Transmission electron micrographs of untreated human stratum corneum a and stratum corneum after 24 h of in vitro exposure to PBS b , Posintro TM c , or Posintro TM + hydration d . The specimens were fi xed in 3% ( v / v ) glutaraldehyde and 1% ( w / v ) osmium tetroxide followed by dehydration in a gradient of graded ethanol solutions and in propylene oxide with gradually increasing amounts of Epon. Ultrathin sections were collected on formvar-coated grids and counterstained with uranyl acetate and lead citrate. SG stratum granulosum, SC stratum corneum; black arrows indicate examples of the spherical-like domains. Size bar 1 μm 

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... was not signi fi cantly different from the P app in Posintro TM nanoparticles with 25% DC-cholesterol. These results show that the Posintro TM nanoparticles increase the permeability of methyl nicotinate, a small lipophilic molecule (Mw 137 Da, log P 3.1) across human skin. The increased permeability seems to be caused by the components of the Posintro TM nanoparticles equally to the nanoparticulate structure, yet the effects of the individual components seem not to be additive. The objective of this study was to investigate the effect of ISCOM nanoparticles on the penetration of model compounds into and across human skin in vitro and to investigate the mechanism by which this might occur. In the present study, the penetration depth and the penetration pathway of acridine – Posintro TM in human skin was visualized (Figs. 5 and 6). Permeation of acridine through the stratum corneum was observed following in vitro exposure of acridine – Posintro TM to the surface of human skin (Fig. 5). The penetration occurred primarily via the intercellular pathway between the corneocytes, which ties in with the fact that acridine is a very hydrophobic molecule that can be incorporated into the lipid-based nanoparticles and there- fore would be expected to distribute to the lipid-rich intercellular space (21,22). The same penetration pro fi le was also observed after incorporation of another hydrophobic fl uorophore, DiD-PE, into the Posintro TM nanoparticles. DiD-PE was observed to penetrate via the intercellular pathway through the stratum corneum into epidermis after 48 h of application (data not shown). Application of acridine in PBS with 10% mega-10 showed only minor penetration of the fl uorescent label into the stratum corneum, indicating that the Posintro TM nanoparticles or their components increased the extent of acridine permeation. To elucidate whether the penetration of acridine was dependant on the incorporation into Posintro TM nanoparticles or merely caused by coadministration, the control of choice would be to coadministrate free acridine with Posintro TM nanoparticles. However, dissolution of acridine in aqueous buffer requires addition of detergents, which would interrupt the cage-like structures of the nanoparticles and thereby change the nanoparticle characteristics. The CLSM technique can produce valuable indications on skin penetration of various kinds of particles and the mechanism of penetration. Dubey et al. previously demon- strated, using CLSM, that rhodamine red loaded in elastic liposomes was able to penetrate much deeper than the probe loaded in conventional rigid liposomes (23). Furthermore, CLSM was used to show that Quantum dots penetrate through the most super fi cial stratum corneum layers and are located near hair follicles (9,24). Despite the obvious advantages of the CLSM technique, a disadvantage of the method is that penetration of the fl uorophore only is observed and that merely hypothetical conclusions regarding the penetration depth and pathway of the particles as such can be made. Data from the CLSM studies indicated that Posintro TM nanoparticles increased the permeation of acridine through the stratum corneum. However, it could not be determined from these experiments whether Posintro TM nanoparticles penetrate as intact or disintegrated particles or whether they merely cause an increase in the permeability of the released acridine. To further elucidate the mechanism of penetration of the Posintro TM nanoparticles, TEM micrographs were recorded to evaluate the effect of cutaneous treatment with Posintro TM nanoparticles on human skin and possibly to localize intact nanoparticles in the skin. Spherical-like domains were clearly observed in the intercorneocyte space after exposure to Posintro TM nanoparticles (Fig. 8b) and not observed in the control samples. Similar expanded intercellular spaces in the stratum corneum have also been observed with topical application of various aliphatic and aromatic hydrocarbons from jet fuel to porcine skin in vivo (25) and after selective lipid extraction from the skin (26). A possible explanation for presence of the spherical-like domains is agglomeration of nanoparticles or particle remnants in clusters between the corneocytes increasing the intercorneocyte spacing locally. The increased spacing between the corneocytes caused by the Posintro TM nanoparticles could enhance the penetration of the associated or coadministered molecule via the intercellular route. The spherical-like domains might in reality also be remnants of lamellar bodies budded off from the stratum granulosum, but as the