Harry E. Boddé

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.

Visualization of in vitro percutaneous penetration of mercuric chloride; transport through intercellular space versus cellular uptake through desmosomes

The aim of the present study was to visualize the routes of penetration of mercuric chloride through human skin in vitro at the ultrastructural level, and to find out to what extent intra- and extracellular space contribute to the percutaneous transport of the model compound. Dermatomed human skin was subjected to in vitro mercuric chloride diffusion experiments in a bicompartmental polycarbonate diffusion cell. Upon interrupting a diffusion experiment, skin samples were treated with ammonium sulfide vapour to precipitate the mercury as mercuric sulfide, then processed for transmission electron microscopy. The presence of mercury in the precipitates was verified using X-ray microanalysis. An additional series of experiments involved the immersion of freshly excised human plantar callus in mercuric chloride solutions, followed by the same tissue processing protocol as used for the dermatomed skin samples; the mercury treated callus samples were likewise subjected to transmission electron microscopy. The results indicate that the intercellular route of transport through the stratum corneum predominates, but that after longer transport times, apical corneocytes tend to take up material, leading to a bimodal distribution of mercury: in the apical region of the stratum corneum there is mercury both in- and outside the cells; in the medial and proximal region intercellular transport prevails. There were no signs of a discontinuity in the in-depth distribution of mercury in that an almost unmountable barrier would exist in the lower region of the stratum corneum, as suggested by e.g. Sharata and Burnette [1,2]. However, there was evidence of the presence of two types of cells: apical corneocytes, which tend to take up mercuric ions relatively easily, and medial and proximal corneocytes, which are less capable of doing so. Interestingly, the results furthermore indicated that intracellular mercury uptake in the apical, squamous region of the stratum corneum occurred preferentially via the desmosomes. This was also the case in the callus immersion studies. These results suggest that the desmosomes may serve to channel material into corneocytes, especially in the squamous region where the desmosomes are beginning to disintegrate and hence the cellular lipid envelopes are leaky. A reservoir function for the apical zone of the stratum corneum is being suggested.

A Surface Energy Analysis of Mucoadhesion: Contact Angle Measurements on Polycarbophil and Pig Intestinal Mucosa in Physiologically Relevant Fluids

The possible role of surface energy thermodynamics in mucoadhesion was investigated with Polycarbophil and pig intestinal mucosa. In separate experiments, the surface energy parameters of the substrate (mucosa) and the adhesive (polymer film) were determined by contact angle measurements on captive air/octane bubbles in three physiologically relevant test fluids (isotonic saline, artificial gastric fluid, and artificial intestinal fluid). Whereas the swollen Polycarbophil films were relatively hydrophilic as indicated by small water contact angles (22, 23, and 16°), the water contact angles measured on mucosal tissue were significantly larger (61, 48, and 57°). Hence, mucus was found to possess an appreciable hydrophobicity. The measured adhesive performance (force of detachment) between Polycarbophil and pig small intestinal mucosa was highest in non-buffered saline medium, intermediate in gastric fluid, and minimal in intestinal fluid. In agreement with this trend, the mismatch in surface polarities between substrate and adhesive, calculated from the contact angle data, increased in the same order.

Estradiol Permeation from Nonionic Surfactant Vesicles Through Human Stratum Corneum in Vitro

The permeation of estradiol from vesicular formulations through human stratum corneum was studied in vitro. The vesicles were composed of nonionic n-alkyl polyoxyethylene ether surfactants (CnEOm). The thermodynamic activity of estradiol present in each formulation was kept constant by saturating all formulations with estradiol. The effects of both the particle size and the composition of the formulation on estradiol permeation across excised human stratum corneum were investigated. Stratum corneum that was pre-treated with empty surfactant carriers allowed for significantly higher estradiol fluxes compared with untreated stratum corneum. However, estradiol fluxes obtained in these pretreatment experiments appeared to be significantly lower than those obtained by the direct application of the estradiol-saturated carrier formulation on top of the stratum corneum. Furthermore, in the case of pretreatment of the stratum corneum, an increase in carrier size resulted in a decrease in estradiol flux. For direct application the opposite was found. Two mechanisms are proposed to play an important role in vesicle–skin interactions, i.e., the penetration enhancing effect of surfactant molecules and the effect of the vesicular structures that are most likely caused by adsorption of the vesicles at the stratum corneum–suspension interface.

