Prevention Of Binding Of VEGF165 To VEGFR2 By Gold Nanoparticle: An AFM Study

OBJECTIVE:Research has shown that gold nanoparticles can prevent the binding of VEGF165 to receptor VEGFR2, thereby inhibiting the proliferation of VEGF165-mediated vascular endothelial cell. The molecular mechanism, however, is unclear. Atomic force microscopy(AFM) is a powerful tool which is widely applied in research of Molecular Biology. In this study, AFM were utilized in investigating the molecular interaction between gold nanoparticles, VEGF165 and VEGFR2, and the significance of the underlying mechanism.Methods:1,Before and after incubation with VEGF165 for 12h in 4℃,GNP were screened by the integrated tools including UV-vis absorption spectroscopy, particle size analysis and AFM under near-physiological condition.2,Three different concentrations of GNP (5 nmol/L,2 nmol/L and 0.5 nmol/L)were incubated with 100μl0.02μg/ml VEGF165 for 12h in 4℃respectively, then treated with starved vascular endothelial cells. AFM was used to examine the ultrastructural changes of vascular endothelial cell surface.3,VEGFR2 were immobiled on glassclip by APTES-GA followed by treatment with VEGF165 and GNP-VEGF165 respectively. Before and after the procedure, AFM was applied for screening, changes of the size of molecules and the molecules topogram were documented and analyzed.Results:1,After treated with VEGF165, the GNP absorption peak revealed a slight red shift, and the size distribution of GNP was increased from 20 nm to 30 nm. By AFM imaging, GNP was 22.05±1.52 nm average in diameter while the average diameter of GNP-VEGF165 was 33.91±2.61nm. Bounding of GNP and VEGF165 and the formation of core-shell structure was indicated.2,AFM screening showed changes of ultrastructure on vascular endothelial cells surface. The group of VEGF165 displayed signs of cell proliferation. Granulation of cell surface, increase in cell-to-cell contact, formation of pseudopodia and appearance of membranes pores were all documented. Signs of cell proliferation were negative in GNP-VEGF165 group.3,AFM imaged single molecules of VEGFR2 with an average diameter of 80.10±9.13 run. After incubation with VEGF165, screening of AFM showed protein complexs accompanied with small protrusions around. Measuring of the protein complex revealed with an average long diameter of 246.17±14.84 nm, and an short diameter in average of 150.10±8.84 nm. After incubation with GNP-VEGF165, AFM revealed protein complex with an average long diameter of 182.89±6.81 nm, and an average short diameter of 103.17±3.63 nm. The complex was observed 30 nm particles around.Conclusions:1,GNP binded with VEGF165 through chemical bonding and form "core-shell" complexs in near-physiological conditions.2,vascular endothelial cells proliferation, induced by VEGF165, could be inhibited by GNP.3,GNP could prevent binding of VEGF165 to VEGFR2 and hence preventing the formation of VEGFR2 dimer, resulting in inhibition of signal transduction of VEGF165.4,AFM is a powerful tool in research of Molecular Biology.