Dual Contributions of Analyte Adsorption and Electroosmotic Inhomogeneity to Separation Efficiency in Capillary Electrophoresis of Proteins.

Improving separation efficiency in capillary electrophoresis (CE) requires systematic study of the influence of the electric field (or solute linear velocity) on plate height for a better understanding of the critical parameters controlling peak broadening. Even for poly(diallyldimethylammonium chloride) (PDADMAC)/poly(sodium styrenesulfonate) (PSS) successive multiple ionic-polymer layer (SMIL) coatings, which lead to efficient and reproducible separations of proteins, plate height increases with migration velocity, limiting the use of high electric fields in CE. Solute adsorption onto the capillary wall was generally considered as the main source of peak dispersion, explaining this plate height increase. However, experiments done with Taylor dispersion analysis and CE in the same conditions indicate that other phenomena may come into play. Protein adsorption with slow kinetics and few adsorption sites was established as a source of peak broadening for specific proteins. Surface charge inhomogeneity was also identified as a contribution to plate height due to local electroosmotic fluctuations. A model was proposed and applied to partial PDADMAC/poly(ethylene oxide) capillary coatings as well as PDADMAC/PSS SMIL coatings. Atomic force microscopy with topography and recognition imaging enabled the determination of roughness and charge distribution of the PDADMAC/PSS SMIL surface.

Taylor Dispersion Analysis to support lipid-nanoparticle formulations for mRNA vaccines.

Lipid nanoparticles (LNPs) are currently the most advanced non-viral clinically approved messenger ribonucleic acid (mRNA) delivery systems. The ability of a mRNA vaccine to have a therapeutic effect is related to the capacity of LNPs to deliver the nucleic acid intact into cells. The role of LNPs is to protect mRNA, especially from degradation by ribonucleases (RNases) and to allow it to access the cytoplasm of cells where it can be translated into the protein of interest. LNPs enter cells by endocytosis and their size is a critical parameter impacting their cellular internalization. In this work, we studied different formulation process parameters impacting LNPs size. Taylor dispersion analysis (TDA) was used to determine the LNPs size and size distribution and the results were compared with those obtained by Dynamic Light Scattering (DLS). TDA was also used to study both the degradation of mRNA in the presence of RNases and the percentage of mRNA encapsulation within LNPs.

Taylor dispersion analysis discloses the impairment of Aß peptide aggregation by the presence of a fluorescent tag.

The use of fluorescently tagged amyloid peptides, implicated in Alzheimer's disease, to study their aggregation at low concentrations is a common method; however, the fluorescent tag should not introduce a bias in the aggregation process. In this work, native amyloid peptides Aß(1-40) and Aß(1-42) and fluorescein-5-isothiocyanate (FITC), tagged ones, were studied using Taylor dispersion analysis coupled with a simultaneous UV and light-emitting diode-induced fluorescence detection, to unravel the effect of FITC on the aggregation process. For that, a total concentration of 100 µM of peptides consisting of a mixture of native and tagged ones (up to 10% in moles) was applied. Results demonstrated that FITC had a strong inhibition effect upon the aggregation behaviour of Aß(1-42), whereas for Aß(1-40), only a retardation in kinetics was observed. It was also shown that when mixed solutions of Aß(1-40) and Aß(1-42) are used, the Aß(1-42) alloform was the leading peptide in the aggregation process, and when the latter was tagged, the aggregation kinetics decreased but the lifetime of potentially toxic oligomers was drastically increased. These results confirmed that the hydrophilicity of the N-terminus part of the peptide plays a major role in the aggregation process.

Size and Charge Characterization of Lipid Nanoparticles for mRNA Vaccines.

