Selenium-Sulfur-Doped Carbon Dots with Thioredoxin Reductase Activity

Open AccessCCS ChemistryCOMMUNICATION14 Jul 2022Selenium-Sulfur-Doped Carbon Dots with Thioredoxin Reductase Activity Luo Zhang, Chenxing Sun, Yizheng Tan and Huaping Xu Luo Zhang Key Lab of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084 Tsinghua-Peking Joint Center for Life Sciences, Beijing 100084 Google Scholar More articles by this author , Chenxing Sun Key Lab of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084 Google Scholar More articles by this author , Yizheng Tan Key Lab of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084 Google Scholar More articles by this author and Huaping Xu *Corresponding author: E-mail Address: [email protected] Key Lab of Organic Optoelectronics and Molecular Engineering, Department of Chemistry, Tsinghua University, Beijing 100084 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.021.202101203 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail Thioredoxin reductase (TrxR) is an essential enzyme for regulating the redox balance in cells, promoting cell proliferation, and inhibiting cellular apoptosis. The biochemical pathway of TrxR metabolism involves a series of selenium-sulfur (Se-S) dynamic chemistry. Theoretically, nanomaterials with Se-S dynamic bonds could exhibit TrxR-mimetic activities to tune TrxR activity, affecting cellular activities. Herein, we report the fabrication of Se-S-doped carbon dots (Se-S-CDs), synthesized by a facile hydrothermal method. The Se-S-CDs exhibited good stability and solubility in an aqueous solution, with a quantum yield of 13.27%. The doping of Se-S bonds endowed the Se-S-CDs with great capability of enhancing the TrxR activity, and consequently, a remarkable promotion of cell viability. The significance of Se-S bonds, as well as CDs’ role as matrices, are discussed. Moreover, the Se-S-CDs could also revive the cells from the damage induced by oxidative stress. The above-mentioned properties demonstrated the potential of the Se-S-CDs for cells and tissues culturing, solving the poor cell viability issue, which is desperately needed for organ transplantation and revitalization of prematurely aging cells, especially those exposed to oxidative stress. Download figure Download PowerPoint Introduction Thioredoxin reductase (TrxR) is a selenoprotein1 that has attracted much interest, as it intimately links to many cellular events, including regulating the intracellular redox balance, promoting cell proliferation, and inhibiting cellular apoptosis.2–4 The selenocysteine residue (Sec) and cysteine residue (Cys) conserved in a C-terminal (–Gly–Cys–Sec–Gly) of TrxR constitute the redox-active site of the enzyme and had been proved to be essential for its catalytic activity and functions.5,6 A wide variety of investigations have been conducted toward a series of selenium-sulfur (Se-S) dynamic chemical conversions of TrxR.7–10 Despite the success of the studies on the structure and mechanism of TrxR, researchers mainly focused on inhibiting the TrxR activity to breaking the intracellular redox balance, aimed at inducing cellular apoptosis as a strategy for cancer therapy.11–15 However, the benefit of promoting cellular proliferation by TrxR was scarcely reported, which can be applied to cells and tissues culturing to solve poor cell viability issues, crucial for organ transplantation. To this end, the discovery of artificial nanozymes opens opportunities for enhancing the enzymatic properties of cells.16–18 Compared with natural enzymes, nanozymes have higher operational stability, lower cost, facile synthesis, and great robustness against stringent conditions.19–22 A lot of nanoparticles exhibit enzymatic activities; these include Fe3O4 nanoparticles, noble metal nanoparticles, transition-metal dichalcogenide nanoparticles, and carbon nanomaterials.19,23–25 Among the nanomaterials, zero-dimensional carbon dots (CDs) have been proved to be promising candidates for bioapplications, attributed to the low cytotoxicity, high water solubility, excellent biocompatibility, and sufficient functional groups on the surface for modifications.26–30 New synthetic approaches or modifications for CDs have been constantly developed with growing interest.31–38 Particularly, heterogeneous atoms doping has been documented as an effective method for impacting the intrinsic attributes of CDs by interfering with the overall electrical distribution, contributing to the functional maturation of CDs toward non-toxic metal-free catalysts for biochemical processes.23,39–42 For instance, Huang and Qu groups43–45 reported peroxidase-like activity of CDs and used it as antibacterial agents. Chen et al.46 enhanced the peroxidase-like activity by doping iron and nitrogen atoms into CDs. Our group47 reported the successful synthesis of