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2102178 (1 of 11) © 2021 Wiley-VCH GmbH www.small-journal.com RESEARCH ARTICLE Cu,Zn Dopants Boost Electron Transfer of Carbon Dots for Antioxidation Sheng Xue,* Tingwei Zhang, Xiaokai Wang, Qinhua Zhang, Siyang Huang, Liwei Zhang, Liyu Zhang, Wenjie Zhu, Yin Wang, Mingbo Wu, Qingshan Zhao, Peifeng Li, and Wenting Wu* Dr. S. Xue, S. Huang, L. Zhang, L. Zhang, W. Zhu, Dr. Y. Wang, Prof. P. Li Institute for Translational Medicine The Affiliated Hospital of Qingdao University College of Medicine Qingdao University Qingdao 266021, China E-mail: [email protected] T. Zhang, X. Wang, Q. Zhang, Prof. M. Wu, Q. Zhao, Prof. W. Wu State Key Laboratory of Heavy Oil Processing College of Chemical Engineering Institute of New Energy China University of Petroleum (East China) Qingdao 266580, China E-mail: [email protected] DOI: 10.1002/smll.202102178 superoxide dismutase (SOD), [2] frequently followed by amount of secondary ROS (e.g., toxic H 2 O 2 ) further converted into highly oxidizing ·OH with the assistant of peroxidase, which is a kind of more pathogenic ROS. [3] In this respect, the SOD, no panacea, cannot perfectly resist the oxidation alone. Therefore, it is highly desired to fabricate multi-antioxidant enzyme-based defense system, especially combining catalase (CAT), another most important enzyme to ultimately reduce the subsequent influence of H 2 O 2 and subsequent ·OH in enzyme therapies. Great attempts have been made to find efficient antioxidant enzyme mimics, such as molecular complexes (e.g., metal salen complexes) [4] and nanoparticles (e.g., carbon, platinum, ceria, manganese, and vanadium). [5] However, they seldom pro- cess both SOD and CAT activities. One of the limited examples was Mn salen com- plexes, [6] which process the SOD like activities close to that of natural enzymes under mild condition, but the best CAT activi- ties from the modified Mn salen complex was only 32.4%. [7] Like myocardial ischemia, many diseases can cause progressive H + generation and intra-/extracellular pH fall due to increased anaerobic metabolism, net hydrolysis, and CO 2 retention. [8] But for the multi-functional nanoparticles, especially for the metal oxide and metal nanoparticles, most of them switch from CAT to peroxidase mimics as the pH reduces to acidic condition. [1e,9] The metal active site with more electrons promotes the HO-OH (H 2 O 2 ) heterolysis producing O 2 and H 2 O (CAT), otherwise the homolysis can happen to generate •OH (peroxidase). [10] As the pH reducing, H + makes the active sites lack electrons resulting in the increasing peroxidase activity. [1f,11] Therefore, nano materials with abundant electrons and powerful electron transfer properties may quickly balance H + influence on elec- tron distribution. Carbon dots (CDs), giant electron reservoirs, are low cost, low toxic, and excellent biocompatibility, [12] which provide potential candidates for both SOD/CAT mimics. In order to improve the electron transfer (both eletron donating and accepting) abili- ties, various single and dual metals (e.g., Mn, Fe, Co, Ni, Cu, and Zn) were covalently doped into CDs. Just like nature selec- tion for Cu/Zn SOD, Cu and Zn co-doping CDs (CuZn-CDs) Enzyme-mimicking nanomaterials for antioxidative therapy is a promising star to treat more than 200 diseases or control their progressions through scavenging excessive reactive oxygen species (ROS), such as O 2 and H 2 O 2 . However, they can inversely produce stronger ROS (e.g., •OH) under many disease conditions (e.g., low pH for myocardial ischemia). Herein, a biocom- patible -Cu-O-Zn- bimetallic covalent doped carbon dots (CuZn-CDs) pro- cessing both catalase (CAT) and superoxide dismutase activities are reported, mainly because of their abundant electrons and the excellent electron transfer abilities. In addition, Cu dopant helps to balance the positive charge at Zn dopant resulting from low pH, enabling CuZn-CDs to still process CAT ability rather than peroxidase ability. Benefiting from it, CuZn-CDs exhibit sufficient in vitro ROS scavenging ability and cardiomyocyte protective effect against ROS-induced damage. In vivo results further demonstrate that CuZn-CDs can protect the heart from ischemia-reperfusion injury. In addition to antioxidative therapy, the rapid renal clearance and low toxicity properties of CuZn-CDs in animal model reveal high biocompatibility which will facilitate clinical use. The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smll.202102178. 1. Introduction Reactive oxygen species (ROS) and oxidative stress widely contribute to more than 200 diseases development, such as cardiovascular ischemia, Alzheimer's disease, metabolic disor- ders, and cancer. [1] Generally, O 2 is considered to be the main initial ROS, and it can be catalyzed into O 2 and H 2 O 2 by the Small 2021, 2102178