Messenger RNA vaccines have come into the spotlight as a promising and adaptive alternative to conventional vaccine approaches. The efficacy of mRNA vaccines relies on the ability of mRNA to reach the cytoplasm of cells, where it can be translated into proteins of interest, allowing it to trigger the immune response. However, unprotected mRNA is unstable and susceptible to degradation by exo- and endonucleases, and its negative charges are electrostatically repulsed by the anionic cell membranes. Therefore, mRNA needs a delivery system that protects the nucleic acid from degradation and allows it to enter into the cells. Lipid nanoparticles (LNPs) represent a nonviral leading vector for mRNA delivery. Physicochemical parameters of LNPs, including their size and their charge, directly impact their in vivo behavior and, therefore, their cellular internalization. In this work, Taylor dispersion analysis (TDA) was used as a new methodology for the characterization of the size and polydispersity of LNPs, and capillary electrophoresis (CE) was used for the determination of LNP global charge. The results obtained were compared with those obtained by dynamic light scattering (DLS) and laser Doppler electrophoresis (LDE).

Generation and characterization of air micro-bubbles in highly hydrophobic capillaries.

The generation of air microbubbles in microfluidic systems or in capillaries could be of great interest for transportation (single cell analysis, organite transportation) or for liquid compartmentation. The physicochemical characterization of air bubbles and a better understanding of the process leading to bubble generation during electrophoresis is also interesting in a theoretical point of view. In this work, the generation of microbubbles on hydrophobic Glaco™ coated capillaries has been studied in water-based electrolyte. Air bubbles were generated at the detection window and the required experimental parameters for microbubbles generation have been identified. Generated bubbles migrated against the electroosmotic flow, as would do strongly negatively charged solutes, under constant electric field. They have been characterized in terms of dimensions, electrophoretic mobility, and apparent charge.

Molecular Determinants of Fibrillation in a Viral Amyloidogenic Domain from Combined Biochemical and Biophysical Studies.

The Nipah and Hendra viruses (NiV and HeV) are biosafety level 4 human pathogens classified within the Henipavirus genus of the Paramyxoviridae family. In both NiV and HeV, the gene encoding the Phosphoprotein (P protein), an essential polymerase cofactor, also encodes the V and W proteins. These three proteins, which share an intrinsically disordered N-terminal domain (NTD) and have unique C-terminal domains (CTD), are all known to counteract the host innate immune response, with V and W acting by either counteracting or inhibiting Interferon (IFN) signaling. Recently, the ability of a short region within the shared NTD (i.e., PNT3) to form amyloid-like structures was reported. Here, we evaluated the relevance of each of three contiguous tyrosine residues located in a previously identified amyloidogenic motif (EYYY) within HeV PNT3 to the fibrillation process. Our results indicate that removal of a single tyrosine in this motif significantly decreases the ability to form fibrils independently of position, mainly affecting the elongation phase. In addition, we show that the C-terminal half of PNT3 has an inhibitory effect on fibril formation that may act as a molecular shield and could thus be a key domain in the regulation of PNT3 fibrillation. Finally, the kinetics of fibril formation for the two PNT3 variants with the highest and the lowest fibrillation propensity were studied by Taylor Dispersion Analysis (TDA). The results herein presented shed light onto the molecular mechanisms involved in fibril formation.

Antigen-Adjuvant Interactions in Vaccines by Taylor Dispersion Analysis: Size Characterization and Binding Parameters.

Vaccine adjuvants are immunostimulatory substances used to improve and modulate the immune response induced by antigens. A better understanding of the antigen-adjuvant interactions is necessary to develop future effective vaccine. In this study, Taylor dispersion analysis (TDA) was successfully implemented to characterize the interactions between a polymeric adjuvant (poly(acrylic acid), SPA09) and a vaccine antigen in development for the treatment of Staphylococcus aureus. TDA allowed one to rapidly determine both (i) the size of the antigen-adjuvant complexes under physiological conditions and (ii) the percentage of free antigen in the adjuvant/antigen mixture at equilibrium and finally get the interaction parameters (stoichiometry and binding constant). The complex sizes obtained by TDA were compared to the results obtained by transmission electron microscopy, and the binding parameters were compared to results previously obtained by frontal analysis continuous capillary electrophoresis.

Unraveling the Speciation of ß-Amyloid Peptides during the Aggregation Process by Taylor Dispersion Analysis.

