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The adverse vascular effects of multi-walled carbon nanotubes (MWCNTs) to human vein endothelial cells (HUVECs) in vitro: role of length of MWCNTs
© The Author(s) 2017
Received: 8 June 2017
Accepted: 6 November 2017
Published: 10 November 2017
Increasing evidences indicate that exposure to multi-walled carbon nanotubes (MWCNTs) could induce adverse vascular effects, but the role of length of MWCNTs in determining the toxic effects is less studied. This study investigated the adverse effects of two well-characterized MWCNTs to human umbilical vein endothelial cells (HUVECs).
The internalization and localization of MWCNTs in HUVECs were examined by using transmission electron microscopy (TEM). The cytotoxicity of MWCNTs to HUVECs was assessed by water soluble tetrazolium-8 (WST-8), lactate dehydrogenase (LDH) and neutral red uptake assays. Oxidative stress was indicated by the measurement of intracellular glutathione (GSH) and reactive oxygen species (ROS). ELISA was used to determine the release of inflammatory cytokines. THP-1 monocyte adhesion to HUVECs was also measured. To indicate the activation of endoplasmic reticulum (ER) stress, the expression of ddit3 and xbp-1s was measured by RT-PCR, and BiP protein level was measured by Western blot.
Transmission electron microscopy observation indicates the internalization of MWCNTs into HUVECs, with a localization in nuclei and mitochondria. The longer MWCNTs induced a higher level of cytotoxicity to HUVECs compared with the shorter ones. Neither of MWCNTs significantly promoted intracellular ROS, but the longer MWCNTs caused a higher depletion of GSH. Exposure to both types of MWCNTs significantly promoted THP-1 adhesion to HUVECs, accompanying with a significant increase of release of interleukin-6 (IL-6) but not tumor necrosis factor α (TNFα), soluble ICAM-1 (sICAM-1) or soluble VCAM-1 (sVCAM-1). Moreover, THP-1 adhesion and release of IL-6 and sVCAM-1 induced by the longer MWCNTs were significantly higher compared with the responses induced by the shorter ones. The biomarker of ER stress, ddit3 expression, but not xbp-1s expression or BiP protein level, was significantly induced by the exposure of longer MWCNTs.
Combined, these results indicated length dependent toxic effects of MWCNTs to HUVECs in vitro, which might be associated with oxidative stress and activation of ER stress.
Multi-walled carbon nanotubes (MWCNTs) are among the most popular carbonaceous nanoparticles (NPs) with great uses not only in microelectronics and energy storage, but also biotechnology . In biomedicine, MWCNTs have been shown great potential with many important applications, such as drug delivery and biomedical imaging [2, 3]. These biomedical applications could increase the contact of human blood vessels to MWCNTs. Therefore, it is necessary to assess the potential toxicity of MWCNTs to vascular systems in order to understand the potential adverse effects of MWCNTs entering circulation as well as to ensure the safe use of MWCNTs in nanomedicine [4, 5]. Indeed, convincing data indicated that exposure to carbonaceous NPs, including MWCNTs, could induce adverse health effects to vascular system both in vivo and in vitro [6–8], but the mechanisms remain unclear.
Multi-walled carbon nanotubes are high aspect ratio materials characterized with a high length to width ratio. Increasing evidences have shown that their potential toxicity is correlated with the physicochemical properties, e.g., length, composition and surface chemistry [9, 10]. Particularly, the length of carbon nanotubes (CNTs) has been shown to be crucial to influence their toxic potential. For example, convincing data showed that longer CNTs were more potent to induce inflammatory responses and thus promote the development of pulmonary fibrosis, which is likely associated with frustrated phagocytosis induced by longer fibers [11, 12]. Similarly, Kim et al.  found that longer MWCNTs were more cytotoxic to Chinese hamster ovary cells, but neither longer MWCNTs nor shorter MWCNTs induced genotoxicity in vivo or in vitro. In contrast, Han et al.  showed that the shorter MWCNTs were more toxic to C6 rat glioma cells, which was associated with an increase of oxidative stress. Combined, previous studies showed a crucial role of length of CNTs in determining the toxicity, which could be dependent on the endpoints evaluated and the models used. However, the association between length of MWCNTs and vascular effects is less studied at present. Cao et al.  showed that exposure to longer MWCNTs was associated with a stronger response in endothelial activation in vitro and plaque progression in vivo, but the longer and shorter MWCNTs used in that study had indeed similar characteristics. Donkor and Tang et al.  showed length dependent uptake and retention of MWCNTs in endothelial cells, but the toxicity of different MWCNTs was not further assessed in that study.
