Jean-Michel Siaugue

Dye removal from aqueous solution by magnetic alginate beads crosslinked with epichlorohydrin.

Innovative magnetic alginate beads are used to remove organic pollutants from aqueous solution under different experimental conditions. These alginate beads (EpiMAB) are prepared by an extrusion technique and crosslinked with epichlorohydrin. They contain both magnetic nanoparticles and activated carbon (AC). With the addition of magnetic properties, the beads can be easily recovered or manipulated with an external magnetic field. Their capacity to adsorb pollutants is linked to encapsulated AC and to active sites coming from both magnetic nanoparticles and alginate. The efficiency of the beads as biosorbent for the removal of dyes is assessed using methyl orange (MO) and methylene blue (MB) as model molecules. The dye uptake is found to vary with the initial concentration and the charge of the adsorbed molecule. The Langmuir equation fits well the adsorption data with maximum adsorption capacities of 0.02 mmol/g for MO and 0.7 mmol/g for MB. Kinetics experiments are performed to evaluate the equilibrium time; the pseudo-second-order kinetic model adequately describes the experimental data. The influence of the pH of the solution on adsorption is also investigated and a comparison with alginate beads crosslinked by calcium ions is made.

Co(II) removal by magnetic alginate beads containing Cyanex 272

In this study, a series of batch experiments is conducted to investigate the ability of magnetic alginate beads containing Cyanex 272 to remove Co(II) ions from aqueous solutions. Equilibrium sorption experiments show a Co(II) uptake capacity of 0.4 mmol g(-1). The data are successfully modelled with a Langmuir equation. A series of kinetics experiments is then carried out and a pseudo-second order equation is used to fit the experimental data. The effect of pH on the sorption of Co(II) ions is also investigated. Desorption experiments by elution of the loaded beads with nitric acid at pH 1 show that the magnetic alginate beads could be reused without significant losses of their initial properties even after 3 adsorption-desorption cycles.

Charge-based characterization of nanometric cationic bifunctional maghemite/silica core/shell particles by capillary zone electrophoresis.

In view of employing functionalized nanoparticles (NPs) in the context of an immunodiagnostic, aminated maghemite/silica core/shell particles were synthesized so as to be further coated with an antibody or an antigen via the amino groups at their surface. Different functionalization rates were obtained by coating these maghemite/silica core/shell particles with 3‐(aminopropyl)triethoxysilane and 2‐[methoxy(polyethyleneoxy)propyl]‐trimethoxysilane at different molar ratios. Adequate analytical performances with CE coupled with UV‐visible detection were obtained through semi‐permanent capillary coating with didodecyldimethyl‐ammonium bromide, thus preventing particle adsorption. First, the influence of experimental conditions such as electric field strength, injected particle amount as well as electrolyte ionic strength and pH, was evaluated. A charge‐dependent electrophoretic mobility was evidenced and the separation selectivity was tuned according to electrolyte ionic strength and pH. The best resolutions were obtained at pH 8.0, high ionic strength (ca. 100 mM), and low total particle volume fraction (ca. 0.055%), thus eliminating interference effects between different particle populations in mixtures. A protocol derived from Kaisers original description was performed for quantitation of the primary amino groups attached onto the NP surface. Thereafter a correlation between particle electrophoretic mobility and the density of amino groups at their surface was established. Eventually, CE proved to be an easy, fast, and reliable method for the determination of NP effective surface charge density.

Interactions between giant unilamellar vesicles and charged core-shell magnetic nanoparticles.

This work combined two tools, giant unilamellar vesicles (GUVs) and core-shell magnetic nanoparticles (CSMNs), to develop a simplified model for studying interactions between the cell membrane and nanoparticles. We focused on charged functionalized CSMNs that can be either cationic or anionic. Using optical, electron, and confocal microscopy, we found that giant vesicle-nanoparticle interactions did not result from a simple electrostatic phenomenon because cationic CSMNs tended to bind to positively charged bilayers, whereas anionic CSMNs remained inert.

