a shear rate of 7.34 s-1. Morphology Measurement Morphology of the LNE was characterized by freeze-fracture transmission electron microscopy (FF-TEM). Samples were immersed rapidly into liquid ethane cooled by liquid nitrogen. They were then transferred into liquid nitrogen after about 5 s. The samples, after being transferred into the chamber of the freeze-etching apparatus (BALZERS BAF-400D), were fractured at -120 C and 3 9 10-7 mbar. After being etched for 1 min, Pt–C was sprayed onto the fracture face at 45 , and then C was sprayed at 90 . The replicas were taken out of the chamber and placed on a copper grid mesh after washing with hexane. After processing, morphology data from the samples were detected under a transmission electron microscope (PHILIPS-FEI TECNAI20). 123