Elevated Cellular Uptake of Succinimide- and Glucose-Modified Liposomes for Blood-Brain Barrier Transfer and Glioblastoma Therapy.

The uptake of four liposomal formulations was tested with the murine endothelial cell line bEnd.3 and the human glioblastoma cell line U-87 MG. All formulations were composed of DPPC, cholesterol, 5 mol% of mPEG (2000 Da, conjugated to DSPE), and the dye DiD. Three of the formulations had an additional PEG chain (nominally 5000 Da, conjugated to DSPE) with either succinimide (NHS), glucose (PEG-bound at C-6), or 4-aminophenyl ß-D-glucopyranoside (bound at C-1) as ligands at the distal end. Measuring the uptake kinetics at 1 h and 3 h for liposomal incubation concentrations of 100 µM, 500 µM, and 1000 µM, we calculated the liposomal uptake saturation S and the saturation half-time t1/2. We show that only succinimide has an elevated uptake in bEnd.3 cells, which makes it a very promising and so far largely unexplored candidate for BBB transfer and brain cancer therapies. Half-times are uniform at low concentrations but diversify for high concentrations for bEnd.3 cells. Contrary, U-87 MG cells show almost identical saturations for all three ligands, making a uniform uptake mechanism likely. Only mPEG liposomes stay at 60% of the saturation for ligand-coated liposomes. Half-times are diverse at low concentrations but unify at high concentrations for U-87 MG cells.

Increased Cellular Uptake of ApoE3- or c(RGD)-Modified Liposomes for Glioblastoma Therapy Depending on the Target Cells.

As effective treatment of glioblastoma is still an unmet need, targeted delivery systems for efficient treatment are of utmost interest. Therefore, in this paper, surface modifications with a small peptide c(RGD) or physiological protein (ApoE3) were investigated. Cellular uptake in murine endothelial cells (bEnd.3) and different glioma cells (human U-87 MG, rat F98) was tested to elucidate possible differences and to correlate the uptake to the receptor expression. Different liposomal formulations were measured at 1 and 3 h for three lipid incubation concentrations. We calculated the liposomal uptake saturation S and the saturation half-time t1/2. An up to 9.6-fold increased uptake for ApoE3-modified liposomes, primarily in tumor cells, was found. Contrarily, c(RGD) liposomes showed a stronger increase in uptake in endothelial cells (up to 40.5-fold). The uptake of modified liposomes revealed enormous differences in S and t1/2 when comparing different tumor cell lines. However, for ApoE3-modified liposomes, we proved comparable saturation values (~25,000) for F98 cells and U-87 MG cells despite a 6-fold lower expression of LRP1 in F98 cells and a 5-fold slower uptake rate. Our findings suggest that cellular uptake of surface-modified liposomes depends more on the target structure than the ligand type, with significant differences between cell types of different origins.

Emulsifying mechanisms of phospholipids in high-pressure homogenization of perfluorocarbon nanoemulsions.

Phospholipids are the most ubiquitous emulsifiers in foods, beverages, pharmaceuticals, and human physiology, but their emulsifying properties are extremely complex. Differential analyses of mechanisms contributing to their functionality are presented in a modular approach. Addition of cholesterol to a natural phospholipid blend disturbs emulsification beyond specific thresholds for size, polydispersity and formation of emulsifying monolayers. Beyond a ratio of lipid concentration to dispersed volume of 1 mM per 1% (v/v) of perfluorocarbon (PFC), phospholipids no longer form monolayers but instead form triple layers that emulsify the PFC. Using synthetic saturated phospholipids, it can be shown that emulsification is most successful for fatty acids closely below their main transition temperature. Phospholipid head groups are more effective for emulsification the more they increase the area per molecule or the zeta potential. Including a comparison with literature results, it can be shown that high molecular weight emulsifiers like proteins are not dependent on the ratio of viscosities η of the dispersed phase to the continuous phase, ηD/ηC. In contrast, smaller molecular weight emulsifiers like phospholipids show a mild increase in effectiveness with rising ηD/ηC, although this increase is not as strong as that observed for low molecular weight detergents. Ruptures of highly resistant emulsifying interfacial layers obviously lead to direct droplet break-up, irrespective of the resistance of a high-viscosity droplet. The lower the break-up resistance of an emulsifier, the more is it governed by the bulk viscosity of the dispersed phase. Our results allow the preparation of a phospholipid-stabilized emulsion with optimized emulsification settings for pharmaceutical applications.

