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Toxicity of β-blockers is one of the most common causes of poison-induced cardiogenic shock throughout the world. Therefore, methodologies for in vivo removal of the drugs from the body have been under investigation. Intralipid emulsion (ILE) is a common commercial lipid emulsion used for parenteral nutrition, but it has also been administered to patients suffering from drug toxicities. In this work, a set of β-blockers of different hydrophobicity's (log K D values ranging from 0.16 to 3.8) were investigated. The relative strength of the interactions between these compounds and the ILE was quantitatively assessed by means of binding constants and adsorption constants of the formed β-blocker-ILE complexes. The binding constants were determined by capillary electrokinetic chromatography and the adsorption constants were calculated based on different adsorption isotherms. Expectedly, the binding constants were strongly related to the log K D values of the β-blockers. The binding and adsorption constants also show that less hydrophobic β-blockers interact with ILE, suggesting that this emulsion could be useful for capturing such compounds in cases of their overdoses. Thus, the use of ILE for treatment of toxicities caused by a larger range of β-blockers is worth further investigation., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Susanne Wiedmer reports financial support was provided by the Finnish Society of Sciences and Letters. Torgny Fornstedt reports financial support was provided by Swedish Knowledge Foundation. Vaclav Kasicka reports financial support was provided by the Czech Academy of Sciences. Jorgen Samuelsson reports financial support was provided by Swedish Knowledge Foundation., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Our study demonstrates that nanoplasmonic sensing (NPS) can be utilized for the determination of the phase transition temperature (T m ) of phospholipids. During the phase transition, the lipid bilayer undergoes a conformational change. Therefore, it is presumed that the T m of phospholipids can be determined by detecting conformational changes in liposomes. The studied lipids included 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). Liposomes in gel phase are immobilized onto silicon dioxide sensors and the sensor cell temperature is increased until passing the T m of the lipid. The results show that, when the system temperature approaches the T m , a drop of the NPS signal is observed. The breakpoints in the temperatures are 22.5 °C, 41.0 °C, and 55.5 °C for DMPC, DPPC, and DSPC, respectively. These values are very close to the theoretical T m values, i.e., 24 °C, 41.4 °C, and 55 °C for DMPC, DPPC, and DSPC, respectively. Our studies prove that the NPS methodology is a simple and valuable tool for the determination of the T m of phospholipids.

Owing to their unique properties and unlimited structural combinations, the ubiquitous use of ionic liquids (ILs) is steadily increasing. The objective of the present work is to shed light onto the effects of amidinium- and phosphonium-based ILs on phospholipid vesicles using a nanoplasmonic sensing measurement technique. A new and relatively simple method was developed for the immobilization of large unilamellar vesicles on two different hydrophilic surfaces composed of titanium dioxide and silicon nitride nanolayers. Among the pretreatment conditions studied, vesicle attachment on both substrate materials was achieved with HEPES buffer in the presence of sodium hydroxide and calcium chloride. To get an understanding of how ILs interact with intact vesicles or with supported lipid bilayers, the ILs 1,5-diazabicyclo(4.3.0)non-5-enium acetate ([DBNH][OAc]), tributyl(tetradecyl)phosphonium acetate ([P 14444 ][OAc]), and tributylmethylphosphonium acetate ([P 4441 ][OAc]) were introduced into the biomimetic system, and the characteristics of their interactions with the immobilized vesicles were determined. Depending on the IL, in situ real-time IL binding and/or phospholipid removal processes were observed. Although [DBNH][OAc] did not have any significant effect on the phospholipid vesicles, the strongest and the most significant effect was observed with [P 14444 ][OAc]. The latter caused clear changes in the phospholipid bilayer: the ILs interacted with the bilayers, resulting in deformation of the vesicles most probably due to the formation of vesicle-IL aggregates. Only a mild effect was observed when [P 4441 ][OAc], at a very high concentration, was exposed to the intact vesicles. In general, these results led to new insights into the effects of ILs on phospholipid vesicles, which are of great importance to the overall understanding of the harmfulness of ILs on biomembranes and biomimicking systems. In addition, the present work highlights the pivotal role of this highly surface-sensitive indirect biosensing technique in scrutinizing and dissecting the integrity and architecture of phospholipid vesicles in the nanoscale range.

