Cationic PAMAM Dendrimers Aggressively Initiate Blood Clot Formation

Abstract

Poly(amidoamine) (PAMAM) dendrimers are increasingly studied as model nanoparticles for a variety of biomedical applications, notably in systemic administrations. However, with respect to blood contacting applications, amine-terminated dendrimers have recently been shown to activate platelets and cause a fatal, disseminated intravascular coagulation (DIC)-like condition in mice and rats. We here demonstrate that, upon addition to blood, cationic G7 PAMAM dendrimers induce fibrinogen aggregation, which may contribute to the in vivo DIC-like phenomenon. We demonstrate that amine-terminated dendrimers act directly on fibrinogen in a thrombin-independent manner to generate dense, high-molecular-weight fibrinogen aggregates with minimal fibrin fibril formation. In addition, we hypothesize this clot-like behavior is likely mediated through electrostatic interactions between the densely charged cationic dendrimer surface and negatively charged fibrinogen domains. Interestingly, cationic dendrimers also induced aggregation of albumin, suggesting that many negatively charged blood proteins may be affected by cationic dendrimers. To investigate this further, zebrafish embryos (ZFE) were employed to more specifically determine the speed of this phenomenon and the pathway- and dose-dependency of the resulting vascular occlusion phenotype. These novel findings show that G7 PAMAM dendrimers significantly and adversely impact many blood components to produce rapid coagulation and strongly suggest that these effects are independent of classic coagulation mechanisms. These results also strongly suggest the need to fully characterize amine-terminated PAMAM dendrimers in regards to their adverse effects on both coagulation and platelets, which may contribute to blood toxicity.

Relation between biophysical properties of nanostructures and their toxicity on zebrafish

“…Finally, Jones et al (2012) demonstrated that G7 PAMAM dendrimers, with amine terminal groups, induce fibrinogen aggregation after injection in zebrafish larvae of 96 hpf, which may contribute to a disseminated intravascular coagulationlike phenomenon. In this way, once again, the results obtained by these authors emphasize the importance of studying nonlethal side effects of positively charged dendrimers.…”

“…To our knowledge, only four publications describe the use of the zebrafish model to analyze the toxicity of diverse dendrimer types (Bodewein et al 2016;Heiden et al 2007;Oliveira et al 2014;Pryor et al 2014) (Table 3). Another two papers have used the zebrafish model to test the toxicity of drug-dendrimer complexes (Prieto et al , 2014; and another one to test the biocompatibility of dendrimers with zebrafish red blood cells (Jones et al 2012). Heiden et al (2007) exposed zebrafish embryos of 6 h post-fertilization (hpf) to G3.5 and G4 PAMAM dendrimers and found that G4 PAMAM dendrimers, with amine terminal groups, were toxic in lethal and sublethal parameters in a dose-and time-dependent way, and that G3.5 PAMAM dendrimers, with carboxylic terminal groups, were not toxic at any of the concentrations tested (no increase in mortality and no sublethal signs of toxicity or malformations).…”

Comparative toxicological assessment of PAMAM and thiophosphoryl dendrimers using embryonic zebrafish

“…9,[20][21][22][23] Recent studies in mice and zebrafish (Danio rerio) have also shown polyamidoamine (PAMAM) dendrimers with amine terminated groups affected blood-clot formation when administered intravenously, while neutral and anionic dendrimers of the same generation demonstrated no observable vascular complications. 24,25 The authors suggest that these effects are driven by the high affinity amine for platelets or vascular endothelium. 24 The observed toxicity with dendrimers however, is likely due to a combination of generation and charge, as the net charge is dependent on the generation.…”

“…24,25 The authors suggest that these effects are driven by the high affinity amine for platelets or vascular endothelium. 24 The observed toxicity with dendrimers however, is likely due to a combination of generation and charge, as the net charge is dependent on the generation. 11,18 In contrast with PAMAM dendrimers, the limited research on thiophosphoryl dendrimers has demonstrated minimal inherent toxicity in vitro.…”

Nanoparticles in relation to peptide and protein aggregation

“…The reduced zeta potential in hydrogenated complexes facilitates the interaction between peptide molecules, resulting in conversion of Aβ40 from random coils to β-sheet structures. 79 Very recently, Jones et al 80 demonstrated that fibrinogen, and other anionic blood proteins, undergo rapid and extensive aggregation by densely charged cationic G7 dendrimers through a thrombinindependent and cellular activation-free mechanism. The acidic nature of these proteins and their high concentration in blood predispose these elements to aggregation with oppositely charged dendrimers.…”