Nanoparticle‐Mediated Delivery of Bleomycin

The occurrence of side effects induced by poor distribution of antitumor agents is still an important problem in cancer treatment. The challenge consists of both improving the tumor bioavailability of drugs and confining them as closely as possible to their biological targets. The use of nanoparticles as vectors for drug delivery has been intensively documented during the last two decades. Some of them (ironand gold-based nanoparticles, quantum dots) are at the same time agents for imaging, thus allowing the simultaneous follow-up of the treatment efficiency. They can also take part in the treatment: magnetic nanoparticles can be used for hyperthermia, while gold-based nanoparticles can be used for photothermal therapy. The use of nanometric vectors brings some answers to the problem of bioavailability. Considering their large surface-tovolume ratio, they offer the possibility of transporting major quantities of drugs. Thanks to passive and active targeting, they ensure limited harmful systemic distribution. Indeed, taking into account the enhanced permeation and retention (EPR) effects, suitable nanoparticles can carry drugs into solid tumors. Moreover, the grafting of targeting moieties, such as antibodies or folic acid, onto the particles surface allows active targeting, thus decreasing the interaction with healthy cells. The efficiency of treatments is also limited because of intracellular drug resistance mechanisms: only a small amount of an agent reaches its biological target. Significant improvement in drug efficiency could be obtained through the development of therapeutic strategies to pass drugs across the biological barriers. From this point of view, nanoparticles appear to be good candidates for drug delivery, because they are internalized in cells mainly by endocytosis pathways. Nevertheless, nanoparticles suffer from two major limitations: the alteration of their surface in biological media and their in vivo stealth. To cope with these two main problems, hybrid systems have been designed, which combine inorganic cores with organic or inorganic shells. Therapeutic molecules can either be inserted into the shell or grafted onto it. One should also mention the association with polymers such as polyethylene glycol that are able to ensure both stealth and in vivo stability. The strategy we propose herein is based on the use of multifunctional core–shell nanoparticles made of gFe2O3@SiO2-PEG-NH2 (PEG = polyethylene glycol), which allow covalent anchoring of biomolecules. Compared with a nanometric system incorporating drugs, the grafting of an antineoplastic agent at the surface allows a drastic reduction in the uncontrolled release of the agent and thus in side effects in healthy tissues. We have chosen in this work to study the grafting of bleomycin-A5 (BLM-A5), an anticancer drug that chelates metals such as Fe and catalyzes the formation of single-stranded (ss) or double-stranded (ds) DNA lesions in the presence of oxygen. The therapeutic efficiency of bleomycin (BLM) is severely limited because of its side effects, notably strong pulmonary toxicity. Dispensing the drug at lower doses near the biological target could lead to its wider use in oncology. Moreover, the delivery of this drug has been poorly studied, mainly using micrometric systems, such as glass beads or polyvinylpyridine microgels. The nanoplatforms we propose are core–shell magnetic nanoparticles (CSMNs) obtained using a procedure developed by our group (Figure 1A). The core consists of citrate-coated maghemite nanoparticles (g-Fe2O3, diameter around 7 nm). The shell is a layer of silica, twice functionalized by PEG chains, to hide the nanoparticles from the reticuloendothelial system (RES), and by amino groups to ensure BLM-A5 anchoring. Following van Blaaderen s method, fluorescent CSMNs can be synthesized by addition of rhodamine isothiocyanate-derived 3-aminopropyltriethoxysilane (APTS) during the coating of the magnetic cores by silica. These core–shell particles are characterized by a mean hydrodynamic diameter of 40 nm, and transmission electron microscopy (TEM) images display spherical particles with a mean physical diameter of 35 nm (Figure 1B). These particles are positively charged (+ 14 mV), and the surface density of amino groups can be tuned by varying the APTS to 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (PEOS) ratio. Such particles, with different surface densities of amino groups, have been reported previously and have been fully characterized using capillary zone electrophoresis. The magnetic properties of the maghemite cores, which contain from two to three g-Fe2O3 nanoparticles, are not modified by encapsulation. These CSMNs can be dispersed in 150 mm 3-(N-morpholino)propanesulfonic acid (MOPS) buffer at pH 7.4 or in other media with high concentrations of salts and/or proteins, while maintaining colloidal stability at room temperature for at least six months. BLM-A5 (Figure 1C) was covalently anchored onto these platforms [*] T. Georgelin, Dr. J.-M. Siaugue, Prof. V. Cabuil Physicochimie des Electrolytes, Collo des et Sciences Analytiques (PECSA), UMR 7195 UPMC-CNRS-ESPCI-ENSCP Universit Pierre et Marie Curie Univ Paris 06, CC 51 4 place Jussieu, 75252 Paris Cedex 05 (France) Fax: (+ 33)1-4427-3228 E-mail: jean-michel.siaugue@upmc.fr Homepage: http://www.pecsa.upmc.fr

Magnetogenetic Control of Protein Gradients Inside Living Cells with High Spatial and Temporal Resolution

Tools for controlling the spatial organization of proteins are a major prerequisite for deciphering mechanisms governing the dynamic architecture of living cells. Here, we have developed a generic approach for inducing and maintaining protein gradients inside living cells by means of biofunctionalized magnetic nanoparticles (MNPs). For this purpose, we tailored the size and surface properties of MNPs in order to ensure unhindered mobility in the cytosol. These MNPs with a core diameter below 50 nm could be rapidly relocalized in living cells by exploiting biased diffusion at weak magnetic forces in the femto-Newton range. In combination with MNP surface functionalization for specific in situ capturing of target proteins as well as efficient delivery into the cytosplasm, we here present a comprehensive technology for controlling intracellular protein gradients with a temporal resolution of a few tens of seconds.