Aggregation mechanisms of amyloid ß peptides depend on multiple intrinsic and extrinsic physicochemical factors (e.g., peptide chain length, truncation, peptide concentration, pH, ionic strength, temperature, metal concentration, etc.). Due to this high number of parameters, the formation of oligomers and their propensity to aggregate make the elucidation of this physiopathological mechanism a challenging task. From the analytical point of view, up to our knowledge, few techniques are able to quantify, in real time, the proportion and the size of the different soluble species during the aggregation process. This work aims at demonstrating the efficacy of the modern Taylor dispersion analysis (TDA) performed in capillaries (50 µm i.d.) to unravel the speciation of ß-amyloid peptides in low-volume peptide samples (∼100 µL) with an analysis time of ∼3 min per run. TDA was applied to study the aggregation process of Aß(1-40) and Aß(1-42) peptides at physiological pH and temperature, where more than 140 data points were generated with a total volume of ∼1 µL over the whole aggregation study (about 0.5 µg of peptides). TDA was able to give a complete and quantitative picture of the Aß speciation during the aggregation process, including the sizing of the oligomers and protofibrils, the consumption of the monomer, and the quantification of different early- and late-formed aggregated species.

Screening for pancreatic lipase natural modulators by capillary electrophoresis hyphenated to spectrophotometric and conductometric dual detection.

The search for novel pancreatic lipase (PL) inhibitors has gained increasing attention in recent years. For the first time, a dual detection capillary electrophoresis (CE)-based homogeneous lipase assay was developed employing both the offline and online reaction modes. The hydrolysis of 4-nitrophenyl butyrate (4-NPB) catalyzed by PL into 4-nitrophenol and butyrate was monitored by spectrophotometric and conductimetric detection, respectively. The assays presented several advantages such as economy in consumption (few tens of nanoliters for online assays to few tens of microliters for offline assays), no modification of lipase, rapidity (<10 min) and versatility. Tris/MOPS (10 mM, pH 6.6) was used as the background electrolyte and the incubation buffer for enzymatic reactions. We confirmed that in the conditions of the study (small substrate 4-NPB, 37 °C, pH 6.6), the PL was active even in the absence of dipalmitoylphosphatidylcholine (DPPC) vesicles, generally used to mimic the lipid-water interface. This was confirmed by the maximum velocity (Vmax) and the Michaelis-Menten constant (Km) values that were the same order of magnitude in the absence and presence of DPPC. The developed method was used to screen crude aqueous plant extracts and purified compounds. We were able to identify the promising PL inhibition of hawthorn leaf herbal infusions at 1 mg mL-1 (37%) and PL activation by fresh and dry hawthorn flowers (∼24%). Additionally, two triterpenoids purified from extracts of oakwood were identified for the first time as potent PL inhibitors demonstrating 51 and 58% inhibition at 1 mg mL-1, respectively.

Quantification of Adsorption and Optimization of Separation of Proteins in Capillary Electrophoresis.

The improvement of separation efficiency for protein analysis in capillary electrophoresis (CE) is a challenging topic in which protein adsorption onto the capillary wall plays a crucial role. In this work, a simple method allowing the quantification of the adsorption of proteins onto the coated or untreated inner surface of the fused silica capillary was developed based on the determination of the retention factor by measuring separation efficiency of individual proteins at different separation voltages (i.e., different linear velocities). This approach was applied to the quantification of the residual adsorption of four test proteins on five-layer polyelectrolyte coatings and bare fused silica capillary. It allows to get a fair ranking of the coating performances toward protein adsorption, whatever their apparent electrophoretic mobilities (migration times) are. Due to the existence of (even low) residual adsorption, the electrophoretic operating conditions (electric field, capillary length, and internal diameter) can be optimized to improve the separation performances resulting in experimental separation efficiency up to ∼600 000 plates.m-1 in conditions compatible with MS coupling. This approach represents a crucial step in the course to get antifouling coatings for protein separation in CE. It can be used for the evaluation and ranking of virtually any coating (neutral or charged) in CE.

Capillary Zone Electrophoresis-Top-Down Tandem Mass Spectrometry for In-Depth Characterization of Hemoglobin Proteoforms in Clinical and Veterinary Samples.

