2021 Volume 46 Issue 6 Pages 273-282
Quantum dots (QDs) are new types of fluorescent nanomaterials which can be utilized as ideal agents for intracellular tracking, drug delivery, biomedical imaging and diagnosis. It is urgent to understand their potential toxicity and the interactions with the toxin-susceptible vascular system, especially vascular endothelial cells. In this study, we intended to explore whether the cytotoxicity of CdTe (cadmium telluride) QDs was partly induced by nitrosative stress in vascular endothelial cells. Our results showed that the intracellular amount of CdTe QDs was gradually increased in a dose- and time-dependent manner, and a concentration-dependent decrease in viability were observed when incubated with CdTe QDs of 20-80 nM. The peroxynitrite level was significantly up-regulated by QDs treatment, which indicated the nitrosative stress was activated. Furthermore, nitrotyrosine level was increased after 24 hr CdTe QDs exposure in a dose-dependent manner, which suggested that CdTe QDs-induced nitrosative stress was associated with tyrosine nitration in EA.hy926. In addition, CdTe QDs induced EA.hy926 apoptosis, and the percentage of cells with low Δψm was increased after CdTe QDs treatment, indicating the mitochondrion depolarization was induced. The increased ROS fluorescence was observed in a QDs dose-dependent manner, which suggested that the oxidative stress was also involved in the CdTe QDs-induced endothelial cytotoxicity. Our work provided experimental evidence into QDs toxicity and potential vascular risks induced by nitrosative stress for the future applications of QDs.
Quantum dots (QDs) are new types of fluorescent nanomaterials and utilized as ideal agents for intracellular tracking, drug delivery, biomedical imaging and diagnosis (Jahangir et al., 2019; Gil et al., 2021). It has been known that QDs offer unique luminescent advantages over existing fluorescent dyes, which make them useful for applications in biological and medical fields (Kamila et al., 2016). For example, QDs have excellent photoluminescence quantum yield, greater photostability, broader excitation ranges with narrower emission spectra, and size-tunable fluorescent peaks. Besides these exceptional properties, shell coatings may also be useful for attaching conjugates to trace therapeutic and diagnostic macromolecules, receptor ligands, or antibodies (Wang et al., 2018; Babu and Paira, 2017; Zhou et al., 2015; Alaghmandfard et al., 2021).
Over the past decade, with increasingly large scale production and application, the likelihood of human exposure to QDs is highly possible (Utkin, 2018). In particular, these nanocrystals have been found in environments as degradation products. To date, the potential risks of QDs on human health have not been well explored yet, as our knowledge of QDs in vitro and in vivo toxicities remain elusive (Hu et al., 2021). The systematic toxicity evaluation of QDs is of critical importance for their practical biological and biomedical applications. Although the potential toxicity of QDs remains unresolved in biomedical applications and a challenge for clinical studies thus far, a number of toxicological studies on QDs have been carried out for this purpose (Zou et al., 2019; Mirnajafizadeh et al., 2019; Li et al., 2020).
Accumulating evidence implicates that the exposure pathway of QDs is related to the target of toxic effects (Paesano et al., 2016). In the application process, QDs often entered the body through the skin, respiratory system, or blood vessels. Also, the equivalent sub-points are used for in vivo fluorescent imaging agents or drug tracers, usually with intravenous injection (Zou et al., 2019). It has been reported that most QDs can penetrate the skin after 24 hr of perfusion, and some of the QDs penetrated into the dermis even within 8 hr (Volkova et al., 2018). In these approaches, endothelial cells are identified as the first barrier to blood vessels and play a vital role in maintaining vascular homeostasis. The QDs directly contact with the endothelial cells on the inner surface of the vascular lumen, which may cause damage to endothelial cells, and further injure tissues and organs through blood circulation (Yan et al., 2016). Therefore, to safely use QDs, it is urgent to understand their potential toxicity and the interactions with the toxin-susceptible vascular system, especially endothelial cells, as the surface of blood vessels may be the primary attack sites during QDs-caused vascular injury.
