Effect of engineered TiO2 and ZnO nanoparticles on erythrocytes, platelet-rich plasma and giant unilamelar phospholipid vesicles

Abstract

Background: Massive industrial production of engineered nanoparticles poses questions about health risks to living beings. In order to understand the underlying mechanisms, we studied the effects of TiO2 and ZnO agglomerated engineered nanoparticles (EPs) on erythrocytes, platelet-rich plasma and on suspensions of giant unilamelar phospholipid vesicles.

Results: Washed erythrocytes, platelet-rich plasma and suspensions of giant unilamelar phospholipid vesicles were incubated with samples of EPs. These samples were observed by different microscopic techniques. We found that TiO2 and ZnO EPs adhered to the membrane of washed human and canine erythrocytes. TiO2 and ZnO EPs induced coalescence of human erythrocytes. Addition of TiO2 and ZnO EPs to platelet-rich plasma caused activation of human platelets after 24 hours and 3 hours, respectively, while in canine erythrocytes, activation of platelets due to ZnO EPs occurred already after 1 hour. To assess the effect of EPs on a representative sample of giant unilamelar phospholipid vesicles, analysis of the recorded populations was improved by applying the principles of statistical physics. TiO2 EPs did not induce any notable effect on giant unilamelar phospholipid vesicles within 50 minutes of incubation, while ZnO EPs induced a decrease in the number of giant unilamelar phospholipid vesicles that was statistically significant (p < 0,001) already after 20 minutes of incubation.

Conclusions: These results indicate that TiO2 and ZnO EPs cause erythrocyte aggregation and could be potentially prothrombogenic, while ZnO could also cause membrane rupture.

Figure 1

TiO₂and ZnO agglomerated engineered nanoparticles (dissolved in 0.3M glucose solution) imaged by transmission electron microscopy (A,C) and by scanning electron microscopy (B,D). Concentration of nanoparticles was 10 μg/ml.

Figure 2

Relative number of agglomerated engineered nanoparticles distributed over size. The height of the column represents the number of agglomerated engineered nanoparticles pertaining to a given size interval, divided by the number of all agglomerated engineered nanoparticles measured. Left: the relative number of glucose-suspended TiO₂ agglomerated engineered nanoparticles, right: the relative number of glucose-suspended ZnO agglomerated engineered nanoparticles, as measured by the dynamic light scattering method.

Figure 3

Effect of PBS-citrate (control) (A, B), TiO₂ (C, D) and ZnO (E, F) agglomerated engineered nanoparticles on populations of washed human erythrocytes as observed by phase contrast optical microscopy. Agglomerated engineered nanoparticles were suspended in phosphate buffer saline.

Figure 4

Effect of PBS-citrate (control) (A, B), TiO₂ (C, D) and ZnO (E, F) agglomerated engineered nanoparticles on washed human erythrocytes as observed by scanning electron microscopy. Agglomerated engineered nanoparticles were suspended in phosphate buffer saline. Arrows point to very large agglomerates of nanoparticles.

Figure 5

Effect of PBS-citrate (control) (A, B), TiO₂ (C, D) and ZnO (E, F) agglomerated engineered nanoparticles on populations of washed human erythrocytes as observed by scanning electron microscopy. Agglomerated engineered nanoparticles were suspended in phosphate buffer saline.

Figure 6

Effect of PBS-citrate (control) (A-C), TiO₂ (D-F) and ZnO (G-I) agglomerated engineered nanoparticles on human platelet rich plasma as observed by scanning electron microscopy. Samples were incubated with agglomerated engineered nanoparticles dissolved in phosphate buffer saline for 1 hour (A,D, G), 3 hours (B, E, H) and 24 hours (C, F, I).

Figure 7

Effect of PBS-citrate (control), TiO₂ and ZnO agglomerated engineered nanoparticles on washed canine erythrocytes. Effect of PBS-citrate (control) (A), TiO₂ (B) and ZnO (C) agglomerated engineered nanoparticles on populations of washed canine erythrocytes. An erythrocyte with pores (pores are marked by black arrows) in an untreated sample (D). Binding of agglomerates to washed canine erythrocytes: large TiO₂ agglomerate (marked by a black arrow) (E) and numerous ZnO aggregates on the surface of echinocyte (marked by a white arrow) (F). Agglomerated engineered nanoparticles were suspended in phosphate buffer saline.

Figure 8

Effect of PBS-citrate (control) (A), TiO₂ (B) and ZnO (C) agglomerated engineered nanoparticles on canine platelet-rich plasma. Platelet-rich plasma was incubated with agglomerated engineered nanoparticles dissolved in phosphate buffer saline for 1 hour. Activated platelets (white arrow) and larger number of erythrocytes (gray arrow) including echinocytes can be seen in the sample incubated with ZnO (C).