Gebrenegus Yohannes

Asymmetrical flow field-flow fractionation technique for separation and characterization of biopolymers and bioparticles

Field-flow fractionation (FFF) is one of the most versatile separation techniques in the field of analytical separation sciences, capable of separating macromolecules in the range 10³-10¹⁵ g mol⁻¹ and/or particles with 1 nm-100 μm in diameter. The most universal and most frequently used FFF technique, flow FFF, includes three types of techniques, namely symmetrical flow FFF, hollow fiber flow FFF, and asymmetrical flow FFF which is most established variant among them. This review provides a brief look at the theoretical background of analyte retention and separation efficiency in FFF, followed by a comprehensive overview of the current status of asymmetrical flow FFF with selected applications in the field of biopolymers and bioparticles.

Thermal aggregation of bovine serum albumin studied by asymmetrical flow field-flow fractionation.

The use of asymmetrical flow field-flow fractionation (AsFlFFF) in the study of heat-induced aggregation of proteins is demonstrated with bovine serum albumin (BSA) as a model analyte. The hydrodynamic diameter (d(h)), the molar mass of heat-induced aggregates, and the radius of gyration (R(g)) were calculated in order to get more detailed understanding of the conformational changes of BSA upon heating. The hydrodynamic diameter of native BSA at ambient temperature was approximately 7 nm. The particle size was relatively stable up to 60 degrees C; above 63 degrees C, however, BSA underwent aggregation (growth of hydrodynamic diameter). The hydrodynamic diameters of the aggregated particles, heated to 80 degrees C, ranged from 15 to 149 nm depending on the BSA concentration, duration of incubation, and the ionic strength of the solvent. Heating of BSA in the presence of sodium dodecyl sulfate (1.7 or 17 mM) did not lead to aggregation. The heat-induced aggregates were characterized in terms of their molar mass and particle size together with their respective distributions with a hyphenated technique consisting of an asymmetrical field-flow fractionation device and a multi-angle light scattering detector and a UV-detector. The carrier solution comprised 8.5 mM phosphate and 150 mM sodium chloride at pH 7.4. The weight-average molar mass (M(w)) of native BSA at ambient temperature is 6.6x10(4) g mol(-1). Incubation of solutions with BSA concentrations of 1.0 and 2.5 mg mL(-1) at 80 degrees C for 1 h resulted in aggregates with M(w) 1.2x10(6) and 1.9x10(6) g mol(-1), respectively. The average radius of gyration and the average hydrodynamic radius of the heat-induced aggregate samples were calculated and compared to the values obtained from the size distributions measured by AsFlFFF. For comparison static light scattering measurements were carried out and the corresponding average molar mass distributions of solutions with BSA concentrations of 1.0 and 2.5 mg mL(-1) at 80 degrees C for 1 h gave aggregates with M(w) 1.7x10(6) and 3.5x10(6) g mol(-1), respectively.

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.

Conformational changes of apoB-100 in SMase-modified LDL mediate formation of large aggregates at acidic pH

During atherogenesis, the extracellular pH of atherosclerotic lesions decreases. Here, we examined the effect of low, but physiologically plausible pH on aggregation of modified LDL, one of the key processes in atherogenesis. LDL was treated with SMase, and aggregation of the SMase-treated LDL was followed at pH 5.5–7.5. The lower the pH, the more extensive was the aggregation of identically prelipolyzed LDL particles. At pH 5.5–6.0, the aggregates were much larger (size >1 µm) than those formed at neutral pH (100–200 nm). SMase treatment was found to lead to a dramatic decrease in α-helix and concomitant increase in β-sheet structures of apoB-100. Particle aggregation was caused by interactions between newly exposed segments of apoB-100. LDL-derived lipid microemulsions lacking apoB-100 failed to form large aggregates. SMase-induced LDL aggregation could be blocked by lowering the incubation temperature to 15°C, which also inhibited the changes in the conformation of apoB-100, by proteolytic degradation of apoB-100 after SMase-treatment, and by HDL particles. Taken together, sphingomyelin hydrolysis induces exposure of protease-sensitive sites of apoB-100, whose interactions govern subsequent particle aggregation. The supersized LDL aggregates may contribute to the retention of LDL lipids in acidic areas of atherosclerosis-susceptible sites in the arterial intima.

Ultrathin polyelectrolyte multilayers: in situ ESR/UV-Vis-NIR spectroelectrochemical study of charge carriers formed under oxidation

Redox cycling of self-assembled polyelectrolyte multilayers, consisting of well-defined water-soluble electronically conducting poly-3-(3′-thienyloxy)propyltriethylammonium (P3TOPA) and insulating PSS (poly(sodium-4-styrenesulfonate)) on indium–tin oxide (ITO) electrode, or poly-3-(3′-thienyloxy)propanesulfonate) (P3TOPS) and insulating poly(diallyldimethylammonium chloride) (PDADMA) on Au electrode, were investigated using in situ ESR/UV-Vis-NIR cyclovoltammetry and modulated electroreflectance. The spectroelectrochemical responses of (P3TOPA/PSS)n multilayers on ITO were compared with the spectroelectrochemistry of P3TOPA dissolved in aqueous or DMSO solutions. We found that both in solution and in the film the radical cationic structures (polarons) were primarily formed upon oxidation in chains of high conjugation length. However, they fully disproportionate into neutral and dicationic segments and the spinless charge carriers dominate, similarly to recent results with well defined long chain oligothiophenes and ladder type indenofluoranthene oligomers. A similar spectroelectrochemical behaviour was observed for the (P3TOPS/PDADMA)n system and its electroreflectance data indicate that the generation of polarons was markedly slower than the production of bipolarons. We compare these new results on well-defined polymer chains with the previous in situ spectroelectrochemical studies of the electrochemically prepared conducting polymer layers (PT, PMeT, PPy, PANI) in order to find a general scheme to describe the redox behaviour of different films with various degree of structure homogenity.

