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

Justyna Hajtuch,1 Eliza Iwicka,1 Anna Szczoczarz,1 Damian Flis,1 Elżbieta Megiel,2 Piotr Cieciórski,2 Marek Witold Radomski,3 Maria Jose Santos-Martinez,4 Iwona Inkielewicz-Stepniak1 1Department of Pharmaceutical Pathophysiology, Medical University of Gdansk, Gdansk, Poland; 2Faculty of Chemistry, University of Warsaw, Warsaw, Poland; 3Department of Anatomy, Physiology and Pharmacology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; 4School of Pharmacy and Pharmaceutical Sciences and School of Medicine, Trinity College Dublin, Dublin, Dublin 2, IrelandCorrespondence: Iwona Inkielewicz-Stepniak, Tel +48 58 349 1516, Fax +48 58 349 1517, Email iwona.inkielewicz-stepniak@gumed.edu.plPurpose: 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.Graphical Abstract: Keywords: drug delivery, antiplatelet, RGD, aggregation, coagulation system, biocompatibility