Figure 4.

siRNA-3WJ Nanotherapeutics Specifically and Significantly Knock Down Expression of Target Genes

(A) qPCR assays showing the knockdown of Gfap, Vim, and Lcn2 target mRNA by 5 nM doses of their respective siRNA-3WJ nanostructures. Downregulation measured 48 hr after transfection, under resting conditions. Mock (Lipofectamine-only) or non-targeted negative control 3WJs were used as negative controls. Gapdh was the reference gene. (B) Densitometric quantification of Vim (54 kDa) and GFAP (50 kDa) western blots showing siRNA-3WJ-induced protein knockdown in preventative activation conditions (20 DIV) and a representative image. Quantification is expressed relative to β-actin and normalized to non-transfected control samples. (C) Representative immunofluorescence micrographs depicting the decrease in GFAP and Vim immunoreactivity upon G10- or V10-3WJ treatment, respectively, in preventative activation conditions, compared with non-treated and N03-3WJ-treated controls (GFAP and Vim: green, DAPI-stained nuclei: blue). (D) Quantification and representative image of Lcn2 (23 kDa) western blot under resting, therapeutic, and preventative activation conditions upon L12-3WJ treatment (16 DIV), compared with non-treated controls. Quantification as per (B). (E) Lcn2 immunofluorescence, as per (C), with quantification of intensity density (n = 3 biological replicates, 2 coverslips per replicate, 10 fields of view per coverslip) of L12- and N03-3WJ-treated samples versus non-treated controls (Lcn2: red; DAPI-stained nuclei: blue). Scale bars, 100 μm. (F) Schematic of the LGV1-3WJ, a single 3WJ core functionalized with the siRNA moieties of each of the L12-, G10-, and V10-3WJs. In vitro transcribed RNA strands LGVa, LGVb, and LGVc are annealed together; black circles indicate a wobble base pair. (G) Agarose gel (3%, TBM buffer) illustrating the stepwise assembly and retarded mobility of the extended LGV1-3WJ construct relative to single- and double-stranded intermediates. Ultra-low dsDNA ladder is for reference. (H) qPCR assays showing the knockdown of Gfap, Vim, and Lcn2 target mRNA by a 5 nM dose of the LGV1-3WJ nanostructure, as per the single-siRNA 3WJs in (A). Quantitative results expressed as the mean of n ≥ 3 biological replicates ± SD. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; or ****p ≤ 0.0001, relative to control samples of the same activation condition (two-way ANOVA [qPCR] or one-way ANOVA [western blot] with Dunnett’s multiple comparison test).