Growing evidence indicates that endothelial cells are the primary targets of nitrosative stress in cell damage (Tao et al., 2014). In the process of nitrosative stress, peroxynitrite (ONOO-) is a highly reactive nitrogen species generated from the reaction between nitric oxide (NO) and superoxide (O2.-) under pathological conditions, which exert a contributory effect by participating in nitrating tyrosine signaling (Maiti et al., 2017). It has been demonstrated that ONOO- is more frequently regarded as deleterious due to its nitrosative damage to lipids, proteins and DNA (Wen et al., 2015). What is more, ONOO- has been implicated in various redox-related diseases (Chandrashekaran et al., 2017; ben Anes et al., 2014). Despite significant advances in understanding of the pathological role of ONOO-, the relationship of CdTe (cadmium telluride) QDs and ONOO- to the cytotoxicity of endothelial cell damage still remains to be established.
In this study, we sought to investigate the potential endothelial nitrosative stress cytotoxicity of CdTe QDs. We applied biochemical and fluorescence evaluation to characterize the involvement of nitrosative stress cytotoxicity in human endothelial cells. Cytotoxicity of CdTe QDs was evaluated by CCK8 methods. Confocal microscopy and flow cytometry were used to investigate CdTe QDs-induced peroxynitrite. The expression of nitrotyrosine was analyzed by western blotting. Our work provided experimental evidence and new insights into CdTe QDs toxicity and revealed the potential vascular risks of nitrosative stress for the future applications of QDs.
Human umbilical vein endothelial cell line, EA.hy926 was purchased from American Type Culture Collection (ATCC) and cultured as previously reports (Li et al., 2015). Briefly, EA.hy926 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), penicillin (100 U/mL, Thermo Fisher Scientific), and streptomycin (100 U/mL, Thermo Fisher Scientific). The cultures were maintained at 37°C in a 95% humidified atmosphere with 5% CO2.
Cell viability detected by CCK8 assayThe CdTe QDs used in this research were obtained from Zhongke Wuyan company (Beijing, China). The EA.hy926 cells were treated with several concentration of CdTe QDs (0, 20, 40, 60, 80, 120, 160 nM), which were allowed an incubation period of 24 hr. The cytotoxicity was evaluated by morphological observation of cells under bright-field microscopy and CCK8 assay (Beyotime Institute of Biotechnology, Suzhou, China). After introducing 10 μL of CCK8 solution for 1 hr, the absorption at 450 nm was measured by Microplate Spectrophotometer (MD I3X). Each experiment was repeated three times and the average values were taken in analyses.
The uptake of CdTe QDs by EA.hy926The uptake efficiency of the EA.hy926 cells treated with CdTe QDs was determined by flow cytometry analysis. For analysis of the dose-dependent uptake of CdTe QDs by EA.hy926 cells, CdTe QDs of 20 nM, 40 nM, 80 nM, 120 nM and 160 nM were added to EA.hy926 cells and incubated for 24 hr. For analysis of the time-dependent uptake of CdTe QDs, cells were treated with 40 nM CdTe QDs for 1 hr, 3 hr, 6 hr, 12 hr, 24 hr and 48 hr. After that, cells were washed with PBS three times to remove the CdTe QDs. Then the cells were harvested and resuspended in 500 μL PBS, analyzed by flow cytometry (BD-FACS-Verse). The fluorescence was monitored at 488 nm. Each plot represented 10,000 viable cells, and non-viable cells were excluded by appropriate gating. All data analyses were carried out using FCS express V3.0.
