Helmut W. Meyer

DQAsomes: A Novel Potential Drug and Gene Delivery System Made from Dequalinium™

AbstractPurpose. 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.

Small-angle X-ray scattering and electron microscopy of crude dispersions of swelling lipids and the influence of the morphology on the repeat distance

From X-ray measurements in different lipid systems it is concluded that the repeat distance measured in lipids with limited swelling in the lamellar liquid crystalline state exhibits qualitatively the same dependence on the water concentration. Four regions of water concentrations with different structural and morphological changes can be distinguished. Their existence is qualitatively explained. The morphology of samples with water concentrations near the boundary between the single lamellar Lα phase and the two phase region α + water has a drastic influence on the repeat distance measured. A procedure of how this boundary can be determined is proposed.

The protective effect of free and membrane-bound cryoprotectants during freezing and freeze-drying of liposomes

Liposomes were prepared from natural (EPC) and hydrogenated (HEPC) egg phosphatidylcholine, with and without cholesterol (CHOL), from sucrose fatty acid ester (SPS7; sucrose-palmitate/stearate) with CHOL and dicetylphosphate (DCP) or from EPC and HEPC with the mono-, di- and tri-ester of SPS7. The cryoprotective activity of sucrose or membrane-bound sucrose fatty esters was assessed. Vesicles were frozen and thawed, or freeze-dried and reconstituted, and retention of the encapsulated marker 5,6-carboxyfluorescein (CF) was monitored. CF retentio n decreased with decreasing freezing temperature, while increasing concentrations of sucrose provided increasing cryoprotection during freezing and thawing. SPS7 vesicles were fully protected by 0.6 M sucrose, whereas equimolar mixtures of EPC and HEPC with SPS7 required 1 M sucrose for complete protection. EPC/CHOL liposomes retained maximally 85% and HEPC/CHOL liposomes 95% marker at the highest sucrose concentration. Lyophilized liposomes without sucrose or in mixture with the SPS mono- or diester retained <10% CF. Lyophilization of EPC and HEPC liposomes in the presence of 0.4 M sucrose resulted in 75% retention of originally encapsulated marker. Differential scanning calorimetry showed a significant reduction of the transition temperature (Tc) of lyophilized HEPC liposomes in the presence of sucrose and the SPS monoester. Infrared spectroscopy indicated sucrose and the SPS monoester forming strong hydrogen bonds with phosphate head groups which supports the water replacement of ‘pseudohydration’ hypothesis.

Ultrastructural characterization of peptide-induced membrane fusion and peptide self-assembly in the lipid bilayer.

The peptide sequence B18, derived from the membrane-associated sea urchin sperm protein bindin, triggers fusion between lipid vesicles. It exhibits many similarities to viral fusion peptides and may have a corresponding function in fertilization. The lipid-peptide and peptide-peptide interactions of B18 are investigated here at the ultrastructural level by electron microscopy and x-ray diffraction. The histidine-rich peptide is shown to self-associate into two distinctly different supramolecular structures, depending on the presence of Zn(2+), which controls its fusogenic activity. In aqueous buffer the peptide per se assembles into beta-sheet amyloid fibrils, whereas in the presence of Zn(2+) it forms smooth globular clusters. When B18 per se is added to uncharged large unilamellar vesicles, they become visibly disrupted by the fibrils, but no genuine fusion is observed. Only in the presence of Zn(2+) does the peptide induce extensive fusion of vesicles, which is evident from their dramatic increase in size. Besides these morphological changes, we observed distinct fibrillar and particulate structures in the bilayer, which are attributed to B18 in either of its two self-assembled forms. We conclude that membrane fusion involves an alpha-helical peptide conformation, which can oligomerize further in the membrane. The role of Zn(2+) is to promote this local helical structure in B18 and to prevent its inactivation as beta-sheet fibrils.

Hydration of DMPC and DPPC at 4°C produces a novel subgel phase with convex–concave bilayer curvatures

Abstract Hydration of dimyristoyl- and dipalmitoylphosphatidylcholines at 4°C results in the formation of a characteristic subgel phase designated Pcc. Examination of the phase by freeze-fracture electron microscopy shows convex–concave deformations of the planar bilayer which are of two types. A smaller type with a radius of curvature of about 20 nm predominates in DMPC, and a larger type with about 70 nm radii of curvatures dominates in DPPC. The Pcc phase can also be formed in samples hydrated at temperatures above the main phase transition if the dispersion is frozen slowly and subsequently incubated at 4°C for several days. The subgel Pcc phase was distinguished from the subgel Lc phase by the temperature of transition, packing of the acyl chains on the basis of wide-angle X-ray diffraction, and 2H-NMR spectra characteristic of a ‘solid-ordered’ phase. Vibrational spectra of the carbonyl and phosphate regions are consistent with a partially reduced hydration state. The origin of the convex–concave bilayer deformation is believed to result from constraints imposed by limiting hydration of the headgroup and a frustration arising from the spontaneous curvature of both monolayers.

Pretransition-ripples in bilayers of dipalmitoylphosphatidylcholine: undulation or periodic segments? A freeze-fracture study

Freeze-fracture analysis of ripple structures of 1,2-dipalmitoylphosphatidylcholine bilayers leads to the conclusion that the asymmetric ripple is the basic structure formed by periodic segments with different tilt direction. The molecules are tilted by about 30 degrees from the bilayer normal but arranged in two positions. Symmetric ripples are also formed by an alternation in tilt direction of the segments but the succession is more complex. A ridge in their valleys or a cleft at their crests may indicate structures formed or deformed during preparation (replication, etching). The freeze-fracture method reveals transition structures in ripple formation which are helpful in interpretation, but does not support a model consisting of an undulation of the bilayer by periodic fluid-like and gel-like domains.