The effects of ionic liquids on model phospholipid membranes were studied by small-angle X-ray scattering, dynamic light scattering (DLS) and zeta potential measurements. Multilamellar 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine liposomes and large unilamellar vesicles composed of l-α-phosphatidylcholine (eggPC) and l-α-phosphatidylglycerol (eggPG) (80:20mol%) or eggPC, eggPG, and cholesterol (60:20:20mol%) were used as biomimicking membrane models. The effects of the phosphonium-based ionic liquids: tributylmethylphosphonium acetate, trioctylmethylphosphonium acetate, tributyl(tetradecyl)-phosphonium acetate, and tributyl(tetradecyl)-phosphonium chloride, were compared to those of 1-ethyl-3-methyl-imidazolium acetate. With multilamellar vesicles, the ionic liquids that did not disrupt liposomes decreased the lamellar spacing as a function of concentration. The magnitude of the effect depended on concentration for all studied ionic liquids. Using large unilamellar vesicles, first a slight decrease in the vesicle size, then aggregation of vesicles was observed by DLS for increasing ionic liquid concentrations. At concentrations just below those that caused aggregation of liposomes, large unilamellar vesicles were coated by ionic liquid cations, evidenced by a change in their zeta potential. The ability of phosphonium-based ionic liquids to affect liposomes is related to the length of the hydrocarbon chains in the cation. Generally, the ability of ionic liquids to disrupt liposomes goes hand in hand with inducing disorder in the phospholipid membrane. However, trioctylmethylphosphonium acetate selectively extracted and induced a well-ordered lamellar structure in phospholipids from disrupted cholesterol-containing large unilamellar vesicles. This kind of effect was not seen with any other combination of ionic liquids and liposomes., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Phospholipid vesicles were covalently attached to iminoaldehyde-coated fused silica capillaries and applied to the separation of model steroids by open-tubular CEC (OT-CEC). The effects of reducing the formed Schiff's base with sodium borohydride and of the liposome composition on the stability of the coating were investigated. In addition, the studies were focused on the optimization of running conditions (pH values and composition of BGE solution) when CEC, using capillaries covalently bound with liposome dispersions, was coupled to MS. The effect of cholesterol in the liposome dispersion on the binding of model analytes was studied, using liposome dispersions comprising 80/20 mol% zwitterionic 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC) and the negatively charged phospholipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) and 40/40/20 mol% POPC/POPS/cholesterol. Cholesterol in liposomes (greatly) enhanced the stability of the capillaries by making the coatings more rigid, resulting in lower retention factors for all the studied model steroids. Although most of the studies were carried out by open tubular CEC-UV Vis, the applicability of the capillaries to on-line CEC-MS was demonstrated as well. On-line CEC-MS studies on model steroids proved the suitability of coated capillaries for analyte-lipid membrane interaction studies, and especially for such analytes that are difficult to detect by conventional on-line UV Vis.