Hemoglobin (Hb) constitutes an important protein in clinical diagnostics-both in humans and animals. Among the high number of sequence variants, some can cause severe diseases. Moreover, chemical modifications such as glycation and carbamylation serve as important biomarkers for conditions such as diabetes and kidney diseases. In clinical routine analysis of glycated Hb, sequence variants or other Hb proteoforms can cause interference, resulting in wrong quantification results. We present a versatile and flexible capillary zone electrophoresis-mass spectrometry screening method for Hb proteoforms including sequence variants and modified species extracted from dried blood spot (DBS) samples with virtually no sample preparation. High separation power was achieved by application of a 5-layers successive multiple ionic polymer layers-coated capillary, enabling separation of positional isomers of glycated α- and ß-chains on the intact level. Quantification of glycated Hb was in good correlation with the results obtained in a clinical routine method. Identification and characterization of known and unknown proteoforms was performed by fragmentation of intact precursor ions. N-Terminal and lysine glycation could be identified on the α- and ß-chain, respectively. The versatility of the method was demonstrated by application to dog and cat DBS samples. We discovered a putative new sequence variant of the ß-chain in dog (T38 → A). The presented method enables separation, characterization, and quantification of intact proteoforms, including positional isomers of glycated species in a single run. Combined with the simple sample preparation, our method represents a valuable tool to be used for deeper characterization of clinical and veterinary samples.

Cosolvents in Self-Emulsifying Drug Delivery Systems (SEDDS): Do They Really Solve Our Solubility Problems?

The aim of this study was to investigate the fate and the impact of cosolvents in self-emulsifying drug delivery systems (SEDDS). Three different SEDDS comprising the cosolvents DMSO (FD), ethanol (FE), and benzyl alcohol (FBA) as well as the corresponding formulations without these cosolvents (FD0, FE0, and FBA0) were developed. Mean droplet size, polydispersity index (PDI), ζ potential, stability, and emulsification time were determined. Cosolvent release studies were performed via the dialysis membrane method and Taylor dispersion analysis (TDA). Furthermore, the impact of cosolvent utilization on payloads in SEDDS was examined using quinine as a model drug. SEDDS with and without a cosolvent showed no significant differences in droplet size, PDI, and ζ potential. The emulsification time was 3-fold (FD0), 80-fold (FE0), and 7-fold (FBA0) longer due to the absence of the cosolvents. Release studies in demineralized water provided evidence for an immediate and complete release of DMSO, ethanol, and benzyl alcohol. TDA confirmed this result. Moreover, a 1.4-fold (FD), 2.91-fold (FE), and 2.17-fold (FBA) improved payload of the model drug quinine in the selected SEDDS preconcentrates was observed that dropped after emulsification within 1-5 h due to drug precipitation. In parallel, the quinine concentrations decreased until reaching the same levels of the corresponding SEDDS without cosolvents. Due to the addition of hydrophilic cosolvents, the emulsifying properties of SEDDS are strongly improved. As hydrophilic cosolvents are immediately released from SEDDS during the emulsification process, however, their drug solubilizing properties in the resulting oily droplets are very limited.

Study of Interactions between Antigens and Polymeric Adjuvants in Vaccines by Frontal Analysis Continuous Capillary Electrophoresis.

Vaccine adjuvants are used to enhance the immune response induced by antigens that have insufficient immunostimulatory capabilities. The present work aims at developing a frontal analysis continuous capillary electrophoresis (FACCE) methodology for the study of antigen-adjuvant interactions in vaccine products. After method optimization using three cationic model proteins, namely lysozyme, cytochrome c, and ribonuclease A, FACCE was successfully implemented to quantify the free antigen and thus to determine the interaction parameters (stoichiometry and binding constant) between an anionic polymeric adjuvant (polyacrylic acid, SPA09) and a cationic vaccine antigen in development for the treatment for Staphylococcus aureus. The influence of the ionic strength of the medium on the interactions was investigated. A strong dependence of the binding parameters with the ionic strength was observed. The concentration of the polymeric adjuvant was also found to significantly modify the ionic strength of the formulation, the extent of which could be estimated and corrected.

