Study of RNA interference mediated by lipid-coated calcium phosphate nanoparticle transfection in high-grade gliomas

Gliomas are the most prevalent brain tumor types in adults. The recent discoveries in gene regulation involved in gliomas initiation and development allowed for the generation of a new therapeutic approach using a... [ view full abstract ]

Gliomas are the most prevalent brain tumor types in adults. The recent discoveries in gene regulation involved in gliomas initiation and development allowed for the generation of a new therapeutic approach using a sequence-specific gene silencing to alter gene activities.1,2
Calcium phosphate nanoparticles (CaP NPs) have been shown to be an attractive tool for non-viral gene delivery thanks to their biocompatibility and biodegradability. Indeed, calcium ions form complexes with the nucleic acid backbone, thus stabilizing RNA and DNA structures and, therefore, avoiding their fast degradation by nucleases.3,4
As a model platform useful for the investigation of gene silencing in gliomas, we synthesized CaP NPs for siRNAs delivery in GFP-expressing T794 oligodendroglioma cells isolated from transgenic new born mice induced with high grade brain tumor. This ex vivo model allowed us to study the efficiency of anti-GFP siRNA delivery following GFP fluorescence relating to the relevant gene expression. In order to enhance the circulation half-life in future in vivo experiments and to allow the functionalization of CaP nanoparticles, the core was coated with a cationic asymmetric lipidic bilayer (LCP NPs). LCP NPs were characterized by transmission electron microscopy (14.3±1.8 nm), dynamic light scattering and zeta-potential, showing a mean hydrodynamic diameter of 33.2±3.8 nm and a mean zeta-potential of +31.95±1.3 mV.
Interestingly, both control (empty LCP NPs) and siRNA-loaded LCP NPs did not exert a cytotoxic effect on T794 cells in 0.2-7 nM concentration. Flow cytometry analysis showed huge internalization of LCP NPs (2 nM) by cells up to 24 h of incubation. Treatment with siRNA-loaded LCP NPs confirmed their efficiency by decrease of the mean fluorescence intensity of GFP signal. On the basis of these promising results, we can translate this non-viral gene delivery system to alter other oncogenes supposed to play a pivotal role in gliomas initiation and development, including Sox2.