Fluorescence evaluation of ONOO- level in EA.hy926NP3 is a fluorescent switch-on probe, which can be used to examine the ONOO- formation, as previously reported (Li et al., 2015). For flow cytometry analysis, EA.hy926 cells were first stained with 1 μM NP3 at 37°C for 30 min, and then treated with 20 nM, 40 nM, 60 nM and 80 nM CdTe QDs for 1 hr. The fluorescence of NP3 was monitored at 500-550 nm (λex = 405 nm) by flow cytometry (BD-FACS-Verse). Each plot represented 10,000 viable cells, and non-viable cells were excluded from flow cytometry analysis by appropriate gating. For confocal fluorescent imaging experiments to evaluate NP3 in EA.hy926 cells upon CdTe QDs treatment, EA.hy926 cells were first stained with 1 μM NP3 at 37°C for 30 min. The residual probe was washed three times by PBS, and then treated with 40 nM CdTe QDs for 1 hr. The fluorescence was monitored by laser scanning confocal microscope fluoview FV1000 (Olympus, Tokyo, Japan) at λem = 500-550 nm (λex = 405 nm). Digital images were captured using the FV10-ASW 3.0 viewer software (Olympus). Cell counts were performed using a 60 × objective in at least five fields of view randomly selected from each culture dish. At least three independent experiments were counted. All data analyses were carried out using Image J software (NIH, Bethesda, MD, USA).
Cell apoptosis detection by flow cytometry analysisAnnexin V-fluorescein isothiocyanate (FITC)-propidium iodide (PI) Apoptosis kit (Beyotime Institute of Biotechnology) was used to test cell apoptosis. Briefly, the EA.hy926 cells were seeded into 6-well plates at the density of 1 × 105 cells/well. After the cells well attached, 40 nM CdTe QDs was added to the wells and incubated for 24 hr. Then the cells were harvested and resuspended at a density 1 × 106 cells/mL in 500 μL binding buffer. According to the instruction of kit, 5 μL Annexin V-FITC was added to the samples and incubated at 4°C for 15 min. followed by staining with PI for 5 min. After that, the cells were resuspended in 500 μL PBS and analyzed by flow cytometry (BD-FACS-Verse). The fluorescence was monitored at 488 nm and 405 nm. Each plot represented 10,000 viable cells, and non-viable cells were excluded by appropriate gating. All data analyses were carried out using FCS express V3.0.
Reactive oxygen species (ROS) assayThe level of intracellular ROS was determined with a Reactive Oxygen Species Assay Kit (Beyotime Biotechnology). Briefly, EA.hy926 cells were seeded into 6-well plates at the density of 1 × 105 cells/well. After 24 hr of CdTe QDs treatment, cells were incubated with 10 μL DCFH-DA at 37°C for 30 min. The samples were washed with PBS for 3 times and resuspended in 500 μL PBS. The fluorescence was monitored at 488 nm by flow cytometry. Each plot represented 10,000 viable cells, and non-viable cells were excluded by appropriate gating. All data analyses were carried out using FCS express V3.0.
Evaluation of mitochondrial membrane potentialJC1 staining was used to assess the mitochondrial membrane potential (Δψm) after CdTe treatment in EA.hy926. Briefly, EA.hy926 cells were seeded into 6-well plates at the density of 1 × 105 cells/well. After 24 hr of CdTe QDs treatment, cells were stained with JC1 for 10 min at 37°C. The samples were washed with PBS for three times and resuspended in 500 μL PBS. The fluorescence was monitored at 488 nm and 535 nm by flow cytometry. Each plot represented 10,000 viable cells, and non-viable cells were excluded by appropriate gating. All data analyses were carried out using FCS express V3.0.
Western blotting analysisCells were collected and total protein was extracted after lysing with RIPA buffer (containing 0.2% Triton X-100, 5 mmol/L EDTA, 1 mmol/L PMSF, 10 mg/mL leupeptin, 10 mg/mL aprotinin, added with 100 mmol/L NaF, and 2 mmol/L Na3VO4) and lysing 30 min on ice, and then quantified by BCA protein assay kit (Beyotime Biotechnology). Equal amounts of each sample protein was separated by 8% sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE) and transferred to a 0.45 μm pore size positively charged Polyvinylidene Fluoride (PVDF, Merck, Darmstadt, Germany) and blocked with 5% dry milk in PBS with 0.1% Tween-20 at room temperature. Immunoblotting was carried out with an anti-nitrotyrosine antibodies (Cell Signaling, Boston, MA, USA) in blotto overnight at 4°C, followed by washing three times with PBST (0.1% Tween-20), and challenged with HRP-conjugated goat anti-rabbit secondary antibody (1:1000) in PBST for 1 hr at room temperature. Then the samples were washed with PBST three times, followed by detection with an enhanced chemiluminescent substrate (Beyotime Biotechnology). All experiments were performed for three replicates.
