Myriam Taverna

Specific antioxidant properties of human serum albumin.

Human serum albumin (HSA) has been used for a long time as a resuscitation fluid in critically ill patients. It is known to exert several important physiological and pharmacological functions. Among them, the antioxidant properties seem to be of paramount importance as they may be implied in the potential beneficial effects that have been observed in the critical care and hepatological settings. The specific antioxidant functions of the protein are closely related to its structure. Indeed, they are due to its multiple ligand-binding capacities and free radical-trapping properties. The HSA molecule can undergo various structural changes modifying its conformation and hence its binding properties and redox state. Such chemical modifications can occur during bioprocesses and storage conditions of the commercial HSA solutions, resulting in heterogeneous solutions for infusion. In this review, we explore the mechanisms that are responsible for the specific antioxidant properties of HSA in its native form, chemically modified forms, and commercial formulations. To conclude, we discuss the implication of this recent literature for future clinical trials using albumin as a drug and for elucidating the effects of HSA infusion in critically ill patients.

Hyaluronic acid-bearing lipoplexes: physico-chemical characterization and in vitro targeting of the CD44 receptor.

The mechanism by which hyaluronic acid (HA)-bearing lipoplexes target the A549 lung cancer cell line was evaluated. For this purpose, cationic liposomes targeting the CD44 receptor were designed thanks to the incorporation in their composition of a conjugate between high molecular weight HA and the lipid DOPE (HA-DOPE). Liposomes containing HA-DOPE were complexed at different lipids:DNA ratios with a reporter plasmid encoding the green fluorescent protein (GFP). Diameter, zeta potential, lipoplex stability and DNA protection from nucleases have been determined. Lipids:DNA ratios of 2, 4 and 6 provided a diameter around 250 nm with a zeta potential of -30 mV. The strength of lipids:DNA interaction and the fraction of DNA protected from enzymatic degradation increased with the lipids:DNA ratio. 2D-immunoelectrophoresis demonstrated the low capacity to activate the C3 fraction of the complement system of any of these three ratios, with and without HA-DOPE. Transfection efficiency in the presence of 0, 10 and 15% of HA-DOPE or unconjugated HA, was determined on the CD44-expressing A549 cells by flow cytometry. Lipoplexes at a lipids:DNA ratio of 2 containing 10% (w/w) of HA-DOPE were the most efficient for transfection. The maximal level of GFP expression was obtained after 6h of incubation demonstrating a slow transfection kinetics of lipoplexes. Finally, lipoplex cellular uptake, measured indirectly by the level of transfection using flow cytometry and validated by fluorescence microscopy, was shown to be mediated by the CD44 receptor and caveolae. These results demonstrate the strong specificity of DNA targeting through the CD44 receptor using HA of high molecular weight as a ligand.

Recent innovations in protein separation on microchips by electrophoretic methods: an update.

Lab‐on‐a‐chip electrophoresis is becoming increasingly useful for protein analysis, thanks to recent developments in this field. This review is an update of the review we published at the start of 2008 [Peng, Y., Pallandre, A., Tran, N. T., Taverna, M., Electrophoresis 2008, 29, 156–177]. The superiority of polymers for the manufacture of analytical microchips has been confirmed. This trend implies several modifications to the processes previously used with glass/silicon chips and requires a better understanding of the interfacial phenomena of these materials. Significant progress in chip‐based techniques for protein analysis has been made in the last 2 years. In addition to advances in traditional electrokinetic modes, counter‐flow gradient focusing techniques have emerged as useful methods not only for separation, but also for the online preconcentration of samples. This review, with more than 175 references, presents recent advances and novel strategies for EOF measurement, surface treatment, sample pretreatment, detection and innovations relating to the different modes of separation.

