Polyvinylpyrrolidone-coated gold nanoparticles inhibit endothelial cell viability, proliferation, and ERK1/2 phosphorylation and reduce the magnitude of endothelial-independent dilator responses in isolated aortic vessels

Abstract

Background: Gold nanoparticles (AuNPs) demonstrate clinical potential for drug delivery and imaging diagnostics. As AuNPs aggregate in physiological fluids, polymer-surface modifications are utilized to allow their stabilization and enhance their retention time in blood. However, the impact of AuNPs on blood vessel function remains poorly understood. In the present study, we investigated the effects of AuNPs and their stabilizers on endothelial cell (EC) and vasodilator function.

Materials and methods: Citrate-stabilized AuNPs (12±3 nm) were synthesized and surface-modified using mercapto polyethylene glycol (mPEG) and polyvinylpyrrolidone (PVP) polymers. Their uptake by isolated ECs and whole vessels was visualized using transmission electron microscopy and quantified using inductively coupled plasma mass spectrometry. Their biological effects on EC proliferation, viability, apoptosis, and the ERK1/2-signaling pathway were determined using automated cell counting, flow cytometry, and Western blotting, respectively. Endothelial-dependent and independent vasodilator functions were assessed using isolated murine aortic vessel rings ex vivo.

Results: AuNPs were located in endothelial endosomes within 30 minutes' exposure, while their surface modification delayed this cellular uptake over time. After 24 hours' exposure, all AuNPs (including polymer-modified AuNPs) induced apoptosis and decreased cell viability/proliferation. These inhibitory effects were lost after 48 hours' exposure (except for the PVP-modified AuNPs). Furthermore, all AuNPs decreased acetylcholine (ACh)-induced phosphorylation of ERK1/2, a key signaling protein of cell function. mPEG-modified AuNPs had lower cytostatic effects than PVP-modified AuNPs. Citrate-stabilized AuNPs did not alter endothelial-dependent vasodilation induced by ACh, but attenuated endothelial-independent responses induced by sodium nitroprusside. PVP-modified AuNPs attenuated ACh-induced dilation, whereas mPEG-modified AuNPs did not, though this was dose-related.

Conclusion: We demonstrated that mPEG-modified AuNPs at a therapeutic dosage showed lower cytostatic effects and were less detrimental to vasodilator function than PVP-modified AuNPs, indicating greater potential as agents for diagnostic imaging and therapy.

Keywords: artery; cell culture; gold; nanoparticles; vascular; vasodilation.

Figure 1

Gold nanoparticle (AuNP) synthesis and characterization. Notes: (A) Transmission electron micrography of spherical monodispersed citrate-stabilized AuNPs (12±3 nm); (B, C) Ultraviolet-visible absorbance spectra of AuNP stability after modification with PVP and mPEG in physiological salt solution (PSS) and culture media. Abbreviations: PVP, polyvinylpyrrolidone; mPEG, mercapto polyethylene glycol.

Figure 2

FTIR spectra for stabilizers and unmodified and modified AuNPs. Note: (A) PVP, (B) AuPVP, (C) mPEG, (D) AumPEG, and (E) AuNPs, illustrating characteristic absorption peaks. Abbreviations: FTIR, Fourier-transform infrared spectroscopy; AuNPs, gold nanoparticles; PVP, polyvinylpyrrolidone; mPEG, mercapto polyethylene glycol.

Figure 3

Gold nanoparticle (AuNP) uptake by BAECs in culture. Notes: Transmission electron micrographs showing the time course of AuNP uptake (citrate-stabilized and mPEG- and PVP-modified) by BAECs. AuNPs are identified as spherical structures inside endosomes within the endothelial cells (arrows). Abbreviations: BAECs, bovine aortic endothelial cells; mPEG, mercapto polyethylene glycol; PVP, polyvinylpyrrolidone.