Silica particles were covalently modified with phospholipids and used as packing material for nano-liquid chromatography (nano-LC). This modification involved aminopropylsilylation of the raw silica particles using 3-(aminopropyl)-triethoxysilane, covalent binding of glutaraldehyde molecules to the aminopropylsilylated particles, and finally covalent binding of different phospholipid vesicles containing primary amino groups to the iminoaldehyde silica particles. Capillaries with an inner diameter of 100μm were packed with phospholipid-coated silica particles using a slurry packing method. The packed capillaries were tested in nano-LC with UV-detection for the separation of acidic, neutral, and basic model analytes. The effect of the buffer ion on the retention factor of the analytes was evaluated using buffer solutions with constant ionic strength and pH. In addition, the effect of the volume of methanol in the mobile phase was studied. The calculated distribution coefficients (logK(D)) of the model compounds were in agreement with those reported in the literature. A good correlation between logK(D) values and octanol/water partitioning coefficients (P(o/w)) for neutral hydrophobic analytes was obtained proving the applicability of the method for predicting partitioning of the compounds with the biomembranes., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Intravenous lipid emulsion is recommended as treatment for local anesthetic intoxication based on the hypothesis that the lipophilic drug is entrapped by the lipid phase created in plasma. We compared a 15.6 mM 80/20 mol% phosphatidyl choline (PC)/phosphatidyl glycerol (PG)-based liposome dispersion with the commercially available Intralipid® emulsion in a pig model of local anesthetic intoxication. Bupivacaine-lipid interactions were studied by electrokinetic capillary chromatography. Multilamellar vesicles were used in the first in vivo experiment series. This series was interrupted when the liposome dispersion was discovered to cause cardiovascular collapse. The toxicity was decreased by an optimized sonication of the 50% diluted liposome dispersion (7.8 mM). Twenty anesthetized pigs were then infused with either sonicated PC/PG liposome dispersion or Intralipid®, following infusion of a toxic dose of bupivacaine which decreased the mean arterial pressure by 50% from baseline. Bupivacaine concentrations were quantified in blood samples using liquid chromatography/mass spectrometry. No significant difference in the context-sensitive plasma half-life of bupivacaine was detected (p=0.932). After 30 min of lipid infusion, the bupivacaine concentration was 8.2±1.5 mg/L in the PC/PG group and 7.8±1.8 mg/L in the Intralipid® group, with no difference between groups (p=0.591). No difference in hemodynamic recovery was detected between groups (p > 0.05)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Interactions between low-molar mass analytes and phospholipid membranes were studied by liposome electrokinetic capillary chromatography (LEKC). The analytes were pesticides, some degradation products, and compounds associated with the manufacture of pesticides. Negatively charged liposome dispersions with different zwitterionic lipids (PC) were applied to the determination of retention factors (k) of 15 charged and uncharged compounds. The liposome dispersions consisted of 80:20 mol% of 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)/POPS, and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/POPS. Retention factors were calculated from the effective electrophoretic mobilities of the analytes under LEKC and CZE conditions and from the effective electrophoretic mobilities of the liposomes, determined by CZE with a polyacrylamide-coated capillary. Determining the liposome mobilities in this way proved to be a good alternative to the conventional method employing a liposome marker compound. The log k values of the analytes for the different liposome dispersed phases were correlated with one another. In addition, correlation curves were determined between log k and calculated octanol-water partition coefficients. The results showed that the zwitterionic phospholipid in the liposome has a major impact on the interactions between the tested compounds and the lipid membranes.

A phospholipid-bovine serum albumin (BSA) coating was developed for chiral capillary electrochromatographic separation of d- and l-tryptophan. Temperature, liposome composition, and liposome-BSA mixing and extrusion were found to have critical effects on the chiral separation of d- and l-tryptophan in terms of resolution, separation efficiency, and migration times. A solution of 0.5mM phosphatidylcholine (PC)-1 mg/ml BSA performed better than a solution of 0.5mM PC/phosphatidylserine (PS) (80:20, mol%)-1 mg/ml BSA as capillary coating; baseline separation of the enantiomers with satisfactory resolution was then achieved. Temperature played a crucial role in the chiral separation, as demonstrated for phospholipid-coated capillaries immobilized with BSA and lysozyme. The d- and l-tryptophans showed a marked difference in separation efficiency on the PC-BSA-coated capillary; the theoretical plate number of l-tryptophan was above 500,000 m(-1), whereas that of d-tryptophan was only about 22,000 m(-1). Immobilized BSA (pI 4.7) showed better chiral separation selectivity for the enantiomers than did immobilized lysozyme (pI 10.5), alpha-chymotrypsin (pI 8.1-8.3), or avidin (pI 10.0-10.5); also resolution was better and analysis time was faster. Hydrophobic interactions played an important role in the BSA-immobilized phospholipid-coated capillaries. The importance of protein net charge and molar mass for its immobilization in phospholipid-coated capillaries is discussed.

