Disturbance of cellular homeostasis as a molecular risk evaluation of human endothelial cells exposed to nanoparticles

Abstract

Even though application of nanoparticles in medicine seems to provide unique solutions for drug delivery and diagnosis diseases, understanding interactions between nanoscale materials and biological systems is imperative. Therefore, this study determined the effect of different types of nanoparticles (NPs) on human endothelial cells and examined the types of toxicity responses they can induce. Four different types of NPs were tested (PLA/MMT/TRASTUZUMAB, PLA/EDTMP, PLGA/MDP, and Pluronic F127 MICELLES), representing three putative areas of application: anticancer therapy, scintigraphy, and cosmetology. The experiments were performed on immortalized human umbilical vein endothelial cells (HUVEC-STs). Light contrast phase microscopy as well as cell viability assays showed that only Pluronic F127 MICELLES decreased the number of HUVEC-STs in contrast to PLA/MMT/TRASTUZUMAB, PLA/EDTMP, and PLGA/MDP NPs, which altered cell morphology, but not their confluency. The tested NPs induced not only DNA strand-breaks and alkali-labile sites, but also internucleosomal DNA fragmentation, visualized as a DNA ladder pattern typical of apoptosis. Moreover, generation of free radicals and subsequent mitochondrial membrane potential collapse showed the significance of free radical production during interactions between NPs and endothelial cells. High concentrations of NPs had different degrees of toxicity in human endothelial cells and affected cell proliferation, redox homeostasis, and triggered mitochondrial dysfunction.

Figure 1

The schematic overview of the four nanoparticles used in the study.

Figure 2

Comparisons of NP cytotoxicity in human endothelial cell line. (A,B) HUVEC-ST cell viability measured by the neutral red assay (left panels) or Alamar Blue assay (resazurin oxidation; right panels) after cell incubation for 72 h with increasing concentrations of NPs. Values are the mean ± SD of three independent experiments with six replicates in each experiment. Statistically significant differences comparing control cells taken as 100%, *p < 0.05 (C) Light microscopy images under contrast phase conditions of HUVEC-STs after treatment for 24 h with 100 μg/mL of PLA/MMT/TRA, PLA/EDTMP, PLGA/MDP, or 0.025 μg/mL of Pluronic F127 Ms. Bar = 50 µm. (D,E) Growth kinetics of HUVEC-STs treated with PLA/MMT/TRA, PLA/EDTMP, PLGA/MDP [100 μg/mL (D) or 250 μg/mL (E)], or Pluronic F127 Ms [0.25 μg/mL (D) or 0.5 μg/mL (E) for 72 h. ***p < 0.001, values are % of those obtained for untreated control (mean ± SD; n ≥ 3).

Figure 3

Genotoxic NP activity in human endothelial cells (A) Schematic overview of DNA damage and its consequences. (B,C) NP genotoxicity in HUVEC-STs. Cells were treated with PLA/MMT/TRA, PLA/EDTMP, PLGA/MDP (100 μg/mL), or Pluronic F127 Ms (0.25 μg/mL) (B) for up to 72 h or incubated only for 72 h with double doses of NPs: PLA/MMT/TRA, PLA/EDTMP, PLGA/MDP (200 μg/mL), or Pluronic F127 Ms (0.5 μg/mL) (C). DNA damage was evaluated by measuring DNA percentage in comet tail by alkaline version of comet assay (pH > 13). *p < 0.05, significant differences between NP-treated and control cells. Fifty images were analysed from each slide. Data are the means ± SD of three independent experiments. (D): ATM and ATR gene transcript expression (relative to HPRT1 housekeeping gene) in HUVEC-STs exposed to NPs for 24 h. Bars represent the means ± SD of three separate experiments (n 6). Asterisks refer to level of significant (*p < 0.05, n = 6) difference in cell transcription treated with investigated NPs compared to untreated cells.

