ABSTRACT
BACKGROUND: Nanoparticle siRNA-conjugates are promising clinical therapeutics as indicated by recent US-FDA approval. In glioma stem cells (GSC), multiple stemness associated genes were found aberrant. We report intracranially injectable, multi-gene-targeted siRNA nanoparticle gel (NPG) for the combinatorial silencing of 3 aberrant genes, thus inhibiting the tumorogenic potential of GSCs. METHODS: NPG loaded with siRNAs targeted against FAK, NOTCH-1, and SOX-2 were prepared by the self-assembly of siRNAs with protamine-hyaluronic acid combination. Electron microscopy, DLS, and agarose gel electrophoresis were used for the physicochemical characterization. Cell transfection and gene-silencing efficiency were studied using human mesenchymal stem cells and rat C6 glioma-derived GSCs. Neurosphere inhibition was tested in vitro using GSCs derived from C6 cell line and glioma patient samples. Patient-derived xenograft model and orthotopic rat glioma model were used to test the effect of NPG on in vivo tumorigenicity. RESULTS: The siRNA nanoparticles with an average size ~ 250 nm and ~ 95% loading efficiency showed cellular uptake in ~95.5% GSCs. Simultaneous gene silencing of FAK, NOTCH-1, and SOX-2 led to the inhibition of neurosphere formation by GSCs, whereas normal stem cells remained unaffected and retained neuronal differentiation capability. GBM PDX models manifested significant impairment in the tumorigenic potential of NPG treated GSCs. Intracranial injection of NPG inhibited tumor growth in orthotopic rat brain tumor model. CONCLUSION: Intracranially injectable n-siRNA NPG targeted to multiple stem-cell signaling impairs glioma initiation capabilities of GSCs and inhibited tumor growth in vivo.
ABSTRACT
Glioma stem cells (GSC) present a critical therapeutic challenge for glioblastoma multiforme (GBM). Drug screening against GSC demands development of novel in vitro and in vivo platforms that can mimic brain microenvironment and support GSC maintenance and tumorigenesis. Here, we report, a 3-dimensionel (3D) biomimetic macro-porous scaffold developed by incorporating hyaluronic acid, porcine brain extra cellular matrix (ECM) and growth factors that facilitates regeneration of GBM from primary GSCs, ex vivo and in vivo. After characterizing with human and rat GBM cell lines and neurospheres, human GSCs expressing Notch1, Sox-2, Nestin, and CD133 biomarkers were isolated from GBM patients, cultured in the 3D scaffold, and implanted subcutaneously in nude mice to develop patient derived xenograft (PDX) models. Aggressive growth pattern of PDX with formation of intratumoral vascularization was monitored by magnetic resonance imaging (MRI). Histopathological and phenotypial features of the original tumors were retained in the PDX models. We used this regenerated GBM platform to screen novel siRNA nanotherapeutics targeting Notch, Sox-2, FAK signaling for its ability to inhibit the tumorigenic potential of GSCs. Current clinical drug, Temozolomide and an anticancer phytochemical, nanocurcumin, were used as controls. The siRNA nanoparticles showed excellent efficacy in inhibiting tumorigenesis by GSCs in vivo. Our study suggests that the brain-ECM mimicking scaffold can regenerate primary gliomas from GSCs in vitro and in vivo, and the same can be used as an effective platform for screening drugs against glioma stem cells.