Regulation of the innate immune system by fragmented heparin-conjugated lipids on lipid bilayered membranes in vitro.

Surface modification with heparin is a powerful biomaterial coating strategy that protects against innate immunity activation since heparin is a part of the proteoglycan heparan sulfate on cell surfaces in the body. We studied the heparinization of cellular and material surfaces via lipid conjugation to a heparin-binding peptide. In the present study, we synthesized fragmented heparin (fHep)-conjugated phospholipids and studied their regulation of the innate immune system on a lipid bilayered surface using liposomes. Liposomes have versatile applications, such as drug-delivery systems, due to their ability to carry a wide range of molecules. Owing to their morphological similarity to cell membranes, they can also be used to mimic a simple cell-membrane to study protein-lipid interactions. We investigated the interaction of complement-regulators, factor H and C4b-binding protein (C4BP), as well as the coagulation inhibitor antithrombin (AT), with fHep-lipids on the liposomal surface. Herein, we studied the ability of fHep-lipids to recruit factor H, C4BP, and AT using a quartz crystal microbalance with dissipation monitoring. With dynamic light scattering, we demonstrated that liposomes could be modified with fHep-lipids and were stable up to 60 days at 4 °C. Using a capillary western blot-based method (Wes), we showed that fHep-liposomes could recruit factor H in a model system using purified proteins and assist in the degradation of the active complement protein C3b to iC3b. Furthermore, we found that fHep-liposomes could recruit factor H and AT from human plasma. Therefore, the use of fHep-lipids could be a potential coating for liposomes and cell surfaces to regulate the immune system on the lipid surface.

Monolayer/Bilayer Equilibrium of Phospholipids in Gel or Liquid States: Interfacial Adsorption via Monomer or Liposome Diffusion?

Phospholipids (PLs) are widely used in the pharma industry and a better understanding of their behavior under different conditions is helpful for applications such as their use as medical transporters. The transition temperature Tm affects the lipid conformation and the interfacial tension between perfluoroperhydrophenanthrene (PFP) and an aqueous suspension of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphatidylcholine (DSPC), as well as a mixture of these PLs with cholesterol. Interfacial tensions were measured with the Du Noüy ring at quasi-equilibrium; the area per molecule was calculated according to the Gibbsian approach and a time-dependent tension gradient. Results show that the time tε to reach quasi-equilibrium was shorter when the temperature was above Tm, indicating a faster adsorption process (tε,DPPC,36 °C = 48 h, tε,DPPC,48 °C = 24 h) for PL in the liquid crystalline state than in the gel state (T < Tm). In addition, concentration-dependent results of the interfacial tension revealed that above the respective Tm and at all concentrations c > 0.1 mM, the average minimum interfacial tension for DPPC and DSPC (14.1 mN/m and 15.3 mN/m) does not differ significantly between those two lipids. Equilibrium between monolayers and bilayers shows that for T < Tm, surface pressures ∏ ≈ 31 mN/m are reached while for T > Tm, ∏ ≈ 41 mN/m. Mixtures with cholesterol only reach ∏ ≤ 31 mN/m Tm, with no significant difference between the two PLs. The higher interfacial tension of the mixture indicates stabilization of the liposomal conformation in the aqueous phase by the addition of cholesterol. The high diffusion coefficients show that adsorption is mainly based on liposomes.

The use of liposomes functionalized with the NFL-TBS.40-63 peptide as a targeting agent to cross the in vitro blood-brain barrier and target glioblastoma cells.

