Journal: International Journal of Pharmaceutics: X

Article Title: Plant-derived extracellular vesicles as a natural drug delivery platform for glioblastoma therapy: A dual role in preserving endothelial integrity while modulating the tumor microenvironment

doi: 10.1016/j.ijpx.2025.100349

Figure Lengend Snippet: Construction of a 3D BTB model and assessment of the barrier-crossing ability of Citrus limon L. -derived EVs. (A) Schematic representation of the 3D BTB model construction and EVs permeability assessment. (B) TEER measurements assessing endothelial barrier formation in hCMEC/D3 cells over seven days. (C) FITC-dextran permeability in the absence (−) or presence (+) of hCMEC/D3 cells. (D) Immunofluorescence staining of the tight junction protein ZO-1 (green) in hCMEC/D3 cells. Nuclei are stained with DAPI (blue). (E) Nanoparticle tracking analysis (NTA) of size distribution and concentration of EVs. (F) Representative transmission electron microscopy (TEM) image of EVs (Scale bar = 100 nm). (G) Size and (H) zeta potential distribution of Citrus limon L. -derived EVs. (I) Fluorescence images showing the uptake of Calcein-AM-labeled EVs (green) by U87 glioblastoma cells. Nuclei are stained with DAPI (blue). scale bar = 100 μm. The magnified images depict high-magnification views of the white boxed areas. (J) Fluorescence intensities of endothelial barrier-crossed EVs were measured using a Cytation 3 Cell Imaging Multi-Mode Reader (BioTek, Winooski, VT, USA). Error bars represent the standard deviation (or standard error) of the mean; however, they are shorter than the height of the symbols and therefore not visible in the figure. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The human brain microvascular endothelial cell line hCMEC/D3 (Catalog #305024) was purchased from Cytion Biosciences.

Techniques: Derivative Assay, Permeability, Immunofluorescence, Staining, Concentration Assay, Transmission Assay, Electron Microscopy, Zeta Potential Analyzer, Fluorescence, Labeling, Imaging, Standard Deviation

Journal: International Journal of Pharmaceutics: X

Article Title: Plant-derived extracellular vesicles as a natural drug delivery platform for glioblastoma therapy: A dual role in preserving endothelial integrity while modulating the tumor microenvironment

doi: 10.1016/j.ijpx.2025.100349

Figure Lengend Snippet: Effects of Citrus limon L. -derived EVs on U87 and hCMEC/D3 Cells. (A, D) Representative live/dead staining images of U87 and hCMEC/D3 cells, respectively, following 48-h treatment with EVs (80 μg/mL). Live cells are stained green (Calcein AM), while dead cells are stained red (PI). (B, E) Cell viability of U87 and hCMEC/D3 cells treated with increasing concentrations of EVs (10, 20, 40, and 80 μg/mL). Data are expressed as the percentage of viable cells relative to the control (mean ± SEM, n = 4). (C, F) Intracellular ROS levels in U87 and hCMEC/D3 cells were normalized to the number of viable cells. (G) Wound healing assay showing hCMEC/D3 cell migration at 0-, 4-, and 24-h post-scratch, (+) with or (−) without EVs. (H) Quantification of migrating cells and hCMEC/D3 cells after scratch wounding. Statistical significance was determined using one-way or two-way ANOVA with Tukey's post hoc test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The human brain microvascular endothelial cell line hCMEC/D3 (Catalog #305024) was purchased from Cytion Biosciences.

Techniques: Derivative Assay, Staining, Control, Wound Healing Assay, Migration

Journal: International Journal of Pharmaceutics: X

Article Title: Plant-derived extracellular vesicles as a natural drug delivery platform for glioblastoma therapy: A dual role in preserving endothelial integrity while modulating the tumor microenvironment

doi: 10.1016/j.ijpx.2025.100349

Figure Lengend Snippet: Evaluation of EVs@TMZ uptake, cytotoxicity, glioblastoma spheroid progression, and VEGF-A secretion in a 3D BTB model. (A) Schematic illustration of EVs@TMZ uptake by U87 in a 3D BTB co-culture model with hCMEC/D3 cells. (B) Confocal microscopy images of 3D U87 cells incubated with EVs@TMZ at 37 °C for 24 h. EVs@TMZ were labeled with calcein-AM (shown in green), actin filaments were labeled with rhodamine phalloidin, and nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (scale bar = 10 μm). The magnified images depict high-magnification views of the white boxed areas. (C) 3D Z -stack image of spatial distribution and uptake of EVs@TMZ in U87 cells. (D) Progressive BTB crossing of EVs@TMZ over 24 h. (E) Cytotoxic effects and (F) ROS levels in hCMEC/D3 cells after 48 h of untreated or treatment with TMZ and EVs@TMZ. (G) Representative stitched microscopic images of 3D U87 in the untreated (Ctrl) group at 24 and 48 h, with arrows indicating aggregates and migration. (H) Representative fluorescence images of 3D U87 after 48 h under three conditions: untreated (Ctrl), TMZ-treated, and EVs@TMZ-treated. (I-L) Quantification of 3D U87, including viable cells (I), ROS levels (J), migrated cells (K), and core density (L). (M) VEGF-A levels in U87, U87 co-cultured with hCMEC/D3 (U87 + hCMEC/D3), TMZ-treated, and EVs@TMZ-treated. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The human brain microvascular endothelial cell line hCMEC/D3 (Catalog #305024) was purchased from Cytion Biosciences.

Techniques: Co-Culture Assay, Confocal Microscopy, Incubation, Labeling, Migration, Fluorescence, Cell Culture