Ultrasmall Glyco-Gold Nanoparticles: Synthesis Optimization, Characterization and Applications in Immune-Cell Targeting

xxii, 245 p.Gold nanoparticles (GNP) are hybrid materials, with excellent physicochemical characteristics, made of a gold core and a corona of organic molecules, amongst them carbohydrates. Ultrasmall GNP (nanoclusters) are usually obtained through a modified Brust-Schiffrin synthesis. The combination of glycoscience and ultrasmall GNP enables wide biotechnological and clinical applications. Using GNP multivalency or multifunctionality properties, carbohydrates can, for instance, trigger the cluster glycoside effect required for the targeting of lectin receptors. To deliver reliable and reproducible GNP material, a tight control of the synthesis and a broad spectrum of analytical techniques are essential. To this end, the passivation step of the aqueous Brust-Schiffrin synthesis was thoroughly studied using different bifunctional platforms made of either positive or negative oligo-PEG and a monosaccharide with a short alkyl side chain with narrow one-parameter variations (pH, temperature). An increased passivation time led, for all the models studied, to an increase of the core and the overall size of the particles, as well as to a gradual decrease of the ligand density. Attributing these changes to the presence of sodium borohydride, the reducing agent that forms the particles, scavenging ion exchange resins were tested. IRA-400 was able to remove most of sodium borohydride from the crude solution and prevent the passivation effects previously observed. Moreover, an extensive characterization was performed to optimize the analytical techniques, compare the data and obtain the most accurate description of the synthesized material. This work demonstrated the sensitivity of techniques such as UV-Vis spectrophotometry and size exclusion chromatography for GNP size evolution monitoring. A novel chromatographic method of corona characterization for weak or non-UV absorbent ligands was developed using charged aerosol detection coupled with mass spectrometry (LC-CAD-MS), accompanied by a new particle etching protocol using tris(2-carboxyethyl)phosphine (TCEP) (to enable complete release of the ligands in the reduced, thiol form). The results were compared to those obtained by 1H NMR. Taking advantage of the mass dependent property of the CAD, the degree of functionalization after post-functionalization reactions was also determined. Results obtained with both techniques were similar and validated the complementarity of the methods. A library of alpha-mannose derivatives together with oligosaccharides were used to decorate GNP through post-functionalization reactions. The different routes to design the alpha-mannose library and functionalize GNP were compared to find the most efficient method considering parameters such as the yield of the final alpha-mannose derivative, the degree of functionalization of the GNP and challenges of characterization. alpha-Mannose derivatives were synthesized with different functional groups: amine, carboxylic acid, azide, alkyne, isothiocyanate. GNP bearing the complementary moieties were then coupled to the carbohydrates, with the goal of achieving the highest degree of functionalization. Non-derivatized oligosaccharides were also bound to an amino-oxy GNP through an oxime link. The success of the non-modified carbohydrate oxime route created a straightforward method for GNP decoration. Biochemical (microarray, biolayer interferometry) and biological (cell uptake) assays were performed to achieve and optimize the targeting of lectins such as DC-SIGN by Glyco-GNP. Biolayer interferometry demonstrated that -mannose and, more significantly, the dimer alpha-mannose1,2 alpha-mannose (and two chemically enhanced mimetics ISh045 and ISh046) were able to effectively bind to DC-SIGN when presented on particles with a 4 nm core (plasmonic), but not when incubated with 2 nm core GNP. These results were in line with the cell uptake assay performed with a dendritic cell (THP-1) model expressing DC-SIGN. Specific uptake was only observed with 4 nm core GNP functionalized with alpha-mannose1,2 alpha-mannose and its mimetics (30-fold increase) and alpha-mannose (6-fold increase). The ability of GNP to quench fluorescence was used to screen a library of lectins with different carbohydrate affinities. A microarray of fluorescent lectins was printed, and the GNP were able to quench the fluorescence by selectively binding to the lectins, discriminating them depending on their spatial orientation and sugar specificity

Direct numerical simulations of mass transfer in square microchannels for liquid-liquid slug flow

Microreactors for the development of liquid-liquid processes are promising technologies since they are supposed to offer an enhancement of mass transfer compared to conventional devices due to the increase of the surface/volume ratio. But impact of the laminar flow should be negative and the effect is still to be evaluated. The present work focuses on the study of mass transfer in microchannels by means of 2D direct numerical simulations. We investigated liquid-liquid slug flow systems in square channel of 50 to 960 μm depth. The droplets velocity ranges from 0.0015 to 0.25 m/s and the ratio between the channel depth and the droplets length varies between 0.4 and 11.2. Droplet side volumetric mass transfer coefficients were identified from concentration field computations and the evolution of these coefficients as a function of the flow parameters and the channel size is discussed. This study reveals that mass transfer is strongly influenced by the flow structure inside the droplet. Moreover, it shows that the confinement of the droplets due to the channel size leads to an enhancement of mass transfer compared to cases where the droplets are not constrained by the walls

