Intracranially injectable multi-siRNA nanomedicine for the inhibition of glioma stem cells

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.

Keywords: cancer stem cells; gene silencing; nanoparticle; neurosphere; self-assembly.

Figure 1.

Physicochemical characterization of n-siRNA. (A) Schematic representation of n-siRNA formed by the self-assembly of siRNA against FAK, NOTCH-1, and SOX-2. (B) DLS showing the size distribution of nanoparticle (average 250nm) at N/P ratio 20. (C) Representative TEM image of n-siRNA. (D) Intracranially injectable n-siRNA-loaded nanoparticle gel (NPG) prepared using hyaluronic acid. (E) SEM image of lyophilized NPG showing macroporous structure. (F) Agarose gel electrophoresis showing complexation of siRNA: lane 1–RNA ladder, lane 2-naked siRNA, lane 3-n-siRNA (N/P-10), lane 4-n-siRNA (N/P-20). (G) siRNA loading efficiency in nanoparticle prepared at N/P:10 and 20. Data are mean ± standard deviation.

Figure 2.

n-siRNA uptake and functional activity in rat C6 glioma cells. (A) Representative confocal images showing n-Cy5-siRNA uptake in (i) C6 cells (red) (ii) corresponding merged image (iii) merged confocal image in C6 neurospheres. (B) Flow analysis of n-siRNA uptake showing (i) unstained, (ii) naked-siRNA, and (iii) n-siRNA (Alexaflour 555) in C6 neurospheres, indicating uptake in 98.7% cells. (C) Relative gene expression levels of FAK, NOTCH-1, and SOX-2 gene analyzed by quantitative real-time PCR after treatment with n-siRNA_FAK, n-siRNA_NOTCH-1 or siRNA_SOX-2 (100 nM each), *P < .05 compared to corresponding untreated. (D) Wound healing assay showing inhibition of cell migration in n-siRNA_FAK treated C6 glioma cells. Data are mean ± standard deviation.

Figure 3.

Neurosphere inhibition assay in C6 glioma cell line. (A) Neurosphere imaged at different time intervals Day (1–14): (i) untreated (ii) n-siRNA_scrambled (iii) TMZ (iv) n-siRNA_FAK (v) n-siRNA_NOTCH-1 (vi) n-siRNA_SOX-2 (vii) n-siRNA_{FAK+NOTCH-1+SOX-2} (FNS) (viii) TMZ treated for 72 h followed by n-siRNA_FNS. (B) Relative percentage spheroid area on day-14 post-treatment, where area of untreated spheres was considered as 100% (data are mean ± SEM) (C) C6 neurosphere viability on day-4, 8, and 14 post-treatment with individual n-siRNA or the final combination of n-siRNA_FNS without TMZ. *P < .05, data are mean ± standard deviation.

Figure 4.

Inhibition of neurospheres in patient-derived glioma stem-like cells. (A) Representative micrographs of neurosphere formation on day-14 post (i) n-siRNA_scrambled (ii) n-siRNA_FAK (iii) n-siRNA_NOTCH-1 (iv) n-siRNA_SOX-2 (v) TMZ (vi) n-siRNA_FNS, and (vii) TMZ+n-siRNA_FNS. (B) Corresponding graphical representation of relative percentage neurosphere area, where the area of n-siRNA_scrambled-treated group is assigned as 100%. Data are mean of 3 sample per patient ± standard deviation, *P < .001 against n-siRNA_scrambled.

Figure 5.

In vivo antitumour effect of intracranial n-siRNA treatment: (A) Representative T2 weighted MR images of Day 3–60 for the untreated, n-siRNAscrambled and n-siRNA_FNS based NPG (3µg) injected into orthotopic rat glioma (C6). Tumour growth can be seen in untreated and n-siRNA_scrambled groups by Day-10 leading to mortality around Day-20, in contrast, n-siRNAFNS treated group (A1-A6) showed no significant tumour growth till 120 days (B) Graph showing quantitative tumour volume in orthotopic glioma in different treatment groups, estimated from MRI (C) H&E of brain sections showing tumour growth in (i) untreated (ii) n-siRNA_scrambled (iii) n-siRNAFNS groups.*P < .001, data are mean ± SEM.