Se-doped CDs with peroxidase mimicking activity for free-radical scavenging. Wu et al.48 synthesized nitrogen-doped CDs as promising oxidase-mimicking nanozyme for photodynamic antimicrobial chemotherapy. Recently, Yang et al.49 demonstrated a topoisomerase I-like activity of the cysteine-derived CDs for mediating topological rearrangement of supercoiled DNA. Herein, in virtue of the special adventure of CDs, together with the Se-S dynamic chemistry pertaining to the active site of TrxR, we report a new enzymatic Se-S-doped CDs (Se-S-CDs) by a facile and robust hydrothermal method. The Se-S bonds doped into the CDs, as well as anchored on the surface of the CDs, making it hypothetically possible to complex with the intracellular components30 for simulating the activity of TrxR and dynamically regulating the intracellular microenvironment, thereby altering specific cellular activities. The Se-S-CDs exhibited uniform size distribution of 5–8 nm and an improved quantum yield of 13.27%. Unlike the Se-doped CDs,47 S-doped CDs,50 or any other dichalcogenide quantum dots,24 the Se-S-CDs could significantly promote cell viability via enhancement of TrxR activity, attributable to the doping of Se-S bonds and the CDs as the matrix. Additionally, the Se-S-CDs could also revive cell damage caused by oxidative stress by enhancing the redox balancing property of TrxR. Collectively, the Se-S-CDs showed promising potential for a series of bioapplications, including rapid cell culturing, tissue culturing for skin grafting, hence, solving the poor cell viability issue after harvesting for organ transplantation, and revitalization of premature aging cells, especially those exposed to oxidative stress. Results and Discussion In a typical synthesis, we mixed selenocystine and cysteine (two essential amino acid residues in TrxR) in a basic solution via vigorous vortexing. Amid reaction, a new amino acid containing Se-S dynamic covalent bond (Sec-Cys mimic) was formed via Se-S metathesis. The products provided a carbon source for the subsequent carbonization process (Scheme 1). The formation of the Se-S containing amino acid was confirmed by mass spectra. This newly formed amino acid could be detected throughout the carbonization process, demonstrating that the Se-S dynamic covalent bond continuously and stably participated in the synthesis at 60 °C ( Supporting Information Figure S1). The Se-S-CDs were successfully synthesized via a mild hydrothermal process and collected after dialysis purification and lyophilization (see Supporting Information for details). Scheme 1 | Schematic diagram of the synthetic route for Se-S-CDs. The spontaneous Se-S metathesis happens when mixing aqueous l-Cysteine and l-Selenocystine solutions via vortex at room temperature, forming the Se-S dynamic covalent bond containing amino acid. The Se-S-CDs were obtained by heating Se-S containing amino acid solution at 60 °C for 48 h. Download figure Download PowerPoint The existence of Se-S bonds in the Se-S-CDs was confirmed by X-ray photoelectron spectroscopy (XPS) spectra (calibrated by the peak position of the C–C bond; Supporting Information Figure S2).51 For the Se3d, we found that the original Se-Se, 55.3 eV in the selenocystine, ( Supporting Information Figure S3a) split into two peaks, Se-Se (55.2 eV) and Se-S (56.1 eV) upon mixing (Figure 1a), manifesting the Se-S metathesis, which well-matched the electrospray ionization mass spectrometry (ESI-MS) result ( Supporting Information Figure S1c). The Se-Se (55.3 eV) and Se-S (56.0 eV) were also identifiable in the final Se-S-CDs, and the ratio of Se-S and S-S was calculated to be 7:3 based on the deconvoluted integral area of Se3d (Figure 1b). For the S2p, the peak position of the C-S bond was eventually shifted from 163.0 eV (cysteine) to 163.4 eV (Se-S-CDs), which was indexed to the C-S-Se bond (Figure 1c and 1d), with a calculated ratio of S-Se and S-S equal to 7:3. Taken together, the results validated the successful doping of Se-S dynamic covalent bonds in the CDs. Figure 1 | XPS spectroscopy analysis: Se3d of (a) the mixture of cysteine and selenocysteine and (b) Se-S-CDs, and S2p of (c) the mixture of cysteine and selenocysteine and (d) Se-S-CDs. Download figure Download PowerPoint We further employed UV–vis spectroscopy and Fourier transform infrared (FT-IR) spectroscopy to differentiate Se-S-CDs from the single element doped CDs (Se-CDs47 and S-CDs).50 From the UV–vis spectra ( Supporting Information Figure S4a), we recognized the apparent changes in the absorbance peak position between the three CDs. The peak position for the π–π* transition of the aromatic sp2 core was shifted to 270 nm in Se-S-CDs, and the n–π* transitions from the surface-/molecule-state region exhibited broad and blurry peak centering at 350 nm.52 Also, the different bonding structures