Penetration of compounds through human stratum corneum as studied by Fourier transform infrared photoacoustic spectroscopy.

The penetration of the lipophilic model permeant, 1-cyanodecane, into isolated human stratum corneum (SC) was followed nondestructively by step-scan Fourier transform infrared (FTIR) photoacoustic spectroscopy (PAS) with phase modulation technique. The uptake of the compound in the SC was quantified by monitoring the alterations in the spectra in the course of the penetration using multivariate analysis. Step-scan technique in conjunction with phase modulation offers the possibility for controllable depth profiling (sampling depth up to 30 microm) during the penetration process. Based on Fick's second law and assuming a virtually layered structure of the membrane, depth-dependent diffusion coefficients were derived by numerical fitting of the spectroscopic data. For 1-cyanodecane, the diffusion coefficient in the inner region of the SC is 1.6-fold that measured in the outer region.

Separation of erythrocytes into age-related fractions by density or size? Counterflow centrifugation.

During the process of aging red blood cells become denser and smaller. Counterflow centrifugation separates particles of lower density and smaller diameter from those of higher density and bigger diameter. Thus, the question arises: which property of the red cells, density or size, governs the age-related separation by counterflow centrifugation? It is shown that it is the size which dominates the balance between sedimentation and streaming. Age-related separation of human red blood cells by counterflow centrifugation (elutriation) was analysed by the standard hematological parameters (hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration), hemoglobin A1c and the membrane protein ratio 4.1a/(4.1a+4.1b). Red blood cells with a high hemoglobin A1c content and a high 4.1a/(4.1a+4.1b) ratio were found in the early fractions of the elutriation. This proves that old cells make up early fractions, while the "youngsters" constitute later ones. The elutriation technique used (yielding human red blood cells in a "healthy state") and the age parameters studied show that the membrane protein ratio 4.1a/(4.1a+4.1b) is another reliable age parameter for the assessment of red blood cell age.

Quantification of lipids, liposomes, amino acids, and proteins by thermal ultramicrodigestion and an ultramicrocoulometric assay, based on the reaction of hypobromite with ammonia.

The quantification of nitrogen in organic material is described. It is based on a novel thermal ultramicrodigestion in combination with an ultramicrocoulometric quantification. The lower detection limit of the coulometric measurement is 0.5 microg nitrogen, which corresponds to 20 microg lipid, 3 microg glycine, or 4 microg protein. Therefore it is as sensitive as the frequently used Lowry method. In contrast to the Lowry protein determination it is not disturbed by detergents and most other interfering substances.

Dequalinium vesicles form stable complexes with plasmid DNA which are protected from DNase attack.

Upon sonication, the antimicrobial and antineoplastic compound dequalinium forms vesicles (DQAsomes, Weissig et al., 1998). Dequalinium (1,1'-(1,10-decamethylene-bis-[aminoquinaldinium])-chloride) was shown to be a fluorophore with an emission maximum at 366 nm. Addition of DNA results in a characteristic quenching of its intrinsic fluorescence. After density gradient centrifugation a band of dequalinium (DQA) tightly associated with DNA is located between the DNA and DQA bands. DQA/DNA-complexes containing plasmid DNA at a molar ratio of DQA/DNA 6:1 are completely protected against DNase activity. Addition of negatively-charged lipids release intact DNA in the same manner as from cationic lipid/DNA complexes. As regards biological effects, DQAsomes show a differential cytotoxicity for normal and sarcoma cell lines. In vitro incubation with fluorescein-labeled oligodeoxynucleotides (5'-fluorescein-[GATC]5) showed an increased uptake of the tagged oligodeoxynucleotide if complexed with dequalinium. We hypothesize that the DQA/DNA complexes are well-suited for 'DQAsomal gene transfer' in vitro and in vivo. Noteworthy, they display an intrinsic antitumor activity manifested by differential cytotoxicity for normal and sarcoma cells.

Reconstructed human skin as model for liposome-skin interaction.