Optimizing Water-Based Extraction of Bioactive Principles of Hawthorn: From Experimental Laboratory Research to Homemade Preparations.

Hawthorn (Crataegus) is used for its cardiotonic, hypotensive, vasodilative, sedative, antiatherosclerotic, and antihyperlipidemic properties. One of the main goals of this work was to find a well-defined optimized extraction protocol usable by each of us that would lead to repeatable, controlled, and quantified daily uptake of active components from hawthorn at a drinkable temperature (below 60 °C). A thorough investigation of the extraction mode in water (infusion, maceration, percolation, ultrasounds, microwaves) on the yield of extraction and the amount of phenolic compounds, flavonoids, and proanthocyanidin oligomers as well as on the Ultra High Performance Liquid Chromatography (UHPLC) profiles of the extracted compounds was carried out. High-resolution Fourier transform ion cyclotron resonance mass spectrometry was also implemented to discriminate the different samples and conditions of extraction. The quantitative and qualitative aspects of the extraction as well as the kinetics of extraction were studied, not only according to the part (flowers or leaves), the state (fresh or dried), and the granulometry of the dry plant, but also the stirring speed, the temperature, the extraction time, the volume of the container (cup, mug or bowl) and the use of infusion bags.

Limits in Size of Taylor Dispersion Analysis: Representation of the Different Hydrodynamic Regimes and Application to the Size-Characterization of Cubosomes.

Taylor dispersion analysis (TDA) is an absolute method (no calibration needed) for the determination of the molecular diffusion coefficient (D) based on the band broadening of a solute in a laminar flow. TDA is virtually applicable to any solute with size ranging from angstrom to sub-micrometer. The higher sizing limit is restricted by the occurrence of possibly two regimes: convective and hydrodynamic chromatography (HDC) regimes, which have different physical origins that should not be confused. This work aims at clearly defining the experimental conditions for which these two regimes can play a role, alone or concomitantly. It also calculates the relative error on D due to the HDC regime according to the solute to capillary size ratio. It is demonstrated in this work that HDC does not significantly affect the TDA measurement as long as the hydrodynamic radius of the solute is lower than 0.0051 times the capillary radius. Experimental illustrations of the occurrence of the two regimes are given taking polystyrene nanoparticles as model solutes. Finally, application of TDA to the sizing of large real-life solutes is proposed, taking cubosomes as new drug nanocarriers of potential interest for drug delivery purposes.

Taylor Dispersion Analysis of Polysaccharides Using Backscattering Interferometry.

Taylor dispersion analysis (TDA) allows the determination of the molecular diffusion coefficient (D) or the hydrodynamic radius (Rh) of a solute from the peak broadening of a plug of solute in a laminar Poiseuille flow. The main limitation plaguing the broader applicability of TDA is the lack of a sensitive detection modality. UV absorption is typically used with TDA but is only suitable for UV-absorbing or derivatized compounds. In this work, we present a development of the TDA method for non-UV absorbing compounds by using a universal detector based on refractive index (RI) sensing with backscattering interferometry (BSI). BSI was interfaced to a capillary electrophoresis-UV instrument using a polyimide coated fused silica capillary and an in-house designed flow-cell assembly. Polysaccharides were selected to demonstrate the application of TDA-BSI for size characterization. Under the conditions of validity of TDA, D and Rh average values and the entire Rh distributions were obtained from the (poly)saccharide taylorgrams, including non-UV absorbing polymers.

On the ionic strength dependence of the electrophoretic mobility: From 2D to 3D slope-plots.

