Electron diffraction provides new information on human stratum corneum lipid organization studied in relation to depth and temperature.

The outermost layer of mammalian skin, the stratum corneum, provides the body with a barrier against transepidermal water loss and penetration of agents from outside. The lipid-rich extracellular matrix surrounding the corneocytes in the stratum corneum is mainly responsible for this barrier function. In this study (cryo-) electron diffraction was applied to obtain information about the local lateral lipid organization in the extracellular matrix in relation to depth in human stratum corneum. For this purpose, stratum corneum grid-strips were prepared from native skin in vivo and ex vivo. It was found that the lipid packing in samples prepared at room temperature is predominantly orthorhombic. In samples prepared at 32 degrees C the presence of a hexagonal packing is more pronounced in the outer layers of the stratum corneum. Gradually increasing the specimen temperature from 30 to 40 degrees C induced a further transition from an orthorhombic to a hexagonal sublattice. At 90 degrees C all lipids were present in a fluid phase. These results are in good agreement with previously reported wide angle X-ray diffraction and Fourier transformed infrared spectroscopy studies. We conclude that the lipids in human stratum corneum are highly ordered throughout the stratum corneum and that electron diffraction allows monitoring of the local lipid organization, which contributes to the understanding of stratum corneum barrier function.     

Antigen-adjuvant nanoconjugates for nasal vaccination: an improvement over the use of nanoparticles?

Entrapment of antigens in mucoadhesive nanoparticles prepared from N-trimethyl chitosan (TMC) has been shown to increase their immunogenicity. However, because of their large size compared to soluble antigens, particles poorly diffuse through the nasal epithelium. The aim of this work was to study whether nasal vaccination with a much smaller TMC-antigen nanoconjugate would result in higher antibody responses as compared to TMC nanoparticles. TMC was covalently linked to a model antigen, ovalbumin (OVA), using thiol chemistry. For comparison, TMC/OVA nanoparticles and solutions of OVA and a physical mixture of TMC and OVA were made. As shown previously for TMC/OVA nanoparticles, TMC-OVA conjugate prolonged the nasal residence time of the antigen. TMC-OVA conjugate diffused significantly better through a monolayer of lung carcinoma (Calu-3) cells than TMC/OVA nanoparticles did. Moreover, nasal immunization of mice with the conjugate resulted in significantly more OVA positive DCs in the cervical lymph nodes as compared to TMC/OVA nanoparticles. Mice nasally immunized with TMC-OVA conjugate produced high levels of secretory IgA in nasal washes and higher titers of OVA-specific IgG than mice immunized with TMC/OVA nanoparticles after a priming dose. Moreover, as compared to TMC/OVA nanoparticles, TMC-OVA conjugate induced a more balanced IgG1/IgG2a response. In conclusion, the TMC-antigen nanoconjugate improves nasal delivery and immunogenicity of the antigen. This suggests that efficient codelivery of antigen and adjuvant to DCs, rather than a particulate form of the antigen/adjuvant combination, is decisive for the immunogenicity of the antigen.     

Microneedle-Based Transcutaneous Immunisation in Mice with N-Trimethyl Chitosan Adjuvanted Diphtheria Toxoid Formulations

Purpose  

The purpose of this study was to gain insight into the delivery and immunogenicity of N-trimethyl chitosan (TMC) adjuvanted diphtheria toxoid (DT) formulations applied transcutaneously with microneedles.     

Interaction of lipophilic moisturizers on stratum corneum lipid domains in vitro and in vivo

Dry skin symptoms such as scaling and itching are often treated with lipophilic moisturizers. The aim of this study was to investigate the effect of lipophilic moisturizers on the stratum corneum (SC) ultra-structure and lipid organization. Lipophilic moisturizers were applied on the forearms of 4 healthy volunteers for 3 h. Subsequently, the application sites were tape stripped, and selected tape strips prepared for Freeze Fracture Electron Microscopy (FFEM), a method to visualize the SC intercellular lipid parallel to the skin surface. To investigate the effect of lipid moisturizers on the lipid lamellae, isolated SC was pretreated with the lipophilic moisturizers for 24 h prior to performing small angle X-ray diffraction (SAXD) measurements. Additionally, the lipid organization of mixtures prepared with ceramides, cholesterol, free fatty acids and lipophilic moisturizer in a 2:1:1:1 molar ratio were studied using SAXD. The FFEM data (in vivo) as well as the SAXD data (in vitro) show that the lipophilic moisturizers do not change the lipid lamellar organization in the SC. Addition of 20% m/m lipophilic moisturizer to the ceramide:cholesterol:free fatty acids mixture did not inhibit the formation of the long periodicity phase, the characteristic lamellar phase in the SC, even though there was clear evidence that two of the three moisturizers were at least partially incorporated in the long periodicity phase. Concluding, all findings suggest that the lipophilic moisturizers investigated in this study do not drastically change the lamellar organization of the SC intracellular lipid matrix, but that the moisturizers form separate domains in the SC, as was visualized by FFEM.     

