In vitro hematological and in vivo vasoactivity assessment of dextran functionalized graphene

Abstract

The intravenous, intramuscular or intraperitoneal administration of water solubilized graphene nanoparticles for biomedical applications will result in their interaction with the hematological components and vasculature. Herein, we have investigated the effects of dextran functionalized graphene nanoplatelets (GNP-Dex) on histamine release, platelet activation, immune activation, blood cell hemolysis in vitro, and vasoactivity in vivo. The results indicate that GNP-Dex formulations prevented histamine release from activated RBL-2H3 rat mast cells, and at concentrations ≥ 7 mg/ml, showed a 12-20% increase in levels of complement proteins. Cytokine (TNF-Alpha and IL-10) levels remained within normal range. GNP-Dex formulations did not cause platelet activation or blood cell hemolysis. Using the hamster cheek pouch in vivo model, the initial vasoactivity of GNP-Dex at concentrations (1-50 mg/ml) equivalent to the first pass of a bolus injection was a brief concentration-dependent dilation in arcade and terminal arterioles. However, they did not induce a pro-inflammatory endothelial dysfunction effect.

Figure 1

Representative (A) Low resolution AFM image of GNP-Dex nanoparticles. (B) A magnified image of a GNP-Dex nanoparticle. Scale Bars (A) 1.25 μm, (B) 60 nm.

Figure 2. Histamine release from activated and induced RBL-2H3cells treated with GNP-Dex (1-10 mg/ml) or dextran only (0.4 mg/ml and 4 mg/ml) formulations for 45 min.

Data are presented as mean ± SD (n = 6 per group). * = p < 0.05 between treatment groups and uninduced control.

Figure 3. Platelet activation assay presented in terms of PF₄ production in whole human blood from two individuals incubated at 1–10 mg/ml GNP-Dex concentrations for 45 min.

Data are presented as mean ± SD (n = 4 per group). * = p < 0.05 between untreated control and treatment group.

Figure 4

(A) Total complement activation assay presented in terms of Sc5b-9 protein production in human whole blood from two individuals treated with various GNP-Dex (1–10 mg/ml) concentrations.(B) Alternate complement pathway activation in terms of Bb protein production in two human whole blood samples treated with various concentrations of dextran only (0.4–4 mg/ml). Data are presented as mean ± SD (n = 4 per group). * = p < 0.05 between untreated control and particular treatment group.

Figure 5

(A) Pro-inflammatory cytokine release assay presented in terms of TNF-Alpha release in whole human whole blood from two individuals treated with various GNP-Dex (1–10 mg/ml) concentrations.(B) Anti-inflammatory cytokine release assay presented in terms of IL-10 release in whole human blood from two individuals treated with various GNP-Dex (1–10 mg/ml) concentrations. Data are presented as mean ± SD (n = 4 per group). * = p < 0.05 between untreated control and particular treatment group.

Figure 6

(A–C) Representative images of blood cells (RBC shown with red arrows and WBC shown with black arrows) treated with 1 mg/ml, 7 mg/ml and 10 mg/ml GNP-Dex, respectively. (D) Representative image of untreated control blood cells. (E) Representative image showing hemolysed cells treated with poly ethylene imine; a known hemolytic agent. (F) Supernatants obtained after centrifuging red blood cell suspensions treated with GNP-Dex formulations, or Triton × 100 for 45 minutes. (G) Absorbance values of the supernatants at 540 nm obtained after conversion of the hemoglobin present to cyanomethemoglobin. Scale bar = 200 μm for images (A–E).

Figure 7

(A) Micrograph from hamster cheek pouch (shown with normal resting tone), depicting the terminal arteriole, and arcade arteriole. The micropipette was placed so that the contents passed over the arterioles of interest and then washed away in the flowing tissue bath as depicted by the dashed arrows. Scale bar = 20 μm. (B) Dose dependent dilation (mean ± sem) to GNP-Dex for arcade and terminal arterioles. The fitted EC50 and maximal dilation are shown in the figure as mean (sem). n = 8. * indicates both arcade and terminal arterioles exhibit a significant dilation, p < 0.05. (C) Diameter change (mean ± sem) before (pre) and 15 minutes after (post) exposure to 50 mg/ml GNP-Dex for arcade and terminal arterioles in response to 10⁻⁴ mol/L ACh (acetylcholine), ADO (adenosine) or PE (phenylephrine). n = 8.