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Journal of Peking University (Health Sciences) ›› 2021, Vol. 53 ›› Issue (3): 439-446. doi: 10.19723/j.issn.1671-167X.2021.03.001

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Effects of length and chemical modification on the activation of vascular endothelial cells induced by multi-walled carbon nanotubes

SHEN Jie,YANG Di,CHEN Meng-yuan,GUO Xin-biaoΔ()   

  1. Department of Occupational and Environmental Health, Peking University School of Public Health, Beijing 100191, China
  • Received:2021-01-07 Online:2021-06-18 Published:2021-06-16
  • Contact: Xin-biao GUO E-mail:guoxb@bjmu.edu.cn
  • Supported by:
    National Natural Science Foundation of China(21477004)

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Abstract:

Objective: To investigate the effects of multi-walled carbon nanotubes (MWCNTs) with different length or chemical modification on endothelial cell activation and to explore the role of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome. Methods: MWCNTs were characterized by dynamic light scattering (DLS) after being suspended in culture medium. The immortalized mouse cerebral microvascular endothelial cell line b.End3 was treated with short MWCNTs (S-MWCNT, 0.5 to 2 μm), long MWCNTs (L-MWCNT, 10 to 30 μm) and the above long MWCNTs functionalized by carboxyl-(L-MWCNT-COOH), amino-(L-MWCNT-NH2) or hydroxyl-(L-MWCNT-OH) modification. Cytotoxicity of MWCNTs in b.End3 cells was determined by cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) release assay, and non-toxic low dose was selected for subsequent experiments. Effects of all types of MWCNTs on the endothelial activation of b.End3 were determined by the measurement of vascular cell adhesion molecule-1 (VCAM-1) concentration in cell supernatant and adhesion assay of human monocytic cell line THP-1 to b.End3.To further elucidate the mechanism involved, the protein expressions of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3) in cells treated with S-MWCNT, L-MWCNT and L-MWCNT-COOH were measured by Western blot. Results: At a higher concentration (125 μg/cm2) and treated for 24 h, all types of MWCNTs significantly inhibited viability of b.End3 cells. At a sub-toxic concentration (6.25 μg/cm2), all types of MWCNTs treated for 12 h significantly induced the activation of b.End3 cells, as evidenced by the elevated VCAM-1 release and THP-1 adhesion. Compared with S-MWCNT, L-MWCNT significantly promoted endothelial cell activation. L-MWCNT and L-MWCNT-COOH activated b.End3 cells to a similar extent. Furthermore, treatment with S-MWCNT, L-MWCNT and L-MWCNT-COOH increased NLRP3 expression in a time-dependent manner at 6.25 μg/cm2. Compared with S-MWCNT, cells treated with L-MWCNT for 4 h and 12 h exhibited significantly increased protein expressions of NLRP3. However, no significant differences were detected in the level of NLRP3 protein in cells treated with L-MWCNT and L-MWCNT-COOH. Conclusion: Compared with the surface chemical modification, length changes of MWCNTs exerted more influence on endothelial cell activation, which may be related to the activation of NLRP3 inflammasome. Our study contributes further understanding of the impact of MWCNTs on endothelial cells, which may have implications for the improvement of safety evaluation of MWCNTs.

Key words: Multi-walled carbon nanotubes, Endothelial cell activation, Length, Inflammasome

CLC Number: 

  • R12

Table 1

The physicochemical information of multi-walled carbon nanotubes (MWCNTs) provided by the manufacturer"

Type Diameter/nm Length/μm Purity/% SSA /(m2/g) Content/%
S-MWCNT 5-15 0.5-2.0 >98 >350 -
L-MWCNT 5-15 10-30 >98 220-300 -
L-MWCNT-COOH 5-15 10-30 >98 220-300 COOH: 3.86
L-MWCNT-NH2 5-15 10-30 >98 >233 NH2: 0.45
L-MWCNT-OH 5-15 10-30 >98 220-300 OH: 5.58

Table 2

Physicochemical characterization of the multi-walled carbon nanotubes (MWCNT)"

Type Dispersive characteristics in DMEM (2% FBS)
Mean hydro-dynamic size/nm Zeta potential/mV PDI
S-MWCNT 229.50±4.14 -11.13±0.06 0.307
L-MWCNT 88.11±1.58 -9.72±0.25 0.291
L-MWCNT-COOH 223.00±3.47 -9.40±0.24 0.286
L-MWCNT-NH2 123.00±0.69 -8.51±0.50 0.280
L-MWCNT-OH 148.93±4.24 -9.40±0.15 0.248

Figure 1

Cytotoxicity of MWCNTs in b.End3 cells Cells were treated with different concentrations of MWCNTs for 24 h, and cell proliferation rate and cell membrane damage were detected by CCK-8 (A) and LDH (B) release assay, respectively. *P<0.05 or ΔP<0.01 vs. controls. #P<0.05 or ▲P<0.01 vs. L-MWCNT group."

Figure 2

Comparison of endothelial activation effects in b.End3 induced by MWCNTs of different lengths After exposure to different concentrations for 24 h, effects of S-MWCNT and L-MWCNT on the endothelial activation of b.End3 were determined by the measurement of soluble vascular adhesion molecule-1 (sVCAM-1) concentration in cell supernatant (A) and adhesion assay of THP-1 cells to b.End3 (B). *P<0.05, ΔP<0.01 vs. untreated controls. # P<0.05, ▲P<0.01 vs. S-MWCNT group."

Figure 3

Comparison of endothelial activation effects in b.End3 induced by MWCNTs of different chemical modification After exposure to different concentrations for 24 h, effects of L-MWCNT, L-MWCNT-COOH, L-MWCNT-NH2 and L-MWCNT-OH on the endothelial activation of b.End3 were determined by the measurement of soluble vascular adhesion molecule-1 (sVCAM-1) concentration in cell supernatant (A) and adhesion assay of THP-1 cells to b.End3 (B).ΔP<0.01 vs. untreated controls."

Figure 4

Time-dependent effects of endothelial activation effects in b.End3 induced by MWCNTs with different length or chemical modification After exposure to 6.25 μg/cm2 MWCNTs for 4 h, 12 h or 24 h, time-dependent effects of S-MWCNTs and L-MWCNTs on the endothelial activation of b.End3 were determined (A, B). Similarly, time-dependent effects of L-MWCNT, L-MWCNT-COOH, L-MWCNT-NH2 and L-MWCNT-OH on the endothelial activation were also evaluated (C, D). sVCAM-1, soluble vascular adhesion molecule-1. *P<0.05, ΔP<0.01 vs. untreated controls. ▲P<0.01 vs. S-MWCNT group."

Figure 5

Effects of length and carboxyl modification of MWCNTs on the expression of NLRP3 in b.End3 cells Cells were treated with 6.25 μg/cm2 S-MWCNT, L-MWCNT or L-MWCNT-COOH for 4 h, 12 h or 24 h, and the protein expression of NLRP3 were determined by western blot analysis. NLRP3, nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3; GADPH, glyceraldehyde 3-phosphate dehydrogenase. *P<0.05,ΔP<0.01 vs. untreated controls. # P<0.05 vs. S-MWCNT group."

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