Characterization of phosphatidylcholine/polyethylene glycol-lipid aggregates and their use as coatings and carriers in capillary electrophoresis.

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.

Cholesterol-rich membrane coatings for interaction studies in capillary electrophoresis: application to red blood cell lipid extracts.

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.

Stabilization of phosphatidylcholine coatings in capillary electrophoresis by increase in membrane rigidity.

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.

Influence of cetyltrimethylammonium bromide on phosphatidylcholine-coated capillaries.

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).

Simple coating of capillaries with anionic liposomes in capillary electrophoresis.

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.