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ReseaRch aRticle

Cu,Zn Dopants Boost Electron Transfer of Carbon Dots for Antioxidation

Sheng Xue,* Tingwei Zhang, Xiaokai Wang, Qinhua Zhang, Siyang Huang, Liwei Zhang, Liyu Zhang, Wenjie Zhu, Yin Wang, Mingbo Wu, Qingshan Zhao, Peifeng Li, and Wenting Wu*

Dr. S. Xue, S. Huang, L. Zhang, L. Zhang, W. Zhu, Dr. Y. Wang, Prof. P. Li Institute for Translational Medicine The Affiliated Hospital of Qingdao University College of MedicineQingdao UniversityQingdao 266021, ChinaE-mail: [email protected]. Zhang, X. Wang, Q. Zhang, Prof. M. Wu, Q. Zhao, Prof. W. Wu State Key Laboratory of Heavy Oil Processing College of Chemical Engineering Institute of New EnergyChina University of Petroleum (East China)Qingdao 266580, ChinaE-mail: [email protected]

DOI: 10.1002/smll.202102178

superoxide dismutase (SOD),[2] frequently followed by amount of secondary ROS (e.g., toxic H2O2) further converted into highly oxidizing ·OH with the assistant of peroxidase, which is a kind of more pathogenic ROS.[3] In this respect, the SOD, no panacea, cannot perfectly resist the oxidation alone. Therefore, it is highly desired to fabricate multi-antioxidant enzyme-based defense system, especially combining catalase (CAT), another most important enzyme to ultimately reduce the subsequent influence of H2O2 and subsequent ·OH in enzyme therapies.

Great attempts have been made to find efficient antioxidant enzyme mimics, such as molecular complexes (e.g., metal salen complexes)[4] and nanoparticles (e.g., carbon, platinum, ceria, manganese, and vanadium).[5] However, they seldom pro-cess both SOD and CAT activities. One of the limited examples was Mn salen com-

plexes,[6] which process the SOD like activities close to that of natural enzymes under mild condition, but the best CAT activi-ties from the modified Mn salen complex was only 32.4%.[7] Like myocardial ischemia, many diseases can cause progressive H+ generation and intra-/extracellular pH fall due to increased anaerobic metabolism, net hydrolysis, and CO2 retention.[8] But for the multi-functional nanoparticles, especially for the metal oxide and metal nanoparticles, most of them switch from CAT to peroxidase mimics as the pH reduces to acidic condition.[1e,9] The metal active site with more electrons promotes the HO-OH (H2O2) heterolysis producing O2 and H2O (CAT), otherwise the homolysis can happen to generate •OH (peroxidase).[10] As the pH reducing, H+ makes the active sites lack electrons resulting in the increasing peroxidase activity.[1f,11] Therefore, nano materials with abundant electrons and powerful electron transfer properties may quickly balance H+ influence on elec-tron distribution.

Carbon dots (CDs), giant electron reservoirs, are low cost, low toxic, and excellent biocompatibility,[12] which provide potential candidates for both SOD/CAT mimics. In order to improve the electron transfer (both eletron donating and accepting) abili-ties, various single and dual metals (e.g., Mn, Fe, Co, Ni, Cu, and Zn) were covalently doped into CDs. Just like nature selec-tion for Cu/Zn SOD, Cu and Zn co-doping CDs (CuZn-CDs)

Enzyme-mimicking nanomaterials for antioxidative therapy is a promising star to treat more than 200 diseases or control their progressions through scavenging excessive reactive oxygen species (ROS), such as O2

•− and H2O2. However, they can inversely produce stronger ROS (e.g., •OH) under many disease conditions (e.g., low pH for myocardial ischemia). Herein, a biocom-patible -Cu-O-Zn- bimetallic covalent doped carbon dots (CuZn-CDs) pro-cessing both catalase (CAT) and superoxide dismutase activities are reported, mainly because of their abundant electrons and the excellent electron transfer abilities. In addition, Cu dopant helps to balance the positive charge at Zn dopant resulting from low pH, enabling CuZn-CDs to still process CAT ability rather than peroxidase ability. Benefiting from it, CuZn-CDs exhibit sufficient in vitro ROS scavenging ability and cardiomyocyte protective effect against ROS-induced damage. In vivo results further demonstrate that CuZn-CDs can protect the heart from ischemia-reperfusion injury. In addition to antioxidative therapy, the rapid renal clearance and low toxicity properties of CuZn-CDs in animal model reveal high biocompatibility which will facilitate clinical use.

The ORCID identification number(s) for the author(s) of this article can be found under https://doi.org/10.1002/smll.202102178.