Reconstructed human skin was prepared from human keratinoblasts. After 1 week of cultivation at the air-liquid interface a stratified layer developed, similar to native human epidermis. Liposomes with an average diameter of 50 nm, made of phosphatidylcholine (PC), phosphatidylserine (PS) and human stratum corneum lipids (hSCL) were applied on top of this culture system. The rate of penetration through the reconstructed human epidermis was 1.38, 0.55 and 0.013 ng lipidh-1cm-2 for PC, hSCL and PS liposomes, respectively. Electron microscopy and confocal laser scanning microscopy showed that PS and hSCL liposomes aggregated at the skin surface, while PC liposomes remained homogeneously dispersed. Fluorescence measurements demonstrated that vesicles, made of native human stratum corneum lipids rapidly mixed with PS liposomes, weakly with hSCL liposomes and did not mix with PC liposomes.

Interaction of UV light-induced alpha-tocopherol radicals with lipids detected by an electron spin resonance prooxidation effect.

The reaction rate constants of the interaction between light-induced alpha-tocopherol radicals with unsaturated lipids in a heterogeneous system compared to a homogeneous system are of the same order of magnitude. The decay rates of compartmentalized alpha-tocopherol radicals were significantly reduced by using negatively charged sodium dodecyl sulfate (SDS) micelles. A partially resolved electron spin resonance (ESR) hyperfine structure was observed under the conditions of both high lipid concentrations in comparison to the alpha-tocopherol concentration and of a regular distribution of alpha-tocopherol molecules inside the heterogeneous lipid structures. Alpha-tocopherol radicals have a considerable prooxidation potential at higher concentrations. Ascorbic acid dissolved in the aqueous medium provokes very fast alpha-tocopherol radical recycling through the boundary layer between the aqueous medium and micelles. By contrast, very slow reactions such as those of alpha-tocopherol radicals with glutathione through this boundary layer are measurable. Despite using the heterogeneous SDS micellar system, the decay kinetics of the alpha-tocopherol radical ESR signal is simply compounded. In addition to the known stabilization effect of cholesterol in membrane systems, cholesterol itself acts as a target molecule attacked by free radicals, e.g. alpha-tocopherol radicals. Using stratum corneum extracts that contain unsaturated lipids and cholesterol the alpha-tocopherol radical can prooxidatively react with these compounds. Using focused UV light generates a high radical yield in a relatively short time compared to the lifetime of the alpha-tocopherol radicals. The decay processes after radical induction can be characterized as consecutive reactions. The compartmentalization of radicals induced in SDS micelles and the close proximity of target molecules are essential if very slow one-electron reductions are to be measured.

Energy-filtered cryotransmission electron microscopy of liposomes prepared from human stratum corneum lipids.

We used cryo-TEM to examine the morphology of vesicles formed from lipids of the human stratum corneum (hSC). Human stratum corneum lipid liposomes (hSCLLs) were prepared in buffer at various pH values, using different preparation methods (film method, extrusion, ultrasonication, detergent dialysis). The morphology of hSCLLs at pH 7.4 differed markedly from that of liposomes formed by phospholipids, showing folds, stacks and membrane thickening. At pH 5.0, corresponding to natural conditions at the skin surface, membrane structures are essentially the same as those prepared at pH 7.4. Sharp edges in hSCLLs, branching membranes and stable membrane stacks were explained by the presence of ceramides, the major components and structural elements of human stratum corneum lipids (hSCLs). Thickened areas in the membranes may be caused by the local accumulation of triacylglycerols and cholesterol esters in the hydrophobic interior of the bilayer.

Energy-filtered cryotransmission electron microscopy of liposomes prepared from human stratum corneum lipids.

