Silica nanoparticle multifunctionalization by solid phase synthesis for biomedical applications

Les nanomatériaux combinant des fonctions de ciblage, d'imagerie, de thérapie et de détection font l'objet de nombreuses recherches dans le domaine de la santé. Les travaux présentés dans cette thèse concernent la multi‐fonctionnalisation de nanoparticules (NPs) par un procédé de synthèse supportée. Le support solide développé dans cette étude est constitué d'un matériau poreux en verre sur lequel sont greffées de manière temporaire des nanoparticules de silice. La fonctionnalisation de la surface des nanoparticules a été réalisée de façon automatisée par une chimie de synthèse dite aux phosphoramidites. Dans un premier temps, cette technique a permis d'obtenir des densités de greffage de l'ordre de 5000 à 7000 oligonucléotides par nanoparticule, ce qui représente une fonctionnalisation 10 à 20 fois supérieure à celles obtenues par des méthodes de greffage en solution. Les brins d'ADN synthétisés sur les NPs ont montré une bonne accessibilité pour l'hybridation avec un brin d'ADN complémentaire, ouvrant la voie à des applications thérapeutiques ou à l'intégration de ces objets dans des systèmes de détection. La deuxième partie de ces travaux est consacrée à la vectorisation d'une protéine thérapeutique, le G‐CSF (facteur de croissance de colonies de granulocytes), par des nanoparticules présentant également des propriétés d'imagerie. Ces nanovecteurs thérapeutiques ont montré des propriétés de stimulation cellulaire in vitro et de ciblage de la rate, organe réservoir de neutrophiles, in vivo. Enfin il a été démontré que la modification de NPs sur support ouvre des perspectives intéressantes pour la préparation d'assemblages complexes de nanoparticules (dimères et NPs dissymétriques)Nanomaterials combining targeting, imaging, therapy and sensing properties are of growing interest for biomedical applications. The work reported in this thesis concerns nanoparticle (NP) multifunctionalization by solid phase synthesis. The solid support developed in this study is composed of a porous glass material on which silica NPs are temporarily grafted. Nanoparticle surface functionalization was performed by automated synthesis using phosphoramidite chemistry. Firstly, high surface loadings from 5000 to 7000 oligonucleotides per NP were achieved, representing a functionalization 10 to 20‐fold greater than those obtained by coupling methods in solution. DNA strands synthesized on NPs showed a good accessibility for hybridization with a complementary DNA strand, paving the way for therapeutic applications or integration of these objects in detection systems. The second part of this work was devoted to the vectorization of a therapeutic protein, GCSF (Granulocyte‐Colony Stimulating Factor) by nanoparticles that also exhibited imaging properties. These therapeutic nanocarriers showed cell stimulating properties in vitro and spleen targeting, which is a reservoir of neutrophils, in vivo. Finally, it was demonstrated that the solid phase modification of NPs opens interesting perspectives for the production of complex nanoparticle assemblies (dimers and asymmetric NPs

Nanoparticles with multiple properties for biomedical applications: A strategic guide

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Methylene blue phosphoramidite for DNA labelling

We thank Oceane Clabaux and Alexandre Escoffier for their contribution to the work.International audienceWe report the first synthesis of a methylene blue (MB) phosphoramidite derivative suitable for DNA solid-phase synthesis. The electrochemical and optical properties of the resulting MB modified oligonucleotides were confirmed. This new molecule is an important breakthrough in the design of new probes labelled with MB

Label-free electrochemical monitoring of protein addressing through electroactivated "click" chemistry on gold electrodes

International audienceIn this work, using electrochemical impedance spectroscopy (EIS), we have, for the first time, label-free monitored protein immobilization on a gold surface through a strategy of electroaddressing, compatible with the production of microarrays for multi-detection. This functionalization is achieved via the alkyne/azide cycloaddition, better known as the "click" reaction. The electroaddressing was applied to a polythiol hexynyl derivative previously grafted onto the gold surface. This compound consists of two dithiol phosphate groups and a hexynyl function and was synthesized through a supported synthesis approach, from a dithiol reagent, phosphoramidite (DTPA), and a hexynyl phosphoramidite. Next, an azide-PEG3-biotin derivative was grafted onto the modified gold surface by electro-chronocoulometry. The "click" reaction was controlled by electrochemical impedance spectroscopy, showing the change in impedance only when the electroaddressing was performed at − 300 mV. No effect on the EIS signal was observed when a positive potential was applied, confirming the specificity of the electroactivation. Biotin-modified electrodes were used to fix streptavidin and the immobilization was monitored using EIS. Fluorescent streptavidin-functionalized silica nanoparticles were also specifically grafted onto the biotinylated gold surface in order to confirm the "click" reaction using fluorescence microscopy. The obtained streptavidin platform was used to detect the surface coverage by biotinylated human serum albumin (HSA). The lowest detectable concentration is 10 pg/mL, and surface saturation is obtained with concentrations higher than 100 ng/m

Impact of immobilization support on colorimetric microarrays performances

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Synthesis and electroactivated addressing of ferrocenyl and azido-modified stem-loop oligonucleotides on an integrated electrochemical device

International audienceWe report a strategy to address stem-loop oligonucleotides on a gold surface in order to develop a robust and ultra-sensitive integrated electrochemical DNA sensor. Probe immobilization relies on the potential-assisted copper-catalyzed alkyne-azide cycloaddition. Firstly, a tetrathiol-hexynyl derivative was used to introduce alkyne functions onto the electrode surface. This anchor has proved its robustness in conditions used for the " click " reaction and in wet storage. Then, different ferrocenyl and azido-modified stem-loop oligonucleotides were synthesized using the solid-phase synthesis technique and their immobilization was studied. Hybridization assays with the DNA target were performed in a complex medium by cyclic voltammetry. The detection sensitivity achieved by our functionalized electrodes was significantly increased, as a detection limit of 10 fM was determined. We also demonstrated that grafting of the stem-loop oligonucleotides via the electroactivated " click " reaction was specific to the gold surface on a microfabricated electro chemical device for the Lab-on-Chip application that fully integrates Au working microelectrodes, Pt counter and Ag reference electrodes.

Granulocyte-Colony Stimulating Factor Nanocarriers for Stimulation of the Immune System (Part II): Dose-Dependent Biodistribution and In Vivo Antitumor Efficacy in Combination with Rituximab

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Granulocyte Colony-Stimulating Factor Nanocarriers for Stimulation of the Immune System (Part I): Synthesis and Biodistribution Studies

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Nanoparticules fluorescentes ciblantes pour l'imagerie médicale

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Oligonucleotide solid-phase synthesis on fluorescent nanoparticles grafted on controlled pore glass

International audienceOligonucleotide solid-phase synthesis is now possible on nano-sized particles, thanks to the use of controlled pore glass-nanoparticle assemblies. We succeeded in anchoring silica nanoparticles (NPs) inside the pores of micrometric glass via a reversible covalent binding. The pore diameter must be at least six times the diameter of the nanoparticle in order to maintain efficient synthesis of oligonucleotides in the synthesizer. We demonstrated that the pores protect NP anchoring during DNA synthesis without decreasing the coupling rate of the phosphoramidite synthons. This bottom-up strategy for NP functionalization with DNA results in unprecedented DNA loading efficiency. We also confirmed that the DNA synthesized on the nanoparticle surface was accessible for hybridization with its complementary DNA strand