The authors have declared that no competing interests exist.
Conceived and designed the experiments: JCD ZWS. Performed the experiments: JCD YBY. Analyzed the data: JCD YBY YL YY. Contributed reagents/materials/analysis tools: ZWS YBL XQZ PLH. Wrote the paper: JCD ZWS.
Silica nanoparticles have become promising carriers for drug delivery or gene therapy. Endothelial cells could be directly exposed to silica nanoparticles by intravenous administration. However, the underlying toxic effect mechanisms of silica nanoparticles on endothelial cells are still poorly understood. In order to clarify the cytotoxicity of endothelial cells induced by silica nanoparticles and its mechanisms, cellular morphology, cell viability and lactate dehydrogenase (LDH) release were observed in human umbilical vein endothelial cells (HUVECs) as assessing cytotoxicity, resulted in a dose- and time- dependent manner. Silica nanoparticles-induced reactive oxygen species (ROS) generation caused oxidative damage followed by the production of malondialdehyde (MDA) as well as the inhibition of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). Both necrosis and apoptosis were increased significantly after 24 h exposure. The mitochondrial membrane potential (MMP) decreased obviously in a dose-dependent manner. The degree of DNA damage including the percentage of tail DNA, tail length and Olive tail moment (OTM) were markedly aggravated. Silica nanoparticles also induced G2/M arrest through the upregulation of Chk1 and the downregulation of Cdc25C, cyclin B1/Cdc2. In summary, our data indicated that the toxic effect mechanisms of silica nanoparticles on endothelial cells was through DNA damage response (DDR) via Chk1-dependent G2/M checkpoint signaling pathway, suggesting that exposure to silica nanoparticles could be a potential hazards for the development of cardiovascular diseases.
Silica nanoparticles have been found extensive applications in biomedical and biotechnological fields
The human umbilical vein endothelial cells (HUVECs) line isolated from the umbilical cord by collagenase digestion has been used for in vitro studies of endothelial cells function
Mammalian cells are frequently at risk of DNA damage from a variety of endogenous and exogenous sources, including reactive oxygen species, ultraviolet light, background radiation and environmental factors
To our best knowledge, this is the first study to illustrate the biological interaction mechanisms between DDR pathways and endothelial cells toxic effect triggered by silica nanoparticles. Prior to undertaking in vitro toxicity experiments, the characterization of silica nanoparticles, which is essential for nanotoxicity studies, was performed by transmission electron microscope (TEM) and dynamic light scattering (DLS) measurements. To investigate the toxic effect mechanisms of endothelial cells induced by silica nanoparticles, we conducted a sequence of assessments including cellular uptake and morphology, cell viability, membrane integrity, intracellular ROS generation, oxidative damage, DNA damage, cell cycle arrest, apoptosis and necrosis after HUVECs exposure to silica nanoparticles for 24 h. We also measured the protein levels of Chk1, Cdc25c, cyclin B1/Cdc2 to analyze whether silica nanoparticles-induced endothelial cells toxic effect was through DDR via Chk1-dependent G2/M checkpoint signaling pathway.
Silica nanoparticles were prepared using the Stöber method
The primary human umbilical vein endothelial cells (HUVECs) line was purchased from the Cell Resource Center, Shanghai Institutes for Biological Sciences (SIBS, China). The cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM) (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 100 U/mL penicillin and 100 µg/mL streptomycin, and cultured at 37°C in 5% CO2 humidified environment. For experiments, the cells were seeded in 6-well plates (except MTT assay using 96-well plates) at a density of 1×105 cells/mL and allowed to attach for 24 h, then treated with silica nanoparticles suspended in DMEM of certain concentrations for another 24 h. Before use, the stock suspensions of silica nanoparticles were sonicated for 5 min. Controls were supplied with an equivalent volume of DMEM without silica nanoparticles. For all experiments, each group had five replicate wells. Data are expressed as means ± S.D. from three independent experiments (*p<0.05).