1. Introduction

Reactive oxygen species (ROS) and oxidative stress widely contribute to more than 200 diseases development, such as cardiovascular ischemia, Alzheimer's disease, metabolic disor-ders, and cancer.[1] Generally, O2

•− is considered to be the main initial ROS, and it can be catalyzed into O2 and H2O2 by the

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exhibits the best intrinsic SOD/CAT-like activity among wide range of metal doping CDs, wherein its SOD activities are close to nature SOD enzyme, and CAT activities come to 95.4%. More importantly, it exhibits stable antioxidation ability under a wide range of pH (from 1.2 to 13.0) and high temperature (150 °C) environment, more convenient transportation, and storage than nature antioxidant enzymes. In addition, CuZn-CDs were used as SOD/CAT mimics for the treatment of myocardial ischemia-reperfusion injury, and can provide ischemic protection after treatment.

2. Results and Discussion

2.1. Synthesis and Characterization of CuZn-CDs

Unlike previous metal ion dopants, metal ions chelated with ethylenediamine-tetraacetic acid (EDTA) can easily form satu-rated salen-like structure, similar to SOD mimics derived from organometallic complex. On the one hand, it prefers square-planar geometries, a flaky graphite conductive structure could be easily formed. On the other hand, this kind of precursors can form covalent metal dopant in CDs to further improve its electron transfer properties.[12c,13] In order to adapt CDs to hypersaline physiological environment and to prevent aggre-gation, CDs derived from the mixture of NaCl and the metal chelated EDTA, namely M1M2-CDs (e.g., CuZn-CDs), were pre-pared through a facile one-step pyrolytic synthesis (Figure 1a), wherein CDs with various sole metal or dual metal dopants were prepared (see the Experimental Section in the Supporting Information for details) and carefully studied their activities vide infra. Among these CDs, CuZn-CDs show best activities as the precursors ratio of EDTA-Cu and EDTA-Zn fixed to 1:1, mostly similar to nature selection.

Transmission electron microscopy (TEM) images clearly showed that CuZn-CDs were uniform with the diameter less

than 3  nm, the most distribution in the range of 1.5–2  nm (Figure  1b), and the height below 4.0  nm (Figure S1, Supporting Information), less than the kidney filtration threshold of 5.5 nm[14] and microvasculature vessel diam-eters (arterioles 10–100  µm, capillaries 4–10  µm and venules 10–100  µm).[15] Compared with non NaCl CDs (u-CuZn-CDs), CuZn-CDs were uniformly dispersed during all experiments, and even stable in the PBS media for at least half year (Figure S1, Supporting Information). In addition, ZnCu-CDs process good crystallinity with a lattice spacing of 0.22  nm corresponding to the lattice fringes of the (100) planes of graphite carbon[16] (Figure  1b inset), which is consistent with Raman and FT-IR results (Figures S2 and S3, Supporting Information). This gra-phene structure may benefit the electron transfer for the anti-oxidation process.

The valence state of Cu/Zn dopants in CuZn-CDs was first studied because it is important for antioxidation process. In electron spin resonance (ESR) spectra, there is no Zn+ ESR signal observed (Figure 1c inset), indicating that there are Zn0 or Zn2+ dopants in CDs. The Cu ESR result (Figure  1d inset) shows that there is hyperfine structure at low fields, which can be assigned to the spin frustrated Cu2+ dopant derived from the d orbital.[17a] For Cu dopants, the g tensor parameter (g) of ZnCu-CDs is 2.090 (< 2.3), indicating the covalent character of the CuII–ligand bond inside the CDs.[12c,17b] In X-ray absorp-tion near-edge structure (XANES) spectra, the Zn absorption K-edge of CuZn-CDs is close to ZnO (Figure  1c). Associated with the ESR results, the valence state of Zn dopants in CDs is positive divalent (Zn2+). The Cu absorption K-edge of CuZn-CDs located between CuO and Na2[Cu(EDTA)] (Figure 1d), sug-gesting that Cu dopants in CuZn-CDs are also nearly positive divalent (Cu2+), which is consistent with Cu ESR results.

Then, the surrounding structure of metal dopants was studied by EXAFS and DFT calculations. The Fourier trans-form (FT) of the Cu extended EXAFS spectra (Figure S4a, Supporting Information) show a peak probably corresponding

Figure 1. Preparation and characterization of CuZn-CDs. a) Schematic preparation of CuZn-CDs and biological experiments. b) TEM image of CuZn-CDs. Insert are the statistical chart of particle size distribution and lattice spacing. c) XANES spectra at the Zn k-edge of CuZn-CDs, Na2[Zn(EDTA)], ZnO, and Zn foil. d) XANES spectra at the Cu k-edge of CuZn-CDs, Na2[Cu(EDTA)], CuO, and Cu foil. e) The front and side view of the structure model for the active metal dopant in ZnCu-CDs.

