Injectable nanoparticle siRNA conjugate mediated gene silencing for glioblastoma stem cells and tumor cells

Abstract

Nanoparticle based siRNA conjugate is a promising therapeutic approach as indicated by recent FDA approval. In this thesis, we have investigated the development and in vitro / in vivo characterization of multiple siRNA-loaded self-assembled nanoparticle gel (NPG) that can be injected locally into the brain (tumor resected cavity); for combinatorial silencing of genes associated with glioma stem cells (GSC). GSCs are characterized by deregulated stem-cell signaling, efficient drug-efflux properties, and the ability to scavenge reactive oxygen species. We used a clinically approved cationic peptide to orchestrate the self-assembly of multiple siRNA sequences targeting FAK, NOTCH-1, and SOX-2 into a single nanoparticle (n-siRNAFAK+NOTCH-1+SOX-2), which is further made into a locally injectable-gel using hyaluronic acid. The nanoparticles showed an average size of ~ 250 nm with ~ 95% siRNA loading efficiency and ~ 95.5% cellular uptake in GSCs characterized by CD133, Nestin, SOX-2, and NOTCH-1 expression. Silencing of FAK, NOTCH-1 or SOX-2 with their respective singular n-siRNA sequence resulted in down-regulation of specific targets but caused compensatory up-regulation of others. Upon simultaneous gene-silencing using n-siRNAFAK+NOTCH-1+SOX-2, ~ 90% GSCs underwent cell death and lost their ability to form GBM neurospheres, whereas normal stem cells remained unaffected while retaining neuronal differentiation capacity. GSCs derived from glioblastoma patients (n=4) showed inability to form neurospheres post nanoparticle treatment whereas the clinical drug; temozolomide, had little effect, indicating the clinical significance of NPG-based treatment. GBM patient derived xenograft (PDX) models exhibiting the characteristics of patient samples also showed significant impairment in the tumorigenic potential of nanoparticle-treated GSCs. In effect, this thesis investigation shows that locally injectable n-siRNA NPG targeted to multiple stem-cell signaling can impair the glioma initiation capabilities of GSCs. newline