Search

β-Blockers belong to a frequently used class of drugs primarily used to treat heart and circulatory conditions. Here we describe the use of lipid vesicles and liposomes as cell membrane biomimicking models in capillary electromigration (CE) and liquid chromatography (LC) techniques for the investigation of interactions between lipid membranes and β-blockers. In addition to liposomes, the use of commercial intravenous lipid emulsions, and their interactions with β-blockers are also discussed. Different CE and LC instrumental techniques designed for these purposes are introduced. Other methodologies for studying interactions between β-blockers and lipid membranes are also briefly discussed, and the different methodologies are compared. The aim is to give the reader a good overview on the status of the use of liposomes and lipids in CE and LC for studying β-blocker interactions., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Susanne K. Wiedmer reports financial support was provided by Finnish Society of Sciences and Letters., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Nanoplasmonic sensing (NPS), based on localized surface plasmon resonance, with sensors composed of glass covered with golden nanodisks and overlaid with a SiO 2 coating was applied in this study. Egg phosphatidylcholine (eggPC), being an easily accessible membrane-forming lipid, was used for preparation of biomimicking membranes. Small unilamellar vesicles with an approximate hydrodynamic diameter of 30 nm, formed by sonication in 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid buffer, were adsorbed within 10 min on the sensor surface either as intact vesicles or as a planar bilayer. The adsorbed biomembrane systems were further utilized for interaction studies with four different well-known surfactants (negatively and positively charged, zwitterionic, and nonionic) and each surfactant was tested at concentrations below and above the critical micelle concentration (CMC). Our results allowed the evaluation of different NPS patterns for every particular supported membrane system, surfactant, and its concentration. The most significant effect on the membrane was achieved upon the introduction of zwitterionic surfactant micelles, which in fact completely solubilized and removed the lipid membranes from the sensor surface. Other surfactant micelles interacted with the membranes and formed mixed structures remaining on the sensor surface. The studies performed at the concentrations below the CMCs of the surfactants showed that different mixed systems were formed. Depending on the supported membrane system and the type of surfactant, the mixed systems indicated different formation kinetics. Additionally, the final water rinse revealed the stability of the formed systems. To investigate the effect of the studied surfactants on the overall surface charge of the biomembrane, capillary electrophoresis (CE) experiments were carried out in parallel with the NPS analysis. The electroosmotic flow mobility of an eggPC-coated fused silica capillary was used to measure the total surface charge of the biomembrane after its treatment with the surfactants. Our results indicated in general good correlation between CE and NPS data. However, some discrepancies were seen while applying either zwitterionic or positively charged surfactants. This confirmed that CE analysis was able to provide additional data about the investigated systems. Taken together, the combination of NPS and CE proved to be an efficient way to describe the nature of interactions between biomimicking membranes and amphiphilic molecules.

The use of bare fused silica capillary in CE can sometimes be inconvenient due to undesirable effects including adsorption of sample or instability of the EOF. This can often be avoided by coating the inner surface of the capillary. In this work, we present and characterize two novel polyelectrolyte coatings (PECs) poly(2-(methacryloyloxy)ethyl trimethylammonium iodide) (PMOTAI) and poly(3-methyl-1-(4-vinylbenzyl)-imidazolium chloride) (PIL-1) for CE. The coated capillaries were studied using a series of aqueous buffers of varying pH, ionic strength, and composition. Our results show that the investigated polyelectrolytes are usable as semi-permanent (physically adsorbed) coatings with at least five runs stability before a short coating regeneration is necessary. Both PECs showed a considerably decreased stability at pH 11.0. The EOF was higher using Good's buffers than with sodium phosphate buffer at the same pH and ionic strength. The thickness of the PEC layers studied by quartz crystal microbalance was 0.83 and 0.52 nm for PMOTAI and PIL-1, respectively. The hydrophobicity of the PEC layers was determined by analysis of a homologous series of alkyl benzoates and expressed as the distribution constants. Our result demonstrates that both PECs had comparable hydrophobicity, which enabled separation of compounds with log Po/w > 2. The ability to separate cationic drugs was shown with β-blockers, compounds often misused in doping. Both coatings were also able to separate hydrolysis products of the ionic liquid 1,5-diazabicyclo[4.3.0]non-5-ene acetate at highly acidic conditions, where bare fused silica capillaries failed to accomplish the separation., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The use of phospholipid vesicles (liposomes) in EKC and CEC, as well as the use of CE for studying liposomes and lipid-analyte aggregates, are briefly reviewed. The works are presented in the order of appearance. The review describes the most common liposome dispersions used for achieving separation of neutral and charged compounds in liposome EKC. The CEC part is divided into two parts, discriminating between liposomes immobilized on capillaries for hindering analytes from interacting with the fused-silica wall and liposomes immobilized on capillaries for achieving chromatographic phases for separation of compounds. Finally, the use of CE for studying liposomes and lipid-analyte aggregates as analytes is discussed in brief.

