Investigating cationic and zwitterionic successive multiple ionic-polymer layer coatings for protein separation by capillary electrophoresis.

Successive multiple ionic-polymer layers (SMILs) have long since proved their worth in capillary electrophoresis as they ensure stable electroosmotic flow (EOF) and relatively high separation efficiency. Recently, we demonstrated that plotting the plate height (H) against the solute migration velocity (u) enabled a reliable quantitative evaluation of the coating performances in terms of separation efficiency. In this work, various physicochemical and chemical parameters of the SMIL coating were studied and optimized in order to decrease the slope of the ascending part of the H vs u curve, which is known to be controlled by the homogeneity in charge of the coating surface and by the possible residual solute adsorption onto the coating surface. SMILs based on poly(diallyldimethylammonium chloride) (PDADMAC) and poly(sodium styrene sulfonate) (PSS) were formed and the effect of each polyelectrolyte molar mass and of the number of polyelectrolyte layers (up to 21 layers) was studied. The use of polyethylene imine as an anchoring first layer was considered. More polyelectrolyte couples based on PDADMAC, polybrene, PSS, poly(vinyl sulfate), and poly(acrylic acid) were tested. Finally, zwitterionic polymers based on the poly(α-l-lysine) scaffold were synthesized and used as the last layer of SMILs, illustrating their ability to finetune the EOF, while maintaining good separation efficiency.

Taylor dispersion analysis using capacitively coupled contactless conductivity detector.

Taylor dispersion analysis (TDA) is a simple and absolute method to determine the hydrodynamic radius of solutes that respond to UV or fluorescence detections. To broaden the application range of TDA, it is necessary to develop new detection modes. This study aims to study capacitively coupled contactless conductivity detector (C4D) for the analysis of charged macromolecules. The detection sensitivities and hydrodynamic radii were compared for a C4D detector and a UV detector on positively or negatively charged polymers responding both to UV and C4D (poly-L-lysine and poly(acrylamide-co-2-acrylamido-1-methyl-propanesulfonate). The influence of the composition of the background electrolyte on the detection sensitivity has been studied and optimized for C4D detection. The influence of the molar mass and of the polymer chemical charge density on the C4D and UV sensitivities of detection have been investigated based on well-characterized copolymers samples of different molar masses and charge densities. The advantages and disadvantages compared to UV detection, as well as the range of applicability of C4D detection in TDA were identified. C4D detection can be an alternative method for sizing charged polymers of reasonable molar mass (typically below 105 g mol-1) that do not absorb in UV. A decline in the sensitivity of detection in C4D was observed for higher molar masses.

Size-based characterization of dendrigraft poly(L-lysine) by free solution capillary electrophoresis using polyelectrolyte multilayer coatings.

Dendrigraft poly(L-lysine) (DGL) constitutes a promising dendritic-like drug vehicle with high biocompatibility and straightforward access via ring-opening polymerization of N-carboxyanhydride in water. The characterization of the different generations of DGL is however challenging due to their heterogeneity in molar mass and branching ratio. In this work, free solution capillary electrophoresis was used to perform selective separation of the three first generations of DGL, and optimized conditions were developed to maximize inter-generation resolution. To reduce solute adsorption on the capillary wall, successive multiple ionic polymer layer coatings terminated with a polycation were deposited onto the inner wall surface. PEGylated polycation was also used as the last layer for the control of the electroosmotic flow (EOF), depending on the PEGylation degree and the methyl-polyethylene glycol (mPEG) chain length. 1 kDa mPEG chains and low grafting densities were found to be the best experimental conditions for a fine tuning of the EOF leading to high peak resolution. Molar mass polydispersity and polydispersity in effective electrophoretic mobility were successfully determined for the three first generations of DGL.

Self-emulsifying drug delivery systems (SEDDS): How organic solvent release governs the fate of their cargo.

Organic solvents are commonly used in self-emulsifying drug delivery systems (SEDDS) to increase payloads of orally administered poorly soluble drugs. Since such solvents are released to a varying extent after emulsification, depending on their hydrophilic nature, they have a substantial impact on the cargo. To investigate this impact in detail, quercetin and curcumin as model drugs were incorporated in SEDDS comprising organic solvents (SEDDS-solvent) of logP < 2 and > 2. SEDDS were characterized regarding size, payload, emulsification time and solvent release. The effect of solvent release on the solubility of these drugs was determined. Preconcentrates of SEDDS-solventlogP < 2 emulsified more rapidly (< 1.5 min) forming smaller droplets than SEDDS-solventlogP > 2. Although, SEDDS-solventlogP < 2 preconcentrates provided higher quercetin solubility than the latter, a more pronounced solvent release caused a more rapid quercetin precipitation after emulsification (1.5 versus 4 h). In contrast, the more lipophilic curcumin was not affected by solvent release at all. Particularly, SEDDS-solventlogP < 2 preconcentrates provided high drug payloads without showing precipitation after emulsification. According to these results, the fate of moderate lipophilic drugs such as quercetin is governed by the release of solvent, whereas more lipophilic drugs such as curcumin remain inside the oily phase of SEDDS even when the solvent is released.

