Superresolution and Fluorescence Dynamics Evidence Reveal That Intact Liposomes Do Not Cross the Human Skin Barrier.

In this study we use the combination of super resolution optical microscopy and raster image correlation spectroscopy (RICS) to study the mechanism of action of liposomes as transdermal drug delivery systems in human skin. Two different compositions of liposomes were applied to newly excised human skin, a POPC liposome and a more flexible liposome containing the surfactant sodium cholate. Stimulated emission depletion microscopy (STED) images of intact skin and cryo-sections of skin treated with labeled liposomes were recorded displaying an optical resolution low enough to resolve the 100 nm liposomes in the skin. The images revealed that virtually none of the liposomes remained intact beneath the skin surface. RICS two color cross correlation diffusion measurements of double labeled liposomes confirmed these observations. Our results suggest that the liposomes do not act as carriers that transport their cargo directly through the skin barrier, but mainly burst and fuse with the outer lipid layers of the stratum corneum. It was also found that the flexible liposomes showed a greater delivery of the fluorophore into the stratum corneum, indicating that they functioned as chemical permeability enhancers

Cartoon depicting the sample preparation.

<p>Samples were labeled non-occlusively. Intact skin samples for imaging were mounted SC side down on a microscope cover glass for imaging. Samples for cryo sectioning were sectioned and mounted as depicted.</p

Hydrophobic Mismatch Triggering Texture Defects in Membrane Gel Domains

The orientational texture of gel-phase lipid bilayers is a phenomenon that can structure membrane domains. Using two-photon polarized fluorescence microscopy and image analysis, we map the lateral variation of the lipid orientation (the texture) in single domains. With this method, we uncover a lipid-induced transition between vortex and uniform textures in binary phospholipid bilayers. By tuning the lipid composition, the hydrophobic mismatch at the domain boundary can be varied systematically as monitored by AFM. Low hydrophobic mismatch correlates with domains having uniform texture, while higher mismatch values correlate with a vortex-type texture. The defect pattern created during early growth persists in larger domains, and a minimal model incorporating the anisotropic line tension and the vortex energy can rationalize this finding. The results suggest that the lipid composition and the domain nucleation process are critical factors that determine the texture pattern of membrane domains

RICS data.

<p>The panels show the CC-RICS data (upper graph) and the fit (lower graph), for RhB-PE and ATTO-647N-PE labeled LUVs (panels A-C) and FLUVs (panels D-F) together with an intensity image of the ATTO-647N-PE channel. A) CC-RICS and fit for LUVs at the SC surface. A clear cross correlation is seen. B and C are CC-RICS and fit for LUVs at 4μm below the SC surface. In 15% of the measurements a cross correlation was found (panel B) while most of the measurements showed no cross correlation (panel C). D) CC-RICS and fit for FLUVs at the SC surface. A clear cross correlation is seen. E and F are CC-RICS and fit for FLUVs at 4μm below the SC surface. In 9% of the measurements a cross correlation was found (panel E) while most of the measurements showed no cross correlation (panel F). The image sizes are 22 x 22μm².</p

STED images of FLUVs on intact skin.

<p>Several vesicles are visible on the surface (A) and at different depths of the skin (B-D). FLUVs are not observed deeper in the skin. In the lower layers, C and D, the FLUVs are generally located at the edge of the corneocytes. Scale bars are 5 μm.</p

Cryo frozen and sectioned skin labelled with the free dye, Atto-488-DPPE, in PBS buffer.

<p>A) A STED image of the sample in which the SC is clearly visible, as the bright u-shaped layer in the center of the image; the width of the lipid layers in the SC are measured to approximately 100–120 nm. A line scan across the marked line is shown in B, and the individual lipid layers are recognizable. C) A confocal cross section (XZ plane) through a skin section. The slice is orientated with the SC to the left. Only the upper surface of the SC, away from the cover glass, is labeled. This is where the SC was directly exposed to the label. In contrast, the SC facing the glass (lower part in the z direction) is unlabeled. This is likely due to barrier properties of the SC towards the dye. In contrast, the SG and SS/SB are labeled all the way through in the z direction. Scale bars are 5 μm.</p

Image of the surface of intact skin.

<p>The skin samples were labeled using LUVs with Atto-488-DPPE. After labeling for 6–8 hours the samples were rinsed and patted dry, removing most of the LUVs from the surface. An intense labeling of the lipids around the corneocytes is observed. A) and B) shows a STED and confocal image, respectively, of a large area where several corneocytes can be distinguished. C) and D) are enlargements of the mark regions in A and B. The resolution difference between confocal and STED can clearly be seen. E) shows an intensity line profile across the line marked in C (blue) and D (green), again it is evident that STED reveals details not resolved in the confocal image. Both the STED and confocal image have been deconvolved. Scale bars are 5 μm for A and B, and 1 μm for C and D.</p