J.A. Bouwstra

The important role of stratum corneum lipids for the cutaneous barrier function.

The skin protects the body from unwanted influences from the environment as well as excessive water loss. The barrier function of the skin is located in the stratum corneum (SC). The SC consists of corneocytes embedded in a lipid matrix. This lipid matrix is crucial for the lipid skin barrier function. This paper provides an overview of the reported SC lipid composition and organization mainly focusing on healthy and diseased human skin. In addition, an overview is provided on the data describing the relation between lipid modulations and the impaired skin barrier function. Finally, the use of in vitro lipid models for a better understanding of the relation between the lipid composition, lipid organization and skin lipid barrier is discussed. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

Intestinal transit of bioadhesive microspheres in an in situ loop in the rat—A comparative study with copolymers and blends based on poly(acrylic acid)

Two commercially available copolymers of acrylic acid, Carbomer (Carbopol® 934P) and Polycarbophil (Carbopol® EX-55 resin), and blends with Eudragit® RL 100 were screened for their mucoadhesive properties by determining the force needed to detach a polymer coated glass plate from porcine intestinal mucosa in vitro. Microspheres of poly(2-hydroxyethyl methacrylate) (PHEMA) were synthesized by suspension polymerization and coated with candidate mucoadhesive polymers in an air-suspension process. A chronically isolated ileal loop model in the rat was used in order to study the intestinal transit of the microspheres. Bioadhesive properties of this potential drug delivery system were evaluated by recording the mean residence time of the microspheres when injected into the in situ perfused gut segment. Polycarbophil showed significantly improved mucoadhesive properties in vitro in comparison to Carbomer. In the in situ model, the residence time of Carbomer-coated microspheres was comparable to the non-coated controls, whereas Polycarbophil-coated spheres initially showed a marked bioadhesion.

Interactions between liposomes and human stratum corneum in vitro: freeze fracture electron microscopical visualization and small angle X-ray scattering studies.

Summary The interactions between three liposomal formulations and human stratum corneum were visualized using freeze fracture electron microscopy. A new replica cleaning method was introduced. Human stratum corneum was submerged for 48 h in liposome suspensions prepared from commercially available phospholipid mixtures. The size, lamellarity and lipid moieties of the liposomes were similar. The main difference between the three phospholipid formulations was the hydrophilicity of the headgroups. The composition dependence of the interactions between these vesicles and human stratum corneum was investigated.

Thermodynamic and structural aspects of the skin barrier

Abstract The effect of water and propylene glycol (PG) on the physical-chemical properties of the human stratum corneum has been studied with small angle X-ray scattering (SAXS) and differential thermal analysis (DTA). Hydration (already published by Knutsen et al. [1]) or pretreatment of the stratum corneum with PG resulted in a shift of two gel-liquid transitions of the lipid bilayers to a lower temperature region indicating that water and PG interacts with the stratum corneum lipids. From SAXS experiments it appeared that the repeat distance between the lamellae found in untreated human stratum corneum is only 6.5 nm. This distance remained unchanged upon water and PG pretreatment suggesting that no swelling of the lipid bilayers occurred. This indicates the neither water nor PG is intercalated into the bilayers. The apparent contradictory results obtained with DTA and SAXS can be explained as follows. It is possible that incorporation of water or PG in the head group regions of the lipids results in an increase in the mean interfacial area per lipid without changing the repeat distance. The interaction of N -alkyl-azocycloheptane-2-one(C n zone, azone derivatives in which n is the number of carbon atoms in the alkyl chain) in combination with PG with human stratum corneum has also been studied using the two techniques mentioned above. The results of DTA experiments indicate that azone derivatives possessing a longer alkyl chain have stronger interactions with the stratum corneum lipid bilayers than shorter alkyl chain azone derivatives do. The former ones probably induce pronounced changes in the bilayer arrangement of stratum corneum lipids, since SAXS experiments showed that C 12 azone in combination with PG resulted in a disappearance of all diffraction peaks.

Wide-angle X-ray Diffraction of Human Stratum Corneum: Effects of Hydration and Terpene Enhancer Treatment

