Fig 1: Purity and specificity of the NBs and NB–conjugates. (a) Purified NBs, NB–Alexa647, and NB–PS conjugates separated by SDS-PAGE. Free dye/PS is observed at the gel front (arrow): (1) purified NBs after PageBlue staining (depicted in black); and (2,3) the fluorescence of Alexa647 and PS detected at 700 nm, respectively (depicted in red). (b) Binding of the unlabeled NBs to the mVEGFR2 protein detected by anti-VHH antibody. (c) Binding of the NB–PS conjugates to the mVEGFR2, hVEGFR2, and mVEGFR1 proteins. Total fluorescence of NB–PS bound to the protein was detected using an Odyssey infrared scanner at 700 nm. (d) Total fluorescence intensity of cell bound/internalized NB–PS conjugates on the murine cell lines after 1-h incubation at 37 °C. (e,f) Fluorescence of NB–Alexa647 conjugates detected by flow cytometry. The murine cell lines were incubated with the conjugates for 1 h at 37 °C followed by trypsinization and FACS analysis. Mean fluorescent intensity (MFI) obtained from flow cytometry. Data are shown as mean ± SD. (g) In vitro competition experiments of the NB–PS conjugates tested on bEnd.3 cells in the presence or absence of ten-times excess of anti-VEGFR2 antibody or unconjugated NBs. Total fluorescence intensity of the associated NB–PS conjugates was detected by an Odyssey infrared scanner at 700 nm (* p < 0.05; ** p < 0.01; *** p < 0.001; t-test).
Fig 2: In vitro nanobody-targeted PDT in co-culture of endothelial and cancer cells. (a) The percentage of cell viability 24 h after illumination relative to the non-treated cells. Murine endothelial cells (MS1) and human cancer cells (OSC) were co-seeded with 1:3 ratio and incubated with different concentrations of VM–PS conjugates targeting VEGFR2 on endothelial cells, and 7D12–PS targeting EGFR on cancer cells for 1 h at 37 °C, then illuminated with 7 mW/cm2 fluence rate and a total light dose of 20 J/cm2 using a 690-nm diode laser through a 600-µm optic fiber. (b) MS1/OSC co-culture treated with VM2–PS (targeting VEGFR2 on MS1) or 7D12–PS (targeting EGFR on OSC cells) or the mixture of both. Dead cells were stained with propidium iodide 24 h after illumination. MS1 and OSC cells shown with red and black arrows, respectively. Scale bar: 30 µm.
Fig 3: Anti-VEGFR2 NBs blocked VEGF-induced proliferation and did not act as receptor agonists. (a) VEGF-A (50 nM) or NBs (1 µM) were added to the serum-starved MS1 cells and incubated for 15 min. VEGFR2 phosphorylation was measured in the total cell lysates by Western blotting: (top) staining of phosphorylated tyrosine 1175 of VEGFR2; (middle) staining of total VEGFR2; and (bottom) staining of actin. (b) Fold changes of P-VEGFR2 in MS1 cells treated with VEGF or nanobodies relative to the non-treated cells (NT). (c) MS1 cells treated with VEGF-A/NBs or both for 72 h followed by viability assay using AlamarBlue® reagent. Data are presented as percent changes in cell proliferation relative to the non-treated cells (mean ± SD).
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