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to the Cu-O/N shell with a distance of 1.94 Å (Table S1, Sup-porting Information), a little shorter than that of CuO and Na2[Cu(EDTA)]. The FT of the Zn extended EXAFS spectra (Figure S4b, Supporting Information) show two peaks. The first peak could correspond to the Zn-O/N shell with a distance of 2.04 Å, shorter than Na2[Zn(EDTA)] (2.11 Å, Table S2, Sup-porting Information), suggesting that the middle O/N may not in the same plane of Zn dopants. The second peak could correspond to the Zn-O-Zn/Cu shell, and there is no Cu-Cu and Zn-Zn signal in Figure S4, Supporting Information, indi-cating that Cu and Zn are covalently dispersed in CuZn-CDs. In addition, the possible structures of the metal active sites were studied by DFT calculations. Among these structures, -Cu-O-Zn- has the lowest total energy (−2319.57  eV, Table S3, Supporting Information and Figure 1e), indicating that it would be the most possible structure. This Cu and Zn structure can stably chelate with carbon structure and hardly run off. The metal content of various CDs was listed in Table S4, Supporting Information. For CuZn-CDs, the Cu and Zn contents are 0.16% and 0.28% respectively, wherein the Cu content is much less than that of nature SOD.[18a] The Cu content of CuZn-CDs used in vivo experiment was only 1‰ of that in human beings.[18b] Therefore, this stable structure and low Cu,Zn content can diminish copper and Zn toxicity.

Compared with pure CDs, CuZn-CDs show stronger visible-light-harvesting ability ranging from 300 to 600 nm (Figure S5, Supporting Information), which could be attributed to the coexistence of Cu/Zn dopants, producing electron transfer from metal to graphene is beneficial to enhance absorption intensity.[12b,19] Interestingly, the absorption intensity of CuZn-CDs is also stronger than that of Cu-CDs and Zn-CDs, indi-cating that there may be electron exchange between Zn and Cu dopants. The color of the CuZn-CDs solution is brown under sunlight, and gives blue emission excited by 365  nm light (Figure S5, Supporting Information inset). After irradiation for 160 min, CuZn-CDs were stable and there was no obvious

photobleaching, which is beneficial for their application in photo induced antioxidation.

2.2. Antioxidation Activities of CuZn-CDs

O2•− and H2O2 as the representative ROS was selected to study

the antioxidation (SOD and CAT) ability. CuZn-CDs in dark condition has the SOD-like activity (38.5%), which gradually increases as enhancing light intensity (Figure S6a, Supporting Information). When the light intensity increased to 1050 W m−2 (1 sun intensity), the SOD-like activity of CuZn-CDs sur-passes that of nature SOD (Figure S6b, Supporting Informa-tion). There is also a concentration-dependent relationship for CuZn-CDs at low concentration range. As the concentration of CuZn-CDs increases to 6 µg mL−1, the SOD activities come to balance and are close to nature SOD activity (Figure S6c–f, Supporting Information).

CuZn-CDs show great tolerance to severe environmental condition. For the pH factor, under extreme acid and alkaline condition, CuZn-CDs keep great SOD activities (Figure S6a, Supporting Information) just like pH close to 7, but the activity of natural SOD obviously decreased in extreme acid and alka-line condition (Figure 2a). For the temperature factor, CuZn-CDs show good thermal stability even when the temperature increases to 150 °C (Figure 2b). Under mild condition (25 °C, pH = 7), the SOD-like activity of CuZn-CDs (61.4%) is close to that of nature SOD (69.9%) with the same amount. Further increasing to 150 °C, the SOD-like activity for CuZn-CDs keeps well around 61%, while nature SOD reduces to only 2.5%. Therefore, CuZn-CDs as a good SOD mimic is easy to storage, transport, and use for further application.

Besides the SOD mimic, it is highly needed a more in-depth service, such as the CAT-like service, to wipe out the secondary ROS generate from O2

•− (e.g., H2O2). The hydrogen peroxide reservation assay (Iodometry method in SI) was first studied

Figure 2. Photocatalytic activity and stability of CuZn-CDs. a) SOD-like activity of CuZn-CDs under pH = 1.2, 7.2, and 13.0, and b) various temperature with the light intensity of 400 W m−2 (0.4 sun intensity). c) CAT ability detected by the iodometry, and d) the O2 production from the decomposition of H2O2 catalyzed by CuZn-CDs, CDs and no catalyst under light irradiation at 0, 5, 10, … and 30 min (pH = 7). e) CAT ability detected under pH = 1.2, 7.2, and 13.0. Data are mean ± s.d. from three independent replicates. **p < 0.01, ***p < 0.001 versus control group; N.S., no significance, Student’s t-test. f) CAT ability detected under various temperatures. XPS analysis of CuZn-CDs g) Cu 2p and h) Zn 2p before and after treatment with H2O2.