LSCM detection: HUVECs were seeded at 1×104 cells in 35 mm-diameter glass bottom cell culture dish and were cultured in DMEM as above. After 24 h of cell attachment, the cells were treated with Ruthenium (II) hydrate (Ru(phen)32+) interior-labeled silica nanoparticles (50 µg/mL) for 24 h at 37°C in serum-free medium. These red fluorescent silica nanoparticles were prepared by a modified Stöber method and characterized as described before
TEM detection: After HUVECs incubated for 24 h with silica nanoparticles (50 µg/mL), the cells were washed with PBS and then centrifuged at 2000 r/min for 10 min. The supernatants were removed. The cell pellets were fixed in a 0.1 M PBS solution containing 2.5% glutaraldehyde and 4% paraformaldehyde for 3 h. They were then washed with 0.1 M PBS, embedded in 2% agarosegel, postfixed in 4% osmium tetroxide solution for 1 h, washed with distilled water, stained with 0.5% uranyl acetate for 1 h, dehydrated in a graded series of ethanol (30%, 60%, 70%, 90%, and 100%), and embedded in epoxy resin. The resin was polymerized at 60°C for 48 h. Ultrathin sections obtained with a ultramicrotome were stained with 5% aqueous uranyl acetate and 2% aqueous lead citrate, air dried, and imaged under a transmission electron microscope (TEM) (JEOL JEM2100, Japan).
Cultured HUVECs were treated with various concentrations (25, 50, 75, and 100 µg/mL) of silica nanoparticles for 24 h. Cell morphology was observed by optical microscope (Olympus IX81, Japan).
The cell viability was measured using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) reduction assay. MTT assay is the most common employed for the detection of cytotoxicity or cell viability following exposure to toxic substances. MTT is a water soluble tetrazolium salt, which is converted to an insoluble purple formazan by cleavage of the tetrazolium ring by succinate dehydrogenase within the mitochondria. The formazan product is impermeable to the cell membranes and therefore it accumulates in healthy cells. The absorbance of formazan was measured at 492 nm using a microplate reader (Themo Multiscan MK3, USA).
The lactate dehydrogenase leakage assay (LDH), which is based on the measurement of lactate dehydrogenase activity in the extracellular medium, was determined using a commercial LDH Kit (Jiancheng, China) according to the manufacturer's protocols. The loss of intracellular LDH and its release into the culture medium is an indicator of irreversible cell death due to cell membrane damage. After HUVECs treated with different concentrations (25, 50, 75, and 100 µg/mL) of silica nanoparticles for 24 h, the supernatants were collected for LDH measurement. 100 µL cell medium was used for LDH activity analysis and the absorbance at 440 nm was measured by a UV-visible spectrophotometer (Beckman DU-640B, USA).
Apoptosis in endothelial cells was measured using the Annexin V-propidium iodide (PI) apoptosis detection kit (KeyGen, China). The kit contains Annexin V conjugated to the flurochrome FITC. This complex displays a high affinity to the membrane phospholipid phosphatidylserine, which undergoes externalization in the earlier stages of apoptosis. To distinguish early apoptotic cells from dead cells resulted from late apoptosis or necrosis, the vital dye PI was used. In this way, FITC negative and PI negative were designated as live cells in the lower left quadrant; FITC positive and PI negative as apoptotic cells in the upper left quadrant; FITC positive and PI positive as necrotic cells in the upper right quadrant; and FITC negative and PI positive as large nuclear fragments in the lower right quadrant
The cytotoxicity effects might occur through the induction of oxidative stress and apoptosis with possible involvement of overproduction of reactive oxygen species (ROS). In this regard, the production of intracellular ROS was measured by flow cytometry using the 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) (Beyotime, China) as an oxidation-sensitive probe. Briefly, 10 mM DCFH-DA stock solution was diluted 1000-fold in cell culture medium without serum or other additive to yield a 10 µM working solution. After the exposure of HUVECs to series dosages (25, 50, 75, and 100 µg/mL) of silica nanoparticles for 24 h, the cells in 6-well plates were washed twice with PBS and incubated in 2 mL working solution of DCFH-DA at 37°C in dark for 30 min. Then the cells were washed twice with cold PBS and resuspended in the PBS for analysis. Fluorescent intensity and percentage of positive cells were measured by flow cytometry (Becton-Dickison, USA). For each sample, at least at least 1×104 cells were collected.
In addition to the analysis of cytotoxicity and ROS levels, the malondialdehyde (MDA) content was measured as an end product of lipid peroxidation. The defense systems against free radical attack were assessed by the measurement of both the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px). After HUVECs exposure to different concentrations (25, 50, 75, and 100 µg/mL) of silica nanoparticles for 24 h, washed once with ice-cold PBS, and lysed in ice-cold RIPA lysis buffer containing 1 mM phenylmethylsulphonyl fluoride (PMSF) (DingGuo, China) and phosphatase inhibitor for 30 min. After centrifuging the lysates at 12,000 rpm, 4°C for 10 min, the supernatants were collected for measurements of the production of MDA, the activities of SOD and GSH-Px. All examinations were carried out using commercially available kits (Jiancheng, China) according to the manufacturer's instructions. The protein concentrations of these extracts were determined by performing the bicinchoninic acid (BCA) protein assay (Pierce, USA).
