The Pharmacological Effects of Silver Nanoparticles Functionalized with Eptifibatide on Platelets and Endothelial Cells

Abstract

Purpose: In the search for new drug delivery platforms for cardiovascular diseases and coating of medical devices, we synthesized eptifibatide-functionalized silver nanoparticles (AgNPs-EPI) and examined the pharmacological activity of AgNPs-EPI on platelets and endothelial cells in vitro and ex vivo.

Methods: Spherical AgNPs linked to eptifibatide were synthesized and characterized. Cytotoxicity was measured in microvascular endothelial cells (HMEC-1), platelets and red blood cells. Platelet mitochondrial respiration was measured using the Oxygraph-2k, a high-resolution modular respirometry system. The effect of AgNPs-EPI on the aggregation of washed platelets was measured by light aggregometry and the ex vivo occlusion time was determined using a reference laboratory method. The surface amount of platelet receptors such as P-selectin and GPIIb/IIIa was measured. The influence of AgNPS-EPI on blood coagulation science was assessed. Finally, the effect of AgNPs-EPI on endothelial cells was measured by the levels of 6-keto-PGF1alpha, tPa, cGMP and vWF.

Results: We describe the synthesis of AgNPs using eptifibatide as the stabilizing ligand. The molecules of this drug are directly bonded to the surface of the nanoparticles. The synthesized AgNPs-EPI did not affect the viability of platelets, endothelial cells and erythrocytes. Preincubation of platelets with AgNPs-EPI protected by mitochondrial oxidative phosphorylation capacity. AgNPs-EPI inhibited aggregation-induced P-selectin expression and GPIIb/IIIa conformational changes in platelets. AgNPs-EPI caused prolongation of the occlusion time in the presence of collagen/ADP and collagen/adrenaline. AgNPs-EPI regulated levels of 6-keto-PGF1alpha, tPa, vWf and cGMP produced in thrombin stimulated HMEC-1 cells.

Conclusion: AgNPs-EPI show anti-aggregatory activity at concentrations lower than those required by the free drug acting via regulation of platelet aggregation, blood coagulation, and endothelial cell activity. Our results provide proof-of-principle evidence that AgNPs may be used as an effective delivery platform for antiplatelet drugs.

Keywords: RGD; aggregation; antiplatelet; biocompatibility; coagulation system; drug delivery.

Graphical abstract

Figure 1

UV-vis spectra recorded during synthesis and purification of AgNPs-EPI. The bands with a maximum at 271 nm correspond to eptifibatide, and the bands with a maximum >400 nm correspond to surface plasmon resonance AgNPs.

Figure 2

A representative TEM image of AgNPs-EPI (A) a histogram of size distribution obtained from the image: (B) and the results of DLS measurements: (C).

Figure 3

FTIR spectra recorded for synthesized conjugate and eptifibatide not bound with nanoparticles (samples prepared as pellets with KBr in a ratio of 1:200 (w/w)).

Figure 4

TGA curves recorded for AgNPs-EPI and eptifibatide (solid lines) and corresponding derivatives with temperature (dotted lines).

Figure 5

Effects of AgNPs-EPI on washed platelets (WP), endothelial cells (HMEC-1) viability following 24 h incubation and % Hb release from RBCs after 12 h incubationData are mean ±SD, n = 3. **p < 0.01.

Figure 6

Inhibition of collagen-induced washed platelet (WP) aggregation by AgNPs-EPI and EPI, (A) – representative light aggregometry tracings showing the anti-aggregator effects of AgNPs-EPI and EPI both at 50 µg/mL. Collagen (black line) was used as a positive control; stirred platelets in the absence of NPs/drug were used as negative control (red line); platelet incubated with AgNPs-EPI with EPI (50µg/mL) and stimulated with collagen (blue line); platelet incubated with EPI (50µg/mL) and stimulated with collagen (green line). The brown arrow indicates the addition of NPs/drug, the green arrow indicates the addition of collagen. (B) – the statistical analysis of AgNPs-EPI and EPI effects on collagen-induced platelet aggregation. Data are mean ± standard deviation. **p < 0.001, ***p < 0.001 versus WP’ (positive control).

Figure 7

Inhibition flow-induced platelet aggregation with collagen/ADP and collagen/epinephrine cartridges by AgNPs-EPI, c – control closure time. Data are mean ±SD. *p < 0.05, **p < 0.005 versus control.

Figure 8

Inhibition of collagen-induced platelet receptor (GPIIb/IIIa and P-selectin) abundance by AgNPs-EPI and EPI. The statistical analysis of the effects of AgNPs-EPI and EPI. Data are expressed as mean ± standard deviation. *p < 0.05, **p < 0.001, ***p < 0.001 versus WP’ (positive control).

Figure 9

Effects of collagen-induced platelet aggregation on leak respiration (A) and OXPHOS coupling efficiency (B) in the presence or absence of AgNPs-EPI and EPI. Data are mean ±SD. *p < 0.05. Hatched bars show the effects of AgNPs-EPI and EPI in the presence of collagen (2 µg/mL).

Figure 10

Inhibition of thrombin-induced release of 6-keto-PGF1alpha, tPA, vWF, and cGMP by AgNPs-EPI but not EPI. Preincubation of HMEC-1 cells with AgNPs-EPI inhibited the release of 6-keto-PGF1α, tPA, vWf, and cGMP. Data are mean ±SD. *p < 0.05, **p < 0.01, ***p < 0.001 to control or as indicated.