Ferry Spies

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.

Enhancement of in vitro permeability of porcine buccal mucosa by bile salts: kinetic and histological studies

Abstract In this study the enhancing effects of di-and tri-hydroxy bile salts on buccal penetration were investigated using fluorescein isothiocyanate (FITC) as a model permeant. The permeability of the porcine buccal mucosa to this compound in the presence and absence of bile salts was determined in vitro. In conjunction with kinetic studies, histological studies were carried out to investigate the interaction between the bile salts and the buccal epithelium at the light and electron microscopic level. In the presence of the bile salts at 0.l M concentration, the permeability of buccal mucosa to FITC increased by a 100–200 fold compared to FITC alone. Calculation of enhancement ratios provided normalization of the permeability data for the enhancer treated tissue with respect to its own control. No significant difference was observed between the enhancing effects of di-hydroxy and tri-hydroxy bile salts. After the bile salt treatment significant morphological and ultrastructural changes occurred. At LM level, partial loss of superficial cells was observed as well as separation of the epithelium from the basal lamina. Upon a 4 hour bile salt treatment, two important changes were observed with FFEM: (1) the fracture plane preferentially ran across the cytoplasmic proteins and through the cytoplasmic space domains; (2) abnormally structured deposits were found in the cytoplasm. Similar to the kinetic studies, no differences were found between the histological effects of di- and tri-hydroxy bile salts.

Interactions of non-ionic surfactant vesicles with cultured keratinocytes and human skin in vitro: a survey of toxicological aspects and ultrastructural changes in stratum corneum

Abstract The inhibition of cell proliferation of SV-40 transformed human keratinocytes was used as a model substrate to determine the dermal toxicity of formulations containing non-ionic surfactant vesicles (NSVs). Both the effects of the molecular structure of the surfactant (polyoxyethylene alkyl ethers/ esters) and the incorporation of cholesterol on the cell proliferation were investigated. The alkyl chain length as well as the length of the polyoxyethylene headgroup had only a minor effect on the proliferation of the keratinocytes. However, the bond by which the alkyl chain was linked to the polyoxyethylene headgroup (i.e. an ether or an ester bond) had a strong influence on the cell proliferation. The concentration of the surfactants that inhibited cell proliferation by 50% was for the ether surfactants 10-fold lower than for the ester surfactants. The cholesterol content of the bilayers had no effect on the cell proliferation. These results are compared with those obtained in earlier studies, where the effect of surfactants on the ciliary beat frequency was determined (a model used to determine the toxicity of intranasal formulations). Furthermore, the interactions between NSVs and excised human skin were determined after 48 h of incubation under occlusion. Freeze-fracture electron microscopy revealed that the vesicles were able to introduce changes in the ultrastructure of the intercellular lipid lamellae of the stratum comeum down to a depth of ~ 10 μm.

ELECTROPERTURBATION OF THE HUMAN SKIN BARRIER IN VITRO : II. EFFECTS ON STRATUM CORNEUM LIPID ORDERING AND ULTRASTRUCTURE

In transdermal iontophoresis, drugs can be driven across the skin by electrorepulsion, but their transport can also be enhanced by electrical perturbation of the skin barrier. Our objective was to study perturbing effects of electrical current on human stratum corneum lipid fine structure combining techniques including freeze‐fracture electron microscopy. Human stratum corneum was subjected to pulsed constant currents, varying from 0.013–13 mA.cm−2. The voltage across the stratum corneum was high‐frequency‐sampled and s.c. impedance values derived from it. Upon termination of the current, skin samples were rapidly frozen and processed for freeze‐fracture electron microscopy or subjected to X‐ray diffraction analysis.

MORPHOLOGICAL OBSERVATIONS ON LIPOSOMES BEARING COVALENTLY BOUND PROTEIN :STUDIES WITH FREEZE-FRACTURE AND CRYO ELECTRON MICROSCOPY AND SMALL ANGLE X -RAY SCATTERING TECHNIQUES

The appearance of protein bound to the surface of intact and microfluidized liposomes and its possible influence on their morphology was examined by freeze-fracture electron microscopy, cryo electron microscopy and small angle X-ray scattering (SAXS) techniques. Results obtained by the two microscopy techniques were in agreement with one another in terms of vesicle size and localization of protein (tetanus toxoid or immunoglobulin G) on the surface of vesicles. Surface-bound protein was observed as particles (10-12 nm diameter) by freeze-fracture electron microscopy and was confirmed by immunogold cryo microscopy. SAXS was shown to be a suitable means to further characterize liposomes with, or without bound protein.