Thus, in this study we evaluated the toxicity of MWCNTs with different lengths to human umbilical vein endothelial cells (HUVECs). These MWCNTs were well-characterized that they are shorter (code XFM22) and longer (code XFM19) MWCNTs. HUVECs were selected as the in vitro model because they are among the most popular models used to evaluate the bio-effects of NPs to endothelium . The cytotoxicity, oxidative stress, release of cytokines and monocyte adhesion induced by the two types of MWCNTs was compared. These endpoints were selected because they were considered as the key events associated with early development of atherosclerosis [4, 8]. To investigate the possible mechanism, the activation of endoplasmic reticulum (ER) stress biomarkers, namely the expression of ddit3 (DNA damage-inducible transcript 3; also known as chop, C/EBP homologous protein) and xbp-1s (spliced X-box binding protein 1) as well as the protein level of BiP (binding immunoglobulin protein; also know as GRP78, 78 kDa glucose-regulated protein), were determined by real-time RT-PCR and Western blot, respectively. The activation of ER stress was evaluated because it has been suggested to be involved in endothelial activation [17, 18], and some recent studies indicated that NP exposure might promote dysfunction of endothelial cells through the activation of ER stress pathway .
Human umbilical vein endothelial cell (passage 1; purchased from ScienCell Research Laboratories, Carlsbad, CA) were cultured in supplemented endothelial medium and used at passage 3–6 as we described earlier . THP-1 monocytes (ATCC) were cultured in supplemented RPMI 1640 medium (Gibco, USA) and used within 2 months as we described elsewhere .
MWCNT characterization and exposure
The physicochemical properties of XFM22 and XFM19 (supplier information)
Special surface area (m2/g)
Electric conductivity (s/cm)
Outer 20–30 nm; inner 5–10 nm
Tap density 0.28 g/cm3; true density: ~ 2.1 g/cm3
Outer 20–30 nm; inner 5–10 nm
Tap density 0.28 g/cm3; true density: ~ 2.1 g/cm3
Transmission electron microscopy (TEM)
For ultrastructural observations, cells were seeded at 5 × 105 on 60 mm diameter cell culture Petri dishes and grown for 2 days before exposure. The cells were exposed to 32 μg/mL XFM22 or XFM19 for 24 h, rinsed, and then scratched by using a cell scraper. After centrifuge, the cells were fixed with 2.5% glutaraldehyde in PBS overnight, post-fixed with 1% OsO4 for 3 h, dehydrated in a graded series of ethanol, and embedded in epoxy Resin (Epon 812). The samples were then sectioned using an ultramicrotome at 70 nm, placed on carbon film supported by copper grids, stained with uranyl acetate and lead citrate, and observed under a TEM (JEM-1230, JEOL Ltd., Tokyo, Japan) operated at 80 kV.
The cytotoxicity of various concentrations of MWCNTs to HUVECs was measured by water soluble tetrazolium-8 (WST-8), lactate dehydrogenase (LDH) and neutral red uptake assays using commercial kits (Beyotime, Nantong, China). WST-8 assay could reflect the viability of mitochondria in living cells because it could be converted to water-soluble yellow formazan by mitochondria. LDH could be used to indicate the integrity of membrane. And neutral red uptake assay could be used to indicate the integrity of lysosomes as it could accumulate in intact lysosomes. For the assays, 4 × 104/well HUVECs were seeded on 24-well plates and grown for 2 days before exposure. Cells were then incubated with 0 μg/mL (control), 2, 4, 8, 16 and 32 μg/mL XFM22 or XFM19 for 24 h, and the cytotoxicity assays were done according to manufacturer’s instructions (Beyotime, Nantong, China). The products were read by an ELISA reader (Synergy HT, BioTek, Woburn, MA, USA). The images of HUVECs after 0 μg/mL (control), 8 and 32 μg/mL XFM22 or XFM19 exposure and neutral red staining were also taken by a light microscope (Olympus, Japan) to indicate morphological changes. For comparison, HUVECs were also exposed to various concentrations of XFI15 for 24 h, followed by WST-8 and neutral red uptake assays to indicate cytotoxicity.