Fast, simple and calibration-free size characterization and quality control of extracellular vesicles using capillary Taylor dispersion analysis.

This study reports the development of a Taylor Dispersion Analysis (TDA) method for the size characterization of Extracellular Vesicles (EVs), which are highly heterogeneous nanoscale cell-derived vesicles (30-1000 nm). Here, we showed that TDA, conducted in uncoated fused silica capillaries (50 µm i.d.) using a conventional Capillary Electrophoresis instrument, is able to provide absolute sizing (requiring no calibration) of bovine milk-derived EVs in a small sample volume (∼ 7 nL) and over their entire size range, even the smallest ones (< 70 nm) not accessible via other techniques that provide nanoparticle sizing in suspension. TDA size measurements were repeatable (RSD < 10%) and the average EV sizes were found in the range of 120-210 nm, in very good agreement with those measured with Nanoparticle Tracking Analysis, commonly used for EV characterization. TDA allowed quantitative estimation of EVs for concentrations ≥ 2 × 1011 EVs/mL. Furthermore, TDA was able to detect minor changes in EV size (i.e. by ∼25 nm upon interaction with specific anti-CD9 antibodies of ∼150 kDa), and to highlight the impact of extraction methods (i.e. milk pretreatment: freezing, acid precipitation or centrifugation; the type of size-exclusion chromatography column) and of fluorescent labeling (i.e. intravesicular or surface labeling) on the isolated EV population size. In parallel to EV sizing, TDA allowed to detect molecular contaminants (average sizes ∼1-13 nm) present within the sample, rendering this method a valuable tool to assess the quality and quantity of EV isolates.

Critical parameters for highly efficient and reproducible polyelectrolyte multilayer coatings for protein separation by capillary electrophoresis.

Since the introduction of polyelectrolyte multilayers to protein separation in capillary electrophoresis (CE), some progress has been made to improve separation efficiency by varying different parameters, such as buffer ionic strength and pH, polyelectrolyte nature and number of deposited layers. However, CE is often overlooked as it lacks robustness compared to other separation techniques. In this work, critical parameters for the construction of efficient and reproducible Successive multiple ionic-polymer layers (SMIL) coatings were investigated, focusing on experimental conditions, such as vial preparation and sample conservation which were shown to have a significant impact on separation performances. In addition to repeatability, intra- and inter-capillary precision were assessed, demonstrating the improved capability of poly(diallyldimethylammonium chloride) / poly(sodium styrene sulfonate) (PDADMAC / PSS) coated capillaries to separate model proteins in a 2 M acetic acid background electrolyte when all the correct precautions are put in place (with run to run%RSD(tm) < 1.8%, day to day%RSD(tm) < 3.2% and cap to cap%RSD(tm) < 4.6%). The approach recently introduced to calculate retention factors was used to quantify residual protein adsorption onto the capillary wall and to assess capillary coating performances. 5-layer PDADAMAC / PSS coatings led to average retention factors for the five model proteins of ∼4×10-2. These values suggest a relatively low residual protein adsorption leading to reasonably flat plate height vs linear velocity curves, obtained by performing electrophoretic separations at different electrical voltages (-10 to -25 kV).

Modifying last layer in polyelectrolyte multilayer coatings for capillary electrophoresis of proteins.

