Ischemic stroke, as one of the most common causes of death, represents an important health issue. The pathology consists of the occlusion of an artery in the brain leading to an acute inflammatory process. Post-stroke inflammation usually results in irreversible secondary brain tissue damage. To date, the clinical application of anti-inflammatory treatments has been either negative or inconclusive. For a better understanding of this complex physiological process and development of efficient treatment, there is an urgent need to develop performant in-vivo diagnostic tools.

In that context, we proposed to design a multimodal hybrid nanoprobe for enhancing the contrast in three different clinical and pre-clinical imaging modalities. The ability of this probe to enhance contrast in MRI (Magnetic Resonance Imaging) and a recently developed spectral photon counting scanner computed tomography (SPCCT) is intrinsic to the inorganic GdF₃ core. The inorganic nanoparticle size and morphology was optimized for the biological application. The third modality, two-photon imaging, provides high spatial resolution, high sensitivity, and allows real-time imaging. To make GdF₃ nanoparticles visible by two-photon microscopy, a specially designed organic moiety is added to the nanoplatform.

The inorganic nanoparticles are synthesized by the original solvothermal method developed in our group. Surface modifications with different PEG derivatives confer to the GdF₃ nanoparticles high stability in physiological media (such as blood), biocompatibility, and stealth. The two-photon active chromophore synthesized in our laboratory is grafted to the particle surface via a thermally activated (catalyst-free) alkyne-azide click reaction.

Toxicity of the nanoobjects has been assessed by using two different tests on four human-derived cells, and no cytotoxic effect of the particles was found.

After the demonstration of the multimodality of the particles, pre-clinical in vivo experiments were performed. We evidenced that the particles successfully enhance SPCCT, MRI contrast in the brain of the small animal via a T2-effect and provide a high-intensity two-photon signal for in-vivo microscopy. Besides, the nanoparticles revealed to be stable and long-circulating in the blood, which favored their cross through the altered blood-brain barrier. Their phagocytose by activated immune cells offered the possibility to follow cell-trafficking.

Keywords: multimodal contrast agent, hybrid nanoparticle, gadolinium fluoride, surface modification of nanoparticles, bisphosphonate PEG, solvothermal nanoparticle synthesis, microwave-assisted nanoparticle synthesis, ischemic stroke, neuroinflammation, brain imaging, MRI, SPCCT, Two-photon microscopy