The oxidative stress in HUVECs after exposure to various concentrations of MWCNTs was indicated by the measurement of intracellular glutathione (GSH) and ROS. The intracellular GSH was measured by using a fluorescence probe monochlorobimane (MCB; Sigma-Aldrich). MCB can enter the cells freely and form a fluorescent GSH-MCB adduct catalyzed by GSH S-transferase . The intracellular ROS was measured by using DCFH-DA, which can form a fluorescent product upon its reaction with a variety of ROS inside the cells . Both of the assays were done as previously described [21, 22]. Briefly, 1 × 104/well HUVECs were seeded on 96-well black plates and grown for 2 days prior to exposure to various concentrations of MWCNTs. After that, the cells were rinsed once, and then incubated with 50 μM MCB or 10 μM DCFH-DA in serum-free medium for 30 min. After rinsed once again, the fluorescence was read at Ex 360 ± 44 nm and Em 460 ± 40 nm (for GSH) or Ex 485 ± 20 nm and Em 528 ± 20 nm (for ROS) by an ELISA reader (Synergy HT, BioTek, Woburn, MA, USA). For comparison, HUVECs were also exposed to various concentrations of XFI15 for 24 h, and intracellular GSH and ROS were determined as indicated above.
The supernatants from WST-8 or neutral red uptake assays were collected and stored at − 20 °C within 1 month before analysis. The release of tumor necrosis factor α (TNFα), interleukin-6 (IL-6), soluble intercellular cell adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule 1 (sVCAM-1) was determined by an ELISA kit according to manufacturer’s instruction (Neobioscience Technology Co., Ltd., China). The detection limits are TNFα 7.8 pg/mL, IL-6 3.9 pg/mL, sICAM-1 0.24 pg/mL and sVCAM-1 15.6 pg/mL. The concentrations of cytokines in all the samples are higher than the detection limits. All of the products were read by using an ELISA reader (Synergy HT, BioTek, Woburn, MA, USA), and the concentrations of each cytokine were plotted according to the standard curve.
The adhesion of THP-1 monocytes to HUVECs was done as previously described . Briefly, HUVECs on 96-well black plates were exposed to various concentrations of MWCNTs for 24 h. To indicate the possible role of ER stress, HUVECs were also co-exposed to an ER stress inducer thapsigargin (TG; Sigma-Aldrich, USA) before adhesion assay. TG was used as 1 μM because it has been shown before that TG at this concentration could activate ER stress pathway in HUVECs . THP-1 monocytes were labeled with 10 μM CellTracker™ Green CMFDA (5-chloromethylfluorescein diacetate, Invitrogen, Carlsbad, CA), and 5 × 104/well labeled THP-1 cells were incubated with the exposed HUVECs for another 1 h for adhesion. After that, the unbound THP-1 cells were washed away, and the green fluorescence from the adherent THP-1 cells was read at Ex 485 ± 20 nm and Em 528 ± 20 nm by an ELISA reader (Synergy HT, BioTek, Woburn, MA, USA).
