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Overall Approach and Methodology
Introduction
The emphasis of this project is on the specific functionalisation and characterization of GNPs. The basic materials and technologies are available or relatively easy to expand, but the integrated application for specific aims is innovative. In particular oligosaccharide synthesis and isolation are very labour intensive. The major research activities towards the project objectives and goal are summarized in the following paragraphs.
Providing materials
The carbohydrate epitopes required in this project, are to a large extent already available in the partner laboratories or are commercially available. The methodology for the synthesis or isolation from natural sources of oligosaccharides that are not readily available is well developed and available within the network. The synthesis of neoglycoconjugates for the preparation of specifically functionalized gold GNPs requires the assembly of the spacered oligosaccharide moiety using protecting group patterns and strategies, which are compatible with the thiol group chemistry needed for GNP generation. Furthermore, the choice of the linker to attach the oligosaccharide to the gold surface is of major importance: length, hydrophobicity and/or hydrophilicity have to be carefully designed. This may require developing some new chemistry in order to optimize the synthesis and properties of specific GNPs for specific purposes.
Various (non-GNP) interacting partners are available commercially or within the network. Additional purification will be done using well-established techniques. Cryptococcal and pneumococcal polysaccharides and derived oligosaccharides are accessible to the network partners. Lectins will be isolated and purified from natural sources using chromatographic techniques. Monoclonal antibodies against S. pneumoniae are available, and several labs are equipped for the preparation and purification of additional (monoclonal) antibodies. Various mutated protein libraries and antibody phage display libraries are available for screening purposes. Here, tagged GNPs or other (established) screening systems can be used to find new protein binders. Selected recombinant antibody fragments, or other wild type or mutated carbohydrate-binding protein can be produced and purified from Escherichia coli using established methods
GNP generation, purification and characterisation
Within the proposed network the know-how and the infrastructure to prepare specific GNPs are already present in some groups and will be transferred to other partners as well. This methodology allows for control on ligand density, ratio of ligands coupled to the GNPs, solubility, stability and size. Purification techniques such as dialysis, membrane filtering and gel exclusion chromatography permit to obtain highly homogeneous and pure GNP samples.
State-of-the-art complementary analysis techniques are required for a complete characterization of the GNP. Microscopic techniques provide information of the overall size and shape. The spectroscopic techniques (FTIR, UV/VIS, fluorescence, NMR, MS, XPS, etc) reveal details of the behaviour and chemistry of the GNPs.
Identification of novel receptors for GNPs
For the search of novel molecular interacting partners, several strategies will be followed. GNPs and hybrid GNPs (e.g. labelled with fluorescent tags) will be tested in assays against lectin / (recombinant) antibody libraries and complex biological samples, using traditional techniques. A special approach involves the combination of GNP technology and a recently introduced novel glycoprobe technology developed by one of the partners in the network. In the glycoprobe technology use is made among others of a lysyl digoxin tag and a photoreactive crosslinker. This is the field of expertise of the Croatian partner. Multimeric presentation of ligands on the hybrid GNPs will be used to enable formation of strong noncovalent complexes between GNPs and specific receptors. Receptors picked up in this way will be purified on affinity columns and identified by MS and MS/MS analysis.
In vitro / ex vivo studies of the molecular basis of specific interactions
Biophysical and biochemical methods are available to study the specific interactions of GNPs with their interacting partners. These include TEM, AFM (direct interaction forces), SPR (affinity), MS, and NMR. Expertise already exists in different laboratories within the network and will be propagated through the network.
Immunological assays, of use in the defined projects, to study molecule-molecule and cell-molecule interactions, including inhibition assays, are already available within the scientific network. Cell binding of fluorescently labelled GNPs will be traced using the FACS technique.
In vivo studies
For the hybrid GNPs vaccine research focused on pathogenic bacteria, S. pneumoniae vaccination mice models are available within the network. Here the focus is mainly on the generation and classification of antibodies.
To study what will happen with GNPs decorated with glycans that recognize specific carbohydrate-binding proteins when administered to the organism, including the mode of administration (per os, i.v. or i.m), needs detailed investigations of tissue material. For these studies use will be made of hybrid GNPs containing fluorescent tags to be used in fluorescent microscopy and ELISA-type assays. Tissues that are identified as targets will be further analysed by electron microscopy. Since certain carbohydrate-lectin interactions appear to be particularly important in the brain, effects of GNP administration will also be studied in animals with experimentally disrupted blood-brain barrier. Usually, nanoparticles are not able to cross the blood-brain barrier. For example, less that 10% of the commercially available magnetic nanoparticles Resovist cross this barrier, which represents a problem for brain imaging. We are trying to solve this problem and are confident that glyconanoparticles will decisively contribute to solve this problem. In addition another approach, making use of radiolabelled GNPs will be worked out. Images, made on a gamma camera over a period of time after injection into experimental animals, make the measurement of the rate of clearance of GNPs possible as well as many other biomedically relevant parameters. Macro- and microautoradiography can be used to look at the GNP distribution within the tissues.
To our knowledge in-vivo localization and clearance monitoring of GNPs by NMR imaging has not yet been explored. Glycogold foresees possibilities in this area. Metallic particles, as small as they may be, are expected to induce a detectable disturbance in the local magnetic field.
Modelling Approaches.
Molecular modelling using recently emerged hybrid quantum mechanics-molecular mechanics (QM/MM) approaches is a suitable tool for the understanding of carbohydrate-protein interactions and for the prediction of their affinities taking into account the flexibility of both the protein and ligand. The statistical thermodynamic origin of the multivalency effect relies on radial distribution of distances between bound and pendant ligands obtained from molecular dynamics. The QM/MM method provides a quantum mechanics calculation on certain parts of the system (the covalent gold-glycoconjugate linkage and gold cluster) while the remainder of the system is treated with a molecular mechanics force field. The results obtained with the QM/MM method will also provide necessary structural and energetic data to parameterize the force fields used for carbohydrates.
In order to obtain an atomic description of the interactions of protein with the carbohydrate antigen whose presentation is dictated by the core of the GNPs (i.e. the density and the length of the functionalised branches) dedicated computer programs will be developed: the Nano-Particle Builders. They will allow exhaustive computational modelling of the interactions (both in terms of structures and of energy of binding) from which quantitative structure-properties relationships will be established providing guidelines to the design of new GNPs tailored for a given application.