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

Unveiling the Extracellular Space of the Brain: From Super-resolved Microstructure to In Vivo Function

International audienc

Nanoscale and functional heterogeneity of the hippocampal extracellular space

Summary: The extracellular space (ECS) and its constituents play a crucial role in brain development, plasticity, circadian rhythm, and behavior, as well as brain diseases. Yet, since this compartment has an intricate geometry and nanoscale dimensions, its detailed exploration in live tissue has remained an unmet challenge. Here, we used a combination of single-nanoparticle tracking and super-resolution microscopy approaches to map the nanoscale dimensions of the ECS across the rodent hippocampus. We report that these dimensions are heterogeneous between hippocampal areas. Notably, stratum radiatum CA1 and CA3 ECS differ in several characteristics, a difference that gets abolished after digestion of the extracellular matrix. The dynamics of extracellular immunoglobulins vary within these areas, consistent with their distinct ECS characteristics. Altogether, we demonstrate that ECS nanoscale anatomy and diffusion properties are widely heterogeneous across hippocampal areas, impacting the dynamics and distribution of extracellular molecules