The protein level of BiP (GRP78) was determined by Western blot, using α-Tubulin as internal control. 2 × 105/well HUVECs were seeded in 6-well plates and grown for 2 days before exposure to 0 μg/mL (control) and 32 μg/mL XFM22 or XFM19 for 24 h. After exposure, the cells were rinsed once by Hanks solution and proteins were extracted by using cell lysis buffer for Western and IP (Beyotime, Nantong, China) following instructions. A total of 20 μL/well protein was then dissolved in NuPAGE™ 4–12% Bis–Tris protein gels and transferred to a nitrocellulose membrane (0.2 µm pore size) using iBlot® 2 Gel Transfer Device (Thermo-Fisher, USA). The membrane was blocked by QuickBlock™ Blocking Buffer for Western Blot (Beyotime, Nantong, China), and then incubated with 1:1000 diluted first antibodies at 4 °C. The first antibodies are BiP rabbit monoclonal antibody (Cell Signalling Technology, USA) and α-Tubulin rabbit polyclonal antibody (Beyotime, Nantong, China). After that, the membrane was washed three times with Western wash buffer, followed by the incubation with 1:2000 diluted secondary antibody [HRP-labeled Goat Anti-Rabbit IgG (H + L); Beyotime, Nantong, China]. After 1 h incubation, the membrane was washed three times with Western wash buffer, and stained by BeyoECL Plus for 2 min (Beyotime, Nantong, China). The membrane was imaged by using FluorChem FC2 (Alpha Innotech, USA), and the relative density was determined by ImageJ (NIH).
Quantitative real-time RT-PCR
The mRNA level of ddit3 (chop) and xbp-1s were determined by quantitative real-time RT-PCR, using gapdh as internal control. 2 × 105/well HUVECs were seeded in 6-well plates and grown for 2 days before exposure to 0 μg/mL (control) and 32 μg/mL XFM22 or XFM19 for 24 h. After exposure, the cells were rinsed once by Hanks solution and total mRNA was extracted using TRI Reagent® following manufacturer’s instructions (Sigma-Aldrich, USA). The cDNA was synthesized by using HiFiScript cDNA Synthesis Kit following manufacturer’s instructions (Cwbiotech, Beijing, China). The quantitative real-time PCR was done using UltraSYBR Mixture (Cwbiotech, Beijing, China) on PikoReal™ qPCR system (Thermo-Fisher, USA). The primers for each gene are as follows: gapdh ACAGCCTCAAGATCATCAGC (forward primer) and GGTCATGAGTCCTTCCACGAT (reverse primer), ddit3 GGAAACAGAGTGGTCATTCCC (forward primer) and GGAAACAGAGTGGTCATTCCC (reverse primer), xbp-1s CCGCAGCAGGTGCAGG (forward primer) and GAGTCAATACCGCCAGAATCCA (reverse primer). The mRNA levels were expressed as the ratio between the mRNA level of the target genes and the internal control gene using the comparative 2−∆Ct method.
All the data were expressed as mean ± SD (standard deviation) of means of 3–5 independent experiments for statistical analysis . Two-way ANOVA followed by Tukey HSD test was used to compare the difference in R 3.3.3 (categorical factors concentrations and lengths of MWCNTs); p value < 0.05 was considered to be statistically significant.
Characteristics of XFM22 and XFM19
The physicochemical properties of XFM22 and XFM19 (measured in this study)
BET surface area (m2/g)
Hydrodynamic size (nm)
Zeta potential (mV)
Diameter 17.6 ± 5.6 nm (range 10–30 nm); length 282.1 ± 155.5 nm (range 100–600 nm)
171.4 ± 4.4
0.9 ± 0.1
Diameter 17.4 ± 4.6 nm (range 10–25 nm); length 883.6 ± 501.7 nm (range 200–2000 nm)
190.6 ± 2.4
− 18.9 ± 0.8
Internalization of MWCNTs and ultrastructural changes of HUVECs
For comparison, exposure to up to 32 μg/mL XFI15 did not significantly affect cytotoxicity as assessed by WST-8 and neutral red uptake assays (p > 0.05; Additional file 1: Figure S4).
For comparison, exposure to various concentrations of XFI15 did not significantly affect intracellular GSH or ROS (p > 0.05; Additional file 1: Figure S5).