MMP was detected by using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi dazo-lylcarbocyanide iodine (JC-1) (Sigma, USA). This probe can selectively enter into mitochondria and reversibly change color from red to green as the membrane potential decreased. The ratio of green to red expresses the change of MMP. Cells were treated with silica nanoparticles (25, 50, 75, and 100 µg/mL) for 24 h. After washing with PBS, the cells were incubated with 10 µg/mL working solution of JC-1 for 20 min. Then the cells were washed with PBS twice and analyzed by flow cytometry (Becton-Dickison, USA). The green fluorescence intensity was determined at an excitation wavelength of 488 nm and an emission wavelength of 525 nm, whereas the red fluorescence intensity determined at an excitation wavelength of 488 nm and an emission wavelength of 590 nm. For each sample, at least at least 1×104 cells were collected.
Comet assay, also known as single cell gel electrophoresis (SCGE), is a visual and sensitive technique for measuring DNA breakage in individual mammalian cells. The DNA damage induced by silica nanparticles was performed by Single cell gel electrophoresis kit (Biolab, China). HUVECs were collected and resuspended in PBS. 20 µL of the cells suspension and 80 µL of low melting agarose were mixed and 80 µL of the suspension pipetted onto a comet-slide. The slides were placed flat in dark at 4° C for 10 min for the mixture to solidify. Then the slides were placed in pre-chilled lysing solution at 4° C for 2 h. Slides were removed from lysing solution, tapped on a paper towel to remove any excess lysis solution and immersed in alkaline solution for 45 min in dark at room temperature. The slides were washed twice for 5 min. Then the slides were electrophoresed at low voltage (300 mA, 25 V) for 30 min. Slides were removed from the electrophoresis unit after the designated time, tapped to remove excess buffer at room temperature. Subsequently, the air-dried slides were stained with DNA-binding dye propidium iodide (PI) and evaluated under a fluorescence microscope (Olympus IX81, Japan). To prevent additional DNA damage, all the steps described above were conducted under dimmed light or in the dark. The data were analyzed by CASP software based on 100 randomly selected cells per sample. The percentage of tail DNA, tail length and Olive tail moment (OTM) were selected as indicators of DNA damage.
The cell cycle detection kit (KeyGen, China) was used to determine the DNA index (DI) and cell-cycle phase distributions. The method involved dissolving of the cell membrane lipids, eliminating the cell cytoskeleton with trypsin, digesting the cellular RNA and stabilizing the chromatin with spermine. HUVECs were exposed to various concentrations of silica nanoparticles for 24 h, washed with PBS three times and trypsinized. After centrifuged at 1000 rpm for 5 min, the cells were washed one time in PBS and fixed in 70% ethanol for at least 24 h. The fixed cells were washed twice with PBS and treated with 100 µL RNase A at 37°C for 30 min. Finally, the cells were stained with 400 µL propidium iodide and incubated in dark for 30 min. A total of at least 1×104 cells for each sample were analyzed by flow cytometry (Becton Dickinson, USA).
To analyze whether silica nanoparticles influence the expression of G2/M DNA damage checkpoint regulators, we measured the protein levels of Chk1, Cdc25c, cyclin B1/Cdc2 in HUVECs by western blot analysis. Total cellular protein extracts were determined by performing the bicinchoninic acid (BCA) protein assay (Pierce, USA). The equal amounts of lysate proteins (40 µg) were loaded onto SDS-polyacrylamide gels (12% separation gels) and electrophoretically transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA). After blocking with 5% nonfat milk in Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBST) for 1 h at room temperature, the membrane was incubated with Chk1, Cdc25C, Cdc2, cyclin B1 [1∶1000, rabbit antibodies, Cell Signaling Technology (CST), USA] overnight at 4°C, washed with TBST, and incubated with a horseradish peroxidase-conjugated anti-rabbit Ig G secondary antibody (CST, USA) for 1 h at room temperature. After washed three times with TBST, The antibody-bound proteins were detected using the ECL chemiluminescence reagent (Pierce, USA).
