The cytotoxicity evaluation of magnetic iron oxide nanoparticles on human aortic endothelial cells

Abstract

One major obstacle for successful application of nanoparticles in medicine is its potential nanotoxicity on the environment and human health. In this study, we evaluated the cytotoxicity effect of dimercaptosuccinic acid-coated iron oxide (DMSA-Fe2O3) using cultured human aortic endothelial cells (HAECs). Our results showed that DMSA-Fe2O3 in the culture medium could be absorbed into HAECs, and dispersed in the cytoplasm. The cytotoxicity effect of DMSA-Fe2O3 on HAECs was dose-dependent, and the concentrations no more than 0.02 mg/ml had little toxic effect which were revealed by tetrazolium dye assay. Meanwhile, the cell injury biomarker, lactate dehydrogenase, was not significantly higher than that from control cells (without DMSA-Fe2O3). However, the endocrine function for endothelin-1 and prostacyclin I-2, as well as the urea transporter function, was altered even without obvious evidence of cell injury in this context. We also showed by real-time PCR analysis that DMSA-Fe2O3 exposure resulted in differential effects on the expressions of pro- and anti-apoptosis genes of HAECs. Meanwhile, it was noted that DMSA-Fe2O3 exposure could activate the expression of genes related to oxidative stress and adhesion molecules, which suggested that inflammatory response might be evoked. Moreover, we demonstrated by in vitro endothelial tube formation that even a small amount of DMSA-Fe2O3 (0.01 and 0.02 mg/ml) could inhibit angiogenesis by the HAECs. Altogether, these results indicate that DMSA-Fe2O3 have some cytotoxicity that may cause side effects on normal endothelial cells.

Figure 1

The TEM images of HAECs incubated with 0.02 mg/ml of DMSA-Fe₂O₃ for 24 h. (a) HAEC without DMSA-Fe₂O₃ (×8,000). (b) HAEC without DMSA-Fe₂O₃ (×30,000). (c) HAEC incubated with DMSA-Fe₂O₃ (×5,000). (d) HAEC incubated with DMSA-Fe₂O₃ (×30,000). Abbreviations: n, nucleus; v, vesicle; Arrows denote the DMSA-Fe₂O₃ or particulate matter.

Figure 2

The viability of HAECs incubated with DMSA-Fe₂O₃. Data are expressed as mean ± SD from independent experiments. Control values from HAECs incubated without DMSA-Fe₂O₃ were defined as 1. (a) HAECs were incubated with DMEM containing the gradient concentrations of DMSA-Fe₂O₃ for 24 h (0.001, 0.01, 0.02, 0.05, 0.1, 0.2 mg/ml), n = 7. (b) HAECs were incubated with DMEM containing 0.05 mg/ml DMSA-Fe₂O₃ for the indicated time (4, 24, 48, 72 h). n = 5. *p < 0.05 vs. control; **p < 0.01 vs. control.

Figure 3

Levels of injury marker, LDH, and endocrine factors in supernatant of HAECs. Incubated with 0.02 mg/ml DMSA-Fe₂O₃ for 24 h. Ratios relative to the control cells (without DMSA-Fe₂O₃) are shown. *p < 0.05 vs. control; **p < 0.01 vs. control.

Figure 4

Fold changes in gene expression: apoptosis, adhesion molecules, ER stress, oxidative stress, and calcium-handling proteins. The changes of HAECs incubated with 0.02 mg/ml DMSA-Fe₂O₃ for 24 h to control the cells (HAECs without DMSA-Fe₂O₃) were analyzed by the 2^-ΔΔCT method. Gene symbols and corresponding encoded proteins: MAP3K5, apoptosis signal-regulating kinase 1 (ASK1); TRAF2, tumor necrosis factor receptor-associated factor 2 (TRAF2); DAB2IP, ASK1-interacting protein (AIP1); MAPK8, mitogen-activated protein kinase 8 (JNK1); MAPK9, mitogen-activated protein kinase 9 (JNK2); MAPK14, mitogen-activated protein kinase 14 (p38 MAPK α); ERN1, endoplasmic reticulum to nucleus signaling 1 (IRE1); BCL2, B-cell lymphoma 2 (Bcl-2); BAX, Bcl-2-associated X protein (Bax); NKRF, nuclear factor-κB repressing factor; TXN, thioredoxin; CTSB, cathespin B; CYCS, cytochrome C; CASP9, caspase-9; CASP3, caspase-3; EIF2AK3, eukaryotic translation initiation factor 2α kinase 3 (PERK); ATF4, activating transcription factor 4; DDIT3, DNA-damage-inducible transcript 3 (CHOP); EIF2A, eukaryotic translation initiation factor 2α; NOS3, nitric oxide synthase 3 (eNOS); SOD1, super oxide dismutase 1 (SOD-1); SOD2, super oxide dismutase 2 (SOD-2); ROMO1, reactive oxygen species modulator 1; PTGS1, cyclooxygenase 1 (COX-1); PTGS2, cyclooxygenase 2 (COX-2); VCAM1, vascular cell adhesion molecule 1 (VCAM-1); ICAM1, intercellular adhesion molecule 1(ICAM-1); ICAM2, intercellular adhesion molecule 2 (ICAM-2); SELE, endothelial-leukocyte adhesion molecule 1 (E-selectin); PLCG1, phospholipase C γ1; PLCG2, phospholipase C γ2; ITPR1, inositol 1,4,5-trisphosphate receptor type 1; ITPR2, inositol 1,4,5-trisphosphate receptor type 2; ITPR3, inositol 1,4,5-trisphosphate receptor type 3; CALM1, calmodulin 1.

Figure 5

Effect of DMSA-Fe₂O₃ on tube network formed by HAECs cultured on Matrigel within 14 h. (a) HAECs can form a capillary-like network on Matrigel-coated wells within 14 h. (b) An obvious failure to form networks by HAECs in the presence of 0.01 mg/ml DMSA-Fe₂O₃. (c) Few tube networks by HAECs in the presence of 0.02 mg/ml DMSA-Fe₂O₃. (d) The high urea solution (6M urea) was used as a positive control for the inhibition of tube formation.

Figure 6

Length of tube networks formed by HAEC cultured on Matrigel. Image-Pro plus 6.0 for Windows software was used to measure the length of tube networks (pixels). The stained cells were inspected under a light microscope at ×100 magnification and captured more than three pictures from different fields. The average data from the same well was calculated as its quantitative value. Data are expressed as mean ± SD. **p < 0.01 vs. control.