Phase Behavior of Stratum Corneum Lipid Mixtures Based on Human Ceramides: The Role of Natural and Synthetic Ceramide 1

In a recent study the lipid phase behavior of mixtures of human ceramides, cholesterol, and free fatty acids has been examined. We observed in cholesterol: human ceramide mixtures a prominent formation of the 12.8 nm lamellar phase (referred to as the long periodicity phase). Addition of free fatty acids promoted the formation of a 5.6 nm lamellar phase (referred to as the short periodicity phase) and increased the subpopulation of lipids forming a fluid phase. In this study we focused on the role of human ceramide 1, as the presence of this ceramide appeared to be crucial for proper lipid phase behavior in mixtures prepared with ceramide isolated from pig stratum corneum. In order to do this, mixtures of cholesterol and free fatty acids were prepared with human ceramides, in which natural human ceramide 1 was replaced by either synthetic CER1-linoleate (CER1-lin), or CER1-oleate (CER1-ol), or CER1-stearate (CER1-ste). After substitution of natural human ceramide 1 by synthetic ceramide 1 the following observations were made. (i) In the presence of synthetic CER1-ste no long periodicity phase and no liquid phase could be detected. (ii) In the presence of HCER1-ol a liquid phase was more prominently formed than in the presence of HCER1-lin. (iii) In cholesterol:human ceramide mixtures in the presence of CER1-lin the long periodicity phase was more prominently present than in the presence of CER1-ol. (iv) In the presence of CER1-ste neither a long periodicity phase nor a liquid lateral packing could be detected. The results of these studies further indicate that for the formation of the long periodicity phase a certain (optimal) fraction of lipids has to form a liquid phase. When the fraction forming this liquid phase is either too low or too high, the formation of the short periodicity phase is increased at the expense of the formation of the long periodicity phase. Based on the results of this and previous studies we offer an explanation for the deviation in lipid organization in diseased and in dry skin compared to normal skin

On-Line Diffusion Profile of a Lipophilic Model Dye in Different Depths of a Hair Follicle in Human Scalp Skin

In skin and hair research, drug targeting to the hair follicle is of great interest in the treatment of skin diseases. The aim of this study is to visualize on-line the diffusion processes of a model fluorophore into the hair follicle at different depths using fresh human scalp skin and confocal laser scanning microscopy. Up to a depth of 500 μm in the skin, a fast increase of fluorescence is observed in the gap followed by accumulation of the dye in the hair cuticle. Penetration was also observed via the stratum corneum and the epidermis. Little label reached depths greater than 2000 μm. Fat cells accumulated the label fastest, followed by the cuticular area and the outer root sheath of the hair follicle. Sweat glands revealed very low staining, whereas the bulb at a depth of 4000 μm was visualized only by autofluorescence. From this study, we conclude that on-line visualization is a promising technique to access diffusion processes in deep skin layers even on a cellular level. Furthermore, we conclude that the gap and the cuticle play an important role in the initial diffusion period with the label in the cuticle originating from the gap

Stratum Corneum Lipid Composition and Structure in Cultured Skin Substitutes is Restored to Normal after Grafting onto Athymic Mice

Restoration of an epidermal barrier is a definitive requirement for wound closure. Cultured skin substitutes grafted onto athymic nude mice were used as a model for a long-term study of stratum corneum barrier lipid metabolism and organization. Samples of stratum corneum collected after 12 and 21 d in vitro and 6, 11, and 24 mo postgrafting were examined for their lipid and fatty acid composition, and their lipid organization and structure using electron microscopy and small angle X-ray diffraction, respectively. All of these methods confirm the impaired barrier function of cultured skin substitutes in vitro, as judged from the deviations in lipid composition and from poor organization of the stratum corneum lipids that show no lamellar structure. At 6 mo postgrafting, the total stratum corneum lipid profiles of the epidermal grafts is close to that of the human stratum corneum with the exception of the presence of mouse specific lipids. The increase of ceramides 4–7 in cultured skin substitutes after grafting indicates restored activity of processes involved in the hydroxylation of fatty acids and sphingoid bases. Conversely, the ceramide profile still reveals some abnormalities (elevated content of ceramide 2 and slightly lower content of ceramide 3) and the content of long-chain fatty acids remains below its physiologic level at 6 mo postgrafting, but normalizes by 2 y postgrafting. The ultramicroscopic observations revealed the formation of lamellar extracellular lipid domains by 4 mo postgrafting. Despite these findings, the X-ray diffraction showed differences in the diffraction pattern at 2 y after grafting, suggesting that the organization of stratum corneum lipids in all epidermal grafts differs from that of the native skin. Journal of Investigative Dermatology Symposium Proceedings 3:114–120, 199