In Vitro Human Skin Barrier Modulation by Fatty Acids: Skin Permeation and Thermal Analysis Studies

AbstractPurpose. This study aims to elucidate the skin permeation enhancement and the skin perturbation effects of a number of fatty acids, i.e. straight-chain saturated (SFA), monounsaturated (MUFA) and polyunsaturated acids (PUFA). Methods. The skin permeation enhancement effects were studied using human stratum corneum (SC) and p-aminobenzoic acid (PABA) as a model permeant. The fatty acids in propylene glycol (FA/PG) were applied according to a pre-treatment/co-treatment protocol. The perturbation effects were studied using differential thermal analysis (DTA) on SC after pretreatment with FA/PG. Results. SFA with 6 to 12 carbons exhibit a parabolic correlation between enhancement effect and chain-length, with a maximum at nonanoic-decanoic acids (with 9 and 10 carbons). Nonanoic and decanoic acids exert barely noticeable effects on the thermal behaviour of SC, suggesting that they easily mix with the skin lipids. All cis-6-, 9-, 11- or 13-octadecenoic acids (MUFA) enhance the permeation of PABA to the same extent. DTA revealed that the cis-9- and 13-isomers form a separate domain containing mostly the pure fatty acids within the SC lipids and suppress the lipid transitions at 70°/80°C. PUFA—linoleic (LA), α-linolenic (ALA) and arachidonic acids—enhance PABA permeation stronger than MUFA but additional double bonds do not further increase the degree of enhancement. LA and ALA form separate domains but do not completely suppress the SC lipid transitions at 70°/ 80°C. Increase in the enthalpy changes of 70°/80° transitions linearly correlates to the decrease in the permeability coefficients, suggesting that an increased perturbation of the skin lipids not necessarily has to yield an increased PABA permeation. Conclusions. The enhancement effects of fatty acids on the PABA penetration through SC are structure-dependent, associated with the existence of a balance between the permeability of pure fatty acids across SC and the interaction of the acids to skin lipids.

Visualization of diffusion pathways across the stratum corneum of native and in-vitro-reconstructed epidermis by confocal laser scanning microscopy.

Confocal laser scanning microscopy is a technique that permits the direct visualization in unfixed material of diffusion pathways and the cellular distribution of fluorescent markers after topical applications. This approach, in which the tissue specimen is optically sectioned, allows the study of changes in distribution pattern of applied compounds depending on the vehicle, time and depth without the interference of chemical alterations induced by most of the current techniques used for such studies. Using this technique the permeability properties of in-vitro-reconstructed epidermis were compared with those of the native counterpart. The epidermis was reconstructed by culturing human adult keratinocytes at the air-liquid interface either on fibroblast-populated collagen or on de-epidermized dermis. A fluorescent probe — Nile red (NR) — was applied in three different vehicles — polyethylene glycol (PEG) with a molecule mass of 400 (Da), propylene glycol (PG) and dimethyl sulphoxide (DMSO) — which perturb the SC barrier function to different extents. When NR was applied in PEG and PG on native epidermis, the amount of NR penetrating into and through the SC was very low, but was markedly increased when NR was applied in DMSO. Unlike native epidermis, the reconstructed epidermis allowed rapid NR penetration after the application in any of the solvents used. Furthermore, NR applied on reconstructed epidermis, was distributed quite homogeneously between the cellular and the intercellular spaces throughout the SC, suggesting that not only intercellular lipid structures but also the properties of the cornified envelopes differed markedly from those found in native epidermis. The differences in transport pathways between reconstructed and native epidermis may be partially ascribed to the culture conditions used, since incubation of freshly isolated epidermis under the same culture conditions as used for the reconstruction of the epidermis also leads to profound changes in the NR diffusion pathways.

Effect of N-alkyl-azocycloheptan-2-ones including azone on the thermal behaviour of human stratum corneum

In this study the thermal behaviour of human stratum corneum is investigated using N-alkyl-azacycloheptan-2-one with a varying number of carbon atoms in the alkyl chain. Throughout this article these substances will be referred to as Cn azones, in which n stands for the number of C atoms in the alkyl chain. The experiments have been carried out using differential thermal analysis (DTA). Untreated stratum corneum shows 4 transitions of which 3 are reversible and one is irreversible. The latter is due to protein denaturation, the former are due to gel-liquid state transitions of the lipids. Treatment with Cn azones in combination with propylene glycol shifts the lipid transition peaks, normally found at 345K and 360K, to lower temperatures; treatment with C12 azone decreases the enthalpy involved in the transitions, whereas treatment with C6 azone affects only the temperature of two transitions and not the enthalpy. A decrease in enthalpy is related to an increase in fluidity of the lipid bilayers; there seems to be a close parallel between the change in enthalpy and the change in permeability of stratum corneum.