The uses of MWCNTs in nanomedicine could increase the interactions of human blood vessels with MWCNTs, and it is necessary and urgent to evaluate the toxicity of MWCNTs to endothelial cells. More importantly, it is crucial to assess how the physicochemical properties contribute to MWCNT induced adverse effects in order to design biocompatible MWCNTs [4, 5]. In this study, we investigated the toxicity of MWCNTs of different lengths to HUVECs. The MWCNTs were well-characterized as shorter (average length about 282 nm) and longer MWCNTs (average length about 884 nm; Fig. 2 and Table 2). The shorter MWCNTs showed a larger Zeta potential compared with the longer ones, which could be due to different dispersibility of MWCNTs contributed by the lengths as suggested before [26, 27]. The results indicated that the toxicity of MWCNTs was length dependent that exposure to longer MWCNTs was associated with more morphological changes of HUVECs (Fig. 3) and relatively higher cytotoxicity to HUVECs (Fig. 4). These observations are in agreement with some previous studies showing that longer MWCNTs were more cytotoxic than the shorter ones by using different cell lines [28, 29]. However, it should be noticed that the sensitivity to MWCNTs with different length has also been suggested to be cell-type dependent [30, 31]. But we did not further try to use different types of cells to test this hypothesis. In contrast to MWCNTs, exposure to conductive carbon black was not associated with significant cytotoxicity (Additional file 1: Figure S4) or oxidative stress (Additional file 1: Figure S5), which could indicate a role of fiber-like structure in determining the toxicity of carbonaceous NPs. The longer MWCNTs (XFM19), but not the shorter ones (XFM22), significantly reduced neutral red staining in HUVECs (Fig. 4c and Additional file 1: Figure S3). Our recent studies showed that exposure to ZnO NPs significantly reduced neutral red staining without an effect on cellular viability in macrophages [20, 32]. Previous studies also showed that CNTs could accumulate into lysosomes and induce damages to lysosomes [30, 33, 34]. Therefore, the significantly reduced neutral red staining could indicate that the longer MWCNTs were more toxic to lysosomes in HUVECs.
Previous studies have shown that direct contact of engineered NPs with endothelial cells may promote endothelial activation in vitro [4, 8]. In this study, we also observed that both types of MWCNTs were capable of inducing THP-1 monocyte adhesion (Fig. 8) and release of IL-6 (Fig. 6). This is consistent with previous observations that direct exposure of endothelial cells to carbon-based NPs could induce endothelial activation [15, 35–38]. The results may also support the observations that exposure of laboratory animals to CNTs could impair the function of vascular system [15, 38–40], although the amount of translocation of CNTs into circulation remains unknown. However, in this study we did not find significantly increased release of soluble adhesion molecules after exposure to neither types of MWCNTs (Fig. 7). In our recent studies, we also found that THP-1 adhesion to HUVECs could be induced without the release of soluble adhesion molecules [41, 42]. It remains unclear if MWCNTs induced the expression of adhesion molecules without the release of soluble adhesion molecules, or if MWCNTs promoted monocyte adhesion in an adhesion molecule independent way. Interestingly, the longer MWCNTs induced a stronger response in endothelial activation in terms of THP-1 adhesion as well as release of IL-6 and sVCAM-1 (Figs. 6, 7 and 8). A previous study showed that the longer MWCNTs induced higher VCAM-1 expression in HUVECs in vitro as well as plaque progression in atherosclerotic mice in vivo . However, it should be noticed that the MWCNTs used in previous study had similar characteristics, particularly when they were in suspensions . Herein, by using two well-characterized MWCNTs we clearly showed that MWCNT induced endothelial activation in vitro could be length dependent.
Atherosclerosis is a disease associated with oxidative stress, and exposure to NPs has been suggested to promote endothelial activation through oxidative stress both in vivo and in vitro [6, 43]. To this end we measured intracellular GSH and ROS to indicate oxidative stress, and results showed significantly decreased intracellular GSH particularly after exposure to XFM22. However, ROS was unaltered after exposure to both types of MWCNTs (Fig. 5). Previous studies have found a crucial role of oxidative stress in MWCNT generated health effects, showing as inhibited antioxidant system (i.e., decreased antioxidant levels and inhibited antioxidant enzyme activities) and/or increased ROS . Some studies also showed that the presence of ROS scavenger could alleviate MWCNT induced adverse effects to endothelial cells [35, 45], which further confirmed the role of oxidative stress. It is interesting to notice that the results from this study showed that the longer MWCNTs induced a higher depletion of intracellular GSH (Fig. 5), which may be responsible for higher toxicity of the longer MWCNTs to HUVECs.
