Effect of different silver nanoparticles on haemostasis

Cilj istraživanja: Zbog svoga biocidnoga djelovanja, najviše upotrebljavane nanočestice u medicini su nanočestice srebra (nanoAg). S obzirom na nedostatak spoznaja o njihovim učincima na hemostazu, cilj ovoga istraživanja bio je ispitati učinke nanoAg-a ovisno o njihovoj površinskoj funkcionalizaciji. Nacrt studije: In vitro studija na uzorcima krvi dobivenim od dobrovoljnih zdravih davatelja koji su tretirani s različitim vrstama nanoAg-a. Svaki pokus je uključivao negativnu kontrolu prema kojoj su rezultati normalizirani. Materijal i metode: Sintetizirani su nanoAg-i s različitim stabilizacijskim omotačima: natrijevim bis(2-etilheksil)sulfosukcinatom (AOT), polivinilpirolidonom (PVP), poli-L-lizinom (PLL) i goveđim serumskim albuminom (BSA). Karakterizacija nanoAg-a napravljena je s DLS-om, ELS -om i TEM-om. Na uzorcima krvi zdravih dobrovoljaca ispitani su hemolitički učinci nanoAg-a Drabkinovom metodom, agregacija trombocita sa i bez agonista metodom impedancijske agregometrije, koagulacija plazme PV-om, APTV-om i TV-om, i analizirano je stvaranje trombina u plazmi TGT-om. Učinci nanoAg-a na viskoelastična svojstva pune krvi tijekom zgrušavanja ispitana su tromboelastometrijom. Ulazak nanoAg-a u trombocite vizualiziran je TEM-om i metodom protočne citometrije (PCM) mjerenjem bočnog rasapa svjetla. Citotoksičnost i oksidativni stres ispitani su fluorometrijski. Izražaj aktivacijskih biljega na trombocitima (P-selektin i PAC-1) određen je metodom PCM. Rezultati: Sve vrste nanoAg-a izazivaju hemolizu ovisno o dozi. Najintenzivniju hemolizu izazivaju PLL-nanoAg, vjerojatno zbog njihovoga pozitivnoga naboja koji omogućuje jaču interakciju s membranom eritrocita. PLL-nanoAg pokazuju antikoagulantna svojstva u svim testovima osim u testu agregacije trombocita bez agonista. Ostale nanoAg (PVP-nanoAg, AOT- nanoAg i BSA-nanoAg) pokazuju antikoagulantno svojstvo samo u nekim testovima u plazmi, ali u testovima provedenim u punoj krvi pokazuju prokoagulantno djelovanje. Rezultati ispitivanja interakcija nanoAg-a s trombocitima pokazali su da su sve vrste citotoksične, da ulaze u stanice te da izazivaju blagi oksidativni stres i blagu aktivaciju trombocita tijekom 30 minuta. Najtoksičnije djelovanje je potvrđeno kod PLL-nanoAg vrste. Zaključak: Učinci nanoAg-a na hemostazu, trombocite i eritrocite razlikuju se ovisno o površinskoj funkcionalizaciji. Učinci na hemostazu nisu jednoznačni te zahtijevaju uporabu više različitih metoda, standardizaciju metoda, vrste uzorka i predanalitičkih uvjeta.Objectives: Silver nanoparticles (AgNPs) are among the most used nanomaterials for medical devices due to their biocidal activity, however detailed knowledge on their haemostatic effects is still lacking. This study aimed to evaluate AgNPs effect on haemostasis, in particular considering different types of nanosurface stabilization. Study Design: This is in vitro study, using blood drawn from healthy donors, which was treated with different AgNPs. Every experiment included negative control and results were normalised to negative control. Material and Methods: Differently stabilized AgNPs were prepared using sodium bis(2-ethylhexyl) sulphosuccinate (AOT), polyvinylpyrrolidone (PVP), poly-L-lysine (PLL) and bovine serum albumin (BSA) as coating agents. The careful characterization of each AgNPs was conducted by DLS, ELS and TEM. Venous blood was drawn from healthy volunteers not taking any drugs which might affect the haemostasis and platelets function. The study included haemolysis assay, which was performed according to the modified Drabkin’s method, followed by impedance aggregometry to study the impact of AgNPs on spontaneous or induced platelet aggregation. The effect of AgNP on plasma coagulation activity was studied by measuring the clotting times in tests PT, APTT and TT, as well as the effect of AgNP on plasma potential to produce thrombin using thrombin generation test. Rotational thromboelastometry method was used to study effect of AgNPs on viscoelastic properties of whole blood clotting process. Furthermore, citotoxicity assay and oxidative stress in platelets were evaluated using fluorescent dyes. Additionally, platelet activation markers (P-selectin and PAC-1) were evaluated by flow cytometry (FCM). Uptake of AgNPs in platelets was visualised by transmission electron microscopy and determined using side-scattered light by FCM. Results: All AgNPs were found to cause a dose-dependent haemolysis. The highest extent of haemolysis was observed for PLL-AgNPs, which may be explained by favoured interaction of positively charged AgNPs and erythrocyte’s membrane. PLL-AgNPs are shown to be anticoagulants in all tests, except for platelet aggregation, whereas PVP-AgNPs, AOT- AgNPs and BSA-AgNPs displayed anticoagulant effects only when tested in plasma samples, but procoagulant effects in viscoelastometric whole blood tests. The results, studying interactions of AgNP with platelets, showed uptake of all AgNP in the cells and that they are cytotoxic, causing mild oxidative stress and mild platelet activation. The most toxic effect was confirmed in PLL-AgNP species. Conclusion: The effect of nanoparticles on haemostasis, platelets and erythrocytes is different, depending on nanosurface stabilization. However, the effect on haemostasis is not unambiguous and requires multimethodological approach. Overall, the results demonstrate the need to thoroughly assess the haemostatic effects of AgNPs, using a combination of different techniques, at the same time highlights the need for a standardised approach in sampling and procedure