Biocompatibility Studies of Gadolinium Complexes with Iminodiacetic Acid Derivatives

Abstract

Apart from using as radiopharmaceuticals, iminodiacetic acid derivatives, after complexation with gadolinium, have been also tested as MRI CAs (magnetic resonance imaging contrast agents) since they show high affinity to hepatocytes and therefore provide high-resolution MRI of the liver. The purpose of this study was to evaluate the biocompatibility of four gadolinium complexes with iminodiacetic acid (IDA) derivatives differing in substituent in aromatic ring by estimating their influence on plasma hemostasis, integrity of erythrocyte membrane, and toxicity towards human umbilical vein endothelial cells (HUVECs). The influence of gadolinium-based CAs on plasma hemostasis was evaluated by measuring PT (prothrombin time), APTT (activated partial tromboplastin time), and TT (thrombin time). The effects of tested compounds on RBCs (Red Blood Cells) were assessed using hemolysis assay and microscopy studies. The influence of gadolinium complexes on the barrier properties of HUVECs was assessed by means of real-time method based on the measurements of the impedance changes of the cells. Gadolinium complexes did not affect significantly PT and TT. APTT measurements revealed significant prolongation in the presence of all tested gadolinium complexes at the concentration higher than 0.5 μmol/mL. Hemolysis assay showed that compounds with alkyl substituents in benzene ring without halogen atom (1-3) do not exert unfavorable effect on the integrity of erythrocyte membrane over the entire concentration range. All gadolinium complexes at 1.0 μmol/mL contribute to the decrease in HUVEC viability and integrity. To conclude, the study describes biocompatibility studies of gadolinium-based CAs, provides additional insight into their potential toxicity associated with systemic administration, and underscores the necessity for further research.

Keywords: Biocompatibility; Gadolinium; Iminodiacetic acid; Magnetic resonance imaging.

Fig. 1

Gadolinium complexes 1–4: sodium salt of N-(4-methylacetanilide)iminodiacetate gadolinium–1, sodium salt of N-(2,4-dimethylacetanilide)iminodiacetate gadolinium-2, sodium salt of N-(2,4,6-trimethylacetanilide)iminodiacetate gadolinium-3, sodium salt of N-(3-bromo-2,4,6-trimethylacetanilide)iminodiacetate gadolinium–4. The sythesized complexes were obtained in a form of hexahydrates

Fig. 2

The effects of gadolinium complexes a compounds 1 and 2; b compounds 3 and 4 on prothrombin time (PT) (mean ± SD; n = 5) after 3-min incubation in plasma; final volume 160 μL. Compound 2 at concentration of 0.75 μmol/mL and above significantly prolonged PT. Significant changes are marked with red frame

Fig. 3

The effects of gadolinium complexes a compounds 1 and 2; b compounds 3 and 4 on activated partial thromboplastin time (APTT) (mean ± SD; n = 4) after 3-min incubation in plasma; final volume 160 μL. Exposure to the tested compounds even at the concentrations of 0.5 μmol/mL and above was shown to significantly influence the value of APTT. Statistically significant changes are marked with red frame (p < 0.05)

Fig. 4

The effects of gadolinium complexes a compounds 1 and 2; b compounds 3 and 4 on thrombin time (TT) (mean ± SD; n = 4) after 2-min incubation in plasma; final volume 210 μL. All tested compounds apart from the highest concentration of compounds 1 and 2 did not affect TT. Compounds 1 and 2 at 1.5 μmol/mL significantly prolonged TT. Statistically significant changes are marked with red frame (p < 0.05)

Fig. 5

Percentage of hemolysis obtained from the interaction of gadolium complexes with iminodiacetic acid derivatives with 2% RBC (red blood cell) suspension, compared to the positive control Triton X-100 at 0.2% (100% hemolysis) (mean ± SD; n = 4), *p < 0.05 vs. control. A statistically significant increase in the rate of hemolysis was documented for the highest concentrations of compound 2; however, the value of hemolysis did not exceed 5%. Therefore, it might be concluded that compounds 1–3 do not show adverse effects on the integrity of RBCs membrane. Compound 4 even at the concentration of 0.5 μmol/mL contributed to the erythrocyte hemolysis exceeding 10%, which is regarded as clinically important

Fig. 6

a Effect of gadolinium complexes (1, 2) with iminodiacetic acid derivatives on erythrocyte morphology. Two percent erythrocyte suspension was treated at 37 °C for 60 min with indicated concentrations (0.1–1.5 μmol/mL) of compounds 1–2. Representative phase-contrast images are shown (magnification of 400 times). Compounds 1 and 2 at the highest concentration tested contributed to the formation of echinocytes (marked with arrows). b Effect of gadolinium complexes (3, 4) with iminodiacetic acid derivatives on erythrocytes morphology. Two percent erythrocyte suspension was treated at 37 °C for 60 min with indicated concentrations (0.1–1.5 μmol/mL) of compounds 3–4. Representative phase-contrast images are shown (magnification of 400 times). Single echinocytes were recognized in the case of compound 3

Fig. 7

a The influence of gadolinium complexes (1–4) at the concentration of 1 μmol/mL on the viability and integrity of HUVEC cells expressed as Normalized Cell Index. Light blue–control, dark blue–compound 1, red–compound 2, green–compound 3, pink–compound 4. b The effects of gadolinium complexes on the value of Normalized Cell Index at selected time points. All tested complexes in every time point significantly decreased the value of nCI; *p < 0.05

Fig. 8

The effect of gadolinium complexes with iminodiacetic acid derivatives on morphology of HUVECs. HUVECs cultured in monolayer on 48-well plates were stimulated with indicated concentrations (0.5–1.5 μmol/mL) of compounds 1–4. Representative phase-contrast images are shown (magnification of 100 times). Compounds 1 and 3 over the entire concentration range did not contribute to the changes in cell morphology. Compound 2 at 1 μmol/mL caused cell membrane disintegration, while compound 4 even at the lowest concentration contributed to the lysis of cells