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by UV-Vis spectra, wherein the higher absorption intensity at 352  nm, resulting from the product of I− and H2O2, means the higher H2O2 amount. As shown in Figure  2c, there is a slight decrease in the absorption intensity at 352 nm after the addition of CuZn-CDs, while another 10 min light irradia-tion results in a 36% decrease, indicating that CuZn-CDs can reduce H2O2 amount, especially under light irradiation. From another perspective, the CAT abilities, the decomposition of H2O2 into non-toxic O2 and H2O, were further analyzed by the O2 production test. As the addition of CuZn-CDs increases to 60  µg mL−1, the CAT activities come to balance (Figure S7, Supporting Information). After 30 min light irradiation (less than thoracotomy time), the O2 amount comes to 4.72  mmol with the efficiency of 95.4% (Figure 2d). And it is easy to observe the O2 evolution (Video S1, Supporting Information). Without the light irradiation, O2 amount catalased by CuZn-CDs was only 1.48  mmol. Importantly, there are little highly oxidizing ·OH species derived from the H2O2 decomposition (Figure S8, Supporting Information). With or without the pure CDs, almost no O2 produce under similar conditions (Figure  2d). These results indicate that the photoexcited unsaturated metal dopants are key active sites for CAT performance.

As confirmed by the hydrogen peroxide reservation assay, O2

•− more easily couple with proton and form secondary ROS (H2O2) in neutral and acidic conditions than that in alkaline conditions (Figure S9, Supporting Information), which is sim-ilar to that sub-health or myocardial ischemia-reperfusion injury under acidic condition easily cause oxidative stress occurrence. Therefore, pH was another non-negligible factor. As the pH extremely reduces to 1.2, the CAT ability of CuZn-CDs comes to 82.7% (Figure 2e), which is higher than that of nature CAT (48.2%). In addition, CuZn-CDs also show excellent CAT ability in severe environmental condition. As the temperature increase to 80 °C, the CAT activity of nature CAT began to decrease, but there was no significant change for CuZn-CDs. Still increasing to 150 °C, CuZn-CDs keep 33.5% CAT ability while nature CAT is almost inactivated (Figure 2f).

After 5 cycles of repetitive tests, there is no obvious decrease in CAT ability indicating the excellent stability of CuZn-CDs (Figure S10, Supporting Information). CuZn-CDs before and after treated with H2O2 were tested by the X-ray photo electron spectroscopy (XPS) spectra. For CuZn-CDs, two intense peaks at 932.8 and 952.5  eV for Cu 2p could be assigned to the binding energies of Cu 2p3/2 and Cu 2p1/2 from Cu2+ species (Figure 2g). Two intense peaks at 1021.4 and 1044.6 eV for Zn 2p could be assigned to the binding energies of Zn 2p3/2 and Zn 2p1/2 from Zn2+ species (Figure 2h). Upon interaction with H2O2, the positions of these main peaks did not shift and no new peaks appeared, indicating that metal active sites of CuZn-CDs itself were stable.

2.3. Antioxidation Mechanism of CuZn-CDs under Severe Condition

CuZn-CDs were first studied by thermogravimetric-infrared spectra. H2O is the main substrate released from CuZn-CDs when the temperature below 150 °C (Figure S11, Sup-porting Information). Associated with XPS results (Figure S12,

Supporting Information), the characterization of the surface groups, the mass loss may be resulting from the hydroxyl group and H2O absorbed on the surface of CDs. At the same time, there is no obvious CO, CO2, and CH4 release, indi-cating that the main body of CuZn-CDs themselves are hardly destroyed. Associated with the results of its SOD and CAT activities (Figure  2b,f), the main body of CuZn-CDs (metal dopants and graphene structure) instead of surface groups are the active sites for antioxidation, which favors its thermal sta-bility even at 150 °C. Since SCN− can coordinate with metal site and poison them,[20] the decreasing CAT activities as the addi-tion of KSCN further confirms that metal dopants were the actually active sites (Figure S13, Supporting Information).

In order to confirm the function of metal dopants, various metal doped CDs were prepared. Associated with the redox reaction mechanism of SOD[21] and CAT[11b] (Figure 3a,b), the electron donating/accepting ability of various CDs VS their SOD/CAT activities were examined by fluorescence quenching test (Figures S14–S19, Supporting Information).[12c,e] As shown in Figure 3c,d, both of SOD and CAT activities have the positive relations with the electron donating/accepting abilities. Bime-tallic doped CDs show better performance than those of single metal doped CDs and pure CDs, especially for CuZn-CDs which have the highest electron transfer properties and SOD/CAT abilities among these CDs. The electron transfer ability was also tested directly by transient photocurrent response (Figure S20, Supporting Information). Although the metal dopant of CuZn-CDs is lower than other CDs, CuZn-CDs show obvious advantages for SOD and CAT performance. It can be speculated that the electron transfer properties are greatly influ-enced by metal dopants, and Cu,Zn co-dopants are crucial to wipe out wide kinds of the surplus ROS.

It is interesting that there is only a little pH influence on CuZn-CDs for antioxidation. In situ ESR spectroscopy (ESR) was used to get insight into Cu and Zn dopants under various pH conditions. Under alkaline condition (pH = 11), the O2

•− is the main ROS. Cu2+ dopant can be reduced into Cu+, resulting in the gradual decrease of Cu2+ signal (g  = 2.118, Figure  3e), and O2

•− itself may become harmless O2. Similarly, there is a weak signal (g  = 1.996, Figure  3e inset), and its intensity and shape of the ESR signal are highly symmetrical, which can be assigned to the unpaired electron from s orbital of Zn+.[1e] The unsaturated Cu and Zn dopants act as electron acceptors to pro-mote the conversion of O2

•− into O2. Under netural condition (pH = 7), it can be seen as the mixed ones of acidic and alkaline conditions, and there are both O2

•− and H2O2. The Cu2+ signal reduced and there is no Zn+ signal (Figure 3f and inset).