Statistical analysisSPSS 19 software was used for statistical analyses. All data were presented as the mean ± S.E.M. for the indicated number of separate experiments. Three independent experiments were carried out for each study. Comparisons of differences in the quantitative data among groups were used One-way ANOVA analysis. P < 0.05 was considered to indicate a statistically significant difference.
CdTe QDs with carboxylic acid groups were used in this study. The diameter of the QDs was approximately 4 nm (Fig. S1A). The emission spectrum scanning results revealed that the emission maximum was near 625 nm with a full width at half maximum around 50 nm (Fig. S1B). We first determined the cytotoxic effects of CdTe QDs on endothelial cells, using a range of CdTe QDs concentrations. Briefly, EA.hy926 cells were incubated with several concentration of CdTe QDs (0, 20, 40, 60, 80 nM) for 24 hr, and the cytotoxicity was evaluated by morphological observation of cells under bright-field microscopy and CCK8 assay. As the results shown in Fig. 1, the cells treated with CdTe QDs exhibited morphological changes with more indistinct intercellular boundary, compared with the control cells. In addition, the cells showed a concentration-dependent decrease in viability up to 160 nM with prolonged treatment time of 24 hr. The TC50 values after 24 hr exposure to EA.hy926 cells were 40 nM. This indicated that the CdTe QDs had obvious cytotoxicity on endothelial cells, even at a very low concentration.
The cytotoxicity study of CdTe QDs was assessed in human endothelial cells EA.hy926. CdTe QDs of 20 nM, 40 nM, 60 nM and 80 nM were added to cells, and they were allowed to incubate for 24 hr. The cell phenotype was captured by inverted microscope. After introducing CCK8 solution for 1 hr, the absorption at 450 nm was measured by Microplate Spectrophotometer (MD I3X). Scale bar = 25 μm.
To determine the intracellular uptake of CdTe QDs in endothelial cells, EA.hy926 cells were incubated with different concentrations of QDs solution (20-160 nM), and the cellular concentration of QDs was determined using flow cytometry analysis. As shown in Fig. 2, the intracellular amount of CdTe QDs was increased in a dose-dependent manner linearly with incubation concentrations from 20 nM to 160 nM (Fig. 2). Meanwhile, we evaluated the uptake efficiency of EA.hy926 cells treated with 40 nM CdTe QDs for different time, and the similar results that a time-dependent cellular uptake of QDs shown in Fig. S2 was observed. These suggested that CdTe QDs were able to enter the endothelial cells in the dose- and time-dependent manner.
The uptake efficiency of the EA.hy926 cells treated with CdTe QDs determined by flow cytometry analysis. (A, B) CdTe QDs of 20 nM, 40 nM, 80 nM, 120 nM and 160 nM were added to cells and incubated for 24 hr. Representative pictures of the flow cytometry analysis. (C) Quantification of fluorescence intensity in B. The fluorescence of CdTe QDs was monitored at 615-645 nm (λex = 488 nm). Each plot represented 10,000 viable cells (non-viable cells were excluded by appropriate gating). Data were expressed as mean ± S.E.M., ***P < 0.001 versus control.
