228 formulations of F2, F5, F8, F11 and F14 are shown in Fig. 2. From all of the ﬁve images, only the formulation with no glycerol (Fig. 1a) exhibited an irregular spherical shape, and the distribution of the droplet size was uneven with a large PI value (0.43), as conﬁrmed by the PCS. Furthermore, agglomeration of the droplet was present in Fig. 1a. When 25% glycerol was added to the aqueous phase, the sphere shape of the nanoemulsion became more regular and smaller droplets were dominant (Fig. 1b). When the concentration of glycerol increased sequentially, the formulation exhibited a standard spherical shape, and the distribution of droplet size became more homogenous. In Fig. 1e, the droplet was in a standard spherical shape in pure glycerol, and the distribution of droplet size was very homogenous, as certiﬁcated by the PCS with a 0.09 PI value. Form the above discussion, all the formulations were in a spherical shape, and the extent was affected by the glycerol concentration. As the concentration of glycerol increased, the morphology of the LNE was more spherical and the monodispersity of the LNE droplets was improved. Stability of LNE Droplet size was used as a stability index for all formula- tions stored at 25 C for 180 days (Fig. 2). Most of the formulations revealed an increase in droplet size with the increasing concentration of glycerol. However, the size change became undistinguishable when the concentration of glycerol reached 100%. The formulation (F1–F5) mean droplet size was signiﬁcantly increased after 90 and 180 days of storage. Oswald ripening, the growth in the size of droplets as the contents of one drop diffuses into another, primarily affects nanoemulsions and is the most serious instability concern for nanoemulsions. If there is Nanoscale Res Lett (2010) 5:224–230 any diffusion of the contents of the dispersed phase, large droplets grow larger at the expense of smaller droplets, and the average size of the droplet distribution will continually increase. For an o/w nanoemulsion, the rate of ripening is directly related to the water solubility of the oil. In an ideal situation with a perfectly monodispersed distribution, there should be no ripening due to the lack of differences in the solubility of droplets based on size. Thus, narrow distri- butions will be more resistant to Oswald ripening than broader distributions [21, 29, 30]. Skin Hydration of LNE The results of the skin hydration assay are shown in Fig. 3. After application, all the formulations increased the skin hydration level. However, the formulation that contained LNE improved the hydration more signiﬁcantly than the formulation without LNE, and changes in hydration levels increased with the increasing concentration of LNE from 0.5 to 10%. After 150 min, changes in skin hydration were 24.5, 31.9, 39.3, 43.6 and 72.8% for formulations A, B, C, D and E, respectively. One explanation for these effects is that maybe nanoemulsion droplets adhere to the skin and form a dense ﬁlm that inhibits water evaporation from the skin. Notably, nanoemulsions exhibit distinct adhesive properties to skin surfaces. Hence, as the water evaporated from the nanoemulsion dispersion area, a ﬁlm formed of nanoemulsion droplets stayed on the skin. The capillary forces of the nanometer pores between the nanoemulsion droplets are contractive and promote fusion and dense ﬁlm formation, which is also promoted by the application of pressure [8, 31]. The absorption of some topically applied drugs and cosmetics may be improved due to increased skin hydration [32, 33]. Fig. 2 Stability of LNE size during storage at 25 C (mean ± SD, n = 3) Fig. 3 Effect of LNE on changes in skin hydration after incorporated into o/w cream (mean ± SD, n = 3) 123