Hyaluronan molecular weight: effects on dissolution time of dissolving microneedles in the skin and on immunogenicity of antigen

Biomaterials used as matrix for dissolving microneedles (dMNs) may affect the manufacturing process as well as the potency of the active pharmaceutical ingredient, e.g. the immunogenicity of incorporated vaccine antigens. The aim of this study was to investigate the effect of the molecular weight of hyaluronan, a polymer widely used in the fabrication of dMNs, ranging in molecular weight from 4.8 kDa to 1.8 MDa, on the dissolution of microneedles in the skin in time as well as the antibody response in mice and T-cell activation in vitro. Hyaluronan molecular weight (HA-MWs) did not affect antibody responses (when lower than 150 kDa) nor CD4+ T-cell responses against model antigen ovalbumin. However, the HA-MWs had an effect on the fabrication of dMNs. The 1.8 MDa HA was not suitable for the fabrication of dMNs. Similarly, the 4.8 kDa HA generated dMN arrays less robust compared to the other HA-MWs requiring optimization of the drying conditions. Finally, higher HA-MWs led to longer application time of dMN arrays for a complete dissolution of microneedles into the skin. Specifically, we identified 20 kDa HA as the optimal HA-MW for the fabrication of dMNs as with this MW the dMNs are robust and dissolve fast in the skin without affecting immunogenicity

Development of PLGA nanoparticle loaded dissolving microneedles and comparison with hollow microneedles in intradermal vaccine delivery

Skin is an attractive but also very challenging immunisation site for particulate subunit vaccines. The aim of this study was to develop hyaluronan (HA)-based dissolving microneedles (MNs) loaded with PLGA nanoparticles (NPs) co-encapsulating ovalbumin (OVA) and poly(I:C) for intradermal immunisation. The NP:HA ratio used for the preparation of dissolving MNs appeared to be critical for the quality of MNs and their dissolution in ex vivo human skin. Asymmetrical flow field-flow fractionation and dynamic light scattering were used to analyse the NPs released from the MNs in vitro. Successful release of the NPs depended on the drying conditions during MN preparation. The delivered antigen dose from dissolving MNs in mice was determined to be 1 µg OVA, in NPs or as free antigen, by using near-infrared fluorescence imaging. Finally, the immunogenicity of the NPs after administration of dissolving MNs (NP:HA weight ratio 1:4) was compared with that of hollow MN-delivered NPs in mice. Immunization with free antigen in dissolving MNs resulted in equally strong immune responses compared to delivery by hollow MNs. However, humoral and cellular immune responses evoked by NP-loaded dissolving MNs were inferior to those elicited by NPs delivered through a hollow MN. In conclusion, we identified several critical formulation parameters for the further development of NP-loaded dissolving MNs

Diffusion profile of macromolecules within and between human skin layers for (trans)dermal drug delivery

Delivering a drug into and through the skin is of interest as the skin can act as an alternative drug administration route for oral delivery. The development of new delivery methods, such as microneedles, makes it possible to not only deliver small molecules into the skin, which are able to pass the outer layer of the skin in therapeutic amounts, but also macromolecules. To provide insight into the administration of these molecules into the skin, the aim of this study was to assess the transport of macromolecules within and between its various layers. The diffusion coefficients in the epidermis and several locations in the papillary and reticular dermis were determined for fluorescein dextran of 40 and 500 kDa using a combination of fluorescent recovery after photobleaching experiments and finite element analysis. The diffusion coefficient was significantly higher for 40 kDa than 500 kDa dextran, with median values of 23 and 9 µm2/s in the dermis, respectively. The values only marginally varied within and between papillary and reticular dermis. For the 40 kDa dextran, the diffusion coefficient in the epidermis was twice as low as in the dermis layers. The adopted method may be used for other macromolecules, which are of interest for dermal and transdermal drug delivery. The knowledge about diffusion in the skin is useful to optimize (trans)dermal drug delivery systems to target specific layers or cells in the human skin

Topically Applied Ceramides Interact with the Stratum Corneum Lipid Matrix in Compromised Ex Vivo Skin

Drug Delivery Technolog