Abstract— Wide‐angle X‐ray‐diffraction experiments were used to investigate the molecular organization of barrier components of human stratum corneum. Diffraction lines related to the side‐by‐side lipid packing arrangements in the intercellular bilayers were identified as were patterns arising from secondary protein structures in intracellular keratin. Reflections were also identified which may be produced by proteins in the corneocyte envelopes. The effects of hydration on stratum corneum structure were monitored using 0, 20–40, 40–60, 60–80 and approximately 300% hydrated samples. The packing arrangements in the intercellular lipid bilayers remained the same over the entire hydration range, as did keratin structures. A new diffraction ring, attributable to liquid water, was produced by 300% hydrated samples with a repeat spacing of 0.35 to 0.30‐0.29 nm. The effects of three terpene enhancers, (+)‐limonene, nerolidol and 1,8‐cineole, on stratum corneum structure were monitored. Treatment with each of the terpenes produced additional reflections which were attributed to the presence of the respective liquid enhancers within the stratum corneum. (+)‐Limonene produced an additional reflection at 0.503‐0.489 nm, nerolidol, an additional reflection at 0.486‐0.471 nm and 1,8‐cineole, an intense reflection at 0.583–0.578 nm. Reflections characteristic of gel‐phase lipids and crystalline lipids also remained after all terpene treatments. These results provide no clear evidence of lipid bilayer disruption by the terpenes and suggest that areas of liquid terpene exist within the stratum corneum. The mechanisms underlying propylene glycol synergy with terpene enhancers were investigated. Treatment of stratum corneum with each terpene mixed with propylene glycol gave rise to two additional reflections. One reflection, always positioned at 0.452–0.448 nm, had been observed in control studies following propylene glycol treatment and may have been associated with bilayer structures disrupted by propylene glycol or altered keratin structures. The second reflection was developed by the respective terpene enhancer. For example, treatment with a 1,8‐cineole/propylene glycol mixture produced reflections at 0.457—0.451 nm (propylene glycol‐disrupted lipids or altered keratin) and 0.591—0.578 nm (liquid 1,8‐cineole). Since the reflection at 0.452—0.448 nm was unaffected by co‐application of propylene glycol with terpene enhancers, this study offers no evidence to support the theory that propylene glycol synergy with the terpenes occurs through enhanced lipid disruption.

The role of ceramide composition in the lipid organisation of the skin barrier.

The lipid lamellae in the stratum corneum (SC) play a key role in the barrier function of the skin. The major lipids are ceramides (CER), cholesterol (CHOL) and free fatty acids (FFA). In pig SC at least six subclasses of ceramides (referred to as CER 1, 2-6) are present. Recently it was shown that in mixtures of isolated pig SC ceramides (referred to as CER(1-6)) and CHOL two lamellar phases are formed, which mimic SC lipid organisation very closely [J.A. Bouwstra et al., 1996, J. Lipid Res. 37, 999-1011] [1]. Since the CER composition in SC originating from different sources/donors often varies, information on the effect of variations in CER composition on the SC lipid organisation is important. The results of the present study with mixtures of CHOL including two different CER mixtures that lack CER 6 (CER(1-5) mixtures) revealed that at an equimolar molar ratio their lipid organisation was similar to that of the equimolar CHOL:CER(1-6) and CHOL:CER(1,2) mixtures, described previously. These observations suggest that at an equimolar CHOL:CER ratio the lipid organisation is remarkably insensitive toward a change in the CER composition. Similar observations have been made with equimolar CHOL:CER:FFA mixtures. The situation is different when the CHOL:CER molar ratio varies. While in the CHOL:CER(1-6) mixture the lamellar organisation hardly changed with varying molar ratio from 0.4 to 2, the lamellar organisation in the CHOL:CER(1-5) mixtures appeared to be more sensitive to a change in the relative CHOL content, especially concerning the changes in the periodicities of the lamellar phases. In summary, these findings clearly indicate that at an equimolar CHOL:CER molar ratio the lamellar organisation is least sensitive to a variation in CER composition, while at a reduced CHOL:CER molar ratio the CER composition plays a more prominent role in the lamellar phases. This observation may have an implication for the in vivo situation when both the CER composition and the CHOL:CER molar ratio change simultaneously.

Small angle X-ray scattering: possibilities and limitations in characterization of vesicles.

The use of small angle X-ray scattering (SAXS) for characterization of lipid vesicle dispersions is described. The effect of curvature of the membrane, the presence of proteins in the core and on the surface of the membrane, variations in membrane thickness and distribution in the number of bilayers of the vesicles in the dispersion on the scattering curve is discussed. Concerning unilamellar vesicles, either the membrane curvature of vesicles smaller than 50 nm or variations in membrane thickness result in a disappearance of the first node in the scattering curve, even if the bilayer is symmetric with respect to the electron density distribution. In the case of dispersion in which unilamellar as well as multilamellar vesicles are present it is shown that a small fraction of multilamellar liposomes changes the scattering curve dramatically. Liposomes were prepared from various compositions of dipalmitoylphosphatidylcholine (DPPC) and cholesterol hemisuccinate (CHEMS) by the film method. The electron density profile of the bilayers and distribution in the number of bilayers of the liposome dispersions were determined. The average number of bilayers increased as a function of the decrease in CHEMS content. Liposomes with higher CHEMS content than 10 mol% were unilamellar. It seems that increase in charge intercalated in the bilayers resulted in unilamellar vesicles.