We used cryo-TEM to examine the morphology of vesicles formed from lipids of the human stratum corneum (hSC). Human stratum corneum lipid liposomes (hSCLLs) were prepared in buffer at various pH values, using different preparation methods (film method, extrusion, ultrasonication, detergent dialysis). The morphology of hSCLLs at pH 7.4 differed markedly from that of liposomes formed by phospholipids, showing folds, stacks and membrane thickening. At pH 5.0, corresponding to natural conditions at the skin surface, membrane structures are essentially the same as those prepared at pH 7.4. Sharp edges in hSCLLs, branching membranes and stable membrane stacks were explained by the presence of ceramides, the major components and structural elements of human stratum corneum lipids (hSCLs). Thickened areas in the membranes may be caused by the local accumulation of triacylglycerols and cholesterol esters in the hydrophobic interior of the bilayer.

Aminopeptidase P--a cell-surface antigen of endothelial and lymphoid cells: catalytic and immuno-histotopical evidences.

The physiological function of the GPI-anchored ectoenzyme aminopeptidase P (APP) is still elusive. Most researchers suppose that this enzyme inactivates biologically active peptides like bradykinin, neuropeptide tyrosine (NPY) and others (Vanhoof et al., 1995). We demonstrate by immunohistology with a specific antibody raised in rabbits and measurement of enzymatic activity in suspensions and of confluent monolayers on microscopic coverslips ('monolayer kinetics') that APP is a cell surface enzyme (ectoenzyme) of endothelial and lymphoid cells.

Physicochemical characterisation of human stratum corneum lipid liposomes.

Liposomes were prepared from an extract of all human stratum corneum lipids (hSCL) and characterised in terms of temperature and the presence of Ca2+ by different physicochemical methods. Vesicle aggregation and lateral phase separation were induced by divalent cations with Ca2+ being more efficient than Mg2+. At 24.1 degrees C, i.e. well below physiological temperatures the suspensions consisted of a lamellar phase and crystalline cholesterol. At and above 37 degrees C, this cholesterol surplus was dissolved in the hSCL membranes. However, melting of the hSCL was not completed up to 60 degrees C. The presence of Ca2+ (> or = 9 mM) induced lateral phase separation and fusion of vesicles into extended multilamellar lipid sheets (MLLS) at and above 32.5 degrees C. Upon a subsequent cooling cycle recrystallisation of cholesterol occurred within the MLLS. Finally, membrane mixing of hSCL liposomes with vesicles made of synthetic lipids was investigated. No mixing was observed between either of DPPE/oleic acid, DPPC/DPPE, DPPC/lyso-PC and hSCL liposomes. Mixtures of DPPC/cholesterol hemisuccinate showed a temperature-dependent membrane mixing behaviour, whilst hSCL liposomes and phosphatidylserine liposomes fused temperature-independently with hSCL liposomes.

DQAsomes: a novel potential drug and gene delivery system made from Dequalinium.

PURPOSE: Dequalinium, a drug known for over 30 years, is a dicationic amphiphile compound resembling bolaform electrolytes. The purpose of our work was to determine the state of aggregation of dequalinium in aqueous medium and to investigate both, its ability to bind DNA and its potential to serve as a novel non-viral transfection vector. METHODS: The form of aggregation was determined employing electron microscopic techniques. The DNA binding capacity of dequalinium was assayed using SYBR Green I stain. For in vitro cell transfection experiments plasmid DNA encoding for firefly luciferase was used. RESULTS: Dequalinium forms in aqueous medium liposome-like aggregates, which we term DQAsomes. These dequalinium vesicles bind DNA and they are able to transfect cells in vitro with an efficiency comparable to Lipofectin. CONCLUSIONS: Based on the intrinsic properties of dequalinium such as the in vivo selectivity for carcinoma cells and selective accumulation in mitochondria we propose DQAsomes as a novel and unique drug and gene delivery system.

[Liposomal DNA transfection of human sarcoma cells with p53 alterations]. / Liposomale DNA-Transfektion humaner Sarkomzellen mit p53-Alterationen.

