Production and Use of Gesicles for Nucleic Acid Delivery.

Over-expression of the vesicular stomatitis virus glycoprotein (VSVG) in mammalian cells can induce the formation of VSVG-pseudotyped vesicles (named "gesicles") from membrane budding. Its use as a nucleic acid delivery tool is still poorly documented. Naked-plasmid DNA can be delivered in animal cells with gesicles in presence of hexadimethrine bromide (polybrene). However, little is known about gesicle manufacturing process and conditions to obtain successful nucleic acid delivery. In this study, gesicles production process using polyethylenimine (PEI)-transfected HEK293 cells was developed by defining the VSVG-plasmid concentration, the DNA:PEI mass ratio, and the time of gesicle harvest. Furthermore, parameters described in the literature relevant for nucleic acid delivery such as (i) component concentrations in assembly mixture, (ii) component addition order, (iii) incubation time, and (iv) polybrene concentration were tested by assessing the transfection capacity of the gesicles complexed with a green fluorescent protein (GFP)-coding plasmid. Interestingly, freezing/thawing cycles and storage at + 4 °C, - 20 °C, and - 80 °C did not reduce gesicles' ability to transfer plasmid DNA. Transfection efficiency of 55% and 22% was obtained for HeLa cells and for hard-to-transfect cells such as human myoblasts, respectively. For the first time, gesicles were used for delivery of a large plasmid (18-kb) with 42% of efficiency and for enhanced green fluorescent protein (eGFP) gene silencing with siRNA (up to 60%). In conclusion, gesicles represent attractive bioreagents with great potential to deliver nucleic acids in mammalian cells.

Analysis of intact proteins with capillary zone electrophoresis coupled to mass spectromery using uncoated and coated capillaries.

Although, in general, the application of coated capillaries is recommended for the separation of intact proteins, bare silica capillary is still the most often used capillary due to its simplicity and cheapness. In this work, the performance of bare fused silica capillary for intact protein analysis was compared to that of different (dynamically coated polybrene (PB) and permanently coated linear polyacrylamide (LPA)) coated capillaries using capillary zone electrophoresis - mass spectrometry (CZE-MS). In cases where low pH (pH=1.8) was used in bare silica capillaries, good precision (0.56-0.78 RSD% and 1.7-6.5 RSD% for migration times and peak areas, respectively), minimal adsorption and separation efficiency (N= 27 000/m - 322 000/m) similar to or even better than those obtained with the coated capillaries (created by an intricate multi-step process) was achieved. The PB and the LPA capillaries demonstrated their slightly better resolving power in terms of separating the different forms/variants of the same protein (e.g., hemoglobin subunits). Among the studied capillaries the one with LPA coating showed the most stable separations in the long term (n=25: 0.18-0.49 RSD% and 3.1-4.9 RSD% for migration times and peak areas, respectively). For the separation of a few proteins or even a larger number of proteins in biological samples (e.g., snake venom) the application of the simple and cheap bare fused silica capillary can be considered as an efficient choice.

2-Mercaptoethanol (2-ME)-based IATs or Polybrene method mitigates the interference of daratumumab on blood compatibility tests.

OBJECTIVES: Treating red blood cells (RBCs) with dithiothreitol (DTT) is a wildly-recommended to overcome the interference of the daratumumab (DARA) with blood compatibility testing. Nevertheless, DTT can be hard to obtain in the clinical laboratory, while its use in routine practice may be time-consuming. In the following study, we explored the feasibility of using a commercial 2-mercaptoethanol (2-ME) working solution or the time-saving Polybrene method to mitigate DARA interference. METHODS: Antibody screening and cross-matching were performed using 2-ME or DTT-based indirect antiglobulin tests (IATs) and Polybrene method (with human IgG anti-E same IATs titer as DARA as positive control) on 37 samples. Most clinically important blood group antigens on RBCs were detected after treatment with 2-ME or DTT. RESULTS: Treating RBCs with 2-ME eliminates the DARA interference with the antibody screening or cross-matching; yet, K antigen is denatured during treatment. DARA does not interfere with antibody screening and cross-matching via Polybrene method, while 2+ agglutinations of anti-E antibody with the same titer (IATs method) as DARA could be observed in the positive controls via this method. CONCLUSION: 2-ME-based IATs or Polybrene method could replace DTT-based IATs to mitigate DARA interference.

Transduction Efficiency of Adenovirus Vectors in Endothelial Cells and Vascular Smooth Muscle Cells.