Densitometric analysis of the western blots was performed using Image Lab™ Software (Bio-Rad Version 3.0, USA). The relative values of the samples were measured by normalising all data to the respective control samples of each experiment.
Data were expressed as mean ± S.D. and significance was determined by using one-way analysis of variance (ANOVA) followed by least significant difference (LSD) test to compare the differences between groups. Differences were considered significant at
As shown in
(A) Transmission electron microscopy image: TEM images of silica nanoparticles had a spherical shape with the average diameter of 62 nm. (B) Size distribution: The size distribution measured by ImageJ software showed approximately normal distribution.
Distilled water | DMEM | |||||
Time (h) | Diameter (nm) | PDI | Zeta potential (mV) | Diameter (nm) | PDI | Zeta potential (mV) |
1 | 108.03±0.61 | 0.11±0.01 | −40.33±6.47 | 106.03±0.93 | 0.11±0.02 | −35.27±2.10 |
3 | 106.80±0.63 | 0.10±0.01 | −39.13±5.26 | 105.83±0.90 | 0.10±0.02 | −36.77±2.40 |
6 | 105.60±1.22 | 0.07±0.02 | −41.43±3.29 | 107.27±0.93 | 0.13±0.01 | −36.53±0.64 |
12 | 104.97±0.60 | 0.10±0.02 | −44.10±1.30 | 104.23±1.17 | 0.08±0.01 | −34.37±2.75 |
24 | 104.87±0.64 | 0.08±0.02 | −46.33±3.13 | 104.43±0.21 | 0.10±0.01 | −38.10±0.46 |
Data are expressed as means ± S.D. from three independent experiments.
Since the subcellular localization may play an important role in silica nanoparticles-induced biological effects, we examined the HUVECs uptake of Ru(phen)32+-labeled silica nanoparticles by LSCM. Merged confocal microscopic images of HUVECs (
(A) LSCM images of HUVECs after incubation for 24 h with Ruthenium (II) hydrate labeled silica nanoparticles (50 µg/mL, red) of size 62 nm. The cell skeleton was stained with Phalloidin-FITC (green), and the cell nucleus with 4,6-diamidino-2-phenylindole (DAPI; blue). (B) TEM images of HUVECs exposed to silica nanoparticles for 24 h. Both TEM and LSCM results showed that the silica nanoparticles were internalized into cells compared to control group.
To evaluate the possible toxicity of silica nanoparticles on endothelial cells, cellular morphology and cell viability were determined after exposing HUVECs to silica nanoparticles for 24 h. With the dosages (25, 50, 75, and 100 µg/mL) increasing, the morphological changes of HUVECs became more and more obviously. Cell density reduction, irregular shape and cellular shrinkage were observed (
(A) Morphological changes of HUVECs after exposure to silica nanoparticles for 24 h. Cell density reduction, irregular shape and cellular shrinkage were observed by optical microscope. (B) Cell viability of HUVECs treated with silica nanoparticles was measured by MTT assay after 6 h, 12 h, 24 h exposure. (C) LDH leakage of HUVECs exposed to viarous concentrations of silica nanoparticles for 24 h. The results indicated that silica nanoparticles induced cytotoxicity in a dose- and time-dependent manner. Data are expressed as means ± S.D. from three independent experiments (*p<0.05).
To further analyze the cell death caused by silica nanoparticles, apoptosis and necrosis in HUVECs were measured by flow cytometry. As shown in
(A) Apoptotic and necrotic populations of cells double-stained with PI- and FITC-labled Annexin V were depicted by flow cytometry. FITC negative and PI negative were designated as live cells in the lower left quadrant; FITC positive and PI negative as apoptotic cells in the upper left quadrant; FITC positive and PI positive as necrotic cells in the upper right quadrant; and FITC negative and PI positive as large nuclear fragments in the lower right quadrant. (B) HUVECs exposure to silica nanoparticles caused increase of both necrosis and apoptosis rate. The apoptosis rate was much lower than the necrosis rate. Data are expressed as means ± S.D. from three independent experiments (*p<0.05).
To get a closer insight into cytotoxicity induced by silica nanoparticles, we measured the generation of ROS through fluorescence intensity of dichlorofluorescein (DCF). As shown in
The intracellular levels of ROS and MDA were obviously increased (A, B). While SOD and GSH-Px levels were decreased significantly with a dose-dependent way (C, D). Silica nanoparticles-induced ROS generation caused oxidative damage followed by the production of MDA as well as the inhibition of SOD and GSH-Px. Data are expressed as means ± S.D. from three independent experiments (*p<0.05).
