Article Figures & Data
- Figure 1
Characterisation of PLGA–PAMAM@CircOGDH siRNA NPs. (A) Schematic illustrations of the assembly of PLGA–PAMAM@CircOGDH siRNA NPs. (B) Zeta potential of PLGA, PAMAM, PLGA–PAMAM and PLGA–PAMAM@CircOGDH siRNA NPs. Data are presented as means±SD; n=3 (Mann-Whitney U test). (C) Size distribution of PLGA–PAMAM and PLGA–PAMAM@CircOGDH siRNA NPs. Data are presented as means±SD; n=3 (Mann-Whitney U test). (D) TEM images of PLGA–PAMAM and PLGA–PAMAM@CircOGDH siRNA NPs. Scale bar=200 nm. (E) Electrophoretic mobility of siRNA and PLGA–PAMAM@CircOGDH siRNA NPs on agarose gel. Marker: 1000 bp DNA ladder. Extension time of PLGA–PAMAM@Cy3-CircOGDH siRNA NPs at 0 hour, 24 hours, 48 hours and 72 hours on the UV−Vis (F) and fluorescence spectra (G). CircOGDH, circular RNAs derived from oxoglutarate dehydrogenase; NPs, nanoparticles; PLGA, poly(lactide-co-glycolide); siRNA, small interfering RNA; UV−Vis, ultraviolet–visible; PAMAM, poly amidoamine); TEM, transmission Electron Microscope .
- Figure 2
Cellular uptake of PLGA–PAMAM@CircOGDH siRNA NPs. (A) Representative fluorescent microscope images showing the transfection efficiency of PLGA–PAMAM@Cy3-CircOGDH siRNA NPs (red) in primary cortex neurons at 24 hours. Neurons were stained with Nissl (green), and nuclei were stained with DAPI (blue). Scale bar=50 µm. (B) Cell viability was determined in primary cortical neurons treated with NC, CircOGDH siRNA and PLGA–PAMAM@CircOGDH siRNA NPs. Data were presented as mean ± SD; n=3, Mann-Whitney U test. (C–E) Western blot analysis of BAX, BCL2 and cleaved caspase 3 expression levels in CON and ischaemic-reperfusion (OGD/R) neurons treated with PLGA–PAMAM and PLGA–PAMAM@CircOGDH siRNA NPs. Data were presented as mean±SD; n=3, Mann-Whitney U test. CircOGDH, circular RNAs derived from circular oxoglutarate dehydrogenase; CON, control; NC, normal control; NPs, nanoparticles; OGD/R, oxygen and glucose deprivation reperfusion; PLGA, poly(lactide-co-glycolide); siRNA, small interfering RNA; PAMAM, poly amidoamine; DAPI, 4’,6-Diamidino-2’-phenylindole.
- Figure 3
PLGA–PAMAM@CircOGDH siRNA NPs downregulated CircOGDH expression level in MCAO/R mice. (A) Illustration of the in vivo experimental design. (B) Representative images showing the CBF of mice in the SHAM, MCAO and MCAO/R groups. Units of the colour scale: PUs. (C) and (D) Representative fluorescence imaging showing the cellular uptake of PLGA–PAMAM@Cy3-CircOGDH siRNA NPs (red) in the penumbra tissues of MCAO/R mice 3 days after tail injection. Nuclei were stained with DAPI (blue). Scale bar=50 µm (left), scale bar=10 µm (right). (E) In vivo fluorescence imaging of Cy5.5-labelled PLGA–PAMAM NPs in MCAO mice at 0.5 hour 1.5 hours and 2.5 hours. Units of the colour scale: PUs. (F) RT-qPCR analysis of the CircOGDH expression level in the penumbra tissues from SHAM and MCAO/R mice after tail injection of PLGA–PAMAM and PLGA–PAMAM@CircOGDH siRNA NPs on day 1. Data are presented as means±SD; n=3, Mann-Whitney U test. CBF, Cerebral blood flow; CircOGDH, circular RNAs derived from circular oxoglutarate dehydrogenase; MCAO/R, middle cerebral artery occlusion/reperfusion; NPs, nanoparticles; PLGA, poly(lactide-co-glycolide); PUs, perfusion units; RT-qPCR, quantitative reverse transcription PCR; siRNA, small interfering RNA; PAMAM, poly amidoamine ; DAPI, 4’,6-Diamidino-2’-phenylindole .
- Figure 4
PLGA–PAMAM@CircOGDH siRNA NPs alleviated neuronal apoptosis in MCAO/R mice. PLGA–PAMAM@CircOGDH siRNA NPs improved behavioural recovery in MCAO/R mice 3 days after tail injection, as demonstrated by the adhesive removal test (A), grid-walking test (B) and cylinder test (C). Data are presented as means±SD adhesive removal test: n=4 in each group (Mann-Whitney U test); grid-walking test: n=8 in the SHAM group and the MCAO/R+PLGA–PAMAM@CircOGDH siRNA group, n=6 for MCAO/R+PLGA–PAMAM group (Mann-Whitney U test); cylinder test: n=7 in the SHAM group and the MCAO/R+PLGA–PAMAM@CircOGDH siRNA group, n=6 for the MCAO/R+PLGA–PAMAM group (one-way ANOVA followed by Tamhane T2 test). (D) and (E) Nissl staining showing the number of neurons in the SHAM, MCAO/R+PLGA–PAMAM and MCAO/R+PLGA–PAMAM@CircOGDH siRNA groups 3 days after tail injection. Scale bar=100 µm. For the quantification of results in (E), data were presented as mean±SD; n=3, Mann-Whitney U test. (F)–(H) TUNEL and Nissl staining of brain sections showing TUNEL-positive and intact neurons in the brains of mice in the MCAO/R+PLGA–PAMAM and MCAO/R+PLGA–PAMAM@CircOGDH siRNA groups 3 days after tail injection. White dashed lines (left) indicate potential infarct regions; yellow dashed lines (right) indicate potential penumbra regions. Scale bar=50 µm. Data were presented as mean±SD; n=3, Mann-Whitney U test. CircOGDH, circular RNAs derived from circular oxoglutarate dehydrogenase; MCAO/R, middle cerebral artery occlusion/reperfusion; NPs, nanoparticles; PLGA, poly(lactide-co-glycolide); siRNA, small interfering RNA; PAMAM, poly amidoamine; ANOVA, analysis of Variance; TUNEL, TdT-mediated dUTP nick end labeling.
- Figure 5
PLGA–PAMAM@CircOGDH siRNA NPs exert no significant toxicity in MCAO/R mice. Haematological analysis of AST (A), ALT (B) and CR (C) were evaluated in the SHAM, MCAO+PLGA–PAMAM and MCAO+PLGA–PAMAM@CircOGDH siRNA groups 3 days after tail injection. Data were presented as mean±SD; n=3, Mann-Whitney U test. (D) H&E staining of the heart, liver, spleen, lungs and kidneys in SHAM, MCAO+PLGA–PAMAM and MCAO+PLGA–PAMAM@CircOGDH siRNA mice 3 days after tail injection. Scale bar=100 µm. ALT, alanine transaminase; AST, aspartate transaminase; CircOGDH, circular RNAs derived from circular oxoglutarate dehydrogenase; CR, creatinine; MCAO/R, middle cerebral artery occlusion/reperfusion; NPs, nanoparticles; PLGA, poly(lactide-co-glycolide); siRNA, small interfering RNA; PAMAM, poly amidoamine.
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