Protein adsorption on the inner wall of the fused silica capillary wall is an important concern for capillary electrophoresis (CE) analysis since it is mainly responsible for separation efficiency reduction. Successive Multiple Ionic-polymer Layers (SMIL) are used as capillary coatings to limit protein adsorption, but even low residual adsorption strongly impacts the separation efficiency, especially at high separation voltages. In this work, the influence of the chemical nature and the PEGylation of the polyelectrolyte deposited in the last layer of the SMIL coating was investigated on the separation performances of a mixture of four model intact proteins (myoglobin (Myo), trypsin inhibitor (TI), ribonuclease a (RNAse A) and lysozyme (Lyz)). Poly(allylamine hydrochloride) (PAH), polyethyleneimine (PEI), ε-poly(L-lysine) (εPLL) and α-poly(L-lysine) (αPLL) were compared before and after chemical modification with polyethyleneglycol (PEG) of different chain lengths. The experimental results obtained by performing electrophoretic separations at different separation voltages allowed determining the residual retention factor of the proteins onto the capillary wall via the determination of the plate height at different solute velocities and demonstrated a strong impact of the polycationic last layer on the electroosmotic mobility, the separation efficiency and the overall resolution. Properties of SMIL coatings were also characterized by quartz microbalance and atomic force microscopy, demonstrating a glassy structure of the films.

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.

Successive Multiple Ionic-Polymer Layer Coatings for Intact Protein Analysis by Capillary Zone Electrophoresis-Mass Spectrometry: Application to Hemoglobin Analysis.

Adsorption of analytes, e.g., proteins, often interfere with separation in CE, due to the relatively large surface of the narrow capillary. Coatings often are applied to prevent adsorption and to determine the electroosmotic flow (EOF), which is of major importance for the separation in CE. Successive multiple ionic-polymer layer (SMIL) coatings are frequently used for protein analysis in capillary electrophoresis resulting in high separation efficiency and repeatability. Here, the coating procedure of a five-layer SMIL coating is described using quaternized diethylaminoethyl dextran (DEAEDq) as polycation and poly(methacrylic acid) (PMA) as polyanion. Depending on the analyte, different polyions may be used to increase separation efficiency. However, the coating procedure remains the same.To demonstrate the applicability of SMIL coatings in CE-MS, human hemoglobin was measured in a BGE containing 2 M acetic acid. DEAEDq-PMA coating was found to be the most suitable for hemoglobin analysis due to relatively low reversed electroosmotic mobility leading to increased electrophoretic resolution of closely related proteoforms. Thereby, not only alpha and beta subunit of the hemoglobin could be separated, but also positional isoforms of glycated and carbamylated species were separated within 24 min.

Polyelectrolyte Multilayers in Capillary Electrophoresis.

Capillary electrophoresis (CE) has been proven to be a performant analytical method to analyze both small and macro molecules. Indeed, it is capable of separating compounds of the same nature according to differences in their charge to size ratios, particularly proteins, monoclonal antibodies and peptides. However, one of the major obstacles to reach high separation efficiency remains the adsorption of solutes on the capillary wall. Among the different coating approaches used to control and minimize solute adsorption, polyelectrolyte multilayers can be applied to CE as a versatile approach. These coatings are made up of alternating layers of polycations and polyanions, and may be used in acidic, neutral or basic conditions depending on the solutes to be analyzed. This Review provides an overview of Successive Multiple Ionic-polymer Layer (SMIL) coatings used in CE, looking at how different parameters induce variations on the electro-osmotic flow (EOF), separation efficiency and coating stability, as well as their promising applications in the biopharmaceutical field.

Determination of ultrahigh molar mass of polyelectrolytes by Taylor dispersion analysis.

Taylor dispersion analysis (TDA) was successfully applied to obtain broadly distributed, ultrahigh molar masses of industrial anionic polyacrylamides (IPAMs) up to 25 × 106 g/mol, far beyond the limits of Size Exclusion Chromatography (SEC) (about 7.3 × 106 g/mol for anionic polyacrylamides standards (APAM)). Two protocols of TDA differing in capillary surface and rinsing procedure were employed: (i) bare fused silica capillaries under intensive between-run rinsing with 1 M NaOH, and (ii) fused silica capillaries coated with polyelectrolyte multilayers composed of polydiallyldimethylammonium chloride polycation and sodium polystyrenesulfonate polyanion under simple rinsing with background electrolyte. Both cases led to similar results and in agreement with those obtained by static light scattering, the rinsing capillary step being much shorter in the second case (8 min instead of 30 min). The data processing of the obtained taylorgrams was realized using multiple-Gaussian fitting of the overall taylorgrams, by separating the contribution of low molar mass impurities from the polymeric profiles, and by determining the mean hydrodynamic radii and diffusion coefficients of the polymers. The molar masses of ultra-high molar mass industrial anionic polyacrylamides (IPAM) were derived from the hydrodynamic radii according to logRh versus logMw linear correlation established with APAM standards. Compared to capillary gel electrophoresis for which the size separation was only feasible up to Mw ∼ 10×106 g/mol due to field induced polymer aggregation, TDA largely extended the range of accessible molar mass with easy-to-run and time saving assays.