Determination of alkylphosphonic acids by capillary zone electrophoresis using indirect UV detection

Abstract Capillary zone electrophoresis with indirect UV detection was used for the determination of a series of alkylphosphonic acids. For this purpose, a few UV-absorbing background electrolytes were tested and phenylphosphonic acid, which has a mobility close to that of the analysed compounds, was shown to be the most suitable. The influence of several parameters such as concentration of the UV-absorbing background electrolyte and concentration of borate on both sensitivity and efficiency was investigated. An increase in the borate concentration produced an improvement of the signal-to-noise ratio. Conversely, the sensitivity decreased with increasing concentration of the phenylphosphonic acid. The reproducibility of the method was very satisfactory and limits of detection were less than 0.21 pmol injected.

New method based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) to monitor interaction between nanoparticles and the amyloid-β peptide.

A novel application of capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was proposed to efficiently detect and monitor the interaction between polymeric nanoparticles and the β-Amyloid peptide (Aβ(1-42)), a biomarker for Alzheimers Disease (AD), at concentrations close to physiological conditions. The CE-LIF method allowed the interaction between PEGylated poly(alkyl cyanoacrylate) nanoparticles (NPs) and the soluble Aβ(1-42) peptide monomers to be highlighted. These results were confirmed by surface plasmon resonance (SPR) and confocal laser scanning microscopy (CLSM). Whereas SPR showed an interaction between the NPs and the Aβ(1-42) peptide, CLSM allowed the formation of large aggregates/assemblies at high NP and peptide concentrations to be visualized. All these results suggested that these nanoparticles could bind the Aβ(1-42) peptide and influence its aggregation kinetics. Interestingly, the non-PEGylated poly(alkyl cyanoacrylate) NPs did not alter the aggregation kinetics of the Aβ(1-42) peptide, thus emphasizing the high level of discrimination of the CE-LIF method with respect to NPs.

Recent advances in the capillary electrophoresis of recombinant glycoproteins

Abstract Highly efficient methods are required to analyze recombinant proteins for clinical use. These proteins generally produced from mammalian expression systems are highly glycosylated and consist of a population of glycosylated variants (glycoforms). This review presents the different microscale techniques of capillary electrophoresis (CE) for analyzing the intact recombinant glycoproteins and for monitoring their bioproduction. Because of several advantages such as simplicity, speed and automation, capillary zone electrophoresis (CZE) has been generally employed for the routine analysis of the glycoform populations of intact glycoproteins. Capillary isoelectric focusing (CIEF) is a powerful method for a charge-based separation of the glycoforms. Micellar electrokinetic capillary chromatography (MEKC) represents an alternative method to CZE for the purity control of recombinant glycoproteins, while the sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) with replaceable gel matrices gives an estimation of the glycoform molecular masses. The results from CIEF and SDS-CGE are comparable to those from the corresponding slab gel techniques. The recent advances in the coupling of CZE with mass-spectrometry (MS) offers new perspectives not only for precise molecular mass determinations, but also to better understand the mechanisms involved in the CE separation of glycoforms.

Controlled proteolysis of normal and pathological prion protein in a microfluidic chip.

A microreactor for proteinase K (PK)-mediated protein digestion was developed as a step towards the elaboration of a fully integrated microdevice for the detection of pathological prion protein (PrP). PK-grafted magnetic beads were immobilized inside a polydimethylsiloxane (PDMS) microchannel using a longitudinal magnetic field parallel to the flow direction and a magnetic field gradient, thereby forming a matrix for enzymatic digestion. This self-organization provided uniform pore sizes, a low flow resistance and a strong reaction efficiency due to a very thin diffusion layer. The microreactors performance was first evaluated using a model substrate, succinyl-ala-ala-ala-paranitroanilide (SAAAP). Reaction kinetics were typically accelerated a hundred-fold as compared to conventional batch reactions. Reproducibility was around 98% for on-chip experiments. This microsystem was then applied to the digestion of prion protein from brain tissues. Controlled proteolysis could be obtained by varying the on-chip flow rate, while a complete proteolysis of normal protein was achieved in only three minutes. Extracts from normal and pathological brain homogenates were finally compared and strong discrimination between normal and pathological samples was demonstrated.

