Journal: Journal of Nanobiotechnology
Article Title: Engineering extracellular vesicles to transiently permeabilize the blood–brain barrier
doi: 10.1186/s12951-024-03019-w
Figure Lengend Snippet: Impact of miR-enriched-sEVs in the static human in vitro BBB model. a Schematic representation of the experimental setup. The BBB phenotype was established through 4 days of co-culture in Transwell systems. The modulated sEVs (10 10 sEVs/mL) were incubated for 48 h with the BECs, then the medium was replaced with fresh medium and the ECs monolayer integrity was evaluated through TEER, paracellular permeability and cell viability. b Basal expression of the miRNAs used for the sEVs modulation in the BECs. Values are mean ± SEM (n = 3 independent experiment with 3 technical replicates). c Paracellular permeability (Pe) to lucifer yellow (LY), d TEER values and e BEC viability after 48 h of incubation with miR-enriched sEVs. Cell viability was assessed by a PrestoBlue assay. The values are normalized to the respective controls (miR-scr-sEVs) and expressed as mean ± SEM, n = 2–4 independent experiments with 3 technical replicates (each experiment with a different batch of sEVs, therefore 3 different donors). Unpaired t test between each condition and the BBB model incubated with the miR-scr-sEVs was performed as statistical analysis. *, **, and *** denote statistical significance (p < 0.05, p < 0.01, p < 0.001). f Gene expression analysis of claudin 5, ZO-1, vWF, and occludin in BECs after 48 h incubation with 10 10 miR-383-3p-sEVs/mL. The results are mean ± SEM, n = 4 independent experiments with 3 technical replicates. g Representative confocal images of claudin 5, ZO-1, β-catenin, occludin, and vWF after the incubation with the miR-scr-sEVs and with miR-383-3p-sEVs. Scale bar is 20 μm. h Mean intensity of the immunostaining for claudin 5, ZO-1, β-catenin, occludin, and vWF after incubation with miR-enriched sEVs. The values were normalized by miR-scr-sEVs condition. The data are mean ± SEM, n = 3–6 independent experiments with 2–4 images per experiment. Statistical analysis was performed with an unpaired student’s t -test, *** p < 0.001
Article Snippet: Six hours after, ECs were seeded (80,000 cells/insert) in Transwell ® -Clear Inserts with Polyester (PET) membrane (Corning, 3460) or Transwell ® permeable supports with polycarbonate membrane (Corning, 3401) previously coated with MatrigelTM (Basement Membrane Matrix, 10 mL *LDEV-Free, BD) diluted 1:48 in cold Dulbecco’s Modified Eagle’s Medium (DMEM) for 1 h at room temperature (RT).
Techniques: In Vitro, Co-Culture Assay, Incubation, Permeability, Expressing, Prestoblue Assay, Gene Expression, Immunostaining