Adenoviral vectors are useful tools in manipulating a gene of interest in vitro and in vivo, including in the vascular system. The transduction efficiencies of adenoviral vectors in vascular cells such as endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) are known to be lower than those in epithelial cell types. The effective entry for adenoviral vectors is primarily mediated through the coxsackievirus and adenovirus receptor (CAR), which has been shown to be expressed in both cell types. Cationic liposomes have been used to enhance adenovirus transduction efficiency in nonepithelial cells. Accordingly, the aim of this study is to obtain new information regarding differences in transduction efficiencies, cationic liposome sensitivity, and CAR expression between ECs and VSMCs. Using cultured rat aortic ECs and VSMCs, here, we have compared transduction efficiency of adenoviruses with or without inclusion of liposomes and CAR expression. A significant increase in basal transduction efficiency was observed in ECs compared with VSMCs. Cationic liposome polybrene enhanced transduction efficiency in VSMCs, whereas decreased efficiency was observed in ECs. Western blotting demonstrated expression of the CAR in ECs but not in VSMCs. Proteomic analysis and mouse aorta immunostaining further suggests significant expression of the CAR in ECs but not in VSMCs. In conclusion, adenoviruses can effectively transduce the gene of interest in aortic ECs likely because of abundant expression of the CAR, whereas cationic liposomes such as polybrene enhance the transduction efficiency in VSMCs lacking CAR expression.

Screening of inhibitors against histone demethylation jumonji domain-containing protein 3 by capillary electrophoresis.

Jumonji domain-containing proteins (JMJDs) play an important role in the epigenetic regulation of gene expression. Aberrant regulation of histone modification has been observed in the progression of a variety of diseases, such as neurological disorders and cancer. Therefore, discovery of selective modulators of JMJDs is very attractive in new drug discovery. Herein, a simple capillary electrophoresis (CE) method was developed for screening of inhibitors against JMJD3. A known JMJD3 inhibitor GSK-J1, 5-carboxyfluorescein labeled substrate peptide with an amino acid sequence of KAPRKQLATKAARK(me3)SAPATGG (truncated from histone H3), as well as a small chemical library composed of 37 purified natural compounds and 30 natural extracts were used for method development and validation. The separation of substrate from its demethylated product was achieved by addition of polycation hexadimethrine bromide (HDB) in the running buffer. The enzyme activity was thus assayed accurately through separating the demethylated product from the substrate and then measuring the peak area of the product. The enzyme inhibition can be read out by comparing the peak area of the demethylated product obtained in the present of inhibitors and that of the negative control in the absence of any inhibitor. The merit of the method is proved by discovering two new JMJD3 inhibitors: salvianic acid A and puerarin 6''-O-xyloside.

Capillary zone electrophoresis-native mass spectrometry for the quality control of intact therapeutic monoclonal antibodies.

Therapeutic monoclonal antibodies (mAbs) are complex glycoproteins and ensuring their safety, efficacy and quality is still challenging. Indeed, during their manufacturing process, they are exposed to several stresses that can lead to their denaturation, misfolding or dimerization. We report here a new method based on capillary electrophoresis coupled to native mass spectrometry (MS) with a sheath liquid interface to analyze an intact therapeutic mAb, Infliximab, under non-denaturing conditions that preserve its conformational heterogeneity as well as self-association without inducing further unfolding / denaturation. For capillary zone electrophoresis (CZE) separation, a triple layer coating using polybrene-dextran sulfate-polybrene was employed. A sheath liquid composed of isopropanol - water - acetic acid with a flow rate of 10 µL min-1 and mild MS conditions allowed optimal signal intensities. A specific mass spectrum was obtained for each Infliximab conformation in a "stressed" formulated preparation. This is the first time that within a single analysis different conformational states, i.e. native and unfolded monomers as well as dimers are simultaneously detected. The results and the lack of analytical bias arising from the CZE-MS conditions were confirmed by using atomic force microscopy (AFM) as an orthogonal technique. A middle-up approach combined to CZE-MS analysis of the stressed samples suggested that the dimer formation involved mostly Fab-Fab interactions.

Electrokinetic motion of a micro oil droplet under a glass slide.

Electrokinetic motion of a micro oil droplet beneath a glass slide was experimentally investigated in this paper. The micro oil droplets were released under the glass slide in an aqueous solution and the motion along the glass slide was measured by a microscope. The experimental results indicate that while the electrokinetic mobility increases with the applied electric field, it decreases with the oil droplet size and the ionic concentration of the aqueous solution, respectively. By changing the zeta potential of the glass-liquid interface using polybrene coating from negative to positive, the direction of the electrokinetic mobility is reversed and the absolute value of the electrokinetic mobility increases significantly. Finally, pH effects were also investigated, and it was found that the electrokinetic mobility of the droplets reaches a maximum at pH = 6∼8.