Multiple Routes for Glutamate Receptor Trafficking: Surface Diffusion and Membrane Traffic Cooperate to Bring Receptors to Synapses

Trafficking of glutamate receptors into and out of synapses is critically involved in the plasticity of excitatory synaptic transmission. Endocytosis and exocytosis of receptors have initially been thought to account alone for this trafficking. However, membrane proteins also traffic through surface lateral diffusion in the plasma membrane. We describe developments in electrophysiological and optical approaches that have allowed for the real time measurement of glutamate receptor surface trafficking in live neurons. These include (i) specific imaging of surface receptors using a pH sensitive fluorescent protein, (ii) design of a photoactivable drug to inactivate locally surface receptors and monitor electrophysiologically their recovery, and (iii)application of single molecule fluorescence microscopy to directly track the movement of individual surface receptors with nanometer resolution inside and outside synapses. Altogether, these approaches have demonstrated that glutamate receptors diffuse at high rates in the neuronal membrane and suggest a key role for surface diffusion in the regulation of receptor numbers at synapses

Optimisation of operating conditions in batch for more sustainable continuous process transposition

Implementation of exothermic and fast reactions requires a perfect temperature control to avoid thermal runaway and in most cases to limit by-products production. In order to fit with the heat removal capacity of common devices, expensive strategies are currently used to slow down this kind of reactions in order to avoid a strong temperature increase such as reactants dilution. Within the concept of process intensification, industries could move towards more sustainable process by reducing technology constraints to the benefit of chemistry. For that purpose, a two-step methodology is implemented. The first step consists in the optimisation of the operating conditions only based on stoichio-chemical scheme and kinetic laws. This is carried out by adjusting temperature profile and feeding rate strategy in a batch operation. Then a design for a continuous process is proposed, trying to approach the optimal batch operating conditions. This methodology is applied to the linear alkylbenzene sulfonation

Mono- and Biexponential Luminescence Decays of Individual Single-Walled Carbon Nanotubes

We have studied the exciton recombination dynamics of individual (6,4) and (6,5) single-walled carbon nanotubes embedded in aqueous gels or deposited on glass surfaces. CoMoCat nanotubes systematically display short monoexponential photoluminescence (PL) decays presumably due to defects introduced during their synthesis. In contrast HiPco nanotubes can either display mono- or biexponential PL decays depending on the environmental conditions. Transition from bi- to monoexponential decays can be reproduced by a simple three level model taking into account defect-dependent nonradiative decay mechanisms

Quantum yield optimized fluorophores for site-specific labeling and super-resolution imaging

Single molecule applications, saturated pattern excitation microscopy, or stimulated emission depletion (STED) microscopy demand for bright and highly stable fluorescent dyes1,2. Despite of intensive research the choice of fluorphores is still very limited. Typically a stable fluorescent dyes is covalently attached to the target. This methodology brings forward a number of limitations, in particular, in case of protein labeling. First of all the fluorescent probes need to be attached selectively and site-specifically to prevent unspecific background. This often requires single cysteine mutations for covalent protein modification. Employing quantum dots allows overcoming problems of photo-bleaching3-6. However, the downsides are their large size, rendering the probe inaccessible to spatially confined architectures, issues in biocompatibility due to proper particle coating, and cellular toxicity6-8. Here we propose a new method to overcome the above outlined problems

Intensification of Ester Production in a Continuous Reactor

Numerous continuous intensified reactors are now accessible on the market that offer enhanced thermal performances in a continuous reactor. Such reactors are then particularly suited to fast and highly exothermic reactions. In this paper, the ability to also manage a slow and equilibrated system, the methyl acetate esterification reaction, on condition of intensification in terms of design and operating conditions is presented. To achieve this purpose, a new kinetics model has been developed and validated from experiments carried out in a lab scale batch reactor. Implemented in a simulation framework, this model leads to an intensified design of the reactor and the associated operating conditions. All this intensification methodology has been supported and validated by experimental studies

Influence of solvent choice on the optimisation of a reaction–separation operation : application to a Beckmann rearrangement reaction

In pharmaceutical syntheses, the solvent choice generally represents a complex design step. Traditionally, this choice is operated according to criteria connected with the reaction step and without any consideration on the following separation steps. The purpose of this study is to highlight the benefits of a global approach of optimisation for the solvent determination. In this way, an optimisation framework dedicated to global synthesis is applied to a simple reaction–separation operation integrating a Beckmann rearrangement reaction, leading to interesting solvent choices