PEG-stabilized lipid aggregates are a promising new class of model membranes in biotechnical and pharmaceutical applications. CE techniques, field-flow fractionation, light scattering, quartz crystal microbalance (QCM), and microscopic techniques were used to study aggregates composed of 1-palmitoyl-2-oleyl-sn-glycero-phosphatidylcholine (POPC) and PEG-lipid conjugates. The PEG-lipids, with PEG molar masses of 1000, 2000, and 3000, were 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy-(PEG)] derivatives with either dimyristoyl (DM, 14:0) or distearoyl (DS, 18:0) acyl groups. The 80/20 mol% POPC/PEG-lipid dispersions in HEPES at pH 7.4 were extruded through 100 nm size membranes. Asymmetrical flow field-flow fractionation (AsFlFFF), photon correlation spectroscopy (PCS), and dynamic light scattering (DLS) were used to determine the sizes of POPC and the PEGylated aggregates. All methods demonstrated that the DSPEG-lipid sterically stabilized aggregates were smaller in size than pure POPC vesicles. The zeta potentials of the aggregates were measured and showed an increase from -19 mV for pure POPC to -4 mV for the POPC/DSPEG3000 aggregates. Atomic force microscopy (AFM), electron cryo-microscopy (EM), and multifrequency QCM studies were made to achieve information about the PEGylated coatings on silica. Lipid aggregates with different POPC/DSPEG3000-lipid ratios were applied as capillary coating material, and the 80/20 mol% composition was found to give the most suppressed and stable EOFs. Mixtures of low-molar-mass drugs and FITC-labeled amino acids were separated with the PEGylated aggregates as carriers (EKC) or as coating material (CEC). Detection was made by UV and LIF.

Capillary electrophoretic (CE) methods were used for the quantitative determination of model drugs [salbutamol sulphate (SS), sodium cromoglycate (SCG) and beclomethasone dipropionate (BDP)] in poly(D,L-lactic acid) (PLA) nanoparticles, which were prepared by the nanoprecipitation method. Zeta potential and size distribution of the nanoparticles were determined by electrophoretic mobility determinations and photon correlation spectroscopy, respectively. Interactions between the drugs, the PLA nanoparticles and the fused-silica capillary were investigated by electrokinetic capillary chromatography (EKC). A quantitative CE method was developed for salbutamol sulphate and sodium cromoglycate, and the linearity and repeatability of migration times, peak areas and peak heights were determined. Microemulsion electrokinetic chromatography was used for the quantitative determination of beclomethasone dipropionate. According to this study, the applied electromigration techniques were suitable for the interaction, drug entrapment and dissolution studies of pharmaceutical nanoparticles. The results suggest that even quantitation of the drug located inside the nanoparticles was possible. Encapsulation of the more hydrophilic model drugs (SS, SCG) in the PLA nanoparticles was less efficient than in the case of BDP.

The purpose was to develop a stable biological membrane coating for CE useful for membrane interaction studies. The effect of cholesterol (chol) on the stability of dipalmitoylphosphatidylcholine (DPPC) and sphingomyelin (SM) coatings was studied. In addition, a fused-silica capillary for CE was coated with human red blood cell (RBC) ghost lipids. Liposomes prepared of DPPC/SM with and without chol or RBC ghost lipids were flushed through the capillary and the stability of the coating was measured electrophoretically. Similar mixtures of DPPC/SM with and without chol were further studied by differential scanning calorimetry. The presence of phosphatidylcholine as a basic component in the coating solution of DPPC/SM/chol was found to be essential to achieve a good and stable coating. The results also confirmed the stability of coatings obtained with solutions of DPPC with 0-30 mol% of chol and SM in different ratios, which more closely resemble natural membranes. Finally, the electrophoretic measurements revealed that a stable coating is formed when capillaries are coated with liposomes of RBC ghost lipids.