Under acidic condition (pH = 3), O2•− easily coupled with

proton and formed H2O2 (Figure S9, Supporting Information), at the same time, the proton can also reduce the electron den-sity of metal active sites. In the presence of H2O2, Zn+ signal is hardly observed because Zn+ is very active and rapidly reacts with H2O2. For Cu dopant, the ESR signal intensity of Cu2+ first reduced, and subsequently increased as the irradiation time prolonged from 5 to 10 min, indicating that the gener-ated Cu+ can be gradually oxidized into Cu2+ (Figure  3g). In other words, Zn is more active, and H2O2 prefers to react with it. Generally, the metal active sites with less electrons benefit for the HO-OH homolysis into •OH,[10b,22] which is consistent

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with the previous report that nano materials hardly persist CAT ability under acidic condition. However, CuZn-CDs show the opposite result. DFT calculation was further used to explore the charge distribution of CuZn-CDs under acidic condition. Mul-liken charge of Zn in CuZn-CDs is 1.064, lower than that of Zn in Zn-CDs (1.271). But Mulliken charge of Cu in CuZn-CDs is 0.583, higher than that of Cu in Cu-CDs (0.502). This result indicates that Cu help to balance the electron density of Zn active site and to reduce the proton influence under acid con-dition. Therefore, CuZn-CDs, unlike the previous report, show excellent CAT ability even under acidic condition.

2.4. ROS Scavenging Ability In Vitro

The in vitro antioxidant capacity of CuZn-CDs was investigated. The rat cardiomyocyte-derived H9c2 cells were pretreated with CuZn-CDs and then treated with different concentra-tions of hydrogen peroxide (H2O2). The intracellular ROS level was detected by fluorescence probe DCFH-DA. As shown in Figure 4a,b, an intense fluorescence signal was observed when H9c2 cells were incubated by H2O2 at the concentration of 20, 50, and 100  mm. In the contrast, when H9c2 cells were pre-treated by CuZn-CDs (40  µg mL−1), the fluorescence signal decreased significantly in the presence of H2O2, showing an elimination of ROS by CuZn-CDs. When H9c2 cells were only treated with CuZn-CDs, there was no visible fluorescence signal observed in cells, indicating CuZn-CDs could not increase ROS production in cells.

In order to evaluate cytotoxicity of CuZn-CDs, H9c2 cell via-bility in the presence of CuZn-CDs was determined by MTT assay. As shown in Figure 4c, there was no inhibiting effect on H9c2 cell proliferation observed when incubated with different

concentrations of CuZn-CDs (0–200 µg mL−1) (p >  0.05). This result demonstrated the low cytotoxicity and good in vitro bio-compatibility of CuZn-CDs.

ROS-mediated oxidation in ischemia-reperfusion (I/R) could induce cardiomyocyte apoptosis.[23] Hydrogen peroxide (H2O2) could generate oxidative stress which induces cardiomyocyte apoptosis and has been used in mimicking I/R injury.[24] Cardi-omyocyte apoptosis was detected by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay. A strong green TUNEL-positive signal was observed when H9c2 cells were incubated with 1 mm H2O2 (Figure 4d,e). On the contrary, when pretreated with CuZn-CDs (40 µg mL−1), TUNEL staining was markedly reduced (Figure  4d,e). This result showed CuZn-CDs could reduce the apoptosis induced by ROS.

2.5. In Vivo Biocompatibility of CuZn-CDs

In order to evaluate the in vivo biocompatibility of CuZn-CDs, serum inflammatory cytokine levels, cytokine gene expression in liver and kidney, and major organ histopathology were evalu-ated after CuZn-CDs was intravenously injected into health mouse (0.8 mg kg−1, equal to 20 µg mL−1 for a 25 g mouse with 1  mL of blood, tenfold higher than the concentration used in myocardial cell treatment). As shown in Figure 5a, there was no necrosis, congestion, inflammatory lesions, and tissue damage in heart, kidney, liver, lung, spleen, bladder, cerebral cortex, and hippocampus at 7 days after a single dose intravenous administration of CuZn-CDs. Previous studies showed that intravenously injected CDs can be primarily excreted via urine, which is the major excretion pathway for very small nanopar-ticles (diameter <  5.5  nm).[25] Time-dependent accumulation

Figure 3. Electron donating/accepting abilities and antioxidant mechanism of CuZn-CDs. a) Scheme of CuZn-CDs as SOD mimics and b) CAT mimics. c) SOD activities of various metal doped CDs and pure CDs versus their electron transfer performances. d) CAT activities of the same CDs versus their electron transfer performances. After 0, 5, 10 min light irradiation, the in situ Cu2+ ESR signals from CuZn-CDs at e) pH = 11, f) pH = 7, g) pH = 3 respectively. Insert were the Zn+ ESR signals from CuZn-CDs under relative conditions.