Metabolic Profiling of Urine by Capillary Electrophoresis-Mass Spectrometry Using Non-covalently Coated Capillaries.

In the field of metabolomics, capillary electrophoresis-mass spectrometry (CE-MS) can be considered a very useful analytical tool for the profiling of polar and charged metabolites. However, variability of migration time is an important issue in CE. An elegant way to minimize this problem is the use of non-covalently coated capillaries that is dynamic coating of the bare fused-silica capillary with solutions of charged polymers. In this protocol, an improved strategy for the profiling of cationic metabolites in urine by CE-MS using multilayered non-covalent capillary coatings is presented. Capillaries are coated with a bilayer of polybrene (PB) and poly(vinyl sulfonate) (PVS) or with a triple layer of PB, dextran sulfate (DS), and PB. The bilayer- and triple-layer-coated capillaries have a negative and positive outside layer, respectively. It is shown that the use of such capillaries provides very repeatable migration times.

Simultaneous determination of designer benzodiazepines in human serum using non-aqueous capillary electrophoresis - Tandem mass spectrometry with successive multiple ionic - Polymer layer coated capillary.

A novel non-aqueous capillary electrophoresis - tandem mass spectrometry method for the simultaneous separation, identification and quantification of nine designer benzodiazepines (bentazepam, etizolam, deschloroetizolam, diclazepam, flubromazepam, flubromazolam, nimetazepam, phenazepam, and pyrazolam) was developed. A non-aqueous running electrolyte consisting of 25mM ammonium acetate with 100mM trifluoroacetic acid in acetonitrile was used. The separation was carried out using a semipermanent coated capillary (successive multiple ionic-polymer coating) with a strong anodic electroosmotic flow at a negative separation voltage within twelve minutes. Electrospray ionization with a triple quadrupole mass spectrometry was utilized for the identification and quantification of selected designer benzodiazepines in a positive ionization mode. The developed method was validated and applied on the analysis of spiked serum sample following a simple liquid-liquid extraction. The LODs of the designer benzodiazepines were between 1.5 and 15.0ngmL-1.

Analysis of Somatropin by Double-Injection Capillary-Zone Electrophoresis in Polybrene/Chondroitin Sulfate A Double-Coated Capillaries.

Purity determination of somatropin as a recombinant protein is important to ensure its safety and quality. This is carried out by capillary zone electrophoresis in double-injection mode using polybrene/chondroitin sulfate A double-coated capillaries. Modification of the capillary wall eliminates protein-wall interactions which results in improved accuracy and precision of the determinations. In the double-injection mode two somatropin samples are analyzed within a single electrophoretic run. Prior to the second injection, the first injected plug is electrophoresed for a predetermined time period in order to adjust the inter-plug distance. Here, the principle for the separation of somatropin charge variants is described.

Monitoring antigenic protein integrity during glycoconjugate vaccine synthesis using capillary electrophoresis-mass spectrometry.

A capillary electrophoresis-mass spectrometry (CE-MS) method was developed for the characterization and integrity assessment of the Mycobacterium tuberculosis (MTB) antigens TB10.4 and Ag85B and their chemically produced glycoconjugates, which are glycovaccine candidates against tuberculosis (TB). In order to prevent protein adsorption to the inner capillary wall and to achieve efficient separation of the antigen proteoforms, a polyionic multilayer coating of polybrene-dextran sulfate-polybrene (PB-DS-PB) was used in combination with 1.5 M acetic acid as background electrolyte (BGE). Coupling of CE to high-resolution time-of-flight MS was achieved by a coaxial interface employing a sheath liquid of isopropanol-water (50:50, v/v) containing 0.1 % formic acid. The MTB antigens were exposed to experimental conditions used for chemical glycosylation (but no activated saccharide was added) in order to investigate their stability during glycovaccine production. CE-MS analysis revealed the presence of several closely related degradation products, including truncated, oxidized and conformational variants, which were assigned by accurate mass. Analysis of synthesized mannose conjugates of TB10.4 and Ag85B allowed the determination of the glycoform composition of the neo-glycoproteins next to the characterization of degradation products which were shown to be partly glycoconjugated. Moreover, the selectivity of CE-MS allowed specific detection of deamidated species (protein mass change of 1.0 Da only), indicating that chemical glycosylation increased susceptibility to deamidation. Overall, the results show that CE-MS represents a useful analytical tool for the detailed characterization and optimization of neo-glycoconjugate products. Graphical Abstract Flowchart illustrating Mycobacterium tuberculosis (MTB) antigen glycosylation, glycoconjugate variant and degradation product separation by capillary electrophoresis (CE) and their characterization by intact mass spectrometry (MS).