Anionic phospholipids phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS) were examined for their effect on 1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylcholine (POPC)-containing liposomes used as coating material in capillary electrochromatography. Liposome solvent was N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer at pH 7.4 with and without 3 mM of CaCl2. The background electrolyte solution was HEPES buffer at pH 7.4. The net charge, size, and short-term stability of the liposomes were measured with a Zetasizer. Results showed that calcium interacts with all liposomes but most strongly with POPC/PA. The relative migration times, retention factors, and resolution of the model analytes (one cationic, three uncharged ions, and one anionic) were studied. All liposomes successfully coated the silica capillary. Without calcium the strongest interaction and best separation of the analytes were with the POPC/PI and POPC/PS coatings, while interactions with the POPC/PA coating were weak. Calcium enhanced the interactions of the model analytes with all coatings, and the interactions were then strongest with the POPC/PA coating. In the presence of calcium there appears to be a slight reorganization of the coating with increasing number of runs. Our results indicate strong interactions between calcium and the phosphate groups in phospholipids and demonstrate the significant role of the phospholipid polar head group in phospholipid coatings on silica surfaces.

In circulation the phospholipid transfer protein (PLTP) facilitates the transfer of phospholipid-rich surface components from postlipolytic chylomicrons and very low density lipoproteins (VLDL) to HDL and thereby regulates plasma HDL levels. To study the molecular mechanisms involved in PLTP-mediated lipid transfer, we studied the interfacial properties of PLTP using Langmuir phospholipid monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) to follow the transfer of 14C-labeled phospholipids and [35S]PLTP between lipid vesicles and HDL particles. The AsFlFFF method was also used to determine the sizes of spherical and discoidal HDL particles and small unilamellar lipid vesicles. In Langmuir monolayer studies high-activity (HA) and low-activity (LA) forms of PLTP associated with fluid phosphatidylcholine monolayers spread at the air/buffer interphase. Both forms also mediated desorption of [14C]dipalmitoylphosphatidylcholine (DPPC) from the phospholipid monolayer into the buffer phase, even when it contained no physiological acceptor such as HDL. After the addition of HDL3 to the buffer, HA-PLTP caused enhanced lipid transfer to them. The particle diameter of HA-PLTP was approximately 6 nm and that of HDL3 approximately 8 nm as determined by AsFlFFF analysis. Using this method, it could be demonstrated that in the presence of HA-PLTP, but not LA-PLTP, [14C]DPPC was transferred from small unilamellar vesicles (SUV) to acceptor HDL3 molecules. Concomitantly, [35S]-HA-PLTP was transferred from the donor to acceptor, and this transfer was not observed for its low-activity counterpart. These observations suggest that HA-PLTP is capable of transferring lipids by a shuttle mechanism and that formation of a ternary complex between PLTP, acceptor, and donor particles is not necessary for phospholipid transfer.

The adsorption of anionic phospholipids on silica was investigated by the dissipative quartz crystal microbalance (QCM) technique. Liposomes composed of 1 mM 80:20 mol % of 1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylcholine (POPC)/phosphatidic acid, POPC/phosphatidylglycerol, or POPC/phosphatidylserine in N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) buffer at pH 7.4 (with or without 3 mM of CaCl2) were examined. We have previously demonstrated that similar phospholipid coatings can be used in capillary electrochromatography as a stationary phase for the separation of analytes. In this work, we focus on the formation of the coatings and on the type of lipid structure formed on silica. The QCM investigation comprised qualitative results based on changes in frequency and resistance, and quantitative modeling of the obtained results. The latter was performed using the dissipative QCM, which measures the quartz crystal impedance, combined with equivalent circuit analysis. A previously developed coating and cleaning procedure for phospholipid-coated fused silica capillaries was adopted in this study, and the same silica-coated crystal was used throughout the QCM study. We will demonstrate in this work that the type of lipid structure formed on silica, that is, a rather rigid supported lipid bilayer or a viscoelastic supported vesicle layer (SVL), is highly dependent on the lipid and solvent composition. We also show for the first time that the modeling of the dissipative QCM data can be used to extract a more quantitative picture of an adsorbed SVL, because, so far, published studies have merely used the QCM data in a qualitative sense.

Phospholipid-coated fused-silica capillaries with immobilized avidin were applied in the chiral separation of D,L-tryptophan, D,L-PTH-serine, and D,L-PTH-threonine at pH 7.4 by open-tubular CEC. Liposomes prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Cap biotinyl), or 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(Biotinyl) with different amounts of phosphatidylserine were assessed as phospholipid coating materials. The stability of the coating and the success of the coating procedure were evaluated in terms of the repeatability of the enantiomer migration times and the resolution of enantiomers. The coating procedure itself significantly affected the migration times and resolution of the enantiomers. Reliable chiral separations with high separation efficiencies were achieved through careful choice of the coating method.

