Diamond nanoparticles (nanodiamonds) can transport active drugs in cultured cells as well as in vivo. However, in the latter case, methods allowing to determine their bioavailability accurately are still lacking. Nanodiamond can be made fluorescent with a perfectly stable emission and a lifetime ten times longer than the one of tissue autofluorescence. Taking advantage of these properties, we present an automated quantification method of fluorescent nanodiamonds (FND) in histological sections of mouse organs and tumor, after systemic injection. We use a home-made time-delayed fluorescence microscope comprising a custom pulsed laser source synchronized on the master clock of a gated intensified array detector. This setup allows to obtain ultra-high-resolution images 120 Mpixels of whole mouse organs sections, with subcellular resolution and single-particle sensitivity. As a proof-of-principle experiment, we quantified the biodistribution and aggregation state of new cationic FNDs able to transport small interfering RNA inhibiting the oncogene responsible for Ewing sarcoma. Image analysis showed a low yield of nanodiamonds in the tumor after intravenous injection. Thus, for the in vivo efficacy assay we injected the nanomedicine into the tumor. We achieved a 28-fold inhibition of the oncogene. This method can readily be applied to other nanoemitters with $approx$100 ns lifetime.
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