Glioblastoma is the most common and aggressive brain tumor. Current treatments do not allow to cure the patients. This is partly due to the blood-brain barrier (BBB), which limits the delivery of drugs to the pathological site. To overcome this, we developed liposomes functionalized with a neurofilament-derived peptide, NFL-TBS.40-63 (NFL), known for its highly selective targeting of glioblastoma cells. First, in vitro BBB model was developed to check whether the NFL can also promote barrier crossing in addition to its active targeting capacity. Permeability experiments showed that the NFL peptide was able to cross the BBB. Moreover, when the BBB was in a pathological situation, i.e., an in vitro blood-brain tumor barrier (BBTB), the passage of the NFL peptide was greater while maintaining its glioblastoma targeting capacity. When the NFL peptide was associated to liposomes, it enhanced their ability to be internalized into glioblastoma cells after passage through the BBTB, compared to liposomes without NFL. The cellular uptake of liposomes was limited in the endothelial cell monolayer in comparison to the glioblastoma one. These data indicated that the NFL peptide is a promising cell-penetrating peptide tool when combined with drug delivery systems for the treatment of glioblastoma.

How to Achieve High Encapsulation Efficiencies for Macromolecular and Sensitive APIs in Liposomes.

This research highlights the capacity of a newly introduced centrifugation process to form liposomes from water-in-fluorocarbon nano-emulsions stabilized with phospholipids to incorporate macromolecular and sensitive active pharmaceutical ingredients (API). The encapsulation efficiency of the produced liposomes, incorporating fluorescein-sodium, bovine serum albumin and fluorecein isothiocyanate dextran as model APIs, is determined by applying Vivaspin® centrifugation filtration and quantified by UV-Vis spectroscopy. It was found that higher densities of the fluorocarbons used as the hydrophobic phase enable a higher encapsulation efficiency and that an efficiency of up to 98% is possible depending on the used phospholipid. Among the engineering aspects of the process, a comparison between different membrane substances was performed. Efficiency increases with a higher phospholipid concentration but decreases with the addition of cholesterol. Due to the higher bending modulus, liposome formation is slowed down by cholesterol during liposome closure leading to a greater leakage of the model API. The encapsulation of bovine serum albumin and dextran, both investigated under different osmotic conditions, shows that an efflux negatively affects the encapsulation efficiency while an influx increases the stability. Overall, the process shows the potential for a very high encapsulation efficiency for macromolecules and future pharmaceutical applications.

Liposomes with asymmetric bilayers produced from inverse emulsions for nucleic acid delivery.

Asymmetrical lipid nanoparticles are interesting nanocarriers for charged molecules, like nucleic acids. They promise control over inner and outer charge. High charge density on the inside is favourable for efficient condensation and charge neutralisation of highly charged biopharmaceuticals, while a neutral or slightly negative outer layer promotes biocompatibility. The main goal of this work was the development and characterisation of asymmetric liposomes, prepared using water-in-oil (w/o) nanoemulsions of phospholipids (PLs) and squalene in a centrifugal field. This method enables the control over the lipid composition of each monolayer. Liposomes were prepared by passing PL w/o nanoemulsions through an oil-water interface previously saturated with PLs. We used N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (NBD-PE) or N-(7-Nitrobenz-2-Oxa-1,3-Diazol-4-yl)-1,2-Dihexadecanoyl-sn-Glycero-3- phosphocholine (NBD-PC) as a fluorescent marker for either the inner or outer lipid layer and plasmid DNA (pDNA) as nucleic acid payload. The final liposomes had sizes below 200 nm and polydispersity indexes of 0.3 and had a bilayer asymmetry of 70%, thus shielding the charge of positive PLs in the inner bilayer leaflet. Final formulations were examined using negative staining transmission electron microscopy (TEM). Plasmid encapsulation efficiency of the method was 10-15%. Our results indicate that the w/o nanoemulsion-centrifugation method allows the successful production of liposomes with tailored features for encapsulation of nucleic acid therapeutics.

Thermosensitive liposomes for triggered release of cytotoxic proteins.