Zeta potentials of polydimethylsiloxane surfaces modified by polybrene of different concentrations.

Zeta potential is an important parameter for characterizing the electrokinetic properties of a solid-liquid interface. In this paper, zeta potentials of polydimethylsiloxane surfaces modified by polybrene (PB) solutions of different concentrations in Phosphate buffer solution and pure water were reported. The zeta potentials were measured by an induction current method. The measurements were validated both by a calibration curve based on the data reported in the published papers and by comparing the zeta potential determined by using the Smoluchowski equation and the measured velocity of the electrokinetic motion of particles in a microchannel.

Selective room temperature phosphorescence detection of heparin based on manganese-doped zinc sulfide quantum dots/polybrene self-assembled nanosensor.

A selective system was developed to detect heparin in aqueous solutions by using MPA(3-Mercaptopropionic Acid)-capped Mn-doped ZnS quantum dots (QDs)/polybrene (hexadimethrine bromide) hybrids as a sensitive room temperature phosphorescence (RTP) nanosensor. In this system, the RTP intensity of QDs was remarkably enhanced via electrostatic self-assembly after the addition of polybrene. The addition of heparin into the system was competitively bound to polybrene and enable to deprive it from the surface of QDs, as a result, the RTP intensity of Mn-doped ZnS QDs/polybrene hybrids was reduced with the increased of heparin concentration. Based on this effect, a selective system was proposed to detect heparin. Under the optimal conditions, the change of RTP intensity was proportional to the heparin concentration from 0.05 to 1.4 U mL(-1) (about 0.38-10.76 µg mL(-1)) and the limit of detection (LOD) was 0.021 U mL(-1) (about 0.16 µg mL(-1)). This proposed nanosensor is simple and relatively free of interference from coexisting substances, which can be applied to detect heparin in heparin injection and human serum. In addition, a new pathway was also provided based on the assembly of QDs with other cationic homopolymers for further design of biosensors and detection of biomolecules.

Capillary electrophoresis-based assessment of nanobody affinity and purity.

Drug purity and affinity are essential attributes during development and production of therapeutic proteins. In this work, capillary electrophoresis (CE) was used to determine both the affinity and composition of the biotechnologically produced "nanobody" EGa1, the binding fragment of a heavy-chain-only antibody. EGa1 is an antagonist of the epidermal growth factor receptor (EGFR), which is overexpressed on the surface of tumor cells. Using a background electrolyte (BGE) of 50mM sodium phosphate (pH 8.0) in combination with a polybrene-poly(vinylsulfonic acid) capillary coating, CE analysis of EGa1 showed the presence of at least three components. Affinity of the EGa1 components towards the extracellular domain of EGFR was assessed by adding different concentrations (0-12 nM) of the receptor to the BGE while measuring the effective electrophoretic mobility of the respective EGa1 components. Binding curves obtained by plotting electrophoretic mobility shifts as a function of receptor concentration, yielded dissociation constants (Kd) of 1.65, 1.67, and 1.75 nM for the three components, respectively; these values were comparable to the Kd of 2.1 nM obtained for the bulk EGa1 product using a cellular assay. CE with mass spectrometry (MS) detection using a BGE of 25 mM ammonium acetate (pH 8.0) revealed that the EGa1 sample comprised of significant amounts of deamidated, bisdeamidated and N-terminal pyroglutamic acid products. CE-MS using a BGE of 100mM acetic acid (pH 2.8) in combination with a polybrene-dextran sulfate-polybrene capillary coating demonstrated the additional presence of minor products related to incomplete removal of the signal peptide from the produced nanobody. Combining the results obtained from affinity CE and CE-MS, it is concluded that the EGa1 nanobody product is heterogeneous, comprising highly-related proteins that exhibit very similar affinity towards EGFR.

Analysis of urinary metabolites for breast cancer patients receiving chemotherapy by CE-MS coupled with on-line concentration.