Morphological transitions of brain sphingomyelin are determined by the hydration protocol: ripples re-arrange in plane, and sponge-like networks disintegrate into small vesicles

The phase transition of hydrated brain sphingomyelin occurs at around 35 degrees C, which is close to the physiological temperature. Freeze-fracture electron microscopy is used to characterize different gel state morphologies in terms of solid-ordered and liquid-ordered phase states, according to the occurrence of ripples and other higher-dimensional bilayer deformations. Evidently, the natural mixed-chain sphingomyelin does not assume the flat L beta, phase but instead the rippled P beta, phase, with symmetric and asymmetric ripples as well as macroripples and an egg-carton pattern, depending on the incubation conditions. An unexpected difference was observed between samples that are hydrated above and below the phase transition temperature. When the lipid is hydrated at low temperature, a sponge-like network of bilayers is formed in the gel state, next to some normal lamellae. The network loses its ripples during cold-incubation, which indicates the formation of a liquid-ordered (lo) gel phase. Ripples re-appear upon warming and the sponge-like network disintegrates spontaneously and irreversibly into small vesicles above the phase transition.

The effect of sterols on structures formed in the gel/subgel phase state of dipalmitoylphosphatidylcholine bilayers

The effect of cholesterol and lanosterol on the formation of structures in the gel/subgel phase of 1,2-dipalmitoylphosphatidylcholine was investigated using freeze-fracture electron microscopy and X-ray diffraction. Mixtures with up to 25 mol% sterol were analysed after annealing for between several days and some months at 4 degrees C. Bilayers of DPPC with 5 or 10 mol% sterol showed a domain structure in the gel state. There are rounded or lens-like and often inclined plates within less smooth either plane or striped bilayer areas. The stripes are formed by parallel lines separated by a distance of 30-60 nm. Parallel lines can be induced also in the less smooth but plane areas by warming up to 25 degrees C. X-ray diffraction showed two lamellar repeat spacings at 6.45 and 8.3 nm in both 5 and 10 mol% samples. The plates are interpreted as domains of (nearly) pure DPPC within the sterol containing bilayer. Stripes are present if the concentration of cholesterol is below a critical value (approx. 15 mol%). With time of incubation at 4 degrees C curved deformations appear in parts of the bilayers. Two main types are formed. The small type has a repeat distance of about 100 nm and the large type of about 400 nm. The curved deformations were progressively flattened by warming up to 25-32 degrees C with an accompanying reappearance of stripes but no plates. After prolonged annealing at 4 degrees C there is also the formation of regular ripples. It is concluded that in presence of 5 and 10 mol% sterol in bilayers of 1,2-dipalmitoy-phosphatidylcholine the immiscibility of gel phase and subgel phase changes during prolonged annealing at 4 degrees C. We assume a rearrangement of the molecules into a homogeneous phase state with liquid-ordered properties.

Macroripple-structures induced by different branched-chain phosphatidylcholines in bilayers of dipalmitoylphosphatidylcholine

Abstract Branched-chain phosphatidylcholines with four chains of the length C16, C18 and C20 have been synthesized by dibranching positions C2 or C4 of the acyl chains. Macroripples have been observed by freeze-fracture electron microscopy at concentrations of 5–30 mol% branched-chain phospholipids in DPPC. Macroripples differ from pretransition ripples in structure and dimension but also in their behaviour during incubation at low temperatures for a longer time. Macroripples may survive incubation at 4°C for 14 weeks. Smaller macroripples with initial periodicity of 30–50 nm may widen to twice the primary spacing during this incubation. Macroripples are commonly accompanied by parallel stripes. The stripes, which are most prominent at the crests of the macroripples, are seen on the bilayer fracture-face but not at the bilayer surface revealed by etching. Parallel stripes can also exist without tilting of the bilayer into macroripples. We suggest that phase separation or at least partial demixing of lipids may be a prerequisite for formation of macroripples. The macroripples have a tendency for a second tilting perpendicular to the main tilting. This effect was characteristic for the C2-branched phosphatidylcholines with C18- and C20-chains. A dramatic change from normal straight macroripples into those with second tilt was induced by high-pressure freezing of multilamellar vesicles with C4-branched phosphatidylcholines. The appearance of macroripples was more dependent on chain length than on the location of branching. Branched C16-chains having the same chain length as DPPC were most effective in inducing macroripples. An elongation of the chains by two C-atoms reduced macroripples more than an elongation by four C-atoms. Both types of elongation resulted in a restriction because macroripples are present only in parts of the lamellae. A speciality of the elongated branched-chain phosphatidylcholines is the predominance of macroripples with large periodicities (100 nm and more).

Electron microscopic demonstration of cell surface carbohydrates by means of peroxidase and ferritin complexes of the Lens culinaris lection.

SummaryThe use of Lens culinaris lectin for electron microscopic detection of D-mannose, D-glucose and N-acetyl-D-glucosamine like sites on tumor cells, erythrocytes, erythrocyte ghosts, cultured rat liver cells and various tissues of mice is demonstrated. In addition to Lens culinaris lectin-peroxidase reaction (LcL-po reaction) the preparation of active Lens culinaris lectin-ferritin conjugate are described and the specificity of cytochemical reactions are demonstrated. Furthermore experiments by immuno freeze-etching are reported for topological analysis of the lectin receptors.