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of CuZn-CDs nanoparticles in urine demonstrated that these nanoparticles can be excreted by renal clearance pathway (Figure  5b). There was a peak of concentration of Zn, which was a main component of CuZn-CDs, in urine at 3 h post-injection, following by the concentration coming down to near pre-injection level at 72 h post-injection (Figure 5b). This result revealed a relatively rapid clearance of CuZn-CDs in body.

Additionally, the serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C-reactive protein showed no significant difference (p > 0.05) between control group and CuZn-CDs treated group (Figure 5c–e) at 7 days post-injection. As ultrasmall nanoparticles mainly accumulated in liver and kidney when intravenously injected into mice,[26] the mRNA expression levels of inflammatory cytokines were evaluated. As shown in Figure  5f–k, mRNA expression levels of TNF-α, IL-1α, IL-1β, IL-6, IL-10 and monocyte chemoattractant protein-1

(MCP-1) in liver and kidney of CuZn-CDs group were identical to that in control group at 7 days post-injection. All these results proved that CuZn-CDs would not exhibit obvious toxicity and trigger immune response in mice, revealing the good in vivo biocompatibility of CuZn-CDs.

2.6. In Vivo Therapeutic Efficacy of CuZn-CDs on Myocardial I/R Mice

Intramyocardial injection has been proved to be an effective method to deliver therapeutic agent to heart tissue and rescue the myocardium from acute ischemia.[27] So CuZn-CDs nano-particles were intramyocardial delivered to evaluate the cardiac protective effects in myocardial I/R. To determine the effect of CuZn-CDs nanoparticles on antioxidant I/R therapy, coronary

Figure 4. In vitro ROS scavenging activity and cytotoxicity of CuZn-CDs. a) Effect of CuZn-CDs nanoparticle on H2O2 induced ROS scavenging in H9c2 cardiomyocyte. ROS in H9c2 cells was detected with DCFH-DA probe (green fluorescence) and monitored by fluorescence microscope. Scale bar = 100 µm. b) Statistical analysis of ROS positive H9c2 cells under different treatment conditions. c) Cytotoxicity of CuZn-CDs nanoparticles at different concentrations under light irradiation. d) Fluorescence images of DAPI- and TUNEL-stained H9c2 cells with different treatments. H2O2 induced apoptosis was detected by TUNEL staining (green fluorescence). Scale bar = 20 µm. e) Statistical analysis of the portion of TUNEL staining-positive cells in different groups. Data are mean ± s.d. from three independent replicates. ***p < 0.001 versus control group; N.S., no significance, one-way ANOVA.

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artery ligation-induced myocardial infarction in mice was used in this research. In this ischemia-reperfusion model, C57BL/6 mice underwent a left thoracotomy and the left anterior descending (LAD) coronary artery was temporarily occluded to induce ischemia. The mice that underwent LAD occlusion were randomized to receive intramyocardial injection of CuZn-CDs nanoparticles or saline control alone (IR/saline). After 40 min ischemia, the occlusion was removed to allow ventricle reper-fusion for 15 min. During the ischemia-reperfusion procedure, the heart of mouse was exposed to constant light exposure. To evaluate anti-oxidant, anti-apoptosis, and anti-inflammatory

effects of CuZn-CDs, hearts were collected and examined after reperfusion. The in situ ROS in heart tissue slices was detected with DCFH-DA probe. As shown in Figure 6a,c, obvious DCF fluorescence was measured in hearts from IR/saline group. In contrast, DCF fluorescence was much lower in hearts from sham and CuZn-CDs groups (p  <  0.05). Cardiomyocyte apop-tosis in heart tissue was detected with TUNEL assay. The result showed apoptosis in heart tissue occurred in IR/saline group after reperfusion (Figure 6b,d). On the contrary, cardiomyocyte apoptosis in sham and CuZn-CDs group were significantly reduced (Figure  6b,d, p  <  0.05). In addition, heart cytokine

Figure 5. In vivo biocompatibility evaluation of CuZn-CDs. a) Evaluation of in vivo toxity of CuZn-CDs to major organs (heart, kidney, liver, lung, spleen, bladder, cerebral cortex, and hippocampus) at 7 days after intravenous injection. b) Concentration of Zn in urine at different time points. c–e) Serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and C-reactive protein (CRP) at 7 days post-injection. f–k) mRNA expression levels of inflammatory cytokines TNF-α, IL-1α, IL-1β, IL-6, IL-10 and monocyte chemoattractant protein-1 (MCP-1) in liver and kidney at 7 days post-injection. Data are mean ± s.d. from three or four independent replicates. N.S., no significance as indicated, c–e) Student’s t-test or f–k) one-way ANOVA.