Determining the charge and the nature (small ion, nanoparticle, or polyelectrolyte) of an unknown solute from its electrophoretic characteristics remains a challenging issue. In this work, we demonstrate that, if the knowledge of the effective electrophoretic mobility (µep ) at a given ionic strength is not sufficient to characterize a given solute, the combination of this parameter with (i) the relative decrease of the electrophoretic mobility with the ionic strength (S), and (ii) the hydrodynamic radius (Rh ), is sufficient (in most cases) to deduce the nature of the solute and its charge. These three parameters are experimentally accessible by CZE and Taylor dispersion analysis performed on the same instrumentation. 3D representation of the three aforementioned parameters (µep ; S and Rh ) is proposed to visualize the differences in the electrophoretic behavior between solutes according to their charge and nature. Surprisingly, such 3D slope plot in the case of small ions and nanoparticles looks like a "whale-tail," while polyelectrolyte contour plot represents a rather simple and monotonous map that is independent of solute size. This work also sets how to estimate the effective charge of a solute from a given experimental (S,Rh,µ ep 5 mM ) triplet, which is not possible to obtain unambiguously with only (Rh,µ ep 5 mM ) or (S,µ ep 5 mM ) doublet, where µ ep 5 mM is the effective electrophoretic mobility at 5 mM ionic strength.

A New Robust Estimator of Polydispersity from Dynamic Light Scattering Data.

We introduce a new estimator of particle size polydispersity for dynamic light scattering data, which quantifies the relative width of the intensity-weighted distribution of diffusion coefficients. Simulated dynamic light scattering data are analyzed to (i) compare the accuracy and precision of the new polydispersity indicator to polydispersity measurements from standard cumulant and moment analysis (MA) fits and (ii) establish for each method the optimum data range for fitting. Although MA is preferable at low polydispersity, the new estimator is the most accurate and precise at intermediate and large polydispersities. Finally, we successfully apply the method proposed here to real data from colloidal particles, microgels, and polymer solutions.

Quantifying the Heterogeneity of Chemical Structures in Complex Charged Polymers through the Dispersity of Their Distributions of Electrophoretic Mobilities or of Compositions.

The complexity of synthetic and natural polymers used in industrial and medical applications is expanding; thus, it becomes increasingly important to improve and develop methods for their molecular characterization. Free-solution capillary electrophoresis is a robust technique for the separation and characterization of both natural and synthetic complex charged polymers. In the case of polyelectrolytes, free-solution capillary electrophoresis is in the "critical conditions" (CE-CC): it allows their separation by factors other than molar mass for molar masses typically higher than 20000 g/mol. This method is thus complementary to size-exclusion chromatography (SEC). SEC is widely used to determine molar mass distributions and their dispersities. Utilizing CE-CC, an analogous calculation of dispersity based on the distributions of electrophoretic mobilities was derived and the heterogeneity of composition or branching in different polysaccharides or synthetic polymers was obtained in a number of experimental cases. Calculations are based on a ratio of moments and could therefore be compared to simulations of polymerization processes, in analogy to the work performed on molar mass distributions. Among four possible types of dispersity, the most precise values were obtained with the calculation analogous with the dispersity of molar mass distribution Mw/Mn. In addition, the dispersity value allows conclusions based on a single value: the closer the dispersity is to 1, the more homogeneous the polymer is in terms of composition or branching. This approach allows the analysis of dispersity of important molecular attributes of polymers other than molar mass and aims at improving the overall molecular characterization of both synthetic and natural polymers. The dispersity can also be monitored online while performing a chemical reaction within the CE instrument.

Modelling and predicting the interactions between oppositely and variously charged polyelectrolytes by frontal analysis continuous capillary electrophoresis.

In this work, a systematic study of the interactions between poly(l-lysine) and variously charged statistical copolymers of acrylamide and 2-acrylamido-2-methyl-1-propanesulfonate (PAMAMPS) has been carried out by frontal analysis continuous capillary electrophoresis (FACCE). FACCE was successfully implemented to obtain the interaction parameters (binding constant and stoichiometry) at different ionic strengths and for different PAMAMPS charge densities varying between 15% and 100%. The range of investigated ionic strengths was carefully adjusted according to the PAMAMPS charge density to obtain measurable binding constants by FACCE (i.e. formation binding constant typically comprised between 104 and 106 M-1). The number of released counter-ions during the polyelectrolyte complex formation was systematically quantified via the ionic strength dependence of the binding constant and was compared to the total condensed counter-ion reservoir according to Manning theory on counter-ion condensation. A descriptive and predictive model relating the physico-chemical properties of the two partners, the binding constant and the ionic strength is proposed in the framework of multiple independent interaction sites of equal energy.