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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, η D . In contrast, smaller molecular weight emulsifiers like phospholipids show a mild increase in effectiveness with rising η C . In contrast, smaller molecular weight emulsifiers like phospholipids show a mild increase in effectiveness with rising η D / η C , 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.

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., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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.

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.

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. [ABSTRACT FROM AUTHOR]

Fluorocarbons are one of the most promising hydrophobic phases for future pharmaceutical production processes and various biomedical applications. Yet, because of their specific characteristics such as high density and refractive index similar to water, analysis of water‐in‐fluorocarbon (w/fc) nanoemulsions remains a challenge. The present work examines w/fc nanoemulsions stabilized with phospholipids as natural emulsifiers and tackles the measuring problems of photon correlation spectroscopy (PCS) when used for investigation of fluorocarbon nanoemulsions. These emulsions are suitable to form liposomes via centrifugation and thus, are required to meet certain criteria such as stability and size. The results imply a stability of up to 4 weeks with an average size of 180 nm. The intensity mean diameter gained from PCS measurements shows large scattering directly after sonication which is due to gas bubbles from sonication. The number mean is not influenced by gas bubbles and gives a more accurate depiction of the produced nanoemulsions. These findings are supported by small‐angle X‐ray scattering data, which are additionally applied for liposome analysis measuring a size of approximately 60 nm. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The adsorption of the phospholipid (PL) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) dissolved in the oil phase squalene or squalane was studied at the interface with water. Dynamic interfacial tension was measured by profile analysis tensiometry (PAT). Two different setups were considered, a buoyant oil drop surrounded by water or a pendant water drop in oil. For the proof of methodology the interfacial tensions of water in chloroform and chloroform in water interfaces were measured. From the equilibrium interfacial tensions of different PL concentrations, the Gibbs adsorption isotherm was determined. These isotherms allow the extraction of the minimal areas per lipid molecule as well as critical aggregation concentration (CAC). We found significant differences in the results obtained for both setups. By means of PAT, the CAC determined for water drops in oil was smaller than for oil drops in water. Photon correlation spectroscopy (PCS) and nuclear magnetic resonance (NMR) were used to verify the determined CACs. We concluded the setup-dependent differences to be caused by depletion effects when initially dissolving the surface active component in the small drop volume. We recommend the reversed setup where the surface active component is dissolved in the surrounding bulk phase to avoid corrections of bulk concentrations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: Surface rheology of monolayers of a saturated phospholipid (dipalmitoylphosphatidylcholine, DPPC), an unsaturated phospholipid (dioleoylphosphatidylcholine, DOPC) and cholesterol is studied with axisymmetric drop shape analysis at the argon/water interface. Measurement techniques for lipids are described in detail. Profile analysis tensiometry (PAT) is used to determine the film pressure Π, surface elasticity and surface dilational viscosity of monolayers upon sinusoidal oscillations of the drop surface for various amplitudes a and frequencies f to assess their dependence on these dynamic parameters. It is shown that surface dilational viscosity strongly depends on the frequency and decreases by a factor 2–5 with increasing f in the considered range. Dilational viscosity is higher the more the monolayer approaches a relaxed state. Thus, the molecular interactions are stronger in the relaxed than in the stressed state. Surface elasticity is much less dependent on dynamic conditions. For DPPC a minimum of the dynamic surface elasticity is found for f =12.5mHz (at Π =17.5mNm−1) which coincides well with the relaxation frequencies measured in stress relaxation experiments. The dynamic surface elasticity of DPPC exhibits a plateau in the range 13.5mNm−1 ≤ Π ≤27mNm−1 in good coincidence with the phase boundaries of the coexistence region of micron-sized liquid crystalline domains surrounded by a fluid monolayer phase. In equilibrium measurements (Π/A-isotherms) a plateau of the film pressure is seen at the lower bound and a break at the upper bound of the coexistence region. Film pressure/area isotherms produced by PAT and a Langmuir film balance closely coincide as is shown in a comparison to literature values. However, the surface elasticities measured dynamically with oscillating surfaces widely deviate from those derived from isotherms in the case of DPPC and cholesterol, whereas for DOPC very good agreement can be found. [Copyright &y& Elsevier]