A phospholipid coating with lysozyme as chiral recognition reagent permeated into the phospholipid membrane was developed for the chiral capillary electrophoretic (CE) separation of D- and L-tryptophan. As a kind of carriers, coated as phospholipid membranes onto the inner wall of a fused-silica capillary, liposomes are able to interact with basic proteins such as lysozyme, which may reside on the surface of the phospholipid membrane or permeate into the middle of the membrane. The interaction results in strong immobilization of lysozyme in the capillary. Coatings prepared with liposomes alone did not allow stable immobilization of lysozyme into the phospholipid membranes, as seen from the poor repeatability of the chiral separation. When 1-(4-iodobutyl)-1,4-dimethylpiperazin-1-ium iodide (M1C4) was applied as a first coating layer in the capillary, the electroosmotic flow (EOF) was effectively suppressed, the phospholipid coating was stabilized, and the lysozyme immobilization was much improved. The liposome composition, the running buffer, and the capillary inner diameter all affected the chiral separation of D- and L-tryptophan. Coating with 4 mM M1C4 and then 1 mM phosphatidylcholine (PC)/phosphatidylserine (PS) (80:20 mol%), with 20 mM (ionic strength) Tris at pH 7.4 as the running buffer, resulted in optimal chiral separation with good separation efficiency and resolution. Since lysozyme was strongly permeated into the membrane of the phospholipids on the capillary surface, the chiral separation of D- and L-tryptophan was achieved without lysozyme in the running buffer. The effects of different coating procedures and separation conditions on separation were evaluated, and the M1C4-liposome and liposome-lysozyme interactions were elucidated. The usefulness of protein immobilized into phospholipid membranes as a chiral selector in CE is demonstrated for the first time.

Knowledge of the spatial distribution of lipids in the intraocular lens is important for understanding the physiology and biochemistry of this unique tissue and for gaining a better insight into the mechanisms underlying diseases of the lens. Following our previous study showing the spatial distribution of sphingolipids in the porcine lens, the current study used ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) to provide the whole lipidome of porcine lens and these studies were supplemented by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) of the lens using ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to determine the spatial distribution of glycerophospholipids. Altogether 172 lipid species were identified with high confidence and their concentration was determined. Sphingomyelins, phosphatidylcholines, and phosphatidylethanolamines were the most abundant lipid classes. We then determined the spatial and concentration-dependent distributions of 20 phosphatidylcholines, 6 phosphatidylethanolamines, and 4 phosphatidic acids. Based on the planar molecular images of the lipids, we report the organization of fiber cell membranes within the ocular lens and suggest roles for these lipids in normal and diseased lenses. [ABSTRACT FROM AUTHOR]

Interactions between Intralipid dispersion and local anesthetics (bupivacaine, prilocaine, and lidocaine) were investigated. The amount of bupivacaine (the most cardiotoxic analyte of the local anesthetics studied) entrapped in Intralipid in the presence of plasma was studied using an off-line filtration and solid phase extraction method combined with capillary zone electrophoresis for quantification of free unbound bupivacaine. To confirm interactions between the analytes and Intralipid at lower concentrations, direct injection mass spectrometry was used. The use of immobilized Intralipid chromatography-atmospheric pressure ionization–ion trap mass spectrometry in the study of interactions between drugs and Intralipid dispersion is demonstrated. Finally, interactions between Intralipid dispersion and local anesthetics were investigated by electrokinetic capillary chromatography. The electrophoretic mobility of the Intralipid dispersed phase was calculated using the iterative procedure and a homologous series of alkyl phenyl benzoates (C1–C6), and the retention factors for the analytes were determined. [ABSTRACT FROM AUTHOR]