Visualization studies of the mucoadhesive interface

Abstract Aim of this study was to prove the occurrence of intermixing (“interpenetration”) at the interfacial zone between the mucoadhesive polymer Polycarbophil and intestinal mucus by (electron)-microscopical examination. As both phases are essentially hydrogels, various cryotechniques were tried to achieve dehydration of the samples without destroying the polymeric network structure of the native gels. Freeze fracture replication or sputter-coating of freeze-dried samples for transmission or scanning electron microscopy did not allow to identify the two hydrogel phases by characteristic network structures. Best results were obtained with plastic sections of freeze substituted samples. With light microscopy, mucus glycoproteins could be identified unambiguously by specific histochemical reactions. The mucoadhesive interface appeared as an irregular borderline with many coves and invaginations, but was sharp rather than hazy. There was no evidence for intermixing between mucus and mucoadhesive hydrogel to occur in the μ-range. Interpenetration of free polymer chain ends, however, may still be possible in the nm-range.

Interactions Between non-ionic Surfactant Vesicles and human stratum corneum in vitro.

AbstractThe effects of non-ionic surfactant vesicles (NSVs) on human skin in vitro were studied in relation to the physico-chemical properties of the vesicles. The interactions between NSVs and skin were visualized using both freeze fracture electron microscopy and confocal laser scanning microscopy. The physico-chemical properties of the NSVs were varied in a systematic way, using a broad series of polyoxyethylene monoalkyl ether type surfactants (CnEOm). The number of oxyethylene units (m) was varied between 3, 7 and 10, and the number of carbon atoms (n) was either 12 or 18. Both the effects of liquid state vesicles composed of C12Eo3,7 and C9=9EO10 surfactants and gel state vesicles (C18EO37) were investigated. After the application of the NS V suspension on the stratum corneum surface two essentially different types of vesicle-skin interactions were visualized. Firstly, an interfacial interaction, involving the adsorption of vesicles, and the deposition of bilayer sheets on the outermost layers of th...

Lipid Composition and Barrier Function of Human Skin after Grafting onto Athymic Nude Mice

The barrier properties of human epidermis grafted for 1-3 months onto nude mice are compared with normal human skin. Beside penetration studies with tritiated water and measurements of transepidermal water loss (TEWL), we analyzed the epidermal lipids by high-performance thin layer chromatography and evaluated the ultrastructure of the intercorneocyte lipid arrangement by freeze fracture electron microscopy (FFEM). The permeability of human skin for tritiated water and the TEWL exhibit no significant changes after grafting onto nude mice. FFEM analysis showed that grafted epidermis has the same morphological pattern as normal human epidermis. Regular desmosomes and lamellar lipid structures are present. Grafting did not qualitatively affect the lipid composition of human epidermis. Ceramides which contribute largely to the barrier function, have the same distribution profile.

A novel in-situ model for continuous observation of transient drug concentration gradients across buccal epithelium at the microscopical level

Buccal drug delivery is hindered by the low permeability of the buccal epithelium. The use of a time- and space-resolved visualization technique is inevitable for finding the location of the rate-limiting regions in the epithelium. In this study an in-situ method is presented with which permeation of fluorescent compounds in and across buccal epithelium can be visualized by means of confocal laser scanning microscopy (CLSM) and their topographical distribution quantified. This direct approach to visualize the penetration of fluorescently labelled drugs in thick epithelia avoids artifacts due to fixation, dehydration and embedding and can be executed on a single piece of tissue. Using this method, i.e. visualizing and quantifying the transepithelial drug distribution as a function of time, a rate-limiting zone in porcine buccal epithelium was observed at approximately 150 μm from the surface, in the epithelium. The region where diffusion of fluorescein isothiocyanate (FITC), a hydrophilic marker, was slowed down corresponds with the intermediate strata, the cell layers where membrane coating granules extrude their content in the intercellular space.

The effect of microfluidization of protein-coated liposomes on protein distribution on the surface of generated small vesicles

Tetanus toxoid and immunoglobulin G (IgG), model proteins for vaccines and targeting ligands respectively, were covalently coupled to preformed dehydration-rehydration vesicles (DRV) to produce vesicles with surface-bound proteins (DRV-protein) or to preformed small unilamellar vesicles (SUV) which were used to generate DRV with bound protein [(SUV-protein)DRV]. Of the amount of protein employed for coupling (1 mg), 13.8-45.1% was recovered with the liposomes, depending on the type of preparations and the proteins used. Microfluidization of similar DRV-protein or (SUV-protein)DRV for up to 10 cycles led to the formation of smaller vesicles (98-136 mm diameter) which, however, had modestly reduced (estimated as 8.8-21.7%) bound proteins, again depending on the type of preparation and protein used. Treatment of DRV-protein and (SUV-protein) DRV with proteinase revealed that 32.9-45.6% of the total bound protein was exposed on the liposomal surface. With microfluidized liposomes, the proportion of surface-exposed protein increased to 63.1-76.2%. Incubation of intact and microfluidized DRV-IgG and (SUV-IgG) DRV with a protein A-Sepharose 4B CL gel confirmed the presence of IgG on the liposomal surface (47.1-68.4 and 80.5-82.1% of total bound protein respectively). These studies were supplemented with freeze-fracture electron microscopy of (SUV-toxoid)DRV which demonstrated the presence of protein particles (up to 3; 12-14 nm diameter) on the surface of both intact and microfluidized individual liposomes.