An vital assay allows to optimize liposomal transfection for human tumor cells via FACS. Various cationic lipids were tested to analyse the reporter gene expression (green fluorescent protein, GFP) in different soft tissue sarcoma (STS) cells with known genetic alterations. Furthermore, the cellular uptake of fluorescence-labeled oligodeoxynucleotides (ODN's) was determined. The results obtained with two self-established sarcoma cell lines (LMS6-93, US8-93) were compared with ATCC sarcoma cell lines (Saos-2, A-204, RD) and fibroblast cells. We found maximal 37% cells expressing GFP 24 h post-transfection. All mesenchymal (tumor) cells but not fibroblast cells could be transfected in a cell-specific and lipid-dependent manner. In kinetic studies highest transfection rates were determined between 24 and 48 h, whereas the GFP expression is downregulated after 72 h. Furthermore, we found transfectability is p53 mutation-independent and a relative low toxicity of the new lipids (Lipotaxi and Clonfectin) in comparison to other lipids (Lipofectin, Lipofectamine). By a cell sorting system sarcoma cell lines expressing the reporter gene could be enriched up to 84% of the living cell population. Labeled ODN's were taken up more efficiently (> 90%) when they were mixed with lipids before, but ODN's alone were incorporated into sarcoma cells only in a low percentage (< 10%) and concentration. STS cell cultures showed also a relative high ODN uptake compared with cell lines. We propose the liposomal transfection strategy as an efficient method which can be applied to adherent-growing tumor cells. The method allows simultaneously to study transfection rates, apoptosis and cell cycle alterations in vitro. Furthermore, in future, extension on ex vivo and in vivo transgene expression (xenotransplanted sarcomas) will be evaluated.

Quantitative determination of thiolipids in organic solution, in membranes, and on HPTLC plates.

In the presence of ortho-phthalaldehyde and glucosamine, thiolipids form fluorescent isoindole derivatives. This reaction can be used to quantify single- and double-chain mercaptans in membranes (liposomes) and micellar solutions. The lower detection limit is 100 pmol. In addition, the assay allows the detection of 1.9 nmol thiolipids on HPTLC plates and the fluorescence signal is stable for days. A minor modification of the commonly used DTNB (Ellman's) assay allows the quantification of thiolipids in organic solutions at a concentration down to 3 nmol.

Isolation of ceramide fractions from human stratum corneum lipid extracts by high-performance liquid chromatography.

This report describes a procedure to isolate ceramides from human stratum corneum lipid extracts using preparative liquid chromatography (LC) and semi-preparative high-performance liquid chromatography (HPLC) methods, which is economical in construction and operation. The composition of isolated individual 'peaks' of separated ceramide fractions is reported. These peaks were quantified by scanning of the chromatographic plates stained after high performance thin-layer chromatography (HPTLC). The HPTLC was done by automated multiple development (AMD). The enrichment of the ceramides by liquid chromatography turned out to be necessary for their separation from more polar lipids of the human stratum corneum lipid extract. Only the evaporative light scattering detector gave a stable baseline, reproducible results and eliminated the 'solvent front' in which peaks of interest could coelute. Fluorescence measurements of extracted human stratum lipids indicated lipofuscin-like substances.

Individual variation of human plantar stratum corneum lipids, determined by automated multiple development of high-performance thin-layer chromatography plates.

The stratum corneum lipids are unique in composition and have been used frequently as a model system of the skin's lipid barrier. Automated multiple development (AMD) of high-performance thin-layer chromatography plates in combination with a 25-step gradient, based on methanol, diethyl ether and n-hexane separated the six major human plantar stratum corneum lipids. Post-chromatographic staining of these lipids with a solution of MnCl2-H2SO4 at 130 degrees C or a solution of CuSO4-H3PO4 at 140 degrees C allowed visualization of the lipids and quantification. The MnCl2-H2SO4 solution stained saturated fatty acids less intensely. Therefore, the CuSO4-H3PO4 solution was used for quantification and we found, on average, 2.06% (w/w) cholesterol 3-sulphate, 20.16% (w/w) free fatty acids, 20.25% (w/w) ceramides, 43.53% (w/w) non-esterified sterols, 4.56% (w/w) triacylglycerols and 9.4% (w/w) sterolesters in the human plantar stratum corneum extracts. The concentration of phospholipids was less than 1% (w/w). In addition, the lipid composition of twenty different human plantar stratum corneum extracts was determined. Statistics revealed a correlation between the ratio of free fatty acids and non-esterified sterols (r=0.832, p<0.01, n=20). Several control experiments proved that this correlation is not due to the extraction method, the post-chromatographic staining procedure or bacterial contamination of the stratum corneum.