It was recently shown that ER stress may be involved in NP-induced toxicity to endothelial cells . ER is a crucial organelle involved in proper function of cells. Perturbation of normal function of ER could lead to ER stress, which has been suggested to mediate endothelial activation in metabolic diseases [17, 46]. To indicate the possible role of ER stress in MWCNT-induced toxicity to HUVECs, we measured the biomarkers of ER stress. It was shown that exposure to XFM19, but not XFM22, was associated with significantly increased expression of ddit3 but not that of xbp-1s or BiP protein level (Figs. 8 and 9). A recent study also showed significantly increased DDIT3 protein level in mouse macrophages after exposure to anodic alumina nanotubes, a kind of engineered NPs with high aspect ratio similar to MWCNTs . ddit3 is a transcription factor that could regulate a number of inflammatory cytokines, such as IL-6 [17, 18]. In our recent study, we also showed that stressing HUVECs with ER stress inducer TG significantly promoted IL-6 release from HUVECs . Thus, the fact that XFM19 provoked a stronger response in IL-6 release observed in this study could be due to the activation of ddit3. However, it should be noticed that compared with a previous report showing up to about 15-fold increase of ddit3 expression in ZnO NP exposed HUVECs , the response of ddit3 observed in this study was more modest. Moreover, the expression xbp-1s and BiP protein level was not significantly increased after exposure to XFM22 or XFM19 (Figs. 9 and 10). This is in contrast to previous reports showing the increase of these ER stress biomarkers in ZnO NP, Au NP and CdTe quantum dot exposed HUVECs [49–51]. Therefore, we proposed that exposure to MWCNTs might only induce a modest activation of ER stress. Co-exposure to TG did not further promote MWCNT-induced THP-1 adhesion to HUVECs (Fig. 8), which indicated that HUVECs with ER stress might not be more sensitive to MWCNT exposure.
In this study, the concentrations of MWCNTs were used from 2 to 32 μg/mL. Some studies investigated the toxicity of CNTs following intravenous administration. For example, Ma et al.  recently showed that intravenous injection of mice with 4 mg/kg CNTs (equals to about 50 μg/mL in blood) disrupted iron homeostasis and induced inflammation. Similarly, Zhang et al.  found that injection with 0.5 mg/kg MWCNTs (corresponds to 6.25 μg/mL) induced immunotoxicity in mice, but the PEGylated MWCNTs were less toxic compared with the pristine ones. In contrast, Tang et al.  and Ahmadi et al.  did not find significant toxicological responses in mice even after intravenous administration of CNTs up to 150 μg/mouse (about 75 μg/mL in blood). The different responses observed in different studies could be contributed by the physicochemical properties of CNTs used. For nanomedicinal studies, Wang et al.  and Kafa et al.  injected mice intravenously with 50 μg/mouse MWCNTs (about 25 μg/mL in blood). Both of the studies showed that MWCNTs could accumulate into brains, which indicated that they could be used for drug delivery and bio-imaging. In a different studies, Antaris et al.  used as low as 4 μg/mouse CNTs (about 3.2 μg/mL in blood) for dual imaging/photothermal therapy. The concentrations used in this study were within the concentrations that might happen in vivo.
In summary, the adverse effects of two well-characterized MWCNTs to HUVECs were investigated in this study, and the results indicated that the toxicity of MWCNTs was length dependent. The longer MWCNTs were more cytotoxic and promoted a stronger response in endothelial activation associated with higher depletion of intracellular GSH and ddit3 expression, which suggested a role of oxidative stress and ER stress. The results also suggested that the shorter MWCNTs may be safer for nanomedicinal applications, and it is necessary to limit the contact of longer MWCNTs with human endothelial cells to avoid the adverse health effects.
JL, LL and YC conceived and designed the study; JL, YX, LL and YC performed the experiments and analyzed the data; LL and YC contributed to reagents/materials/analysis tools; YC wrote the paper; All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.
Consent for publication
We give our consent for the manuscript to be published in Journal of Nanobiotechnology.
Ethics approval and consent to participate
Not applicable for this in vitro study.
This work was financially supported by Natural Science Foundation of Hunan Province (Grant No. 2017JJ3348) and the National Natural Science Foundation of China (Grant No. 21707114).
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