Divalent cations affect the stability and structure of phospholipid vesicles and also the binding and immobilization of proteins into phospholipid membranes. The effect of calcium, magnesium, and zinc on zwitterionic phosphatidylcholine (PC) coatings in fused silica capillaries for electrophoresis was the primary interest in this work. In addition, the effect of temperature on the coating stability was investigated by coating 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) liposomes at temperatures above and below the gel- to fluid-state transition. All coatings were performed with PC large unilamellar vesicles (LUV) in 40mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) at pH 7.4 as basic solution. HEPES (40mM) at pH 7.4 was used as background electrolyte (BGE) throughout the study. The stability of the coating was studied by measuring the electroosmotic flow. A molar ratio of 1:3PC/Ca2+ or PC/Mg2+ gave the best coating stability owing to the increased rigidity of the phospholipid membrane furnished by the divalent metal ions. Better results were obtained with DPPC in the more rigid gel state than in the fluid state: the electroosmotic flow was much suppressed and the PC coating was stabilized. Coating the fused silica capillary with PC liposome–metal ion buffer solutions resulted in good electrophoretic separation of basic model proteins (pI-values 7.8–11.0). The electrophoretic results demonstrate the importance of stabilizing the phospholipid coating on fused silica capillaries, either by the addition of divalent metal ions (Ca2+, Mg2+, or Zn2+) or by working in the gel-state region of the phospholipid. [Copyright &y& Elsevier]

The structural resemblance of liposomes to natural cell membranes has caused them to be employed in liquid chromatographic and capillary electromigration techniques to study the interactions between analytes and phospholipid membranes. Phospholipids and liposomes are immobilized in stationary phases for liquid chromatography by a variety of techniques, to which we give special attention. We present selected applications to the separation of analytes. Though still few, applications in capillary electromigration are particularly attractive because of the small amounts of liposomes and samples that are required. We demonstrate the promising use of liposomes as carriers and coating materials in capillary electrophoresis with reference to recent applications. We note future directions for the utilization of phospholipids and liposomes in liquid chromatographic and capillary electromigration techniques. [Copyright &y& Elsevier]

The effect of calcium on phospholipid coatings in fused silica capillaries used in capillary electrophoresis was studied. The anionic liposomes used for the coating consisted of 3 mM 1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylcholine and phosphatidylserine in the ratio 80/20 mol%. Coating was performed as part of the preconditioning, and the capillaries could be used for several runs without the need for liposomes in the background electrolyte solution or for liposome rinses between runs. Phospholipids could easily be flushed away by rinsing with a chloroform-methanol (2:1 v/v) mixture, which made it possible to recoat and reuse the capillaries. A calcium:phospholipid ratio of approximately 3 gave the most stable coating. The stability of the coating and success of the coating procedure were studied by measuring the electroosmotic flow and by separating uncharged steroids, which were used as model compounds. Many parameters that affect the coating, such as preconditioning (with different acids and bases), buffer, temperature during coating, and the physical structures of liposomes, were studied, with and without calcium in the liposome solution. The separation of steroids was improved and was less dependent on coating conditions when calcium was present during the coating. Capillaries optimally coated with anionic phospholipids were applied in the separation of phenols. [ABSTRACT FROM AUTHOR]

Abstract: Anionic phospholipids phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidylserine (PS) were examined for their effect on 1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylcholine (POPC)-containing liposomes used as coating material in capillary electrochromatography. Liposome solvent was N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES) buffer at pH 7.4 with and without 3mM of CaCl2. The background electrolyte solution was HEPES buffer at pH 7.4. The net charge, size, and short-term stability of the liposomes were measured with a Zetasizer. Results showed that calcium interacts with all liposomes but most strongly with POPC/PA. The relative migration times, retention factors, and resolution of the model analytes (one cationic, three uncharged ions, and one anionic) were studied. All liposomes successfully coated the silica capillary. Without calcium the strongest interaction and best separation of the analytes were with the POPC/PI and POPC/PS coatings, while interactions with the POPC/PA coating were weak. Calcium enhanced the interactions of the model analytes with all coatings, and the interactions were then strongest with the POPC/PA coating. In the presence of calcium there appears to be a slight reorganization of the coating with increasing number of runs. Our results indicate strong interactions between calcium and the phosphate groups in phospholipids and demonstrate the significant role of the phospholipid polar head group in phospholipid coatings on silica surfaces. [Copyright &y& Elsevier]