spherical-like domains are much more pronounced in the Posintro TM -exposed skin compared to the control, the nanoparticles seem to have an in fl uence on the occurrence of these domains. The occurrence of the spherical- like domains in the skin was greatly increased in hydrated tissue. The spherical-like domains were observed after 24 h of exposure (Fig. 7) on hydrated skin, but not until 72 h in the absence of hydration. The CLSM studies were performed in the absence of hydration, and thus no penetration of acridine – Posintro TM was observed after 24 h (Fig. 5a), whereas penetration was evident after 72 h of application (Fig. 5c). These CLSM results correlate with the occurrence of the spherical-like domains observed with TEM. The evidence of penetration enhancement of a hydrophobic model compound and the observed structural alterations in the skin caused by the presence of Posintro TM nanoparticles may be explained by either a partial disintegration of the particles or a collapse of the Posintro TM nanoparticulate structure upon interaction with the skin. This interaction could possibly increase the spacing between the corneocytes and make the stratum corneum barrier more permeable to the drugs coadministered or encapsulated. The observed spherical-like domains contained dark electron-dense spots (Fig. 8d). The electron-dense spots presumably consist of lipid-aggregated material originating either from the nanoparticles or the intercorneocyte lipids. Similar electron-dense spots in the lipid areas in the stratum corneum have also previously been observed by van den Bergh et al. after treatment with elastic vesicles and fi xation with osmium tetroxide (11). In that study, the spots were concluded to originate from the vesicle material. Lamellar stacks similar to the contents of lamellar bodies in the stratum granulosum were observed by van den Bergh et al. after application of elastic vesicles onto human and mouse skin (11,13). In the same studies, disorganized intercellular lipids were also observed. The lamellar stacks were found in the upper and lower part of the stratum corneum, and they were either perpendicular to the bilayers of the skin or randomly distributed. The lamellar stacks were suggested to resemble stacks of fl attened elastic vesicles, and these occur similarly to the observed spherical-like domains in the present study, except that in the present study they occur as empty spaces after osmium tetroxide post fi xation. In the studies of van den Bergh et al ., a mixture of osmium and ruthenium tetroxide was used as a fi xation agent. This procedure improves the visualization of lipids between the corneocytes and thus detects the perpendicular bilayers of lipids and disorganized intercellular lipids. A major limitation of ruthenium tetroxide, however, is its high reactivity, which causes severe overall damage of the tissue, and a poor penetration into the stratum corneum and living tissue and thus a poor contrast in TEM imaging (27). Therefore, osmium tetroxide was chosen as a fi xation agent in the present study in order to achieve a better contrast throughout the skin sample. The focus was thus on detection of the effects of the cutaneous treatment rather than localization of the nanoparticle. Osmium tetroxide is normally used as a post fi xation agent of lipids in tissue, but due to the lack of double-bond- containing lipids in the intercorneocyte space, osmium tetroxide cannot fi xate the lipid matrix between the corneocytes (28,29). Thus, con fi rmation of the presence of Posintro TM nanoparticles in the skin was carried out using ruthenium tetroxide as a fi xation agent instead of osmium tetroxide. Electron-dense inclusions similar to the Posintro TM structure were observed between the corneocytes in the upper layers of the stratum corneum after 24 h of application on hydrated skin, but only in very small amounts (data not shown). Since such inclusions were not detected in the control, this could be an indication of the penetration of a few intact nanoparticles into the upper layers of the stratum corneum. The present studies clearly indicate that the Posintro TM nanoparticles have an in fl uence on skin penetration, but no fi nal conclusion can be drawn as to whether Posintro TM nanoparticles penetrate as intact particles. This should be considered along with the ongoing discussions on whether different types of nanoparticles are able to penetrate the skin in their intact form. Schreier and Bouwstra et al. have reviewed the penetration of vesicles, liposomes, and niosomes (21,30,31) and suggested that penetration of intact vesicles in general is unlikely to occur at all. However, Cevc et al. (32) stated that a combination of fl exible vesicles (transfersomes) and nonocclusive application will enable vesicles to penetrate through the stratum corneum under the in fl uence of a “ hydration force ” . In order to evaluate the effect of the nanoparticle structure, traditional permeation cell studies were performed. The human skin permeability of a less hydrophobic model compound, methyl nicotinate, was signi fi cantly increased upon coadministration with Posintro TM nanoparticles in vitro (Fig. 9a). Methyl nicotinate was considered a relatively good model substance as it is reasonably soluble in buffer and easily detectable and ...