Interaction Of Nanogold With VEGF And VEGFR Labeled By Quantum Dots In Near-field Scanning Optical Microscopy

Purpose:Nanogold specially bound VEGF165 with heparin-binding domain, which can highly specific and potent in inhibiting the binding of VEGF165 and VEGF receptor. To visualize the biomolecules interaction at the nanoscale, here we use quantum dots bearing VEGF based on near-field scanning optical microscopy (NSOM) to study the mechanism of nanogold on the inhibitory of VEGF165-VEGFR2 for the first time.Methods:1. To determine the possible inhibitory effect of nanogold on VEGF165-induced endothelial cell proliferation, we examined the viability of endothelial cells by MTT assay, while VEGF121 (which has no heparin binding domain) was set up as control.2. We use atomic force microscope (AFM) to observe the changes of ultrastructure on human umbilical vein endothelial cell surface treated with nanogold or VEGF.3. To characterize the specific binding of QD-VEGF165 with VEGFR2 on endothelial cell surface, the interaction between ligand-receptor was detected by confocal and NSOM.4. HUVECs were serum-starved for 24 hours and treated with VEGF and then with different concentrations of nanogold for five minutes. Phosphorylated PLC-yl protein was detected with Western blot.Results:1. The data clearly showed that nanogold inhibited VEGF165-induced endothelial cell proliferation. As evidenced in control group, nanogold did not inhibit VEGF 121-induced cell proliferation.2. AFM was used to examine the changes of ultrastructure on human umbilical vein endothelial cell surface treated with nanogold or VEGF. After the treatment with VEGF 165, there were many granules or porous channel (about hundreds nanometers in size) on cell surface, the edges of cell extended obviously. In contrast, after the treatment with VEGF165 and nanogold, the number of granules or porous channel on cell surface decreased clearly. There were no significant difference between the group of VEGF121 and VEGF121+nanogold.3. The fluorescence-topographic imaging enables us to observe that the nanogold bound to VEGF165 resulted from inhibiting the binding between VEGF165 and VEGFR2 on the endothelial cell surface.4. When VEGF165 concentration was unchanged (10 μg/L), its inhibitory effect on PLC-yl phosphorylation was obvious as nanogold concentration increased (125,250, and 500 nmol/L). As evidenced in control group, nanogold did not inhibit VEGF121-induced PLC-yl phosphorylation.Conclusions:1. The data clearly showed that nanogold inhibited VEGF165-induced endothelial cell proliferation.2. Nanogold can highly specific and potent in inhibiting the binding of VEGF165 and its receptor. Additionally, we showed that nanogold significantly inhibited the phosphorylation of PLCy, VEGF165-induced cell proliferation in vitro.3. Based on quantum dot labeling, near-field scanning optical microscopy is a powerful tool in study of molecular interactions.