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).

Separation of three strains of polio virus by capillary zone electrophoresis and study of their interaction with aluminum oxyhydroxide.

The development of combination vaccines is essential to reduce the number of injections, shorten vaccination schedules and increase vaccination coverage. Vaccine adjuvants are used to modulate and enhance the immune response induced by the antigens. To support the development of combination vaccines, the study of antigen-adjuvant interactions in the final vaccine formulations is required as interaction competitions may take place between the different antigens. In the present work, a capillary zone electrophoresis (CZE) methodology was firstly optimized on six model proteins, namely bovine serum albumin, ß-lactoglobulin, myoglobin, ribonuclease A, cytochrome C and lysozyme. A cationic dynamic coating (polybrene) and a zwitterionic amino acid additive (ß-alanine) in the background electrolyte were used to reduce the phenomena of protein adsorption on the inner wall of the capillary and thus optimize the separation efficiency of the proteins. The developed methodology was then used to separate three strains from inactivated polio virus, each strain being a whole virus composed of copies of 4 viral proteins and study their interaction with aluminum oxyhydroxide. The antigen-adjuvant interactions could be modulated by addition of phosphate ions playing the role of competitors for the poliovirus.

Taylor Dispersion Analysis and Atomic Force Microscopy Provide a Quantitative Insight into the Aggregation Kinetics of Aß (1-40)/Aß (1-42) Amyloid Peptide Mixtures.

Aggregation of amyloid ß peptides is known to be one of the main processes responsible for Alzheimer's disease. The resulting dementia is believed to be due in part to the formation of potentially toxic oligomers. However, the study of such intermediates and the understanding of how they form are very challenging because they are heterogeneous and transient in nature. Unfortunately, few techniques can quantify, in real time, the proportion and the size of the different soluble species during the aggregation process. In a previous work (Deleanu et al. Anal. Chem. 2021, 93, 6523-6533), we showed the potential of Taylor dispersion analysis (TDA) in amyloid speciation during the aggregation process of Aß (1-40) and Aß (1-42). The current work aims at exploring in detail the aggregation of amyloid Aß (1-40):Aß (1-42) peptide mixtures with different proportions of each peptide (1:0, 3:1, 1:1, 1:3, and 0:1) using TDA and atomic force microscopy (AFM). TDA allowed for monitoring the kinetics of the amyloid assembly and quantifying the transient intermediates. Complementarily, AFM allowed the formation of insoluble fibrils to be visualized. Together, the two techniques enabled us to study the influence of the peptide ratios on the kinetics and the formation of potentially toxic oligomeric species.

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.

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.

Biodegradation of metal-based ultra-small nanoparticles: A combined approach using TDA-ICP-MS and CE-ICP-MS.

The knowledge of the fate of metal-containing nanoparticles in biological media in aqueous media is of utmost importance for the future use of these promising theranostic agents for clinical applications. A methodology based on the combination of TDA-ICP-MS and CE-ICP-MS was applied to study the degradation pathway of AGuIX, a phase 2 clinical ultrasmall gadolinium-containing nanoparticle. Nanoparticle size measurements and gadolinium speciation performed in different media (phosphate buffer, urine and serum) demonstrated an accelerated dissolution of AGuIX in serum, without any release of free gadolinium for each medium.

Chemoprevention with a tea from hawthorn (Crataegus oxyacantha) leaves and flowers attenuates colitis in rats by reducing inflammation and oxidative stress.

The purpose of the study was to determine the effects of a tea from the leaves and flowers of Crataegus oxyacantha in rats with colitis. Colitis was induced by administration of 2,4,6-trinitrobenzene sulfonic acid. Hawthorn tea (HT) (100 mg/kg) was given via gavage for 21 days and the mesalamine drug (100 mg/kg) was administrated during the period of disease onset. HT was rich in total phenolic compounds (16.5%), flavonoids (1.8%), and proanthocyanidins (1.5%); vitexin-2-O-rhamnoside was the main compound detected. Mesalamine and the HT diminished the length of the lesions formed in the colon, in addition to reducing the levels of myeloperoxidase and interleukin-1ß. Mesalamine was able to significantly reverse the body weight loss, while HT improved the activity of glutathione reductase and catalase. Histological scoring was not changed by the interventions, but it was highly correlated with the necrotic area. HT given at 100 mg/kg can be effective against colitis.

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.