Harnessing subcellular-resolved organ distribution of cationic copolymer-functionalized fluorescent nanodiamonds for optimal delivery of active siRNA to a xenografted tumor in mice.

Diamond nanoparticles (nanodiamonds) can transport active drugs in cultured cells as well as in vivo. However, in the latter case, methods allowing the determination of their bioavailability accurately are still lacking. A nanodiamond can be made fluorescent with a perfectly stable emission and a lifetime ten times longer than that 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 tumors, 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 ultra-high-resolution images (120 Mpixels in size) of whole mouse organ sections to be obtained, with subcellular resolution and single-particle sensitivity. As a proof-of-principle experiment, we quantified the biodistribution and aggregation state of new cationic FNDs capable of transporting 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 ≈100 ns lifetime.

Delivery of siRNA to Ewing Sarcoma Tumor Xenografted on Mice, Using Hydrogenated Detonation Nanodiamonds: Treatment Efficacy and Tissue Distribution.

Nanodiamonds of detonation origin are promising delivery agents of anti-cancer therapeutic compounds in a whole organism like mouse, owing to their versatile surface chemistry and ultra-small 5 nm average primary size compatible with natural elimination routes. However, to date, little is known about tissue distribution, elimination pathways and efficacy of nanodiamonds-based therapy in mice. In this report, we studied the capacity of cationic hydrogenated detonation nanodiamonds to carry active small interfering RNA (siRNA) in a mice model of Ewing sarcoma, a bone cancer of young adults due in the vast majority to the EWS-FLI1 junction oncogene. Replacing hydrogen gas by its radioactive analog tritium gas led to the formation of labeled nanodiamonds and allowed us to investigate their distribution throughout mouse organs and their excretion in urine and feces. We also demonstrated that siRNA directed against EWS-FLI1 inhibited this oncogene expression in tumor xenografted on mice. This work is a significant step to establish cationic hydrogenated detonation nanodiamond as an effective agent for in vivo delivery of active siRNA.

Fluorescent Nanodiamond Applications for Cellular Process Sensing and Cell Tracking.

Diamond nanocrystals smaller than 100 nm (nanodiamonds) are now recognized to be highly biocompatible. They can be made fluorescent with perfect photostability by creating nitrogen-vacancy (NV) color centers in the diamond lattice. The resulting fluorescent nanodiamonds (FND) have been used since the late 2000s as fluorescent probes for short- or long-term analysis. FND can be used both at the subcellular scale and the single cell scale. Their limited sub-diffraction size allows them to track intracellular processes with high spatio-temporal resolution and high contrast from the surrounding environment. FND can also track the fate of therapeutic compounds or whole cells in the organs of an organism. This review presents examples of FND applications (1) for intra and intercellular molecular processes sensing, also introducing the different potential biosensing applications based on the optically detectable electron spin resonance of NV- centers; and (2) for tracking, firstly, FND themselves to determine their biodistribution, and secondly, using FND as cell tracking probes for diagnosis or follow-up purposes in oncology and regenerative medicine.

Gymnotic delivery and gene silencing activity of reduction-responsive siRNAs bearing lipophilic disulfide-containing modifications at 2'-position.

Modified oligoribonucleotides used as siRNAs bearing biolabile disulfide-containing groups at some 2'-positions were synthesized following a post-synthesis transformation of solid-supported 2'-O-acetylthiomethyl RNA, previously described. Thus, the reduction-responsive and lipophilic benzyldithiomethyl (BnSSM) modification was introduced at different locations into siRNAs targeting the Ewing sarcoma EWS-Fli1 protein. Thermal stability, serum stability and response to glutathione treatment of modified siRNAs were thoroughly investigated. Among 17 modified siRNAs, significant gene silencing activities were demonstrated for the 8 most stable siRNAs in serum (half-life > 1 h) when using a transfection reagent. Of special interest, two naked 2'-O-BnSSM siRNAs transfection exhibited a remarkable gene silencing activity after 24 h incubation. These inhibitions are consistent with an efficient gymnotic delivery demonstrated by the presence of the corresponding fluorescent siRNAs within cells.