Figure 4

Inhibitory effects of stabilizers and unmodified and modified AuNPs on BAEC proliferation. Notes: **P<0.01; ***P<0.001. Results presented as mean ± SD. Three independent experiments. Abbreviations: AuNPs, gold nanoparticles; BAEC, bovine aortic endothelial cell; mPEG, mercapto polyethylene glycol; PVP, polyvinylpyrrolidone.

Figure 5

The influence of stabilizers and unmodified and modified AuNPs on BAEC viability using flow cytometry. Notes: *P<0.05; **P<0.01. Percentage cell viability of BAECs in the untreated cells and cells treated with stabilizer (PVP), non-modified and modified AuNPs (AuPVP, AumPEG) after 30-minute and 2–24- and 48-hour exposure based on three independent experiments. Results presented as mean ± SD. Abbreviations: AuNPs, gold nanoparticles; BAEC, bovine aortic endothelial cell; PVP, polyvinylpyrrolidone; mPEG, mercapto polyethylene glycol.

Figure 6

Induction of apoptosis and necrosis by stabilizers and unmodified and modified AuNPs in BAECs. Notes: *P<0.05; **P<0.01; ***P<0.001. Apoptosis (A) and necrosis (B) of BAECs in untreated cells and cells treated with stabilizer (PVP) and unmodified and modified AuNPs (AuPVP, AumPEG) after 2- and 24-hour exposure based on three independent experiments. Results presented as mean ± SD. Abbreviations: AuNPs, gold nanoparticles; BAECs, bovine aortic endothelial cells; PVP, polyvinylpyrrolidone; mPEG, mercapto polyethylene glycol.

Figure 7

Modulatory effects of stabilizer and unmodified and modified AuNPs on ACh-induced ERK1/2 phosphorylation in BAECs. Notes: (A) Representative Western blot analysis (A₁) showing the stimulatory effect of 1/10/100 µM ACh on ERK1/2 phosphorylation (p-ERK1/2) in BAECs after 10 minutes’ exposure compared to untreated cells (control); relative expression of p-ERK1/2 calculated as a ratio to ERK1/2 expression, the loading control (A₂). (B) Representative Western blot analysis (B₁) showing the modulatory effects of 10 µM ACh-induced ERK1/2 phosphorylation in the absence or in the presence of stabilizer (PVP) and non-modified and unmodified AuNPs (AuPVP, AumPEG) in BAECs after 10 minutes’ incubation compared to untreated cells (control). The bar graph shows the relative expression of p-ERK1/2 calculated as a ratio to ERK1/2 expression, the loading control (B₂). Abbreviations: AuNPs, gold nanoparticles; ACh, acetylcholine; BAECs, bovine aortic endothelial cells; PVP, polyvinylpyrrolidone; mPEG, mercapto polyethylene glycol; t-ERK, total ERK.

Figure 8

Gold nanoparticle (AuNP) uptake by aortic vessels. Notes: (A) Determination of AuNP uptake using inductively coupled plasma analysis. Concentration of unmodified and polymer-modified AuNPs within aortic vessels after 30 minutes’ exposure. Error bars are SEM. (B) Representative transmission electron micrography illustrating the uptake of PVP-modified AuNPs into endosomal structures (a; b at higher magnification), and also on the surface of endothelial cells lining the aortic vessel (c), within 30 minutes’ exposure. Very few mPEG-modified AuNPs are seen as spherical dense structures within the endothelial cells (d, arrow) when exposed to the higher concentration. Abbreviations: PVP, polyvinylpyrrolidone; mPEG, mercapto polyethylene glycol.

Figure 9

Influence of stabilizers and unmodified and modified AuNPs on dilator responses of aortic vessels. Notes: *P<0.05; **P<0.01; ***P<0.001. Endothelial-dependent (ACh) and independent (SNP) dilator responses of preconstricted aortic vessels after 30 minutes’ exposure ex vivo. Error bars are SEM. Abbreviations: AuNPs, gold nanoparticles; ACh, acetylcholine; SNP, sodium nitroprusside; PVP, polyvinylpyrrolidone; mPEG, mercapto polyethylene glycol.