Nanoscale Res Lett (2010) 5:224–230 Skin Penetration of LNE Images of skin without applying any ﬂuorescent dye revealed a weak ﬂuorescent signal, which was also con- tributed to skin autoﬂuorescence. However, the autoﬂuo- rescence signal was deducted when the photograph of the skin slice (applied with NR dye) was taken. Figure 4a and b depicts representative examples of ﬂuorescence microscopy images of vertically cross-sectioned skin following topical application of the above two formulations for 0.5, 2 and 4 h. Signiﬁcantly, LNE with droplet sizes of about 68 nm clearly promoted penetration of NR into the deeper layers of skin when compared to a GE with a droplet size of about 1,742 nm. The intensity of the NR dye increased with time until 4 h. Quantitative analysis of the dye penetration was obtained from pixel intensities derived from ﬂuorescence Fig. 4 Fluorescent images of skin slices and ﬂuorescent ABU values of the dermis layer at the ﬁxed times of 0.5, 2 and 4 h after the application of the formulation with (2.5 lg/mL) NR dye. a Fluores- cent images of skin slices applied with a formulation of GE; b ﬂuorescent images of skin slices applied with formulations of LNE and c distribution of ﬂuorescent ABU values in the dermis layer (* p \ 0.01, mean ± SD, n = 3) 229 measurements of skin slices. The dermis skin ﬂuorescence measurement of NR was expressed in arbitrary unit (ABU) and is shown in Fig. 4c. A signiﬁcant difference (p \ 0.01) was detected in all the skin dermal layers between the above two formulations. At ﬁxation times of 0.5, 2 and 4 h, the ABU value of ﬂuorescence in the dermis layer that had received the formulation with LNE was 3.7-, 7.3- and 9.9- fold higher than the respective one with GE. The signiﬁcant extent of the skin penetration of the LNE formulation was most likely attributed to the fact that nanoemulsions provide superior skin hydration, which has been shown to be helpful for improving permeation effects [34]. Assuming that the average width of the transepidermal hydrophilic pathway is up to *100 nm, which is similar to the intercorneocyte space, it is conceivable that nanosized droplets are able to traverse through the interconeocyte spaces [35, 36]. An increase in the extent of penetration may result from an alteration in the barrier properties and a greater degree of partitioning of the lecithin nanoemulsion into the stratum corneum [37]. In future studies, the mechanisms of lecithin nanoemulsion penetration, cellular uptake, potential toxic- ity in skin cells and the topical delivery of lipophilic drugs should be investigated. Conclusions The LNE, composed of snake oil, soybean lecithin, glyc- erol and water, was prepared by a HPH method success- fully. Results revealed that LNE not only improved the skin hydration of formulation signiﬁcantly, but also greatly strengthened skin penetration of NR for topical application. LNE may be a promising topical delivery vehicle for lipophilic compounds. Acknowledgments The authors are grateful for FF-TEM support by Shufeng Sun from the Center for Electron Microscope, Institute of Biophysics, Chinese Academy of Sciences, China. References 1. A.C. Williams, B.W. Barry, Adv. Drug Deliv. Rev. 56, 603 (2004). doi:10.1016/j.addr.2003.10.025 2. M.R. Prausnitz, R. Langer, Nat. Biotechnol. 26, 1261 (2008). doi: 10.1038/nbt.1504 3. H.F. Zhou, Q.H. Ma, Q. Xia, N. Gu, Solid State Phenom. 121– 123, 271 (2007) 4. M.J. Lawrence, G.D. Rees, Adv. Drug Deliver. Rev. 45, 89 (2000). doi:10.1016/S0169-409X(00)00103-4 5. M. Changez, M. Varshney, Drug Dev. Ind. Pharm. 26, 507 (2000). doi:10.1081/DDC-100101261 6. H. Rosen, T. Abribat, Nat. Rev. Drug Discov. 4, 381 (2005). doi: 10.1038/nrd1721 7. M. Kreilgaard, E.J. Pedersen, J.W. Jaroszewski, J. Control. Release 69, 421 (2000). doi:10.1016/S0168-3659(00)00325-4 123