Polyethylene glycol-stabilized lipid disks as model membranes in interaction studies based on electrokinetic capillary chromatography and quartz crystal microbalance

Distearoylphosphatidylcholine (DSPC)/cholesterol/distearoylphosphatidylethanolamine (DSPE)-polyethylene glycol 5000 [PEG(5000)] lipid disks, mimicking biological membranes, were used as pseudostationary phase in partial filling electrokinetic capillary chromatography (EKC) to study interactions between pharmaceuticals and lipid disks. Capillaries were coated either noncovalently with a poly(1-vinylpyrrolidone)-based copolymer or covalently with polyacrylamide to mask the negative charges of the fused-silica capillary wall and to minimize interactions between positively charged pharmaceuticals and capillary wall. Although the noncovalent copolymer coating method was faster, better stability of the covalent polyacrylamide coating at physiological pH 7.4 made it more reliable in partial filling EKC studies. Migration times of pharmaceuticals were proportional to the amount of lipids in the pseudostationary phase, and partition coefficients were successfully determined. Because the capillary coatings almost totally suppressed the electroosmotic flow, it was not practical to use the EKC-based method for partition studies involving large molecules with low mobilities. Hence, the applicability of the biomembrane mimicking lipid disks for interactions studies with large molecules was verified by the quartz crystal microbalance technique. Biotinylated lipid disks were then immobilized on streptavidin-coated sensor chip surface, and interactions with a high-molecular-mass molecule, lysozyme, were studied. Cryo-transmission electron microscopy and asymmetrical flow field-flow fractionation were used to clarify the sizes of lipid disks used.

Interaction of phospholipid transfer protein with human tear fluid mucins.

In addition to circulation, where it transfers phospholipids between lipoprotein particles, phospholipid transfer protein (PLTP) was also identified as a component of normal tear fluid. The purpose of this study was to clarify the secretion route of tear fluid PLTP and elucidate possible interactions between PLTP and other tear fluid proteins. Human lacrimal gland samples were stained with monoclonal antibodies against PLTP. Heparin-Sepharose (H-S) affinity chromatography was used for specific PLTP binding, and coeluted proteins were identified with MALDI-TOF mass spectrometry or Western blot analysis. Immunoprecipitation assay and blotting with specific antibodies helped to identify and characterize PLTP-mucin interaction in tear fluid. Human tear fluid PLTP is secreted from the lacrimal gland. MALDI-TOF analysis of H-S fractions identified several candidate proteins, but protein-protein interaction assays revealed only ocular mucins as PLTP interaction partners. We suggest a dual role for PLTP in human tear fluid: (1) to scavenge lipophilic substances from ocular mucins and (2) to maintain the stability of the anterior tear lipid film. PLTP may also play a role in the development of ocular surface disease.

In vitro and in vivo entrapment of bupivacaine by lipid dispersions.

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).

The effect of freeze-drying parameters and formulation composition on IgG stability during drying

The objective of this study was to explore the effects of freeze-drying parameters and formulation composition on polyclonal IgG stability during processing. Samples were freeze-dried with different primary drying pressures and secondary drying heating rates. After drying, changes in IgG in vitro binding activity, monomer recovery, average particle size, and polydispersity were studied from the rehydrated lyophilizates. Significant trends were not observed in binding activities or monomer recoveries, but increases in particle size and polydispersity were observed when using lower primary drying pressure. This effect could no longer be observed when sodium phosphate buffer was removed from the formulation. Altering the secondary drying heating rates did not result in any measurable changes in protein stability.

Sugar treatment of human lipoprotein particles and their separation by capillary electrophoresis.

Entrapment of lipoprotein particles in the extracellular matrix of the arterial intima is a characteristic feature of the development of atherosclerosis, the disease behind myocardial infarction and stroke. In this study, sugars were exploited in the separation of lipoproteins by CE. Monosaccharides, disaccharides and one sugar alcohol used during ultracentrifugal isolation of lipoproteins prevented the strong and unfavorable adsorption of lipoprotein particles on the capillary wall, allowing their selective separation in uncoated fused silica capillary. The effect of ionic strength of the phosphate BGE solution on the separation at physiological pH was clarified. Asymmetrical flow field-flow fractionation and dynamic light scattering showed that sugars affected the structure of lipoproteins by decreasing their sizes. Although in molecular dynamics simulations, only a 19 amino acid peptide of apolipoprotein B-100 and a 15 amino acid peptide of apolipoprotein E were employed, the results also indicated a decrease in lipoprotein size, supporting the asymmetrical flow field-flow fractionation and dynamic light scattering results.