[Show abstract][Hide abstract]ABSTRACT: Membrane interactions of liposomes of ternary phospholipid/cholesterol bilayers are investigated. These interactions lead to discoidal deformations and regular aggregations and are strongly enhanced by the presence of mistletoe lectin (ML), a RIP II type protein. The encapsulation of ML into liposomal nanocapsules is studied with a systematic variation of the lipid composition to monitor its effect on the physical properties: entrapment, mean size, morphology, and stability. Extrusion of multilamellar vesicles through filters 80 nm pore size was used for the generation of liposomes. The mean sizes of liposomes ranged between 120 and 200 nm in diameter with narrow size distributions. The increase in flow rate with pressure for three dioleoylphosphatidylcholine (DOPC)/cholesterol (Chol)/dipalmitoylphosphatidylcholine (DPPC) lipid mixtures was linear and allowed to extrapolate to the minimum burst pressure of the liposomal bilayers. From the minimum pressures P(min), the bilayer lysis tensions gamma(l) were determined. The increase in P(min) and gamma(l) with an increasing content of a saturated phosopholipid (DPPC) indicates that DPPC increases the mechanical strength of lipid bilayers. Apparently, DPPC, like cholesterol, leads to a less compressible surface and a more cohesive membrane. After preparation, vesicle solutions were purified by gel permeation chromatography to separate encapsulated ML from free ML in the extravesicular solution. Purified liposomes were then characterized. The content of entrapped and adsorbed ML was measured using ELISA. Repetitive freezing/thawing cycles prior to extrusion significantly increased ML uptake. On the contrary, adsorption was not affected neither by lipid composition, nor concentration and preparation. Differences in experimental encapsulation efficiency only reflect the differences in the mean vesicle sizes of the different samples as is revealed by a comparison to a theoretical estimate. Cryo-transmission electron microscopy (Cryo-TEM) images show that beside spherical, single-walled liposomes, there is a considerable fraction of discoidally deformed vesicles. Based on our results and those found in the literature, we speculate that the flattening of the vesicles is a consequence of lipid phase separation and the formation of condensed complexes and areas of different bending elasticities. This phenomenon eventually leads to agglomeration of deformed liposomal structures, becoming more pronounced with the increase in the relative amount of saturated fatty acids, presumably caused by hydrophobic interaction. For the same lipid mixture aggregation correlated linearly with the ML content. Finally, tested liposomal samples were kept at 4 degrees C to examine their stability. Only slight fluctuations in diameter and the increase in polydispersity after 3 weeks of storage occurred, with no statistically significant evidence of drug leakage during a time period of 12 days, illustrating physical stability of liposomes.
Article · Aug 2008 · Colloids and surfaces B: Biointerfaces
[Show abstract][Hide abstract]ABSTRACT: Extracts of Viscum album L. produced by a specific homogenization procedure contain viscotoxins (VT) and liposome-like membrane vesicles, formed from cellular membranes. Interactions between these membrane structures and viscotoxins are characterized in this work. Binding properties of viscotoxins with mistletoe extracts or isolated membrane vesicles were analyzed by gel permeation chromatography (GPC) and centrifugation, followed by HPLC/UV for viscotoxin detection. The experiments show that a part of the viscotoxins is bound to membrane vesicles, and that this binding to the membrane structures is reversible. In the case of the vesicles studied from an extract of 100 mg plant material per mL (0.30 mM phospholipids, 244 microg/mL VT), 64 microg/mL VTs are bound to the membranes. The binding properties of the viscotoxin isoforms are different. VTA3 clearly binds more intensively to membrane structures than VTA2 or VTA1. Possible interaction of viscotoxins with DNA, which is also discussed as a mechanism of viscotoxin action, could be shown to be negligible in the framework of these experiments.
[Show abstract][Hide abstract]ABSTRACT: During the preparation of plant extracts by a press-slit technique, membranes of cell walls and cell organelles of the plant material form vesicles, which are colloidally dispersed. It was assumed that chlorophyll-containing green extracts enclose lipoidic structures. Vesicles in aqueous mistletoe extracts (extracts of Viscum album L.) were analyzed by cryo-transmission electron microscopy (cryo-TEM) without fixation. For the first time, it was possible to analyze unfixed vesicles in the mistletoe extract. Micrographs of cryo-TEM showed predominantly unilamellar vesicles of different sizes. The quantification of vesicles was established through the analysis of phospholipids, which are major components of membranes. The method was validated mainly according to ICH guidelines for the validation of analytical methods (Q2A and Q2B). For further characterization of the vesicle size, a method was developed which is based on the separation of the vesicles from low molecular weight substances by size exclusion chromatography. Fractions were collected and average sizes were determined by multi-angle laser light scattering (MALLS). Furthermore, the UV-vis absorbance and phospholipid concentration were analyzed. Phospholipid quantification was in agreement with photometrical data. Sizes determined by cryo-TEM and by light scattering showed consistent results.
Article · Nov 2005 · Colloids and surfaces B: Biointerfaces