OBJECTIVE: This study aimed to explore the relationship between urinary metabolites and clinical chemotherapy response in breast cancer by CE-MS coupled with on-line concentration. DESIGN AND METHODS: Urine samples were obtained from patients with advanced or locally advanced breast cancer (n=21) before and after chemotherapy and healthy volunteers (n=21). A rapid and sensitive hexadimethrine bromide-coating CE-MS method coupled with normal stacking is developed for the determination of organic acids in human urine. Another CE-MS method coupled with pH-mediated sample stacking is used for the analysis of amino acids and organic acids. RESULTS: After receiving chemotherapy, chemotherapy-sensitive patients showed 30% change in metabolite levels compared to healthy people, while chemotherapy-insensitive patients showed only 9% change in metabolite levels compared to healthy people showing recurrence. The extent of energy insufficiency for chemotherapy-insensitive patients was greater than that for chemotherapy-sensitive patients. CONCLUSIONS: Urinary metabolic products may be new potential predictive markers for therapy efficacy. However, more studies with a larger sample size are required to confirm these conclusions.

Separation of somatropin charge variants by multiple-injection CZE with Polybrene/chondroitin sulfate A double-coated capillaries.

The performance of dynamic double-coated fused-silica capillaries with Polybrene and chondroitin sulfate A has been compared with uncoated fused-silica capillaries for the determination of recombinant human growth factor (somatropin) charge variants. The separations were carried out under the same electrophoretic conditions as described in the European Pharmacopoeia, i.e. at pH 6.0 and 30°C. The coating significantly reduced the interactions between the proteins and the surface of the fused-silica capillary. The first five separations performed in a new bare fused-silica capillary were discarded because of very poor separation performance as a result of protein-surface interactions. There was an approximate twofold increase in the interday migration time precision (%RSD ≤ 6.5%) in the double-coated capillaries. The method was successfully transferred to a multiple CZE mode where two samples were analyzed in a single electrophoretic run. The average purity of somatropin certified reference standard was 98.0% (%RSD ≤ 0.3%) determined by using uncoated and coated capillaries.

Enzyme inhibitor screening by CE with an on-column immobilized enzyme microreactor created by an ionic binding technique.

Enzymes are an important class of drug targets. In the early stage of the drug discovery, the major task is to find out the inhibitors of a given enzyme target in a compound library. Herein we describe a method for screening the enzyme inhibitors in the complex mixtures (such as the natural extracts) by capillary electrophoresis with an on-column immobilized enzyme microreactor. The enzyme molecules are immobilized on the capillary wall via ionic binding with the positively charged coating, which was created by simply flushing the column with a solution of polyelectrolyte hexadimethrine bromide. The activities of the immobilized enzymes are assayed by performing the electrophoretic separation and thereafter determining the product of the enzyme-mediated reaction. Enzyme inhibition can be read out directly from the reduced peak area of product in comparison with that in the reference electropherogram obtained in the absence of any inhibitor.

Use of neutral capillaries for the enantioseparation of N-benzoylated amino acids by capillary electrophoresis with bromobalhimycin as chiral selector.

In this study, the partial filling technique on both polycationic polymer hexadimethrine bromide (HDB) modified capillary and eCAP neutral capillary were systematically compared in order to enhance the enantioseparation ability of bromobalhimycin as CE additive. The separation conditions, such as pH, the plug length, and the concentration of bromobalhimycin, etc., were optimized in order to obtain satisfactory separations. As expected, for all tested 28 N-benzoylated amino acids, up to five times higher enantioresolutions were obtained on the eCAP neutral capillary compared to that on the polycationic polymer hexadimethrine bromide modified capillary. Moreover, 26 of 28 tested racemic compounds were almost baseline- resolved without observing any interference from the front of the plug of bromobalhimycin. Although the limitation of longer running time on the neutral capillary, it allows the use of higher content of bromobalhimycin in the running buffer without any interference on the detection of analytes when enantioseparations are more difficult to obtain.

Determination of iodide in seawater by capillary ion chromatography using hexadimethrine bromide modified C30 stationary phases.

A novel and simple capillary ion chromatographic method for the determination of iodide is reported. Separation was achieved on a laboratory-made packed capillary column (100 mm × 0.32 mm i.d.) packed with triacontyl-functionalized silica, followed by a modification with hexadimethrine bromide, where 1 mM sodium chloride-acetonitrile (95:5, v/v) was used as the eluent and UV-absorbing analyte anions were detected at 225 nm. The effects of the eluent composition on the retention behavior of inorganic anions were investigated. The addition of a small amount of an organic substance in an eluent such as acetonitrile increased the retention of iodide, while the addition of methanol decreased its retention. The present analytical method was successfully applied to the rapid and direct determination of iodide in seawater without any preconcentration. Also, this modified column could be used for about two months (6 h operation per day) without hexadimethrine bromide being contained in the eluent.