Safety Aspects of Non‐ionic Surfactant Vesicles: A Toxicity Study Related to the Physicochemical Characteristics of Non‐ionic Surfactants

Abstract— Two different toxicity models were used to assess the relationship between the physicochemical properties of non‐ionic surfactant vesicles (NSVs), and the safety of these vesicles for topical drug administration. The vesicles used in this study consisted of polyoxyethylene alkyl ethers (CnEOm) in which the number of C atoms (n) varied between 12 and 18 and the number of oxyethylene units (m) between 3 and 7. The physicochemical properties of the vesicles are described in terms of hydrophilic‐lipophilic balance (HLB) values, and critical micelle concentrations (CMC), and the rigidity of the bilayers as determined by the gel‐liquid transition temperatures and the cholesterol content of the bilayers. The first toxicity model, comprising the measurement of the ciliary beat frequency, is a tool to assess the safety of intranasally applied formulations. Studies using this ciliotoxicity model revealed that by increasing the length of the alkyl chain of the surfactant, a decrease in toxicity was observed. The opposite correlation was found if the length of the polyoxyethylene headgroup was increased. Furthermore, it was observed that gel‐state vesicles produce less of an effect on the ciliary beat frequency than liquid state vesicles. The second toxicity model, comprising the determination of cell proliferation of human keratinocytes, is a method to assess skin irritancy. In contrast to the ciliotoxicity model the length of the polyoxyethylene headgroup and of the alkyl chains did not seem to have an effect on the safety of the vesicles. However, the bond by which the headgroup is linked to the alkyl chain, showed a very strong effect on the toxicity of the surfactant: oleyl‐EO5 ester vesicles were found to have an effect on the cell proliferation, which was one‐sixteenth that of the oleyl‐EO5 ether vesicles. The cholesterol content did not appear to have an effect on the proliferation of the keratinocytes. Neither the HLB nor the CMC values appeared to have an effect on the safety of the NSV formulations as observed in both toxicity models.

Immune Modulation by Adjuvants Combined with Diphtheria Toxoid Administered Topically in BALB/c Mice After Microneedle Array Pretreatment

PurposeIn this study, modulation of the immune response against diphtheria toxoid (DT) by various adjuvants in transcutaneous immunization (TCI) with microneedle array pretreatment was investigated.MethodsTCI was performed on BALB/c mice with or without microneedle array pretreatment using DT as a model antigen co-administrated with lipopolysaccharide (LPS), Quil A, CpG oligo deoxynucleotide (CpG) or cholera toxin (CT) as adjuvant. The immunogenicity was evaluated by measuring serum IgG subtype titers and neutralizing antibody titers.ResultsTCI with microneedle array pretreatment resulted in a 1,000-fold increase of DT-specific serum IgG levels as compared to TCI. The immune response was further improved by co-administration of adjuvants, showing a progressive increase in serum IgG titers when adjuvanted with LPS, Quil A, CpG and CT. IgG titers of the CT-adjuvanted group reached levels comparable to those obtained after DT-alum subcutaneous injection. The IgG1/IgG2a ratio of DT-specific antibodies decreased in the following sequence: plain DT, Quil A, CT and CpG, suggesting that the immune response was skewed towards the Th1 direction.ConclusionsThe potency and the quality of the immune response against DT administered by microneedle array mediated TCI can be modulated by co-administration of adjuvants.

The influence of two azones and sebaceous lipids on the lateral organization of lipids isolated from human stratum corneum.

The main problem with topical application of compounds to administer drugs to and regulate drug levels in a human body, is the barrier formed by the intercellular lipid matrix of the stratum corneum (SC). In a search for possibilities to overcome this barrier function, a good understanding of the organization and phase behavior of these lipids is required. SC lipid model studies especially provide a wealth of information with respect to the lipid organization and the importance of certain subclasses of lipids for the structure. Previously, we have shown that electron diffraction (ED) provides detailed information on the lateral lipid packing in both intact SC (G.S.K. Pilgram et al., J. Invest. Dermatol. 113 (1999) 403) and SC lipid models (G.S.K. Pilgram et al., J. Lipid Res. 39 (1998) 1669). In the present study, we used ED to examine the influence of two azones and sebaceous lipids on the lateral phase behavior of lipids isolated from human SC. We established that human SC lipids are arranged in an orthorhombic packing pattern. Upon mixing with the two enhancers the orthorhombic packing pattern was still observed; however, an additional fluid phase became more apparent. In mixtures with sebaceous lipids, the presence of the hexagonal lattice increased. These findings provide a basis for the mechanism by which these enhancers and sebaceous lipids interact with human SC lipids.