Fig. 3
Characterization and morphology analysis of poly(D,L-lactic-co-glycolic acid) nanoparticles with p16ink4a siRNA or scrambled siRNA. A Western blot and quantification of the protein levels of p16ink4a after siRNA transfection in BV2 cells. **p < 0.01, versus scrambled siRNA control; unpaired Student’s t test. ACTB: β-actin. B mRNA expression of p16ink4a was quantified by qPCR after transfection in BV2 cells. The data are expressed as the mean ± SEM. ****p < 0.001, versus scrambled siRNA control, unpaired Student’s t test. C,D The size and zeta potential of NPs containing scrambled siRNA (Si) (C) and p16ink4a siRNA (D). E Scanning electron microscope images of NPs. Scale bar: 300 nm. Original magnification: 15,000 × . F MTT cytotoxicity assay of BV2 cells incubated with p16ink4a siRNA encapsulated PLGA NPs (0–200 µg/mL) for 24 h. Data are expressed as mean ± SEM (n = 4) and were analyzed by 1-way ANOVA followed by the Tukey test for multiple comparisons. n.s.: not significant. G The encapsulation efficiency of siRNA NPs as measured by the release of siRNA over 5 days by Nanodrop spectrophotometer. We achieved encapsulation of 31.8% ± 0.1% (mean ± SEM) of the total siRNA into PLGA NPs. H BV2 cells immunostained with anti-Iba1 antibody and 4′,6-diamidino-2-phenylindole (DAPI) after treatment with rhodamine-tagged PLGA NPs for 3 h. Representative images were constructed using Imaris software. Scale bar: 10 µm. I Immunostaining of brain tissues 3 days after injection of AAV-GFP plasmid-loaded PLGA NPs. Tissues were stained with antibodies to Iba1 (microglia marker), GFAP (astrocyte marker), and NeuN (neuronal marker) to visualize the distribution of GFP in the cortex. Representative images were constructed with Imaris software, and GFP fluorescence merged into each cell-type marker was expressed by image sorting. Scale bar: 20 µm. J The volume of merged GFP for each cell-type marker was graphed as a percentage of the total GFP volume. Data are expressed as the mean ± SEM (n = 10 for each group) and were analyzed by 1-way ANOVA followed by the Tukey test for multiple comparisons. ****p < 0.001, Iba1 versus GFAP, and Iba1 versus NeuN