A chemometric approach for optimizing protein covalent immobilization on magnetic core–shell nanoparticles in view of an alternative immunoassay

A chemometric approach was developed to optimize the grafting of a bovine milk allergen: alpha-Lactalbumin (alpha-Lac) on colloidal functionalized magnetic core-shell nanoparticles (MCSNP). Such nanoparticles, functionalized with polyethyleneglycol and amino groups, exhibit a 30nm physical diameter and behave as a quasi-homogeneous system. The alpha-Lac immobilization was achieved through the covalent binding between MCSNP amino groups and alpha-Lac carboxylic moieties using the well-known tandem carbodiimide (EDC) and hydroxysulfosuccinimide (NHS). In this study, a chemometric approach was employed to highlight the parameters influencing the number of grafted proteins on the MCSNP. Three factors were evaluated: the ratio in concentration between EDC and alpha-Lac, between NHS and EDC and the concentration of alpha-Lac. After a first full factorial design to delimit the region of the space where the optimum could be located, a central composite design was then carried out to predict the best grafting conditions. It was established and experimentally confirmed that the optimum parameters are [EDC]/[alpha-Lac]=25; [NHS]/[EDC]=1.55 and alpha-Lac=24.85nmolmL(-1). In these optimal conditions, MCSNP surface was successfully saturated with alpha-Lac (34 alpha-Lac/MCSNP) with a high reproducibility (RSD=2%). The colloidal stability of MCSNP grafted with alpha-Lac as well as the immunological interactions using anti alpha-Lac antibody were then investigated in different buffers. The results emphasized that a 50mM MES buffer (pH 6) allows an efficient immune capture and a satisfying colloidal stability which provide an immunological interaction in homogeneous liquid phase.

Separation of α-lactalbumin grafted- and non-grafted maghemite core/silica shell nanoparticles by capillary zone electrophoresis

The use of nanoparticles (NPs) in immunodiagnostics is a challenging task for many reasons, including the need for miniaturization. In view of the development of an assay dedicated to an original, miniaturized and fully automated immunodiagnostics which aims to mimic in vivo interactions, magnetic zwitterionic bifunctional amino/polyethyleneoxide maghemite core/silica shell NPs functionalized with allergenic α‐lactalbumin were characterized by CE. Proper analytical performances were obtained through semi‐permanent capillary coating with didodecyldimethylammonium bromide (DDAB) or permanent capillary wall modification by hydroxypropylcellulose. The influence of experimental conditions (e.g. buffer component nature, pH, ionic strength, and electric field strength) on sample stability, electrophoretic mobility, and dispersion was investigated using either DDAB‐ or hydroxypropylcellulose‐coated capillaries. Adsorption to the capillary wall and aggregation phenomena were evaluated according to the CE conditions. The proper choice of experimental conditions, i.e. separation under −10 kV in a 25 mM ionic strength MES/NaOH (pH 6.0) with a DDAB‐coated capillary, allowed the separation of the grafted and the non‐grafted NPs.

Kinetic analyses and performance of a colloidal magnetic nanoparticle based immunoassay dedicated to allergy diagnosis

In this paper, we demonstrate the possibility to use magnetic nanoparticles as immunosupports for allergy diagnosis. Most immunoassays used for immunosupports and clinical diagnosis are based on a heterogeneous solid-phase system and suffer from mass-transfer limitation. The nanoparticles’ colloidal behavior and magnetic properties bring the advantages of homogeneous immunoassay, i.e., species diffusion, and of heterogeneous immunoassay, i.e., easy separation of the immunocomplex and free forms, as well as analyte preconcentration. We thus developed a colloidal, non-competitive, indirect immunoassay using magnetic core–shell nanoparticles (MCSNP) as immunosupports. The feasibility of such an immunoassay was first demonstrated with a model antibody and described by comparing the immunocapture kinetics using macro (standard microtiter plate), micro (microparticles) and nanosupports (MCSNP). The influence of the nanosupport properties (surface chemistry, antigen density) and of the medium (ionic strength, counter ion nature) on the immunocapture efficiency and specificity was then investigated. The performances of this original MCSNP-based immunoassay were compared with a gold standard enzyme-linked immunosorbent assay (ELISA) using a microtiter plate. The capture rate of target IgG was accelerated 200-fold and a tenfold lower limit of detection was achieved. Finally, the MCSNP-based immunoassay was successfully applied to the detection of specific IgE from milk-allergic patient’s sera with a lower LOD and a good agreement (CV < 6%) with the microtiter plate, confirming the great potential of this analytical platform in the field of immunodiagnosis.

Human erythrocytes covered with magnetic core-shell nanoparticles for multimodal imaging.

Surface functionalization of human red blood cells (hRBCs) with fluorescent and magnetic silica core-shell nanoparticles is used to design a carrier suitable for multimodal imaging with a long circulating time. The coated magnetic hRBCs show no hemolytic activity, while the advantage of the affinity of proteins for silica allows a further coating.