Mechanism of anti-angiogenic property of gold nanoparticles: role of nanoparticle size and surface charge

Abstract

Discovering therapeutic inorganic nanoparticles (NPs) is evolving as an important area of research in the emerging field of nanomedicine. Recently, we reported the anti-angiogenic property of gold nanoparticles (GNPs): It inhibits the function of pro-angiogenic heparin-binding growth factors (HB-GFs), such as vascular endothelial growth factor 165 (VEGF165) and basic fibroblast growth factor (bFGF), etc. However, the mechanism through which GNPs imparts such an effect remains to be investigated. Using GNPs of different sizes and surface charges, we demonstrate here that a naked GNP surface is required and core size plays an important role to inhibit the function of HB-GFs and subsequent intracellular signaling events. We also demonstrate that the inhibitory effect of GNPs is due to the change in HB-GFs conformation/configuration (denaturation) by the NPs, whereas the conformations of non-HB-GFs remain unaffected. We believe that this significant study will help structure-based design of therapeutic NPs to inhibit the functions of disease-causing proteins.

Figure 1. Effect of gold nanoparticle core size on cell proliferation in HUVECs

[³H] Thymidine incorporation is represented as fold stimulation. (A) Serum starved HU-VECs were stimulated with 10 ng/ml VEGF165 that was preincubated with and without gold nanoparticles (conc = 1nmol/L) (B-D) The effect of dose on HUVEC proliferation with 5nm (B), 10nm (C), and 20nm GNPs (D). The analysis for each nanoparticle were done in triplicate and each C+V = cells stimulated with VEGF165 only. * = P<0.01, ** = P<0.005 as determined by a two-tailed student t-test. Error bars, mean ± SD.

Figure 2. Effect of gold nanoparticle core size on cell proliferation in NIH3T3

[³H] Thymidine incorporation is represented as fold stimulation. Serum starved NIH3T3 were stimulated with 10 ng/ml bFGF (A) or EGF (B) that was preincubated with and without gold nanoparticles (conc = 1 nmol/L). The analysis for each nanoparticle were done in triplicate and each experiment was repeated independently three times. c-b, c-e = only cells, c+b, c+e = cells stimulated with GF. * = P<0.01, ** = P<0.005 as determined by a two-tailed student t-test. Error bars, mean ± SD.

Figure 3. GNPs effect the phosphorylation of VEGF165 receptor

(A) Serum starved HUVECs were stimulated for 5 minutes with VEGF165 (10ng/mL) that was preincubated with or without gold nanoparticles and then immunoblotted with antibodies to phophotyrosine KDR (pKDR) and total KDR levels in the cell extracts. (B) Densitome-tric scanning of phosphotyrosine blots using NIH Image, expressed in percentage. Experiments were performed in triplicate.

Figure 4. Response of calcum signaling in HUVECs in the presence of GNPs

Serum-starved HUVECs were suspended in Ca²⁺ buffer containing Fura-2AM dye and were stimulated with VEGF165 (10ng/mL) that was preincubated with or without gold nanoparticles. The top panel (A) shows the effect of surface size of GNPs on VEGF signaling cascade. (B) Schematic of the GNPs with different surface charges. In panel (C), VEGF165 was preincubated with charge modified GNPs. Experiments were performed in triplicates.

Figure 5. Affinity of VEGF165 binding is dependent on the surface size of GNP

Gold nanoparticles (5nm, 10nm, and 20nm) were incubated with VEGF165. (A) An ELISA was used to quantify the amount of VEGF bound to the nanoparticle surface. Three independently performed experiments showed similar outcomes. Error bars, mean ± SD. (B, C) Far UV-CD spectra was measured from 180 to 250 nm in a 1cm cuvette (B) 0.2mg/mL bFGF in were incubated with and without GNPs in 5mM phosphate buffer (C) 0.15 mg/mL EGF were incubated and without GNPs under similar conditions as listed above. The blanks containing GNPs with same concentration in buffer were subtracted from each data set