Fluorescent molecular nanocrystals embedded in silicate nanoparticles : towards a new type of ultra bright tracers for intravital imaging – ULTRABRIGHT-TRACERS

In tumor biology, imaging of new vessel growths or angiogenesis during tumor developments and changes of the latter after therapy is important in the evaluation and validation of new treatment protocols. All intravital imaging methods lack the spatial resolution to detect microscopic changes at the capillary level; therefore new in vivo imaging protocols are necessary. Two photon microscopy is the only technique that allow in vivo imaging of these vascular changes at a microscopic scale, but to be used, it requires ultrabright fluorescent tracers that can be water-dispersible and colloidally stable. Thus, in this project we plan to carefully control the preparation and properties of nanocomposite particles constituted by a crystallized organic core surrounded by a silicate shell for deep tumor vascular imaging: one millimeter instead of several hundreds of micrometers with the currently available commercial tracers. This silicate shell can be easily functionalized with a tailored surface chemistry by grafting Poly Ethylen Glycols (PEG) chains or peptides molecules at its surface for in vivo targeting of the tumor vascular endothelial cells. We will obtain new hybrid (molecular nanocrystal – amorphous silicate) nanoparticles with sizes ranging between 20 and 100nm. These core-shell particles will constitute a multifunctional nano-platform for the development of stable, biocompatible and ultra-bright biological tracers exhibiting efficient vectorisation properties and high two-photon fluorescence intensitie in the red or near IR for in vivo intravital imaging: laser-scanning fluorescence angiography. In addition, this new type of ultrabright (molecular nanocrystals-silicate shell) labels should be also used for in vitro biological imaging.
The development of this project is first based on the preparation at Néel Institute (MatONLP group) of core-shell nanoparticles constituted by fluorescent molecular nanocrystals surrounded by amorphous silicate shells that can be easily biotargeted. This synthesis from sol-gel solutions involves the accurate control of the confined nucleation and growth of organic nanocrystals in the core of silicate spheres resulting to the hydrolysis and condensation reactions of silicon alkoxides simultaneously introduced in the same initial solution. Several poly(n-trialkoxysilane) organic precursors of the silicate shells will be designed and synthesized by the CMOS group (ICG-Montpellier) in order to obtain nonporous, hydrophilic, biocompatible NPs exhibiting a high colloidal stability in saline solutions and that can be easily post-functionalized. The NP preparation conditions will be adjusted by fine structural characterizations (Néel Institute) obtained from several coupled techniques: scanning and transmission microscopy, diffraction, micro-Raman, Zetametry, Photon Correlated Spectroscopy and XPS in collaboration with ICG-Montpellier. To obtain highly fluorescent particles, very efficient dyes should be involved coming from a specific molecular engineering study done by the molecular chemists of ENS-Lyon (“Chemistry for Optics” group) on organic molecules. Finally, the biological studies, biocompatibility tests and intravitale two-photon microscopy on mice will be carried out by GIN-INSERM U836. We will first test the NPs on the normal cerebral microvasculature followed by in vivo tests on mice bearing gliomas in a dorsal skin fold chamber. In the latter, the NPs should stay inside the vasculature for proper analysis of the blood volume. In the last phase of the project, ligands (cRGD) will be bind on the surface of the NPs for specific targeting of the tumor vascular endothelial cells, collaboration between Néel Institute and GIN.