Genetic analysis identifies potential transmission of low pathogenic avian influenza viruses between poultry farms

Poultry can become infected with low pathogenic avian influenza (LPAI) viruses via (in)direct contact with infected wild birds or by transmission of the virus between farms. This study combines routinely collected surveillance data with genetic analysis to assess the contribution of between‐farm transmission to the overall incidence of LPAI virus infections in poultry. Over a 10‐year surveillance period, we identified 35 potential cases of between‐farm transmission in the Netherlands, of which 10 formed geographical clusters. A total of 21 LPAI viruses were isolated from nine potential between‐farm transmission cases, which were further studied by genetic and epidemiological analysis. Whole genome sequence analysis identified close genetic links between infected farms in seven cases. The presence of identical deletions in the neuraminidase stalk region and minority variants provided additional indications of between‐farm transmission. Spatiotemporal analysis demonstrated that genetically closely related viruses were detected within a median time interval of 8 days, and the median distance between the infected farms was significantly shorter compared to farms infected with genetically distinct viruses (6.3 versus 69.0 km; p < 0.05). The results further suggest that between‐farm transmission was not restricted to holdings of the same poultry type and not related to the housing system. Although separate introductions from the wild bird reservoir cannot be excluded, our study indicates that between‐farm transmission occurred in seven of nine virologically analysed cases. Based on these findings, it is likely that between‐farm transmission contributes considerably to the incidence of LPAI virus infections in poultry.     

In vivo assessment of safety of microneedle arrays in human skin

Microneedle arrays are promising devices for the delivery of drugs and vaccines into or the skin. However, little is known about the safety of the microneedles. In this study we obtained insight in the ability of microneedles to disrupt the skin barrier, which was evaluated by transepidermal water loss (TEWL). We also determined the safety in terms of skin irritation (skin redness and blood flow) and pain sensation. We applied microneedle arrays varying in length and shape on the ventral forearms of 18 human volunteers. An effect of needle length was observed, as TEWL and redness values after treatment with solid microneedle arrays of 400mum were significantly increased compared to 200mum. The blood flow showed a similar trend. Needle design also had an effect. Assembled microneedle arrays induced higher TEWL values than the solid microneedle arrays, while resulting in less skin irritation. However, for all microneedles the irritation was minimal and lasted less than 2h. In conclusion, the microneedle arrays used in this study are able to overcome the barrier function of the skin in human volunteers, are painless and cause only minimal irritation. This opens the opportunity for dermal and transdermal delivery of drugs and vaccines.     

New aspects of the skin barrier organization

In the superficial layer of the skin, the stratum corneum (SC), the lipids form two crystalline lamellar phases with periodicities of 6.4 and 13.4 nm (long-periodicity phase). The main lipid classes in SC are ceramides, free fatty acids and cholesterol. Studies with mixtures prepared with isolated ceramides revealed that cholesterol and ceramides are very important for the formation of the lamellar phases, and the presence of ceramide 1 is crucial for the formation of the long-periodicity phase. This observation and the broad-narrow-broad sequence of lipid layers in the 13.4-nm phase led us to propose a molecular model for this phase. This consists of one narrow central lipid layer with fluid domains on both sides of a broad layer with a crystalline structure. This model is referred to as 'the sandwich model'. While the presence of free fatty acids does not substantially affect the lipid lamellar organization, it is crucial for the formation of the orthorhombic sublattice, since the addition of free fatty acids to cholesterol/ceramide mixtures results in transition from a hexagonal to a crystalline lipid phase. Studies examining lipid organization in SC derived from dry or lamellar X-linked ichthyosis skin revealed that in native tissue the role of ceramide 1 and free fatty acids is similar to that observed with mixtures prepared with isolated SC lipids. From this we conclude that the results obtained with lipid mixtures can be used to predict the SC lipid organization in native tissue.     