A critical comparison of methods to quantify stratum corneum removed by tape stripping

Skin surface stripping with adhesive tape has been used to study the barrier function of the stratum corneum. Usually, the amount of stratum corneum removed by stripping is not linearly proportional to the number of strips removed. The generally accepted quantitative method to determine the amount of stratum corneum material on a tape strip is weighing. This method however has certain drawbacks, it is time consuming and laborious because tape strips have to be weighed twice and sometimes it cannot be used to determine concentration profiles in the skin of active substances in topically applied vehicles. In this paper, the accuracy and reliability of an alternative method to determine the amount of stratum corneum removed by tape stripping of the skin was investigated and compared to weighing. It is based on the spectrophotometric examination of the tape. The light absorption by the proteins on the tape is correlated to the weight of the stratum corneum material. This method was found to be easier and faster than weighing, but it was less reliable because the light scattering of the stratum corneum on the tape largely overshadowed the absorption of the proteins. The light scattering showed a linear increase with an increasing amount of material on the tape, but with a large variability, resulting in calibration curves with correlations of 0.8400. However, direct spectroscopic analysis of stratum corneum tape strips has some distinct advantages even if it cannot be used for the exact quantification of stratum corneum proteins. With direct spectroscopic measurements, a tape strip can be laterally examined to inspect its homogeneity. Direct spectroscopic measurements on tape strips might also be employed to investigate the lateral and in-depth distribution of strongly light-absorbing substances in the stratum corneum.

Effects of bile salts on transport rates and routes of FITC-labelled compounds across porcine buccal epithelium in vitro

Abstract In this study the penetration enhancing effect of bile salts on the transport of hydrophilic macromolecular compounds across porcine buccal mucosa was investigated in-vitro. Coadministration of 100 mM of the trihydroxy bile salts sodium glycocholate (GC) and sodium taurocholate (TC) and the dihydroxy bile salts sodium glycodeoxycholate (GDC) and sodium taurodeoxycholate (TDC) increased the in-vitro transport of fluorescein isothiocyanate (FITC) by a factor of a hundred or more, without a significant difference between the four bile salts. The concentration dependence of the enhancing effect of GDC was studied using FITC-labelled dextrans of increasing molecular weight as permeants (FD4, MW 4400; FD10, MW 9400; FD20, MW 19 600). The maximal enhancement was observed when GDC was coadministered in a concentration of 10 mM, resulting in an enhancement ratio of about 2000 for FD4. Using confocal laser scanning microscopy the effects of bile salts on the penetration pathways of hydrophilic compounds were investigated. The uniform distribution of FITC throughout the epithelium was changed by coadministration of 100 mM of bile salt to an increased amount of the fluorescent probe present in the intercellular domains. The intercellular distribution of both FD4 and FD10 was not changed by a low, but effective, concentration of GDC (2 mM, enhancement ratio of 72 for FD4). Increasing the concentration of GDC to 10 and 100 mM resulted in uptake of the fluorescent probe in the epithelial cells. From these results we conclude that the di- and trihydroxy bile salts studied increase the transport of hydrophilic compounds across buccal epithelium in vitro, below 10 mM by increasing the intercellular transport and at 10 mM and higher concentrations by opening up a transcellular route.

Kinetics, ultrastructural aspects and molecular modelling of transdermal peptide flux enhancement by N-alkylazacycloheptanones

Transdermal delivery is an attractive route to administer peptide drugs. The stratum corneum, however, is a barrier for peptides; hence there is a need for agents to enhance peptide transport. The aim of this study was to investigate the enhancement properties of azacycloheptan-2-ones (Azones) as a function of hydrocarbon chain length. Their ability to enhance the percutaneous penetration of desglycinamide arginine vasopressin (DGAVP) was taken as a criterion. The flux of DGAVP through human stratum corneum was measured. For control experiments the stratum corneum was either untreated or immersed in propylene glycol (PG). The non-enhanced peptide flux through human stratum corneum was 1.6 ± 0.1 nmol/cm2 per h (peptide concentration in the donor 6.0 mM). Pretreatment with PG or hexyl- or octyl-Azone did not change the flux significantly. However, the permeability increased 1.9-fold after pretreatment with decyl-Azone 3.5-fold with dodecyl-Azone, and 2.5-fold with tetradecy]- Azone. Electron micrographs taken from freeze-fracture replicas of skin samples treated with either PG or dodecyl-Azone suggest that these treatments do not drastically change the lamellar appearance of the intercellular lipids. Taken together, the morphological and kinetical data suggest that the enhancing effect of Azone is caused by interference with the packing arrangement of the intercellular lamellar lipids in the stratum corneum, most likely by insertion of Azones into the lipid bilayers. In order to assess the capability of the Azone molecule to perturb the lipid arrangement within the bilayers, the degree of conformational freedom in Azones polar head group was investigated using computer-aided molecular modelling. These calculations indicated that, upon turning the carbonyl moiety towards the interlamellar hydrophilic domain, a ‘soup-spoon’ conformation can be obtained, which is only 1 kcal/mol away from the minimum energy conformation. This ‘soup-spoon’ conformation would be highly favourable in order to accomodate the Azone molecule at the interface between the hydrocarbon and polar head group regions, respectively. This conformation might strongly perturb the lipid structure, making it more permeable to the penetrant. Such an effect apparently optimizes around a chain length of about 12 C-atoms.