It has been demonstrated that peroxynitrite (ONOO-) is more frequently regarded as deleterious due to its nitrosative damage to lipids, proteins, and DNA. What is more, ONOO- has been implicated in various redox-related diseases. Therefore, to evaluate the ONOO- level in the EA.hy926 cells treated with CdTe QDs, confocal microscopy and flow cytometry were used to evaluate the ONOO- level. We employed the ONOO- specific probe NP3, which was developed for the evaluation of nitrosative stress as previously reported (Li et al., 2015). SIN-1 (without 3-morpholinosydnonimine), a donor of ONOO-, was employed as a positive control. As shown in Fig. 3, significant increase of intracellular NP3 fluorescence in EA.hy926 was observed after CdTe QDs treatment, which was blunted by treatment with 200 μM uric acid (UA), an ONOO- scavenger (Hooper et al., 1998). This result was further confirmed by flow cytometric analysis (Fig. 3C, 3D). It is noteworthy that even a low dose of CdTe QDs (20 µM) could enhance the intracellular fluorescence of NP3, indicating that ONOO- level was up-regulated by CdTe QDs treatment, which indicated that the nitrosative stress was activated. Also, the results supported the notion that ONOO- production represents an early marker for nitrosative stress in this cellular system.
Involvement of nitrosative stress induced by CdTe QDs in EA.hy926 cells. (A) Flow cytometry evaluation of nitrosative stress related ONOO- level in the EA.hy926 cells treated with CdTe QDs. Representative pictures of the flow cytometry analysis. The fluorescence of NP3 was monitored at 500-550 nm. (B) Quantification of fluorescence intensity by flow cytometry analysis. (C) Imaging evaluation of ONOO- level in the EA.hy926 cells treated with CdTe QDs. (D) Quantification of image data. A total of 10 cells from a minimum of 3 images for each condition were quantified and averaged. The fluorescence of NP3 was monitored at 500-550 nm. Scale bar = 10 μm. (E, F) Western blotting analysis of nitrotyrosine expression in the EA.hy926 cells upon CdTe QDs treatment. Data were presented as the mean ± S.E.M. of independent experiments performed in triplicate. Statistical significance was set as ***P < 0.001 versus control.
It has been demonstrated that peroxynitrite modifies free tyrosine and tyrosine residues in proteins, which accounts for the effects of endogenously produced ·NO by oxidation and nitration reactions. To further investigate the nitrotyrosine expression following CdTe QDs treatment in endothelial cells, we evaluated the nitrotyrosine expression in the EA.hy926 cells upon CdTe QDs treatment. As shown in Fig. 3E, 3F, significant protein tyrosine nitration increased in a CdTe QDs dose-dependent manner after 24 hr exposure, as detected by Western blot for nitrotyrosine. Our data also revealed that protein tyrosine nitration of a wide range of proteins was significantly increased upon CdTe QDs (60 nM) treatment. Based on these results, data from this part of our study provided convincing evidence that CdTe QDs-mediated endothelial oxidative stress was associated with tyrosine nitration in endothelial cells.
Evaluation of apoptosis and Δψm in the EA.hy926 treated with CdTe QDsThe apoptosis that induced by CdTe QDs in endothelial cell was evaluated by flow cytometry analysis. As shown in Fig. 4A, apoptosis induced by CdTe QDs at the indicated times was quantified and the results showed that CdTe QDs induced significant apoptosis at 24 hr in EA.hy926 cells. The loss of mitochondrial membrane potential (Δψm) indicated the mitochondrial dysfunction which was sensitive to the changes in cellular redox state, and leaded to caspase-dependent cytotoxicity and downstream apoptotic signaling. In order to determine the cause for reduced mitochondrial activity after CdTe QDs treatment (40 nM), we analyzed cells using the mitochondrial membrane potential sensing dye JC1. The results showed a representative time course for the uptake of JC1 by viable endothelial cells, as measured by the increase in monomer and aggregate fluorescence. The temporal changes of Δψm endothelial cells were quantified as seen in Fig. 4B. In contrast to control cells, we found that the percentage of cells with low Δψm was about 20% and 40% in 1 hr and 3 hr, respectively, indicating that the mitochondrion depolarization was induced after CdTe QDs treatment.