The generation of intracellular ROS could cause oxidative damage. Therefore, we measured the production of MDA as well as the activities of SOD and GSH-Px.
The MMP was determined with JC-1 probe by flow cytometry (
The green/red fluorescence intensity ratio was used to express the changes of MMP and the increased ratio indicates decrease of MMP. Data are expressed as means ± S.D. from three independent experiments (*p<0.05).
To investigate the mechanisms of apoptosis induced by silica nanoparticles, DNA damage was measured by comet assay. As shown in
(A) Control group, (B) 25 µg/mL treated group, (C) 50 µg/mL treated group, (D) 75 µg/mL treated group, (E) 100 µg/mL treated group. More severe DNA injury was reflected by larger area of the comet tail. The DNA damage caused by silica nanoparticles was getting more serious with the dosages increasing. The magnification was 200× by fluorescence microscope.
DNA damage | |||
Groups | Tail DNA (%) | Tail Length (µm) | Olive Tail Moment |
Control | 0.40±0.18 | 2.75±0.46 | 0.05±0.02 |
25 µg/mL | 1.72±0.62 | 3.56±0.73 | 0.40±0.10 |
50 µg/mL | 15.99±2.56* | 14.25±4.98* | 6.41±2.62* |
75 µg/mL | 24.76±6.88* | 23.88±9.33* | 11.60±3.99* |
100 µg/mL | 34.21±5.23* | 31.80±5.53* | 15.99±3.65* |
Data are expressed as means ± S.D. from three independent experiments (*p<0.05).
The relevant checkpoints could arrest cell cycle at certain stage as response to DNA damage. Thus, we measured the cell cycle arrest by flow cytometry. As shown in
After exposure to various concentrations of silica nanoparticles for 24 h, flow cytometry were used to determine the cell cycle distribution of HUVECs. The images showed that cell cycle was arrested in G2/M phase. The percentage of cells in G2/M phase increased progressively in a dose-dependent manner, while in G0/G1 and S phase the percentage of cells declined irregular.
Distribution of cell cycle | |||
Groups | G0/G1 | S | G2/M |
Control | 67.19±0.77 | 25.97±0.90 | 6.84±0.99 |
25 µg/mL | 77.70±1.88* | 11.68±1.82* | 10.62±0.85 |
50 µg/mL | 59.54±2.14* | 19.07±1.84* | 21.39±1.41* |
75 µg/mL | 57.41±1.37* | 15.10±1.83* | 27.49±0.66* |
100 µg/mL | 59.91±3.21* | 5.48±1.14* | 34.62±3.14* |
Data are expressed as means ± S.D. from three independent experiments (*p<0.05).
To better understand the G2/M arrest signaling pathway, we examined the expression of G2/M checkpoint regulators in HUVECs by western blot analysis. As shown in
. (A) Effect of silica nanoparticles on the expression of Chk1, Cdc25C, cyclin B1, Cdc2. GAPDH was used as an internal control to monitor for equal loading. (B) Relative densitometric analysis of the proteins bands was performed and presented. Silica nanoparticles induced G2/M arrest through the upregulation of Chk1 and the downregulation of Cdc25C, cyclin B1/Cdc2. Data are expressed as means ± S.D. from three independent experiments (*p<0.05).
Nanoscience has matured significantly during the last decade as it has transitioned from bench top science to applied technology
Currently, cell uptake of nanoparticles is an important issue in designing suitable cell-tracking and drug-carrier nanomaterials systems
It has been confirmed that the LDH release is an indicator of necrosis due to cell membrane damage
In addition, DNA damage could be mediated by oxidative stress depending on the balance between ROS production and antioxidant status
In the current study, we confirmed that silica nanopaticles triggered DDR pathways leading to activate the G2/M cell cycle checkpoint. As shown in
In summary, the present study demonstrates that silica nanoparticles induce ROS generation and DDR, caused endothelial cells toxic effect through Chk1-dependent G2/M DNA damage checkpoint signaling pathway. Thus, our findings suggest that exposure to silica nanoparticles could be a potential hazardous factor for the development of cardiovascular diseases, more studies of relation between silica nanoparticles exposure, adverse effects and biological mechanisms are needed for the safety evaluation and biomedical application of nanoparticles.