Simultaneous analysis by capillary electrophoresis of five amyloid peptides as potential biomarkers of Alzheimer's disease

We report here a CE method for the separation and quantitation of five amyloid peptides (Abeta1-42, 1-40, 1-39, 1-38, and 1-37) considered as potential biomarkers of Alzheimers disease. These amyloid peptides have very similar structures. Sample preparation and storage conditions are critical parameters to ensure their solubility and to avoid the aggregation process in particular for Abeta1-42. Their solubility was found fully dependent on the NH(4)OH concentration that was employed initially to dissolve the lyophilized amyloid peptides. Conditions to achieve a full separation of these peptides were found using a dynamic coating with 1,4-diaminobutane (DAB). The linear decrease of their electrophoretic mobility highlighted an ion-pairing phenomenon between the peptides and DAB. The optimal background electrolyte was a 40 mM borate buffer, pH 9 containing 3 mM of DAB. Under these conditions, resolutions ranged from 1.3 to 2.4 with theoretical plates reaching 300,000. Under the retained conditions, we showed that adsorption of peptides to silica was negligible (recovery over 94.5%) and depletion effect of the background electrolyte was overcome. The method was finally validated in terms of linearity and repeatability and the limits of detection for the five Abeta peptides were estimated. The inter-day repeatability of the migration times was very satisfactory with RSDs less than 1.55%. The RSDs of the peak areas were below 5%. With this CE-UV method, limits of detection of the peptides ranged from 300 to 500 nM. We finally demonstrated that this method can be applied to real biological samples such as CSF.

A study of the binding between polymers and peptides, using affinity capillary electrophoresis, applied to polymeric drug delivery systems.

We have investigated the potential of affinity capillary electrophoresis (ACE) to evaluate binding constants between an anionic polydispersed polymer and four peptides. Nonlinear regression and three current linearization methods, the y‐reciprocal, the x‐reciprocal and the double‐reciprocal, were employed for the estimation of the binding constants. The x‐reciprocal and the double‐reciprocal plots indicated the presence of two portions of straight lines for angiopeptin, triptorelin and the thyrotropin releasing hormone (TRH), and therefore the probable existence of a second‐order interaction which causes the deviation from the 1:1 model. Peptide 1 exhibited a unique binding constant of 2.4×106 M–1. In contrast, angiopeptin, triptorelin and TRH exhibited a K1 of 4.0×106, 5.3×106 and 20.2×106 M–1, respectively, and a K2 of 0.4×106, 0.5×106 and 1.4×106 M–1, respectively. The origin of the high scattering of the data points was further investigated. Neither the viscosity, nor the adsorption of the peptides to the capillary wall appeared to be the determining factor of data scattering. Finally, a possible adsorption of the polymer leading to the electroosmotic flow unstability was supposed.

Comparison of native, alkylated and charged cyclodextrins for the chiral separation of labetalol stereoisomers by capillary electrophoresis

A capillary electrophoresis method for the enantioresolution of labetalol was developed using CDs as chiral selectors and uncoated capillaries. Various native (α-, β- and γ-CD), alkylated (hydroxy propyl-β- and γ-CD, methyl-β-CD) and anionic (sulfated-β-CD and sulfobutylether-γ-CD) cyclodextrins were tested and operational parameters such as buffer pH, concentration of CD were investigated. Propranolol was also studied as a model compound. Uncharged γ-CDs were more effective than β-CDs to separate the enantiomers of labetalol but no complete resolution of the four isomers was obtained. The use of charged cyclodextrins led to a combination of hydrophobic inclusion and ion-pairing interaction in the chiral recognition mechanism. Thus, a complete resolution of the four enantiomers of the labetalol was attained using 7.7 g/l sulfated-β-CD in a 30 mM phosphate buffer, pH 6.5.