Lysolipid-containing thermosensitive liposomes (LTSL) are clinically-relevant drug nanocarriers which have been used to deliver small molecule cytostatics to tumors in combination with local hyperthermia (42 °C) to trigger local drug release. The objective of this study was to investigate the feasibility of LTSL for encapsulation and triggered release of macromolecular drugs such as plant-derived cytotoxins. As therapeutic protein we used Mistletoe lectin-1 (ML1) - a ribosome-inactivating protein with potent cytotoxic activity in tumor cells. Model macromolecules (dextrans, albumin) and ML1 were encapsulated in small unilamellar LTSL with varying lipid compositions by the thin film hydration method and extrusion. LTSLs showed molecular weight dependent heat-triggered release of the loaded cargo. The most promising composition, ML1 formulated in LTSL composed of 86:10:4 %mol DPPC:MSPC:DSPE-PEG2000, was further studied for bioactivity against murine CT26 colon carcinoma cells. Confocal live-cell imaging showed uptake of released ML1 after mild hyperthermia at 42 °C, subsequently leading to potent cytotoxicity by LTSL-ML1. Our study shows that LTSL in combination with localized hyperthermia hold promise as local tumor delivery strategy for macromolecular cytotoxins.

Adsorption of phospholipids at oil/water interfaces during emulsification is controlled by stress relaxation and diffusion.

Adsorption of phosphatidylcholines at oil/water interfaces strongly deviates from spread monolayers at air/water surfaces. Understanding its nature and consequences could vastly improve applications in medical nanoemulsions and biotechnologies. Adsorption kinetics at interfaces of water with different oil phases were measured by profile analysis tensiometry. Adsorption kinetics for 2 different phospholipids, DPPC and POPC, as well as 2 organic phases, squalene and squalane, show that formation of interfacial monolayers is initially dominated by stress-relaxation in the first minutes. Diffusion only gradually contributes to a decrease in interfacial tension at later stages of time and higher film pressures. The results can be applied for the optimization of emulsification protocols using mechanical treatments. Emulsions using phospholipids with unsaturated fatty acids are dominated much more strongly by stress-relaxation and cover interfaces very fast compared to those with saturated fatty acids. In contrast, phospholipid layers consisting of saturated fatty acids converge faster towards the equilibrium than those with unsaturated fatty acids.

Instability Mechanisms of Water-in-Oil Nanoemulsions with Phospholipids: Temporal and Morphological Structures.

Many food preparations, pharmaceuticals, and cosmetics use water-in-oil (W/O) emulsions stabilized by phospholipids. Moreover, recent technological developments try to produce liposomes or lipid coated capsules from W/O emulsions, but are faced with colloidal instabilities. To explore these instability mechanisms, emulsification by sonication was applied in three cycles, and the sample stability was studied for 3 h after each cycle. Clearly identifiable temporal structures of instability provide evidence about the emulsion morphology: an initial regime of about 10 min is shown to be governed by coalescence after which Ostwald ripening dominates. Transport via molecular diffusion in Ostwald ripening is commonly based on the mutual solubility of the two phases and is therefore prohibited in emulsions composed of immiscible phases. However, in the case of water in oil emulsified by phospholipids, these form water-loaded reverse micelles in oil, which enable Ostwald ripening despite the low solubility of water in oil, as is shown for squalene. As is proved for the phospholipid dipalmitoylphosphatidylcholine (DPPC), concentrations below the critical aggregation concentration (CAC) form monolayers at the interfaces and smaller droplet sizes. In contrast, phospholipid concentrations above the CAC create complex multilayers at the interface with larger droplet sizes. The key factors for stable W/O emulsions in classical or innovative applications are first, the minimization of the phospholipids' capacity to form reversed micelles, and second, the adaption of the initial phospholipid concentration to the water content to enable an optimized coverage of phospholipids at the interfaces for the intended drop size.

Interactions between Phospholipids and Organic Phases: Insights into Lipoproteins and Nanoemulsions.