could be identified in the FT-IR spectra ( Supporting Information Figure S4b). The spectra between the three CDs differed, especially in the range of 1400–900 cm−1, ascribed to the different vibrations of C-S and C-Se bonds.53 The distinct O-H and N-H stretching modes (3600–3100 cm−1) indicated that the hydroxyl and amino groups enriched the outer surface of the CDs, facilitating their water solubility and functionalization.54 The morphology and the size distribution of the Se-S-CDs were characterized by images obtained from transmission electron microscopy (TEM) and size measurements by dynamic light scattering (DLS). The Se-S-CDs were nanospheres with a significant size distribution of 5–8 nm (Figure 2a); the nanoscale particles embrace a larger specific surface area. In contrast to the rigorous storage conditions for the natural enzyme, the Se-S-CDs exhibited high stability (up to 300 days) in aqueous solution under ambient conditions ( Supporting Information Figure S5a) and robustness to harsh environments ( Supporting Information Figure S5b). The lattice of the Se-S-CDs was characterized by high-resolution TEM (Figure 2b) together with selected area electron diffraction (SAED; Supporting Information Figure S6). The interplanar spacing of 0.34 nm could be indexed to the (002) facet of graphene, demonstrating that the core of the Se-S-CDs consisted of graphene fragments (a typical structure of CDs).55–58 We further investigated the height of the Se-S-CDs via atomic force microscopy (AFM; Figures 2c and 2d), and the results showed that the height of the Se-S-CDs was in the range of 1.2–2.7 nm, indicating that the core contained ∼4–8 graphene layers. Figure 2 | Morphology and photoluminescence properties of Se-S-CDs: (a) TEM image and DLS size distribution, (b) high-resolution TEM image, (c) AFM topography image, (d) corresponding AFM line-scanning spectra in (c), (e) excitation (black line) and emission (red line) spectra, inset: images of Se-S-CDs aqueous solution without (yellowish) and with (bright blue) excitation at 405 nm, and (f) the slopes of integrated luminescent intensity versus absorbance for fluorescein disodium salt (black line) and Se-S-CDs (red line) at an identical excitation wavelength. The relative quantum yield of Se-S-CDs was calculated based on the ratios of the two slopes. Download figure Download PowerPoint The photoluminescent properties of the Se-S-CDs were investigated using fluorescence spectroscopy. The strongest emission peak position was at ∼480 nm, with an excitation wavelength of ∼400 nm (Figure 2e). The relative quantum yield of the Se-S-CDs was calculated to be 13.27%, benchmarked with the fluorescein disodium salt (95%) at the excitation wavelength of 400 nm (Figure 2f),59 with the Se-S-CDs exhibiting excitation wavelength-dependent luminescence ( Supporting Information Figures S7a and S7b). The emission peak position was redshifted continuously, with an initial increase, followed by decreased luminescent intensity, as the excitation wavelength shifted to red. The strongest emission intensity also rose first and then descended with increasing pH values of the Se-S-CDs solution, effectuating pH-dependent luminescence ( Supporting Information Figures S7c and S7d). To validate the potential biological applications of the Se-S-CDs, we first investigated the endocytosis of the CDs by incubating the human A549 lung cancer cell line in Se-S-CDs containing culture medium at varying times. According to the results obtained from inductively coupled plasma mass spectrometry (ICP-MS; Figure 3a), the intracellular Se content increased to 300% of the original amount after an initial 6-h incubation and remained at the same level as the incubation time was extended to 24 h. This result demonstrated that an efficient endocytosis process was completed within the 6 h incubation period. Attributable to the luminescence of the Se-S-CDs, we also used flow cytometry to verify the endocytosis process (Figure 3b). We observed a dramatic increase in fluorescence intensity of Se-S-CDs, determined at the 6 h datapoint, after which a slight ascent was apparent, consistent with the ICP-MS results. The distribution of the intracellular Se-S-CDs was confirmed visually via confocal microscope images of the A549 cells (Figure 3c). Thus, it is convincing that the Se-S-CDs (blue) were distributed in the cytoplasm but not the nucleus. The confocal images of colocalization experiments showed that the Se-S-CDs neither precisely localize in the mitochondrion (Figure 3d) nor the lysosome (Figure 3e), although a certain amount of the internalized CDs went into both organelles. Figure 3 | Endocytosis and distribution measurements of Se-S-CDs into A549 cells after various incubation duration: (a) ICP-MS analysis, (b) flow cytometry, (c) confocal microscope images of A549 cells