1,2-Dioleyl-3-trymethylammoniumpropane (DOTAP) lipid vesicles were employed as coating precursors to obtain a semipermanent cationic lipid bilayer in silica capillary. The coating procedure was relatively fast and simple. Reliable results for the separation of four basic proteins (alpha-chymotrypsinogen A, ribonuclease A, cytochrome C, lysozyme) were obtained by using an acetate buffer under acidic conditions. The RSDs of the migration times were not higher than 0.5% run-to-run and about 1% day-to-day (3 days), while the RSDs of the peak areas were within 7% day-to-day (3 days). The day-to-day RSD of the EOF mobility of about 1%, confirmed that the DOTAP coating was stable for the separation of basic proteins, under acidic buffers. In addition to basic proteins the DOTAP coating was found suitable under acidic conditions for the repeatable separation of neutral steroids. The potential of DOTAP as a carrier in background electrolyte solution was studied.

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.

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 1 mM of 8:2 mol% phosphatidylcholine (PC)/phosphatidylserine (PS) and 5 mM 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.

Anionic liposomes can be coated on fused-silica capillaries for electrophoresis in the presence of N-(hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) as background electrolyte (BGE) solution. In this work, the interaction of various compounds with zwitterionic and anionic phospholipid coatings was studied with HEPES at pH 7.4 as BGE solution. The chromatographic and electrophoretic behavior of three test sample solutions (anionic, cationic, and neutral) was investigated for evaluation of the phospholipid coatings. Our results show that hydrophobic interactions between analytes and the phospholipid coating are important for the migration of charged analytes. In addition, the performances of other piperazine-based buffers, i.e., N-(2-hydroxyethyl)piperazine-N'-(2-hydroxypropanesulfonic acid), piperazine-N,N'-bis(2-ethanesulfonic acid), and piperazine-N,N'-bis(hydroxypropane sulfonic acid), at pH 7.4, as liposome solvent and BGE solution were evaluated and compared with the performance of HEPES at pH 7.4. The anionic liposome solution comprised 80/20 mol% phosphatidylcholine/phosphatidylserine. A simple test solution was selected and the chromatographic and electrophoretic migration behavior of the analytes was evaluated. The results show that, in addition to HEPES, other piperazine-based buffers at pH 7.4 are suitable for coating of fused-silica capillaries with anionic liposomes.

The effect of pH on the formation and stability of phospholipid coatings in fused-silica capillaries in electrophoresis was investigated. A liposome solution consisting of 3 mM of 80:20 mol% phosphatidylcholine/phosphatidylserine (PC/PS) in N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) buffer was used as coating material. The coating was prepared by a method described earlier and five steroids were used as neutral model analytes. First, the effect of pH of the coating solution on the formation and stability of phospholipid coatings was studied at pH 6.5-8.5. The pH of the background electrolyte (BGE) solution (HEPES) was either kept constant at pH 7.4 or made similar to the pH of the liposome coating solution. Results showed that attachment of the coating on the fused-silica wall mostly depends on the protonation of amines of the phospholipids and HEPES. The ability of the phospholipid coating to withstand changes in pH was then investigated by coating at pH 7.5 and separating steroids with acetic acid, 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), HEPES, or glycine BGE, adjusted to pH between 4.5 and 10.8. The results showed that with use of BGE solution at pH 10.8, the separation of steroids was not successful and the electroosmotic flow was high because of leakage of the phospholipid coating during preconditioning of the capillary with BGE solution. There was no phospholipid leakage with a BGE solution of pH 4.5, indicating that the protonated form of the functional groups of PS and HEPES participating in the attachment of the phospholipid coating to the capillary play an essential role in the success of the coating.

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 40 mM N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) at pH 7.4 as basic solution. HEPES (40 mM) 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 1:3 PC/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.

Large unilamellar vesicles of egg-phosphatidylcholine (eggPC), a naturally occurring phospholipid, were used in capillary electrophoresis (CE) for semi-permanent coating of fused silica capillaries. The stability of the phospholipid coating was tested at different cetyltrimethylammonium bromide (CTAB) concentrations with and without CaCl(2) present in the coating solution. The effect of physical factors influencing the coating stability (e.g. duration of the coating time, storage temperature of the coating solution) were also studied. Standing overnight in background electrolyte (BGE) solution did not alter the eggPC phospholipid coating noticeably. The performance of the coating was tested with a mixture of basic proteins (lysozyme, ribonuclease A and alpha-chymotrypsinogen A). Highest efficiencies (over 200,000 plates m(-1)) were achieved when the capillary was filled for 15 h with a liposome solution containing both CTAB and CaCl(2).

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.

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.

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.