Near-Infrared Fluorescent Organic Nanoparticles for Deep in vivo Imaging – IMAGEin

Inflammation is a major process in many human diseases. The onset phase is characterised by the stimulation of VCAM-1 receptor expression by macrophages/glial cells. Early detection of VCAM-1 expression in chronic inflammation is therefore vital to avoid tissue damage, which will occur in a later phase. Preclinical data on the localisation and density of VCAM-1, obtained with high spatial and temporal resolutions, are essential, which only intra-vital microscopy techniques can provide.
IMAGEin aims to design and study the physico-chemical properties and biological fate of fluorescent organic nanoparticles (FONs), made of fluorophores exhibiting aggregation-induced fluorescence (AIE) properties in the near infrared (NIR) and embedded into a functional polymer shell. These FONs will then be used in practical application in in-vivo two-photon imaging of inflammation through specific targeting of the VCAM-1 receptor.
IMAGEin is structured in 5 major scientific tasks.
The first task will be to obtain NIR-emitting AIE fluorophores by incorporating strong electron-donating heterocycles such as indolizine and azaindolizine into novel structures. One of the challenges will be to shift the emission into the NIR, while maintaining a suitable solid-state fluorescence quantum yield.
The second task concerns the production of functionalized hyaluronic acid (HA) polymers to improve the chemical and biological stability of the FONs and to increase their stealthiness in-vivo. Thus, thiol groups will be introduced in a controlled manner on HA. The modified HA can then be cross-linked by the formation of disulphide bridges. The reactivity of the thiol functions will also allow the introduction of addressing units on the surface of the FONs. Coupling with VCAM-1-specific cAbVCAM1-5 nanobodies will then be performed.
The key step will be the preparation of crystalline FONs, smaller than 50 nm, stable in the colloidal state and rediffusible in water, by a nanoprecipitation/lyophilization procedure. In order to understand the physico-chemical parameters controlling the nanoprecipitation, we will establish the phase diagrams and identify the Ouzo domains, where nanoparticles of sizes below 100nm are generated. Cross-linking for final stability and the addition of Pluronic F68 will promote the crystallisation of the core during freeze-drying. Particular care will be taken to produce solid dispersions of FONs on a gram scale.
The in-vivo fate of the FONs and the FONs/protein interactions will be studied, by analysis of the protein crown formed in vivo. Finally, blood flow and clearance studies, as well as biphoton imaging studies in mice will be conducted. The localisation and density of VCAM-1 will be assessed in a mouse model of neuroinflammation and VCAM-1 targeted substituted FONs.
A strong consortium of 5 partners has been built around this project, bringing their complementary expertise in fluorophore chemistry, glycochemistry, polymer chemistry, nanoprecipitation, nanomedicine, and in vivo microscopy.