treated with Se-S-CDs (blue) for 6 h at the excitation wavelength of 405 nm, and confocal microscope images of colocation of Se-S-CDs (blue) with (d) mitochondrion (green) and (e) lysosome (green) in A549 cells cultured with Se-S-CDs containing culture medium for 6 h at the excitation wavelength of 405 and 488 nm, respectively. Scale bar = 50 μm. ***P < 0.001. Download figure Download PowerPoint The viability of the A549 cells exhibited a remarkable increase (up to ∼45%) after a 24-h incubation in the Se-S-CDs containing culture medium (Figure 4a). The optimum concentration of Se-S-CDs for promoting the cell viability was ∼100 μg/mL, out of which it showed weaker promotion, but no cytotoxicity was manifested as the concentration of the CDs increased to 400 μg/mL. For comparisons, improvement of the cell viability was not observed when the cells were incubated in S-CDs, Se-CDs, and a mixture of S-CDs and Se-CDs, highlighting the importance of Se-S dynamic covalent bonds for TrxR activities ( Supporting Information Figure S8).60,61 Neither the two starting materials nor the mixture of cysteine and selenocystine could promote cell proliferation, indicating the indispensability of the CDs as the matrix for the generation of the Se-S bonds ( Supporting Information Figure S9). To explore the mechanism of the promoted cell viability, we tested the TrxR activity of the cells (Figure 4b). The results revealed a substantial enhancement (up to ∼50%) of the TrxR activity when the concentration of Se-S-CDs reached a range of 100–200 μg/mL, which certified the initially designed function of the Se-S-CDs. Also, it is worth mentioning that a change in the intracellular environment decreased the total thiol group (–SH) by ∼50% as the CDs were endocytosed (Figure 4c). This phenomenon could be attributed to the increasing reducibility induced by the enhancement of TrxR activity and the decrease in the amount of –SH groups to maintain the redox equilibrium. We further proved the enzymatic activity of the Se-S-CDs via catalyzing the 5,5-dithiobis(2-nitrobenzoic) acid (DTNB) reduction reaction by nicotinamide adenine dinucleotide phosphate (NADPH), mimicking the catalytic ability of TrxR. The calculated TrxR activity increased continuously with the ascending concentration of Se-S-CDs, which could confirm the enzymatic activity of Se-S-CDs ( Supporting Information Figure S10). Considering TrxR could facilitate cellular proliferation, the tendencies of enhanced TrxR activity, and an increase of cell viability, was in good agreement, we argued that the cell viability was promoted by the Se-S-CDs-induced enhancement of TrxR activity. Figure 4 | In vitro activities of A549 cancer cells after being incubated at various concentrations of Se-S-CDs containing culture medium for 24 h: (a) cell viability analysis, (b) TrxR activity analysis, Total –SH contents analysis, (d) flow cytometry analysis for ROS changes in A549 cells stained with 2,7-Dichlorodihydrofluorescein diacetate (DCFH-DA) at the excitation wavelength of 488 nm, and (e) anti-inflammatory activity of A549 cells incubated in culture medium containing 200, 400, and 600 μM H2O2 for 1 h before the 24-h incubation in different concentrations of Se-S-CDs containing culture medium. *P < 0.05, **P < 0.01, ***P < 0.001. Download figure Download PowerPoint The variation of cell viability is accompanied by the change in intracellular reactive oxygen species (ROS) levels. The promoted cell viability corresponded to an increasing trend of the intracellular ROS, as shown in Figure 4d. The positive signal of intracellular ROS was mildly increased by 85% when the concentration of Se-S-CDs was increased from 0 to 100 μg/mL, representing the prosperous vitality. Specifically, we tested the concentration of intracellular H2O2 to corroborate the argument, and the amount of intracellular H2O2 per gram of total protein in A549 cells increased from ∼500 to ∼800 μmol/g ( Supporting Information Figure S11). However, the ever-increasing content of ROS would eventually reach the critical value, which could oxidize the Se-S-CDs or damage the cell, thereby stagnating the vigorous proliferation. It is noticeable that the content of ROS increased significantly as the concentration of Se-S-CDs reached 300–400 μg/mL; the calculated positive signals of ROS were three to four times higher, compared with the control group in which cells were incubated in a culture medium without CDs. Consistent with the flow cytometry measurement, the concentration of intracellular H2O2 also increased 2.2 times (∼1100 μmol/g) and 2.6 times (∼1300 μmol/g) after treatment of A549 cells with 300 and 400 μg/mL Se-S-CDs, respectively. Notably, we investigated the ROS change as a function of incubation time at the concentration of 400 μg/mL. The ROS signals in the A549 cells were captured by confocal