Figure 4

DNA damage in HUVEC-STs triggered by NPs is the result of apoptosis. (A) Induction of apoptosis-dependent DNA fragmentation in HUVEC-STs after treatment with NPs [PLA/MMT/TRA, PLA/EDTMP, PLGA/MDP (100 μg/mL), or Pluronic F127 Ms (0.25 μg/mL)] at 24, 48, and 72 h estimated by the TUNEL assay. Results represent the mean ± SD of three independent experiments. *p < 0.05 for significant differences between NP-treated and control cells. (B) DNA fragmentation induced by NPs detected by agarose gel electrophoresis of DNA isolated from human endothelial cells. HUVEC-STs were treated for up to 72 h with 100 μg/mL of PLA/EDTMP, PLGA/MDP, and PLA/MMT/TRA or 0.025 μg/mL of Pluronic F127 Ms; 1—PLA/MMT/TRA, 2—PLA/EDTMP, 3—PLGA/MDP, 4—Control, 5—Pluronic F127 Ms. (C) NP effect (100 μg/mL of PLA/EDTMP, PLGA/MDP, and PLA/MMT/TRA or 0.025 μg/mL of Pluronic F127 Ms) on histone H2AX phosphorylation in human endothelial cells. *p < 0.05, **p < 0.01 for significant differences between NP-treated and control cells. Data are the means ± SD of three independent experiments. (D) Level of mRNA transcripts for histone H2AX gene in HUVEC-STs exposed to NPs (100 μg/mL of PLA/EDTMP, PLGA/MDP, and PLA/MMT/TRA or 0.025 μg/mL of Pluronic F127 Ms) for 24 h. Data are expressed as the means ± SD, (n = 3). Asterisks refer to level of significant (*p < 0.05, ***p < 0.001) difference in mRNA level in NP-treated cells compared to untreated control cells.

Figure 5

Molecular markers of NP-induced apoptosis in human endothelial cells. (A) Double staining with DNA-specific dye (Hoechst 33258 and PI) of HUVEC-STs after NP treatment (100 μg/mL of PLA/EDTMP, PLGA/MDP, and PLA/MMT/TRA or 0.025 μg/mL of Pluronic F127 Ms) for 72 h. Cells were analysed under fluorescence microscope (Olympus IX70, Japan; magnification: 400 ×). (B): Quantification of live, early apoptotic, late apoptotic, and necrotic HUVEC-STs at different times following treatment with NPs in the same conditions as described above. Cells were stained with a mixture of Hoechst 33258/PI. Representative data are shown from three separate experiments. p < 0.05 for significant differences between NP-treated and control cells (C) Time-dependent changes in caspase-3 activity in HUVEC-STs treated with various concentrations of NPs (100 μg/mL of PLA/EDTMP, PLGA/MDP, and PLA/MMT/TRA or 0.025 μg/mL of Pluronic F127 Ms) for 24, 48, and 72 h. Results represent the means ± SD of three independent experiments. *p < 0.05 for significant differences between NP-treated and untreated control cells. (D) Caspase-3 and PARP transcription levels (relative to HPRT1) in HUVEC-STs exposed to examined nanosubstances: PLA/EDTMP, PLGA/MDP, and PLA/MMT/TRA (100 μg/mL) or Pluronic F127 Ms (0.025 μg/mL) for 24 h. Asterisks refer to significant differences (*p < 0.05, **p < 0.01, and **p < 0.001; n = 3) in transcription levels in NP-treated cells compared to untreated cells.

Figure 6

Mitochondrial stress triggered by NPs in HUVEC-STs. (A) Schematic overview of perturbations in mitochondrial functions, which may occur after NP treatment. (B) ROS production in cells treated with NPs (100 μg/mL of PLA/EDTMP, PLGA/MDP, and PLA/MMT/TRA or 0.025 μg/mL of Pluronic F127 Ms) for 24, 48, and 72 h. DCF fluorescence intensity in control cells was set to 100%. Each value represents average ± SD of four independent experiments, *p < 0.05, **p < 0.01, and ***p < 0.001 for significant differences compared to control cells, ^#p < 0.05, ^##p < 0.01 for significant changes compared to samples preincubated with NAC and subsequently incubated with nanosubstances. (C,D): Changes in mitochondrial membrane potential of HUVEC-STs seeded into black 96-well titration microplates and incubated with NPs (100 μg/mL of PLA/EDTMP, PLGA/MDP, and PLA/MMT/TRA or 0.025 μg/mL of Pluronic F127 Ms) for 24, 48, and 72 h (C) or FCCP (D). Fluorescence ratio of JC-1 dimers to JC-1 monomers in control was assumed to be 100%. Results are presented as means ± SD of four experiments. *p < 0.05, **p < 0.01 for significant differences between NP-treated and untreated control cells (taken as 100%); (E) Bax, Bcl2, Cyt c, and AIF gene transcript expression (relative to HPRT1 housekeeping gene) in HUVEC-STs exposed to examined nanosubstances: PLA/EDTMP, PLGA/MDP, and PLA/MMT/TRA (100 μg/mL) or Pluronic F127 Ms (0.025 μg/mL). Asterisks refer to level of significant (**p < 0.01, ***p < 0.001; n = 3) difference in expression in cells treated with investigated NPs compared to untreated cells.