Nanoparticle size and surface charge determine effects of PAMAM dendrimers on human platelets in vitro.

Nanoparticles are finding growing applications in medicine because they may reduce toxicity and improve the solubility, pharmacokinetics and biodistribution profiles1 of traditional pharmaceuticals. In the bloodstream, nanoparticles encounter a very complex environment of plasma proteins and immune cells 2-4. Some nanoparticles intended for drug delivery applications are intentionally engineered so as to reduce their clearance from the bloodstream to extend systemic circulation times and to increase drug delivery to a target site. When blood clearance is fast, nanoparticle interaction with blood components is minimized, however an increase in the circulation time respectively increases the duration of contact with blood components, including those of the coagulation system. Such extended exposure to coagulation factors and thrombocytes may amplify adverse effects such as activation of the blood clotting and occlusion of blood vessels by thrombi. This is why the initial characterization of nanomaterials often includes evaluation of nanoparticle hematocompatibility. Platelets represent cellular components of the blood coagulation system. They are small anucleated cells derived from bone marrow megakaryocytes. Under physiological conditions, 150-450 × 109 platelets per liter circulate in the peripheral blood in 10 days 5. There is also a reservoir of platelets in the spleen which can be rapidly distributed into circulation when needed to maintain hemostasis. Platelets are very sensitive to changes in the blood microenvironment; they can be activated by different physiological agonists such as thrombin, collagen, adenosine-diphosphate (ADP), but also by various microorganisms, immunoglobulins, drugs and some nanomaterials 5. Although several studies have shown that certain nanoparticles, such as, iron-oxide nanoparticles 6, and silver nanoparticles 7, can activate platelets and induce platelet aggregation, no comprehensive structure activity relationship study evaluating the effects of nanoparticle size and surface properties on thrombogenic properties has been conducted. Some trends in particle thrombogenic properties have been described. Interestingly, the reported trends were different for different type of nanoparticles. For example, Koziara et al. have shown anionic cetyl alcohol/polysorbate-based nanoparticles inhibit platelet activation and aggregation, and that this property is decreased when particle surface is modified with polyethylene glycol (PEG), however, the same strongly anionic particles (zeta potential of -40.2mV) neither activated platelets nor induced their aggregation 8. In contrast, Zbiden et al reported that only anionic liposomes activated platelets and induced platelet aggregation 9. In agreement with this, another study reported that platelet activation and aggregation by latex nanoparticles was stronger for most anionic particles and weaker for particles with less negative zeta potentials 10, 11. Both anionic and cationic polystyrene particles activated platelets and induced platelet aggregation, and these effects were stronger with the smaller particles12. These limited and contradictory data do not allow for a clear determination of whether nanoparticle size, surface charge, or composition determines thrombogenic properties. The mechanisms through which nanoparticles induce platelet activation and aggregation are also largely unknown, and the mechanisms may be different for different classes of particles. For example, when various carbon-based particles (fullerene derivatives and nanotubes) and polystyrene nanobeads were studied, only single- walled carbon nanotubes (SWCNT), and multi-walled carbon nanotubes (MWCNT) activated platelets, and this activation could be prevented by inhibitors known to block extracellular calcium influx 13. Another study comparing five different types of carbon-based materials (water soluble fullerene derivative, nanotubes and mixed carbon nanoparticles) reported that all of these particles required activation of glycoprotein integrin receptor GPIIb/IIIa in order to cause platelet aggregation. However, pathways leading to this receptor depended on the size of the particles, with micron-sized particles, but not nanoparticles, requiring protein kinase C (PKC) for the activation of the integrin pathway14. The same study suggested that unlike classical platelet aggregation, carbon-based nanoparticle-induced platelet aggregation did not require thromboxane A2 and ADP release. These data suggest that: 1) nanosized particles may induce platelet aggregation via untraditional pathways, which could render common anti-thrombotic drugs less efficient at reducing aggregation; and 2) platelet activating/aggregating activity and its underlying mechanisms may vary significantly, even within the same class of nanomaterials. One attractive feature of nanotechnology is that nanoparticles can be engineered to either promote platelet aggregation15-17 or inhibit it 18, 19, which can aid in treatment of various blood coagulation disorders. Dendrimers are nanomaterials which have been tested in clinical studies for various applications20. These materials became attractive to drug delivery scientists because they are monodisperse, uniform, and hyperbranched materials with a well-defined and reproducible synthesis and modifiable protein-like structures21. In addition to these properties polyamidoamine (PAMAM) dendrimers are also available through several commercial sources to any laboratory without synthetic capabilities, and can be synthesized and purchased in large quantities. We used 12 formulations of PAMAM dendrimers, varying in size and surface charge, and studied their effects on human platelets in vitro. We verified our findings using several different methods including traditional light transmission aggregometry, and scanning electron microscopy (SEM). The results demonstrated that only large (G4-G6) cationic dendrimers, but not their small (G3) cationic couterparts or anionic or neutral dendrimers, were capable of inducing platelet aggregation. We also showed that platelet aggregation caused by large cationic PAMAM dendrimers is not associated with the release of membrane microparticles, and is not sensitive to a variety of inhibitors known to interrupt different pathways established as triggering platelet activation by physiological agonists. Collectively with published studies reporting dendrimer interaction with and disruption of supported lipid bilayers22-26, and computer simulation 27, our data suggest that large cationic PAMAM dendrimers induce platelet aggregation by disturbing the integrity of cell membranes.