Abstract: Greater stability of liposome coatings and improved resolution of model steroids in capillary electrochromatography (CEC) were sought by adding small diamines (ethylenediamine, diaminopropane, bis-tris-propane, or N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid, HEPES)) to the liposome solution before coating of fused silica capillaries. The phospholipid coatings consisted of 1mM of 8:2mol% phosphatidylcholine (PC)/phosphatidylserine (PS) and 5mM of modifier in buffer solutions (acetate, phosphate, or Tris) at pH 4.0–7.4. The coating was based on a published procedure, and five steroids were used as neutral model analytes in evaluation of the coating. The results showed that under optimal conditions, the small linear diamines increased the packing density of anionic phospholipids, leading to improved separations. In addition, the choice of buffer for the liposome coating and separation appeared to influence the performance of the coatings. While buffers with amino groups take part in the phospholipid bilayer formation, buffers like phosphate may even have negative effect on coating formation. The factors affecting phospholipid coatings with diamines as modifiers are clarified. [Copyright &y& Elsevier]

A new and relatively simple method was developed for coating of capillaries in electrophoresis with liposomes. The liposomes, with a diameter of about 100 nm, are large unilamellar vesicles prepared by extrusion. The liposomes contained 1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylcholine (POPC) or POPC with different proportions of bovine brain phosphatidylserine (PS) and cholesterol. They formed a bilayer structure on the silica surface enabling the separation of neutral compounds. The effectiveness of the coating in separation was evaluated with use of uncharged steroids as model compounds. The coating was also studied by measuring the electroosmotic flow. The best results, taking into consideration both separation and stability, were achieved with anionic 80:20 mol% POPC/PS liposomes. In addition, the effect of coating conditions on the results was investigated. Among the buffers studied [N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), phosphate, tris(hydroxymethyl)aminomethane (Tris) and N-tris(hydroxymethyl)methylglycine (Tricine)], HEPES seemed to have a significant effect on the success of the coating. Successful separation of steroids was achieved only when HEPES buffer was used in the coating procedure and in the background electrolyte solution for the separation. With all other buffers the peaks of the model compounds overlapped. [Copyright &y& Elsevier]

The present work aims at studying the interactions between cholesterol-rich phosphatidylcholine-based lipid vesicles and trioctylmethylphosphonium acetate ([P 8881 ][OAc]), a biomass dissolving ionic liquid (IL). The effect of cholesterol was assayed by using differential scanning calorimetry (DSC) and nanoplasmonic sensing (NPS) measurement techniques. Cholesterol-enriched dipalmitoyl-phosphatidylcholine vesicles were exposed to different concentrations of the IL, and the derived membrane perturbation was monitored by DSC. The calorimetric data could suggest that the binding and infiltration of the IL are delayed in the vesicles containing cholesterol. To clarify our findings, NPS was applied to quantitatively follow the resistance of 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine incorporating 0, 10, and 50 mol% of cholesterol toward the IL exposure over time. The membrane perturbation induced by different concentrations of IL was found to be a concentration dependent process on cholesterol-free lipid vesicles. Moreover, our results showed that lipid depletion in cholesterol-enriched lipid vesicles is inversely proportional to the increasing amount of cholesterol in the vesicles. These findings support that cholesterol-rich lipid bilayers are less susceptible toward membrane disrupting agents as compared to membranes that do not incorporate any sterols. This probably occurs because cholesterol tightens the phospholipid acyl chain packing of the plasma membranes, increasing their resistance and reducing their permeability. [ABSTRACT FROM AUTHOR]

In this study a strategy to immobilize phospholipids onto a polymer-based stationary phase is described. Methacrylate-based monoliths in capillary format (150 × 0.1 mm) were modified by soybean phosphatidylcholine through 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide coupling to obtain stationary phases suitable to mimic cell surface membranes. The covalent coupling reaction involves the phosphate group in phospholipids; therefore, the described methodology is suitable for all types of phospholipids. Immobilization of soy bean phosphatidylcholine on the monolith was confirmed by attenuated total reflectance Fourier transform infrared spectroscopy and gas chromatography-mass spectrometry of the fatty alcohol profile, generated upon reductive cleavage of the fatty acyl side chains of the phospholipid on the monolith surface with lithium aluminium hydride. The prepared stationary phases were evaluated through studies on the retention of low-molar mass model analytes including neutral, acidic, and basic compounds. Liquid chromatographic studies confirmed predominant hydrophobic interactions between the analytes and the synthesized stationary phase; however, electrostatic interactions contributed to the retention as well. The synthesized columns showed high stability even with fully aqueous mobile phases such as Dulbecco's phosphate-buffered saline solution. [ABSTRACT FROM AUTHOR]