images ( Supporting Information Figure S12) and flow cytometry ( Supporting Information Figure S13). From both of these two characterizations, we concluded that a gradually increasing intracellular ROS during the 24-h incubation resulted in an intense 2,7-Dichlorofluorescein (DCF) signal. Thus, both the promotion of cell viability and the enhancement of TrxR activity decreased at this concentration of Se-S-CDs. We further characterized the content of TrxR and thioredoxin (Trx) in A549 cells by western blotting. There was no conspicuous variation for the TrxR content in A549 cells as a function of the concentration of Se-S-CDs containing culture medium ( Supporting Information Figure S14). Contrarily, the content of Trx increased by a large margin as the concentration of Se-S-CDs increased to 300–400 μg/mL, although it only showed slight fluctuation when the concentration was in the range of 0–200 μg/mL. The expression of Trx, which is the actual executor of the reduction reaction, remained at the same level when the concentration of Se-S-CDs was 100–200 μg/mL, implying that the increased ROS was balanced by the enhanced TrxR activity, while the expression of Trx increased, corresponding to the surging amount of ROS at the range of 300–400 μg/mL of Se-S-CDs. To extend the application of promoting the cell viability by the Se-S-CDs at the concentration of 100–200 μg/mL, we tested their anti-inflammatory activity (Figure 4e). Benchmarking against the control group (no H2O2 nor Se-S-CDs), the cell viability was measured after 1 h treatment of 200, 400, and 600 μM H2O2 decreased from 100% to 94%, 79%, and 67%, respectively. In contrast, after a 24-h of A549 cells incubation in 100 μg/mL Se-S-CDs containing medium in the presence of in 200, 400, and 600 μM H2O2, the cell viability went up to 131%, 108%, and 79%, which were increased by 39%, 37%, and 18%, respectively. Similar behavior was identified when changing the concentration of Se-S-CDs from 100 to 200 μg/mL. The changes in intracellular ROS of A549 cells were captured to better understand the role of the Se-S-CDs in anti-inflammatory activity. The intracellular H2O2 was increased from ∼500 to ∼1500 μmol/g after stimulation with 200 μM H2O2 ( Supporting Information Figure S15a), retarding the promotion of cellular proliferation. While the intracellular H2O2 was dragged back to the level in which the cells still exhibited flourishing viabilities after treatment with Se-S-CDs. Similar behaviors were found when the exogenous H2O2 was increased to 400 and 600 μM. We also employed flow cytometry to double-check the changes of intracellular ROS in the anti-inflammatory experiments, and the results were in good agreement with the intracellular H2O2 measurements ( Supporting Information Figures S15b–S15d). These results validated that the presence of Se-S-CDs could make a significant difference in cell viability against the oxidizing environment equivalent to up to 600 μM H2O2 by tuning the TrxR activity to balance the intracellular ROS level. Altogether, the Se-S-CDs could promote cell viability in both normal cells and overoxidized cells by enhancing the TrxR activity. Conclusion We demonstrated a facile and robust synthetic route for fabricating CDs with Se-S dynamic covalent bonds via a hydrothermal method. The Se-S-CDs exhibited uniform size distribution of 5–8 nm and improved the quantum yield to 13.27%. Benefiting from the Se-S bonds and the CDs substrates, the Se-S-CDs embraced great capability of enhancing the TrxR activity, leading to a remarkable promotion of cell viability. Additionally, the Se-S-CDs could revive cells damaged by oxidative stress. Therefore, the Se-S-CDs showed promising potential for biological applications relative to cell culturing, including tissue culturing for skin grafting, promoting cell viability in organ transplantation, and revitalizing premature aging cells, especially those exposed to oxidative stress. This research also sheds light on the feasibility of introducing dynamic chemistry in related biological studies. Supporting Information Supporting Information is available and includes experimental procedures and Figures S1–S15. Conflict of Interest The authors declare no conflict of interest. Funding Information This work was supported financially by the National Basic Research Plan of China (no. 2018YFA208900), the National Natural Science Foundation of China (no. 21734004), and the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (no. 21821001). Acknowledgments The authors wish to acknowledge professor Dongsheng Liu’s group for their assistance with confocal microscopy. References 1. Arner E. S.; Holmgren A.Physiological Functions of Thioredoxin and Thioredoxin Reductase.Eur. J. Biochem.2000, 267, 6102–6109. Google Scholar 2. 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