Nitrogen-doped carbon quantum dot regulates cell proliferation and differentiation by endoplasmic reticulum stress

Abstract

Quantum dots have diverse biomedical applications, from constructing biological infrastructures like medical imaging to advancing pharmaceutical research. However, concerns about human health arise due to the toxic potential of quantum dots based on heavy metals. Therefore, research on quantum dots has predominantly focused on oxidative stress, cell death, and other broader bodily toxicities. This study investigated the toxicity and cellular responses of mouse embryonic stem cells (mESCs) and mouse adult stem cells (mASCs) to nitrogen-doped carbon quantum dots (NCQDs) made of non-metallic materials. Cells were exposed to NCQDs, and we utilized a fluorescent ubiquitination-based cell system to verify whether NCQDs induce cytotoxicity. Furthermore, we validated the differentiation-inducing impact of NCQDs by utilizing embryonic stem cells equipped with the Oct4 enhancer-GFP reporter system. By analyzing gene expression including Crebzf, Chop, and ATF6, we also observed that NCQDs robustly elicited endoplasmic reticulum (ER) stress. We confirmed that NCQDs induced cytotoxicity and abnormal differentiation. Interestingly, we also confirmed that low concentrations of NCQDs stimulated cell proliferation in both mESCs and mASCs. In conclusion, NCQDs modulate cell death, proliferation, and differentiation in a concentration-dependent manner. Indiscriminate biological applications of NCQDs have the potential to cause cancer development by affecting normal cell division or to fail to induce normal differentiation by affecting embryonic development during pregnancy. Therefore, we propose that future biomedical applications of NCQDs necessitate comprehensive and diverse biological studies.

Keywords: ER stress; Nitrogen-doped carbon quantum dot; cell proliferation; differentiation.

Figure 1.

Characterization of NCQDs. A schematic presentation of NCQD Production. (B) absorption of NCQDs by UV-vis. (C) fluorescence spectrum of NCQDs under different wavelengths of excitation (360nm–480 nm), and suspension of NCQDs in DW under UV irradiation at 365 nm. (D) Representative HRTEM image of NCQDs. White arrows indicate NCQDs. (E) X-ray photoelectron spectroscopy (XPS) survey spectrum of NCQDs. The three distinctive peaks indicate oxygen, nitrogen, and carbon. (F) High-resolution XPS spectrum of C1s indicates three binding energy peaks as C-C/C = C, C-N/C-O, and C = O in the core of NCQDs. (G) FT-IR spectrum of NCQDs indicate absorption bands of ν(O-H), ν(N-H), and ν(C = O).

Figure 2.

NCQDs affect cell viability and proliferation. A–B CCK-8 analysis results after 48 h of treatment with OG2(A) and MC3T3-E1 cells (B) with NCQDs at various concentrations (10–160) ug/ml; (C) OG2 treated with NCQD at different concentrations for 3 days, and proliferation effects were assessed by comparing PCNA mRNA expression level using q-RT PCR. The graphs of each fold change were drawn using Prism 6 (GraphPad Software). Data was normalized to control and presented as mean ± SEM of N = 3 experiments. (*p < 0.05, **p < 0.01, compared with non-NCQD treated group). (D) Morphology of OG2 cell treated with NCQD by concentration. Scale bar = 20 μm. (E–F) Wound-healing assay after treatment with each concentration of NCQDs into MC3T3-E1 cells and mouse embryonic fibroblast. The boundaries of each wound were indicated in red line. Scale bar = 200 μm

Figure 3.

NCQD promotes proliferation by activating cell cycle. (A) Schematic diagram of FUCCI system. Cells in G1 phase express RFP through the cdt promoter, and cells in S/G2/M phase express GFP through the geminin promoter. (B) FUCCI-trasnfected HEK29FT cells were treated with NCQD at each concentration (-, 10, 20 μg/ml). 3days after NCQD treatment, cells were observed under a fluorescence microscope. Scale bar = 40 μM (C). The graph depicted values obtained by dividing cells expressing either GFP or RFP alone by the total cell count. The graphs of each fold change were drawn using Prism 6 (GraphPad Software). Data was normalized to control and presented as mean ± SEM of N = 3 experiments.

Figure 4.

NCQDs induce differentiation of mESC. (A) OG2 mESC were seeded and maintained on 2i free media to confirm the differentiation on NCQD. Treated with NCQD at varying concentrations, followed by observation under fluorescence microscopy after 3 days. White arrows indicate cell differentiation, disappeared of GFP. Scale bar = 20 μm. (B) The graph shows the measured GFP intensity through ROI in each image. The graph was drawn using Prism 6 (GraphPad Software). Values presented as mean ± SEM of N = 2 experiments.

Figure 5.

Identification differentiation induced by NCQD. OG2 seeded without 2i was treated with NCQD at different concentrations for 3days, and their cDNA was prepared to compare relative mRNA expression. Expression level of pluripotency (A–C) and differentiation marker (D–H) were analyzed by q-RT PCR. The graphs of each fold change were drawn using Prism 6 (GraphPad Software). Data was normalized to control and presented as mean ± SEM of N = 3 experiments. (*p < 0.05, **p < 0.01, compared with non-NCQD treated group, #p < 0.01 compared with undifferentiated ES cells).

Figure 6.

Analysis of ER stress in response to NCQD treatment. To comprehend differentiation induced by NCQD, expression of representative ER stress marker, Crebzf, Chop, ATF6 (A–C) was examined. The graphs of each fold change were drawn by Prism 6 (GraphPad Software). Data was normalized to control and presented as mean ± SEM of N = 3 experiments. (*p < 0.05, **p < 0.01, compared with non-NCQD treated group, #p < 0.01 compared with undifferentiated ES cells).

Figure 7.

Model of the effect of NCQDs in animal cells. NCQDs transferred into cells by endocytosis may cause ER stress through the IRE1 and PERK signaling pathways of the ER membrane. Depending on the concentration of NCQDs, the effect of inducing ER stress increases and reduces the pluripotency of ESCs, leading to unspecific differentiation.