In circulation the phospholipid transfer protein (PLTP) facilitates the transfer of phospholipid-rich surface components from postlipolytic chylomicrons and very low density lipoproteins (VLDL) to HDL and thereby regulates plasma HDL levels. To study the molecular mechanisms involved in PLTP-mediated lipid transfer, we studied the interfacial properties of PLTP using Langmuir phospholipid monolayers and asymmetrical flow field-flow fractionation (AsFlFFF) to follow the transfer of 14C-labeled phospholipids and [35S]PLTP between lipid vesicles and HDL particles. The AsFlFFF method was also used to determine the sizes of spherical and discoidal HDL particles and small unilamellar lipid vesicles. In Langmuir monolayer studies high-activity (HA) and low-activity (LA) forms of PLTP associated with fluid phosphatidylcholine monolayers spread at the air/buffer interphase. Both forms also mediated desorption of [14C]dipalmitoylphosphatidylcholine (DPPC) from the phospholipid monolayer into the buffer phase, even when it contained no physiological acceptor such as HDL. After the addition of HDL3 to the buffer, HA-PLTP caused enhanced lipid transfer to them. The particle diameter of HA-PLTP was and that of HDL3 ~8 nm as determined by AsFlFFF analysis. Using this method, it could be demonstrated that in the presence of HA-PLTP, but not LA-FLIP, [14C]DPPC was transferred from small unilamellar vesicles (SUV) to acceptor HDL3 molecules. Concomitantly, [35S]-HA-PLTP was transferred from the donor to acceptor, and this transfer was not observed for its low-activity counterpart. These observations suggest that HA-PLTP is capable of transferring lipids by a shuttle mechanism and that formation of a ternary complex between PLTP, acceptor, and donor particles is not necessary for phospholipid transfer. [ABSTRACT FROM AUTHOR]

Abstract: The stability of zwitterionic phosphatidylcholine vesicles in the presence of 20mol% phosphatidyl serine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI), and diacylphosphatidyl glycerol (PG) phospholipid vesicles, and cholesterol or calcium chloride was investigated by asymmetrical flow field-flow fractionation (AsFlFFF). Large unilamellar vesicles (LUV, diameter 100nm) prepared by extrusion at 25°C were used. Phospholipid vesicles (liposomes) were stored at +4 and −18°C over an extended period of time. Extruded egg yolk phosphatidylcholine (EPC) particle diameters at peak maximum and mean measured by AsFlFFF were 101±3nm and 122±5nm, respectively. No significant change in diameter was observed after storage at +4°C for about 5 months. When the storage period was extended to about 8 months (250 days) larger destabilized aggregates were formed (172 and 215nm at peak maximum and mean diameters, respectively). When EPC was stored at −18°C, large particles with diameters of 700–800nm were formed as a result of dehydration, aggregation, and fusion processes. In the presence of calcium chloride, EPC alone did not form large aggregates. Addition of 20mol% of negatively charged phospholipids (PS, PA, PI, or PG) to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC) vesicles increased the electrostatic interactions between calcium ion and the vesicles and large aggregates were formed. In the presence of cholesterol, large aggregates of about 250–350nm appeared during storage at +4 and −18°C for more than 1 day. The effect of liposome storage temperature on phospholipid coatings applied in capillary electrophoresis (CE) was studied by measuring the electroosmotic flow (EOF). EPC coatings with and without cholesterol, PS, or calcium chloride, prepared from liposomes stored at +25, +4, and −18°C, were studied at 25°C. The performances of the coatings were further evaluated with three uncharged compounds. Only minor differences were observed between the same phospholipid coatings, showing that phospholipid coatings in CE are relatively insensitive to storage at +25, +4°C or −18°C. [Copyright &y& Elsevier]