226 Skin Hydration Test The skin hydration effect of LNE was investigated in a blind, placebo-controlled in vivo study. LNE was stirred into o/w cream at 200 rpm evenly at 40 C. Sample 1 was o/w cream without LNE and samples 2, 3, 4 and 5 were o/w cream enriched with 0.5, 2, 5 and 10% LNE (F12, shown in Table 1), respectively. Fifteen volunteers applied the samples on their healthy volar forearm skin. The skin hydration test was conducted at 20 ± 2 C room temper- ature and 50 ± 5% relative humidity. Samples were spread on a 3.14 cm2 area of volar forearm skin with an amount equaling 1.5 lL/cm2. After application, skin hydration was measured at ﬁxed time intervals of 30, 60, 90, 120 and 150 min with a Corneometer CM 825(CK Electronic GmH, Germany). The percent change in skin hydration was calculated with the following equation: D% = (Qt - Q0)/ Q0 9 100. Qt was the hydration value after application time t, and Q0 was the hydration value before application. Skin Penetration Test Experiments were conducted on female Wistar SD rats (180–200 g body weight). The rats were 10 weeks old and were of a similar development stage. They were anesthe- tized by a suitable dose of sodium barbital. The fur on the abdominal area of the rats was carefully removed by an electrical shaver to avoid damage to their stratum corneum. The furless abdominal area was used for in vivo perme- ation studies. In this study, NR, a ﬂuorescent dye, was dissolved into the oil phase during the LNE preparation procedure. The following two samples were used in a blind, placebo-con- trolled in vivo study: one sample was the o/w cream incorporated with NR-loaded LNE (F12, shown in Table 1); the other was the o/w cream incorporated with NR-loaded GE (F12 without HPH). The NR concentration in the o/w cream was 2.5 lg/mL, and the NR-loaded GE or NR-loaded LNE were stirred into the o/w cream at 200 rpm evenly at 40 C. For each set of experiments, 50 mg of sample was applied to the hairless abdominal skin area of *3.14 cm2. At ﬁxed times of 0.5, 2 and 4 h after application, the surplus sample was removed from the skin surface, the skin surface was washed three times with PBS and dried gently under cold air with an electric hair- drier. A 0.5 cm 9 0.5-cm skin piece, which was cut out from the treated area, was embedded in optimal cutting temperature compound and frozen rapidly by liquid nitro- gen. The specimen was removed from liquid nitrogen and was frozen on a metal block. The metal block was then transferred to a cryostat microtome (LE ICACM 1850, Germany) for slicing through vertical cross-sections of skin. Twelve vertical skin sections with a thickness of 123 Nanoscale Res Lett (2010) 5:224–230 25 lm were obtained and stored at 4 C until analyzed microscopically. Skin sections were subjected to ﬂuorescent microscopy using an Olympus CK40 microscope (Olympus, Japan) equipped with a UV source and ﬁlter for ﬂuorescent measurements. Image capture and analysis were carried out by the Image-pro Plus program (Media Cybernetics, USA). The excitation and emission wavelengths were 543 nm and 604 nm for NR, respectively. Images were recorded with a camera integration time of 1/1.8 s, and the same parame- ters were used for the imaging of all samples. The ﬂuo- rescence intensity value was quantiﬁed using Image-pro Plus program. Data Analysis All the data tests were repeated three times and expressed as the mean ± SD. The statistical data were analyzed by a t-test analysis via Origin 7.0. Results and Discussion Particle Size and Viscosity of LNE The mean droplet size of F1-F9 was in the range of 310– 100 nm with a PI value ranging from 0.6 to 0.2, while the mean droplet size of F10-F15 was in the range of 100– 50 nm with a PI value from 0.2 to 0.1 (shown in Table 2). Table 2 Droplet size, polydispersity index (PI) and viscosity of LNE (mean ± SD, n = 3) Formulation F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F12 (without HPH) Droplet size (nm) 309 ± 11 282 ± 9 240 ± 4 238 ± 6 212 ± 4 182 ± 5 163 ± 4 145 ± 2 113 ± 3 92 ± 4 79 ± 2 68 ± 2 71 ± 3 58 ± 2 75 ± 4 1,742 ± 102 PI Viscosity (mPa.s) 0.58 ± 0.09 2.3 ± 0.1 0.43 ± 0.06 5.2 ± 0.4 0.32 ± 0.05 12.1 ± 0.3 0.28 ± 0.04 11.6 ± 0.7 0.26 ± 0.03 15.2 ± 0.5 0.24 ± 0.03 32.3 ± 1.9 0.23 ± 0.04 24.1 ± 1.6 0.25 ± 0.03 43.6 ± 0.8 0.21 ± 0.02 71.2 ± 2.1 0.17 ± 0.03 68.4 ± 3.2 0.15 ± 0.04 83.2 ± 1.9 0.13 ± 0.02 136.7 ± 5.2 0.11 ± 0.01 565.7 ± 13.1 0.09 ± 0.03 989.6 ± 15.2 0.12 ± 0.05 1,574.8 ± 12.5 1.34 ± 0.21 375.2 ± 4.7