Development of an optimal protocol for the ultrastructural examination of skin by transmission electron microscopy

Ifversen

et al. 2009

AAPS J

Abstract. This study aimed to investigate the effect of a novel kind of immune-stimulating complexes (ISCOMs) on human skin penetration of model compounds in vitro to evaluate their potential as a delivery system, ultimately for transcutaneous vaccination. Special focus was on elucidating the mechanisms of penetration. Preparation of ISCOMs was done by dialysis and subsequent purification in a sucrose density gradient. The penetration pathways of acridine-labeled ISCOMs were visualized using confocal laser scanning microscopy (CLSM). Transmission electron microscopy (TEM) was used to evaluate the ultrastructural changes in the skin after application of the ISCOMs with or without hydration. Transcutaneous permeation of the model compound, methyl nicotinate, was evaluated in diffusion cells. The prepared ISCOMs were 42-52 nm in diameter as evaluated by dynamic light scattering with zeta potentials of −33 to −26.1 mV. TEM investigations verified the presence of ISCOM structures. Penetration of acridine into skin was greatly increased by incorporation into ISCOMs as visualized by CLSM. Permeation of methyl nicotinate was enhanced in the presence of ISCOMs. Ultrastructural changes of the intercellular space in the stratum corneum after exposure of ISCOMs were observed on micrographs, especially for hydrated skin. In conclusion, cutaneous application of ISCOMs leads to increased penetration of hydrophobic model compounds through human stratum corneum and thus shows potential as a transcutaneous delivery system. The increased penetration seems to be reflected by a change in the intercellular space between the corneocytes, and the effect is most likely caused by the components of the ISCOMs rather than intact ISCOMs.

Section: Penetration Of Intact Nanoparticles?mentioning

confidence: 99%

In Vitro Cutaneous Application of ISCOMs on Human Skin Enhances Delivery of Hydrophobic Model Compounds Through the Stratum Corneum

Ifversen

et al. 2009

AAPS J

“…Scale bar, 0.5 µm. ids which OsO 4 did not (5,6). The electron opacity observed on the fungal cell wall following postfixing with RuO 4 therefore may be explained by the ability of this fixative to react with polysaccharides and saturated lipids.…”

Section: Discussionmentioning

confidence: 92%

“…RuO 4 has been used mainly to visualize extracellular saturated lipids in ultrastructural studies of stratum corneum from skin. These lipids constitute the dermal permeability barrier (5,6).…”

mentioning

confidence: 99%

Efecto comparativo del tetróxido de osmio y del tetróxido de rutenio como fijadores sobre la ultraestructura de la pared celular de hongos

Torres‐Fernández

Ordóñez

2003

biomedica

The fungal cell wall viewed through the electron microscope appears transparent when fixed by the conventional osmium tetroxide method. However, ruthenium tetroxide post-fixing has revealed new details in the ultrastructure of Penicillium sp. hyphae and Saccharomyces cerevisiae yeast. Most significant was the demonstration of two or three opaque diverse electron dense layers on the cell wall of each species. Two additional features were detected. Penicillium septa presented a three-layered appearance and budding S. cerevisiae yeast cell walls showed inner filiform cell wall protrusions into the cytoplasm. The combined use of osmium tetroxide and ruthenium tetroxide is recommended for post-fixing in electron microscopy studies of fungi.Key words: fungal cell wall, fungal ultrastructure, osmium tetroxide, ruthenium tetroxide, Penicillium sp., Saccharomyces cerevisiae.Efecto comparativo del tetróxido de osmio y del tetróxido de rutenio como fijadores sobre la ultraestructura de la pared celular de hongos Al microscopio electrónico, la pared celular de los hongos es de apariencia translúcida en especímenes procesados mediante la posfijación convencional con tetróxido de osmio. La posfijación con tetróxido de rutenio reveló nuevos detalles ultraestructurales en hifas de Penicillium sp. y en levaduras de Saccharomyces cerevisiae. El aspecto más destacado fue la modificación en la transparencia de la pared celular, característica de la fijación con tetróxido de osmio. El tetróxido de rutenio permite verla como una estructura compuesta por dos o tres capas oscuras de diferente tonalidad. Se observaron también otros dos rasgos importantes: los septos en las hifas de Penicillium sp. presentaban una apariencia trilaminar y en la pared celular de las levaduras de S. cerevisiae, en etapa de gemación, se destacaban unas prolongaciones filiformes que emergían desde la capa interna y se proyectaban hacia el citoplasma. Se recomienda el uso combinado de los dos fijadores en estudios ultraestructurales de los hongos.Palabras clave: pared celular, ultraestructura de los hongos, tetróxido de osmio, tetróxido de rutenio, Penicillium sp., Saccharomyces cerevisiae.

Bulletin of the Korean Chemical Society

“…EM (Electron microscope) and PM analysis: EM analysis was examined with a transmission electron microscope (TEM, Hitachi H-7600). At that time, we introduced RuO4 as a post-fixation agent to preserve these lipids in LC structure by means of the similar method as S.Y Hou et al and B.A.I van den Bergh et al 13,14 At first, a sample was slightly applied on the inner region of a tube. And then, for RuO4 post-fixation, the specimens were post-fixed in 0.25% RuO4 (EMS), the 0.1 M cacodylate buffer for 45 min at room temperature in the dark.…”

Section: Methodsmentioning

confidence: 99%

Lyotropic Behaviors of a Phospholipid-based Lamella Liquid Crystalline Phase Hydrated by Propylene Glycol as a Polar Solvent: Correlation of DSPC vs PG Concentration

Jeong

2007

The lyotropic behaviors to form the structure of distearoylphosphatidylcholine (DSPC)-based liquid crystal (LC) hydrated by only propylene glycol (PG) without water were examined by differential scanning calorimetry (DSC), X-ray diffractions (XRD), polarized microscope (PM) and transmission electron microscope (TEM). By increasing the amount of PG instead of water, it showed the phase transition to be gradually changed from anisotropic structures to other structures more close to isotropic ones and their appearance to be changed from solid-like states to liquid-like ones with more fluidity. Below 50% w/w PG, the mixtures of DSPC and PG resulted in no direct observation of LC structure through PM because they were very close to solid-states. From 55% w/w to 90% w/w of PG, the dense lamella crystalline structures were observed through PM, and their thickness and area decreased as the content of PG increased. Measured by DSC with heating process, the main phase transition from α-lamella phase to isotropic phase appeared from 52.89 °C to 47.41 °C to show linearly decreasing behaviors because PG affects the hydrophobic region of DSPC-based lamella phase. The repeating distance of the lamella phase and the interlayer distance between bilayers were calculated with XRDs and the average number of bilayers related to the thickness in LC structure was approximately estimated by combining with TEM results. The WAXS and DSC measurements showed that all of PG molecules contributed to swelling both the lipid layer in the edge region of lamella phase close to phosphate groups and the interlayer between bilayers below 90% w/w of PG. The phase and thermal behaviors were found to depend on the amount of PG used by means of dissolving DSPC as a phospholipid and rearranging its structure. Instead of water, the inducement of PG as a polar solvent in solid-lamella phase is discussed in terms of the swelling effect of PG for DSPC-based lamella membrane.