Evaluation of mitochondrial membrane potential (Δψm) and apoptosis after CdTe QDs treatment in EA.hy926 cells. (A) CdTe QDs induced apoptosis in EA.hy926 cells. Top: control group without CdTe QDs treatment. Bottom: EA.hy926 cells incubated with 40 nM CdTe QDs. Samples were analyzed on the flow cytometry with 488 nm and 405 nm excitation. (B) Evaluation of Δψm by JC1 staining. Top left: Control group without CdTe QDs treatment. Top right: The EA.hy926 cells incubated with CdTe QDs for 1 hr. Bottom left: The EA.hy926 cells incubated with CdTe QDs for 3 hr. Samples were analyzed on the flow cytometry with 488 nm and 535 nm excitation. Bottom right: Quantification of Δψm by FCM was expressed as the ratio between monomeric and J-aggregate fluorescence (Red/Green).
We next investigated the ROS production following CdTe QDs treatment. The production of ROS was evaluated by the fluorogenic probe DCFH-DA, which forms a fluorescent product when it is oxidized by ROS generated in the cells. As the results shown in Fig. 5, the increased ROS fluorescence was observed in endothelial cells in a CdTe QDs dose-dependent manner, and was significantly increased up to the 80 nM point, which suggested that the oxidative stress was also involved in the CdTe QDs-induced endothelial cytotoxicity.
Flow cytometry evaluation of ROS level in the EA.hy926 cells treated with CdTe QDs. (A) Representative pictures of the flow cytometry analysis. EA.hy926 cells were first stained with 1 μM DCHF-DA at 37°C for 30 min, and then treated with 20 nM, 40 nM, 60 nM and 80 nM CdTe QDs for 1 hr. The fluorescence of DCHF-DA was monitored at 500-550 nm (λex = 488 nm). (B) Quantification of fluorescence intensity by flow cytometry analysis. Each plot represented 10,000 viable cells (non-viable cells were excluded by appropriate gating). Data were expressed as mean ± S.E.M., n = 3. *** P < 0.001 versus control.
The toxicity of QDs is the primary factor that influences the biomedical application of QDs. Thus, it is essential to evaluate their toxicity systematically, and herein the endothelial cell nitrosative stress cytotoxicity induced by CdTe QDs in human endothelial cells was investigated. For the first time, we addressed the pathophysiological relevance of CdTe QDs-induced nitrosative stress following prolonged CdTe QDs stimulation in cultured endothelial cells. We provided several pieces of evidence showing the toxic effects of CdTe QDs at nanomolar concentrations in human endothelial cells: (i) CdTe QDs induced cytotoxicity of nitrosative stress in human endothelial cells, as indicated by the up-regulation of peroxynitrite (ONOO-) level after CdTe QDs treatment, and CdTe QDs-induced endothelial nitrosative stress was associated with tyrosine nitration; (ii) Both nitrosative stress and oxidative stress were involved in the endothelial cytotoxicity induced by CdTe QDs treatment, which contributed to the mitochondrion depolarization and apoptosis.
It has been reported that the pathophysiological relevance of increased nitrosative stress and the mitochondria-dependent apoptotic cascade contributed to diverse cellular stresses in the pathogenesis of endothelial dysfunction (Diers et al., 2013). ONOO- is a highly reactive nitrogen species generated from the reaction between nitric oxide (NO) and superoxide (O2.-) under pathological conditions, which has been implicated in various redox-related diseases. Despite significant advances in understanding of the pathological role of ONOO-, the cytotoxicity of ONOO- in endothelial cell damage remains to be established. In this study, our results showed that nitrosative stress was observed in the endothelial cells treated with CdTe QDs, as indicated by the up-regulated ONOO- even at the low concentration (20 nM) of CdTe QDs. It suggested that nitrosative stress might play an important role in the CdTe QDs-induced endothelial cytotoxicity.