The adsorption of phosphatidylcholines (PCs), dissolved in squalene or squalane as an organic phase, was studied at the interface with water. Using profile analysis tensiometry, the equilibrium adsorption isotherms, minimum molecular interfacial areas, and solubility limits were derived. For squalene, differences in PC solubility and interfacial adsorption were found, depending on PC saturation. Compared to saturated PCs, unsaturated PCs showed a 3-fold-lower interfacial density but up to a 28-fold-higher critical aggregation concentration (CAC). In addition, the solubility limit of unsaturated PC in squalene and in its saturated form squalane diverged by a factor of 739. These findings provided evidence for steric repulsion or π-π interactions of π bonds in both solvent and solute or both effects acting complementarily. In squalane, low solubilities but high interfacial densities were found for all investigated PCs. Changes in fatty acid chain lengths showed that the influence of the increases in entropy and enthalpy on solubility is much smaller than solvent/solute interactions. Oxidation products of squalene lowered the interfacial tension, but increasing concentrations of PC expelled them from the interface. The CAC of saturated PC was increased by oxidation products of squalene whereas that of unsaturated PCs was not. Our findings indicate that the oxidation of triglycerides in lipoprotein cores can lead to increased solubility of saturated phospholipids covering the lipoproteins, contributing to destabilization, coalescence, and terminally the formation of atherosclerotic plaques. The consideration of solvent/solute interactions in molecular modeling may contribute to the interfacial tension and the corresponding kinetic or thermodynamic stability of lipoproteins. Measured areas per molecule prove that PCs form monolayers of different interfacial densities at the squalene/water interface but multilayers at the squalane/water interface. These findings showed that combinations of solvent or solute saturation affect the outcome for nanoemulsions forming either expanded or condensed monolayers or multilayers.

Development and characterization of an innovative heparin coating to stabilize and protect liposomes against adverse immune reactions.

Liposomes have been recognized as excellent drug delivery systems, but when they come in direct contact with different blood components they may trigger an immediate activation of the innate immune system. The aim of the present study was to produce long-circulating, blood-compatible liposomes by developing a construct of liposomes covered by a novel unique heparin complex (CHC; 70 heparin molecules per complex) to avoid recognition by the innate immune system. Unilamellar, cationic liposomes were produced by hand extrusion through a 100-nm polycarbonate membrane. Coating of liposomes with the macromolecular CHC was accomplished by electrostatic interactions. Dynamic light scattering as well as QCM-D measurements were used to verify the electrostatic deposition of the negatively charged CHC to cationic liposomes. The CHC-coated liposomes did not aggregate when in contact with lepirudin anti-coagulated plasma. Unlike previous attempts to coat liposomes with heparin, this technique produced freely moveable heparin strands sticking out from the liposome surface, which exposed AT binding sites reflecting the anticoagulant potentials of the liposomes. In experiments using lepirudin-anticoagulated plasma, CHC-coated liposomes, in contrast to non-coated control liposomes, did not activate the complement system, as evidenced by low C3a and sC5b-9 generation and reduced leakage from the liposomes. In conclusion, we show that liposomes can be successfully coated with the biopolymer CHC, resulting in biocompatible and stable liposomes that have significant application potential.

Mediation of a non-proteolytic activation of complement component C3 by phospholipid vesicles.

Liposomes are becoming increasingly important as drug delivery systems, to target a drug to specific cells and tissues and thereby protecting the recipient from toxic effects of the contained drug. Liposome preparations have been described to activate complement. In this study, we have investigated complement activation triggered by neutral dimyristoyl-phosphocholine (DMPC) liposomes in human plasma and whole-blood systems. Incubation in plasma led to the generation of complement activation products (C3a and sC5b-9). Unexpectedly, investigations of surface-bound C3 revealed contact activated, conformationally changed C3 molecules on the liposomes. These changes were characterized by Western blotting with C3 monoclonal antibodies, and by incubating liposomes with purified native C3 and factors I and H. Quartz crystal microbalance analysis confirmed binding of C3 to planar DMPC surfaces. In addition, we demonstrated that DMPC liposomes bound to or were phagocytized by granulocytes in a complement-dependent manner, as evidenced by the use of complement inhibitors. In summary, we have shown that C3 is activated both by convertase-dependent cleavage, preferentially in the fluid phase, by mechanisms which are not well elucidated, and also by contact activation into C3(H2O) on the DMPC surface. In particular, this contact activation has implications for the therapeutic regulation of complement activation during liposome treatment.