Transdermal Iontophoresis of Rotigotine Across Human Stratum Corneum in Vitro: Influence of pH and NaCl Concentration

PURPOSE: The aim of this study was to characterize the influence of pH and NaCl concentration on the transdermal iontophoretic transport of the dopamine receptor agonist rotigotine across human stratum corneum (HSC). METHODS: Rotigotine transport was studied in vitro in side by side diffusion cells according to the following protocol: 6 h of passive diffusion, 9 h of iontophoresis, and 5 h of passive diffusion. A current density of 0.5 mA cm(-2) was used. The influence of donor phase pH (4, 5, and 6) and different concentrations of NaCl (0.07 and 0.14 M) on rotigotine iontophoretic flux were examined. The acceptor phase was phosphate-buffered saline (PBS) at pH 7.4 except in one series of experiments aimed to study the effects of rotigotine solubility on its iontophoretic transport. In this study, PBS at pH 6.2 was used. In separate studies. 14C-mannitol was used as a marker to determine the role of electro-osmosis during iontophoresis. RESULTS: The estimated iontophoretic steady-state flux (Flux(ss)) of rotigotine was influenced by the pH of the donor solution. At a drug donor concentration of 0.5 mg ml(-1), the iontophoretic flux was 30.0 +/- 4.2 nmol cm(-2) h(-1) at pH 6 vs. 22.7 +/- 5.5 nmol cm(-2) h(-1) at pH 5. However, when the donor concentration was increased to 1.4 mg ml(-1), no significant difference in iontophoretic rotigotine transport was observed between pH 5 and 6. Increase of NaCl concentration from 0.07 M to 0.14 M resulted in a decrease of the rotigotine Flux(ss) from 22.7 +/- 5.5 nmol cm(-2) h(-1) to 14.1 +/- 4.9 nmol cm(-2) h(-1). The contribution of electro-osmosis was estimated less than 17%. Probably due to the lipophilic character of the drug, impeding the partitioning of rotigotine from HSC to the acceptor compartment, steady-state transport was not achieved during 9 h of iontophoresis. CONCLUSIONS: Both pH and NaCl concentration of the donor phase are crucial on the iontophoretic transport of rotigotine. Electro-repulsion is the main mechanism of the iontophoretic transport of rotigotine.     

On-Line Visualization of Dye Diffusion in Fresh Unfixed Human Skin

PURPOSE: The purpose of the current study was to develop a new method to examine the diffusion in fresh unfixed human skin on-line. METHODS: Full thickness skin samples were cut perpendicular to the skin surface (cutting plane facing upwards) with a new cutting device forming part of the final diffusion cell. The donor solution contained 0.1 mg/ml Bodipy FL C5 (moderately lipophilic) dissolved in citric acid buffer, pH 5.0, and the acceptor phase consisted of phosphate-buffered saline, pH 7.4. Images were taken with confocal laser scanning microscopy (CLSM) every 10 min for 8 h. RESULTS: This new method enabled for the first time visualization of concentration profiles in different skin layers simultaneously as a function of time. For this model penetrant, Bodipy FL C5 showed that the lower stratum corneum layer constitutes the greatest barrier to diffusion. Furthermore, there is preferred partitioning of this probe in epidermis vs. either stratum corneum or dermis. CONCLUSIONS: The on-line diffusion cell in combination with CLSM is a promising tool to study diffusion processes of dyes in fresh unfixed skin on-line. The method has the potential to access deeper skin layers as well as to visualize diffusion processes in cells.     

Transdermal Delivery of Pergolide from Surfactant-Based Elastic and Rigid Vesicles: Characterization and in Vitro Transport Studies

Purpose. The aim of this study was to investigate the effect of elastic and rigid vesicles on the penetration of pergolide across human skin. Methods. Vesicles used consisted of the bilayer-forming surfactant L-595 (sucrose laurate ester) and the micelle-forming surfactant PEG-8-L (octaoxyethylene laurate ester), together with the stabilizer sulfosuccinate. A series of L-595/PEG-8-L/sulfosuccinate vesicles were investigated, ranging from very rigid to very elastic. Pergolide-loaded elastic and rigid vesicles were visualized using Cryo-TEM and characterized for size and stability. Transdermal penetration of pergolide from different vesicle compositions was studied in vitro using flow-through Franz diffusion cells. A saturated buffer solution served as the control. Results. Vesicle composition had a major effect on the physico-chemical characteristics, morphology and drug solubility of the vesicular system. L-595/PEG-8-L/sulfosuccinate (70/30/5) elastic vesicles gave the best balance between vesicle stability and elasticity, as well as the highest drug solubility. Transport studies clearly showed that elastic vesicles were superior to rigid vesicles. Elastic vesicles enhanced the drug transport compared to the buffer control, although rigid vesicles decreased the drug transport. The best drug transport was achieved from L-595/PEG-8-L/sulfosuccinate (70/30/5) elastic vesicles, resulting in a steady-state flux of 13.6 ± 2.3 ng/(h*cm²). This was a 6.2-fold increase compared to the most rigid vesicles. Conclusions. This study supports the hypothesis that elastic vesicles are superior to rigid vesicles as vehicles for transdermal drug delivery.