Oxidative damage of human skin lipids. Dependence of lipid peroxidation on sterol concentration.

Photoprotection against sunburn and associated irradiation-induced damages of the human skin is mainly attributed to the darkening of the biochrome melanin by its oxidation. Human skin lipids were examined for an additional protection by sterols. Lipid vesicles prepared from extracted human skin lipids as well as from mixtures of typical lipids of the stratum corneum were irradiated by UV light in the presence and absence of oxygen. The oxidative degradation of various lipids was measured by quantitative HPTLC, by the dichlorofluorescein fluorescent assay, by the thiobarbituric acid assay and a novel luminol-based chemiluminescence technique. Electron spin resonance was used to look for certain radical intermediates. The results indicate, that sterols, mainly free cholesterol, with their high concentration in the lipid barrier of the stratum corneum (up to 50 mol%) effectively compete with the peroxidation of other human skin lipids (ceramides and free fatty acids).

[Human herpesviruses 6 and 7. Basic principles and possible significance for dermatology]. / Die humanen Herpesviren 6 und 7. Grundlagen und mögliche Bedeutung für die Dermatologie.

Primary infection with human herpesviruses 6 and 7 (HHV-6 and HHV-7) during early childhood causes permanent latent infection, usually without any ill effects; only a small percentage of primary infections will lead to exanthem subitum. Like other herpesviruses. HHV-6 and HHV-7 can be reactivated at any time if host defence mechanisms become defective (e.g. in transplant recipients, AIDS, tumour patients). HHV-6 can be reactivated under such conditions and cause a variety of clinical problems, such as exanthems along with interstitial pneumonia or hepatitis for example. In addition, the reactivated virus may influence the course of autoimmune and proliferative diseases such as systemic lupus erythematosus and Hodgkin's disease. While, HHV-7 may be associated with similar disorder, more systematic studies are needed to clarify the clinical implications and the pathogeetic mechanisms of both viruses.

Interaction of phosphatidylcholine liposomes with the human stratum corneum.

The interaction of dimyristoylphosphatidylcholine liposomes with the human stratum corneum was investigated by confocal laser scanning microscopy and differential scanning calorimetry. Human skin is characterized by a high autofluorescence. By introducing appropriate optical filters the autofluorescence of the skin was depressed and the penetration profile of fluorescence labelled vesicles was investigated. From optical sectioning it was obvious that neither the vesicles nor the fluorophore N-(lissamine rhodamine B sulfonyl)diacylphophatidylethanolamine (Rho-PE) penetrates in detectable amounts into the human skin. Differential scanning calorimetry of human stratum corneum revealed, that the peak positions of the human stratum corneum specific endothermic transitions at 10 degrees C, 35 degrees C, 50 degrees C, 62 degrees C, 73 degrees C and 81 degrees C did not change significantly after 18 h of non-occlusive vesicle application. However, the enthalpy of the transitions at 35 degrees C, 50 degrees C, 62 degrees C and 73 degrees C, estimated through peak heights increased, relative to the protein related peak at 81 degrees C. A novel transition at 10 degrees C was observed. From these data we conclude that DMPC liposomes do not penetrate intact into the human skin. We deduce, however, that the vesicles disintegrate at the surface of stratum corneum after non-occlusive application. The individual lipid molecules then interact with the lipid barrier of the stratum corneum and penetrate into the latter, which results in an increase of the enthalpy, related to the lipid components of the SC.