Surface energy of phospholipid bilayers and the correlation to their hydration.

Supported lipid bilayers (SLBs) were prepared on glass and silicon slides grafted with polyethylene glycol (PEG) and covalently bound cholesteryl anchors to fix the lipid bilayer on the surface. Phospholipid bilayers and bilayers modified by addition of covalently bound PEG were investigated. Using contact angle measurements, the surface energy components of bilayer surfaces were analyzed using van Oss' and Owens-Wendt's methods. A quantitative correlation between the polar proton acceptor component of the surface energies and the respective hydration densities was proven for SLBs of pure lipids. We could show that the presence of PEG in the SLB produces a significant change of the proton acceptor component. Regarding the correlation between the surface energies and the hydration densities of SLBs with PEG, we were able to show a dependency on the PEG conformation.

Asymmetric or symmetric bilayer formation during oblique drop impact depends on rheological properties of saturated and unsaturated lipid monolayers.

Bilayer structures are formed by approaching two liquid surfaces with phospholipid monolayers, which are brought into contact by oblique drop impact on a liquid surface. Asymmetric bilayers can be produced by the coupling of drop and target monolayers. In contrast, symmetric bilayers or multilayers are formed by collapse of the compressed target monolayer. We show that under all studied conditions bilayer/multilayer synthesis takes place. The experimental conditions for the synthesis of asymmetric or symmetric bilayers are described quantitatively in terms of the surface rheological (surface elasticity and dilational viscosity) and the hydrodynamical parameters (Weber number and impact angle). The composition and mechanical properties of the phospholipid monolayers strongly influences the patterns of drop impact and the bilayer/multilayer formation. Cholesterol stiffens unsaturated phospholipid monolayers and fluidifies saturated monolayers. All monolayers form asymmetric vesicle-like structures, which are stable in the aqueous medium. Additionally, unsaturated phospholipid monolayers without cholesterol form symmetric vesicles by folding parts of the target monolayer. Sufficient presence of cholesterol in unsaturated phospholipid monolayers inhibits the folding of the target monolayer and the subsequent formation of symmetric bilayers. The rheological properties of saturated and unsaturated phospholipid monolayers and their mixtures with cholesterol are discussed. Based on drop impact results it is shown that the state of a so far undefined region in the DPPC/cholesterol phase diagram is a fluid phase.

Surface rheology and phase transitions of monolayers of phospholipid/cholesterol mixtures.

The dynamic surface elasticity and the surface dilational viscosity of three binary phospholipid/cholesterol mixtures were determined with axisymmetric drop shape analysis on a harmonically oscillating pendent drop. Dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, and dioleoylphosphatidylcholine were used to explore the rheological properties and phase transitions of mixtures of saturated and unsaturated phospholipids with cholesterol. The growth rates for surface dilational viscosity and dynamic elasticity are parallel for all film pressures studied. Characteristic breaks and plateaus could be found for these growth rates, indicating phase transitions. For dipalmitoylphosphatidylcholine/cholesterol and dimyristoylphosphatidylcholine/cholesterol mixtures, phase diagrams with six regions separated by phase boundaries were found, which are in good agreement with phase transitions reported in the literature for static measurements of isotherms and isobars on a Langmuir film balance and from fluorescence microscopy. Some phase boundaries were only found by dynamic, but not by static, elasticity measurements. Imaging methods revealed phase separations produced by the formation of condensed stoichiometric complexes leading to micron-sized and mostly circular domains. The effects of these complexes on monolayer rheology in liquid/liquid phases is described. Furthermore, liquid/solid and solid phase transitions are discussed.

Interactions of viscotoxins with vesicles of genuine plant membranes.

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.

Characterization of membrane vesicles in plant extracts.

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.

Effect of surfactants on the stability of thin liquid film flow on a rotating disk.

The effect of surfactants on surface instabilities of thin liquid film flow on a rotating disk was studied at different flow rates Q (0.5