Development of contrast agents for imaging amyloids in type 2 diabetes – from chemical synthesis to in vivo trials

Les fibres amyloïdes sont associées à un grand nombre de maladies dégénératives, parmi lesquelles le diabète de type 2. Le but de cette thèse de doctorat était donc de développer des agents de contraste permettant d'imager les amyloïdes pancréatiques. Dans un premier temps, la pharmacocinétique de nanoparticules a été déterminée. Les nanoparticules testées étaient composées d'un réseau de silice sur lequel étaient greffés des cycles DOTA complexant des ions gadolinium, permettant l'imagerie par résonnance magnétique, et d'un hexapeptide, ligand des fibres amyloïdes. Suite à ces études, il a été décidé de synthétiser une série de petites molécules dérivées du florbétapir et de la thioflavine. L'affinité de ces molécules pour les fibres amyloïdes pancréatiques a été déterminée. Une étude histologique sur les pancréas d'un potentiel modèle de souris a également été menée. La dernière partie de la thèse n'est pas reliée aux chapitres précédents. Elle porte sur la thermoablation des tumeurs

Multimodal imaging Gd-nanoparticles functionalized with Pittsburgh compound B or a nanobody for amyloid plaques targeting

International audienceGadolinium-based nanoparticles were functionalized with either the Pittsburgh compound B or a nanobody (B10AP) in order to create multimodal tools for an early diagnosis of amyloidoses.MATERIALS & METHODS:The ability of the functionalized nanoparticles to target amyloid fibrils made of β-amyloid peptide, amylin or Val30Met-mutated transthyretin formed in vitro or from pathological tissues was investigated by a range of spectroscopic and biophysics techniques including fluorescence microscopy.RESULTS:Nanoparticles functionalized by both probes efficiently interacted with the three types of amyloid fibrils, with KD values in 10 micromolar and 10 nanomolar range for, respectively, Pittsburgh compound B and B10AP nanoparticles. Moreover, they allowed the detection of amyloid deposits on pathological tissues.CONCLUSION:Such functionalized nanoparticles could represent promising flexible and multimodal imaging tools for the early diagnostic of amyloid diseases, in other words, Alzheimer's disease, Type 2 diabetes mellitus and the familial amyloidotic polyneuropathy

Multivalent glibenclamide to generate islet specific imaging probes

The monitoring of diabetes mellitus, as it develops and becomes clinically evident, remains a major challenge for diagnostic imaging in clinical practice. Here we present a novel approach to beta-cell imaging by targeting the sulphonylurea receptor subtype 1 (SUR1), using multivalent derivatives of the anti-diabetic drug glibenclamide. Since glibenclamide has a high affinity for SUR1 but does not contain a suitable functional group to be linked to an imaging probe, we have synthesized 11 glibenclamide derivatives and evaluated their affinity to SUR1 in MIN6 cells. The most promising compound has been used to obtain multivalent glibenclamide-polyamidoamine (PAMAM) derivatives, containing up to 15 sulphonylurea moieties per dendrimer. The remaining functional groups on the dendrimers can consecutively be used for labeling with reporter groups for different imaging modalities, thus allowing for multifunctional imaging, and for the modification of pharmacokinetic properties. We synthesized fluorochrome-labeled multivalent probes, that demonstrate in cellular assays affinities to SUR1 in the nanomolar range, superior to native glibenclamide. The probes specifically label MIN6 cells, but not HeLa or PANC-1 cells which do not express SUR1. A very low cytotoxicity of the multivalent probes is demonstrated by the persistent release of insulin from MIN6 cells exposed to high glucose concentrations. Furthermore, the probes display positive labeling of beta-cells of primary mouse and human islet-cells ex vivo and of islets of Langerhans in vivo. The data document that multivalent probes based on glibenclamide derivatives provide a suitable platform for further developments of cell-specific probes, and can be adapted for multiple imaging modalities, including those that are now used in the clinics

Gd-nanoparticles functionalization with specific peptides for ß-amyloid plaques targeting

Amyloidoses are characterized by the extracellular deposition of insoluble fibrillar proteinaceous aggregates highly organized into cross-β structure and referred to as amyloid fibrils. Nowadays, the diagnosis of these diseases remains tedious and involves multiple examinations while an early and accurate protein typing is crucial for the patients' treatment. Routinely used neuroimaging techniques such as magnetic resonance imaging (MRI) and positron emission tomography (PET) using Pittsburgh compound B, [11C]PIB, provide structural information and allow to assess the amyloid burden, respectively, but cannot discriminate between different amyloid deposits. Therefore, the availability of efficient multimodal imaging nanoparticles targeting specific amyloid fibrils would provide a minimally-invasive imaging tool useful for amyloidoses typing and early diagnosis. In the present study, we have functionalized gadolinium-based MRI nanoparticles (AGuIX) with peptides highly specific for Aβ amyloid fibrils, LPFFD and KLVFF. The capacity of such nanoparticles grafted with peptide to discriminate among different amyloid proteins, was tested with Aβ(1–42) fibrils and with mutated-(V30M) transthyretin (TTR) fibrils

MOESM1 of Gd-nanoparticles functionalization with specific peptides for ß-amyloid plaques targeting

Additional file 1: Figure S1. Characterization of functionalized nanoparticles. Figure S2. Zeta potential versus pH of AGuIX (black) and AGuIX@PEG (red). Figure S3. Reaction scheme for the synthesis of AGuIX@PEG@[email protected]. Figure S4. Chromatograms obtained with at UV-Visible absorption at λ =295 nm of the AGuIX@PEG, AGuIX@PEG@LPFFD and AGuIX@PEG@KLVFF . Elementary Analyses of grafted nanoparticles: Table S1. Molar ratio deduced from experimental weight percentage; Table S2. Elementary analyses given in weight percent of element in the compound; Table S3. Comparison of the primary amine content evaluated by elementary analysis and TNBS assays. These results allow assessing the molecular formula obtained by elementary analysis. Figure S5. Standard curves of amine quantification in APTES/TEOS equimolar mixture by TNBS assays. Figure S6. The cyanine 5.5 presents characteristic absorption and emission wavelength λexc max Cy5.5 = 675 nm and λem max Cy5.5 = 692 nm. Table S4. The yield of the grafting was calculated thanks to the comparison of the cyanine fluorescence and the DOTA(Gd3+) chelate before and after removal of the ungrafted dye by tangential purification. Figure S7. Effect of nanoparticles on cell viability. Figure S8. PIB derivative synthesis details. Figure S9. Control of the non-binding of Cy5.5 dye to amyloid plaque in brain section of AD mouse model