Fig. 4: G58 peptide promotes EV-mediated siRNA delivery to the brain.

a Confocal microscopy image of Neuro-2a cells after 4 h of incubation with siRNA-loaded G58T EVs. Nuclei of cells were stained with Hoechst 3342. EV surface proteins were labeled with Alexa fluor-633 (red) and siRNA was labeled with Cy3 dye (green). Inset is the magnified image (scale bar: 2 µm) of the marked region showing co-localization of EVs with siRNA (yellow spots). Scale bar:10 µm. The experiment was independently repeated four times. b Silencing of GAPDH gene in N2a cells by EVs engineered with G58T, G58T(tat)2 and G58TF proteins, respectively. Thirty nanomolar of GAPDH siRNA was loaded on to EVs, which were added to cells. Top histogram represents GAPDH mRNA level determined after 48 h of treatment, using probe-based RT-qPCR. Data were normalized with 18 S rRNA. Mock: G58TF-bound EVs alone, Neg. Control: G58TF EVs + non-targeting siRNA. Results shown as mean ± s.d (n = 3 independent biological experiment). Statistical differences were determined by one-way ANOVA, using Dunnett’s multiple comparisons test. During the analysis, saline group was used as control to determined statistical significance. **p = 0.0061, ***p = 0.0001. Bottom western blot shows GAPDH protein level in N2a cells after 72 h of treatment. The experiment was independently repeated three times. c Western blot showing effect of chloroquine (CQ) on silencing of GAPDH protein by G58T and G58TF EVs in N2a cells. (Mock: G58TF EVs alone, Neg. Control: G58TF EVs + non-targeting siRNA, RNAiMax: siRNA with lipofectamine RNAiMAX reagent. Thirty micromolar chloroquine was added to cells along with the EVs. The experiment was independently repeated two times. d In vivo fluorescence images of C57BL/6 mouse brains showing biodistribution of RVG-EVs, G58TF-RVG-EVs and G58T/siRNA-RVG-EVs. Images were taken after 4 h of systemic administration of EVs. Surface proteins of RVG-EVs were labeled with cy5.5-NHS fluorescent dye. Mice injected with saline were used as a negative control. Histogram at the right side shows quantification of the fluorescent signal from the brains of treated mice. (n = 3 mice). Results shown as mean ± s.d. Statistical differences were determined by one-way ANOVA, using Dunnett’s multiple comparisons test. **p = 0.0011, ***p = 0.0001. Animal group treated with saline was used as control to determine the p-value. e In vivo silencing of Htt gene in Q140 HD mouse model after systemic administration of G58TF EVs. Animals received four injections of EVs over four weeks. Seventy two hours after the last dose, animals were sacrificed and sections of brain were analyzed for Htt mRNA level, using probe-based RT-qPCR. Data were normalized with 18 S rRNA (n = 6 mice). Results are shown as mean ± s.d. Statistical differences were determined by one-way ANOVA, using Dunnett’s multiple comparisons test. ns non-significant, **P = 0.0012 when compared to control group (saline). f Immunohistochemistry of cortical regions from the treated animals. Images show mutant HTT protein level and p62-labeled inclusion bodies in the neurons of the cortex of Htt siRNA and Neg. siRNA-treated animals (Scale bar indicated 15 µm). g Histogram representing quantification of p62 inclusion bodies. Results are mean ± s.d, n = 8 slides chosen randomly from each group. Statistical differences were determined using one-way ANOVA (two tails) and post-hoc adjustment using Dunnett’s test. **P = 0.0031 when compared to saline group. Source data are provided as a Source Data file.