Mounting evidence suggests that vascular endothelial cells are attractive from an organic targeting point of view (Duan et al., 2019). Indeed, blood is the first barrier after intravenous nanoparticle application, and endothelial cells transport systems are present throughout the dense network of heart, lung and brain capillaries. Thus, endothelial cells are the primary attack sites during QDs-caused vascular injury. Until now, several lines of studies have been conducted to explore the endothelial cytotoxicity induced by different types of QDs, including CdTe, CdSe, CdTe/ZnS, e.g., and the involvement of ROS associated with oxidative stress has been widely demonstrated. For example, Yan et al. (2016) showed that CdTe QDs could be internalized by HUVECs via both caveolae/raft- and clathrin-dependent endocytosis. Wang et al. (2016) found that QDs influenced endothelial progenitor cell viability and function. Hilger et al. revealed that the cytotoxicity of endothelial cells could be induced by QDs with different nanoparticle structures and functionalization (Landgraf et al., 2015). These studies demonstrated the important role of the biocompatibility of nanoparticles on endothelial cells, which could be concerning with its intended application. In the present study, our findings provided evidence that CdTe QDs-mediated endothelial cytotoxicity was associated with nitrosative stress in EA.hy926 cells, which replenished the systematic cytotoxicity of CdTe QDs during their applying.
Herein, we also demonstrated that CdTe QDs were capable of up-regulating the overabundant intracellular ROS level in endothelial cells, in agreement with the previous studies (Zhang et al., 2015). A number of studies showed that QDs-induced oxidative stress was commonly observed (Duan et al., 2019; Zhang et al., 2015). Lovrić et al. (2005) reported that the toxicity of QDs in PC12 cells had shown involvement of ROS, such as hydrogen peroxide and various hydroperoxide radicals, which impaired the plasma membrane, nucleus, and mitochondria, leading to severe cell dysfunction. Furthermore, they showed that QDs-induced cell damage could be partially prevented or recovered by strong antioxidants, suggesting that ROS was involved in QDs-induced toxicity. Furthermore, we also measured the potential injuries of mitochondrial functions in CdTe QDs treated EA.hy926 and showed the obvious disruption of mitochondrial membrane potential after 24 hr CdTe QDs incubation, suggesting that CdTe QDs-induced stress was tightly associated with their mitochondrial toxicity. Thus, from combination with the results that nitrosative stress cytotoxicity could be induced by CdTe QDs, we concluded that both nitrosative stress and oxidative stress contributed the cytotoxicity of CdTe QDs, and mitochondria may be a major target organelle.
Meanwhile, unlike other cells, vascular endothelial cells obtain most of their energy from anaerobic glycolysis, and the collapse of endothelial mitochondrial membrane potential inhibits mitochondrial production of NO, which can rapidly modulate cellular functions and apoptosis (Giri et al., 2019). It has been demonstrated that the pathophysiological relevance of increased nitrosative stress and the mitochondria-dependent apoptotic cascade contributed to diverse cellular stresses in the pathogenesis of endothelial dysfunction (Han et al., 2011). Therefore, CdTe QDs-induced nitrosative stress damage in endothelial cells may represent an important step in the development of endothelial dysfunction rather than a key role in diminishment of cellular energy production in other cells.
In summary, this is the first study designed to systematically determine the involvement of nitrosative stress cytotoxicity induced by CdTe QDs in human endothelial cells. Although other activation mechanisms such as ROS may be required for complete activation of apoptosis in CdTe QDs-induced cytotoxicity, our current findings indicate that signals from nitrosative stress are critical for triggering of QDs toxicity during the process of CdTe QDs treatment. These findings will greatly help not only when evaluating and assessing the safety of QDs, but also reveal potential nitrosative stress cytotoxicity of nanoparticles in their applications. The data derived from the present study also suggested that ONOO- targeting may be an important strategy to prevent the QDs toxicity and revealed potential vascular risks for the future applications of QDs.
This work was supported in part by Natural Science Foundation of Zhejiang Province (LQY18H310001), Medical Health Science and Technology Project of Zhejiang Provincial Health Commission (2018KY383, 2019RC164).
Conflict of interestThe authors declare that there is no conflict of interest.