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mRNA levels of TNF-α and IL-1α were significantly increased in IR/saline group compared with sham group (Figure  6e,f, p  <  0.01), and there was no statistical difference between

sham group and CuZn-CDs group (Figure  6e,f, p  >  0.05). These results proved CuZn-CDs could play anti-oxidant, anti- apoptosis, and anti-inflammatory role in heart tissue.

Figure 6. In vivo therapeutic evaluation of CuZn-CDs. a) Consecutive transverse cross-section (20 µm) of hearts from different groups 30 min after rep-erfusion. The slices were stained with HE (left) and DCFH-DA (right). Scale bar = 100 µm. b) Fluorescence images of DAPI- and TUNEL-stained heart sec-tion (20 µm) from different groups 30 min after reperfusion. ROS induced apoptosis was detected by TUNEL staining (green fluorescence). Scale bar = 100 µm. c) Statistical analysis of the portion of DCF staining area in different groups. d) Statistical analysis of the portion of TUNEL staining-positive cells in different groups. e,f) mRNA levels of inflammatory cytokines TNF-α, IL-1α in heart tissue 3 h after reperfusion. g) Representative electrocar-diogram recorded at 24 h after reperfusion in mice. h) Bodyweight curve of mice within 3 days after surgery. i) Survival curves in different groups. Data are mean ± s.d. from three independent replicates. * p < 0.05, ** p < 0.01, ***p < 0.001 versus control group; N.S., no significance, one-way ANOVA.

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Electrocardiogram (ECG) was monitored via implanted telemetry device at 24 h after thoracotomy and I/R surgery. There was a typical S-T segment elevation in IR/saline group, and no obvious S-T segment elevation was observed in sham group and CuZn-CDs group (Figure  6g).  The bodyweight of mice in different groups showed a decrease within 3 days after the surgery, and the difference was not statistically different (p > 0.05) (Figure 6h). The overall survival rate (including sur-gical related death) 15 days after surgery was 85.7% in sham group, 73.3% in IR/saline group, and 83.3% in CuZn-CDs group (Figure  6i), with no significant difference among the groups (p > 0.05).

In order to evaluate the protective effect of CuZn-CDs nanoparticles against I/R injury, we calculated the left ventric-ular (LV) infarct size after I/R injury. The LV infarct size was

5.59 ± 1.13% and 4.73 ± 0.51% in CuZn-CDs and sham groups, which was statistically unchanged (p  >  0.05) (Figure 7a,b). However, the infarct size in the control group increased up to 38.43 ± 2.12%, which was statistically higher than that of CuZn-CDs group and sham group (p  <  0.001) (Figure  7a,b). These results revealed CuZn-CDs nanoparticles could protect cardiac myocyte from I/R injury.

In order to examine long-term therapeutic effects of CuZn-CDs, echocardiography was used to noninvasively measure car-diac function at 4 weeks after I/R injury. Compared with mice in sham and CuZn-CDs group, mice in saline control group showed severely impaired anterior wall motion (Figure  7c). Saline control group exhibited significantly increased diastolic (p  <  0.05) and systolic (p  <  0.001) inner chamber dimensions of left ventricular (Figure 7d,e), and ejection fraction (p < 0.001)

Figure 7. Therapeutic efficacy of CuZn-CDs on myocardial I/R mice. a) Representative photographs of Evans blue and TTC double-stained heart sec-tions from different treatment groups. b) Graphic representation of the LV infarct size of different treatment groups. The LV infarct size was expressed as the percentage of infarct area over total AAR in each group (n = 4). c) Representative echocardiograph from different groups. d) LV internal diam-eters of diastole (LVIDd) and e) systole (LVIDs) were measured at the view of long axis from M mode. f) LV ejection fraction (LVEF) and g) fractional shortening (LVFS) were calculated with VisualSonic analysis software (n = 5 mice in each group). h) Representative Masson's trichrome stained heart sections from different groups at 4 weeks post-I/R injury. Scale bar = 1 mm. Data are mean ± s.d., *p < 0.05 versus control group; **p < 0.01 versus control group; ***p < 0.001 versus control group; N.S., no significance as indicated, one-way ANOVA.

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and fractional shortening (p < 0.01) of left ventricular decreased accordingly (Figure  7f,g). On the contrary, the differences between CuZn-CDs and sham groups were not statistically sig-nificant (p > 0.05) (Figure 7c–g). Masson's trichrome staining of heart sections at 4 weeks after I/R injury showed obvious scar formation in saline control group (Figure 7h), but no scar for-mation was found in CuZn-CDs and sham groups (Figure 7h). These results demonstrated the cardiac-protective and thera-peutic function of CuZn-CDs in I/R injury.

3. Conclusion

In summary, we reported the synthesis and evaluation of CuZn-CDs as a novel antioxidant for the therapy of ischemia-reperfusion injury. Benefiting from the excellent electron donating/accepting abilities and electron exchange between the dual metal dopants, CuZn-CDs show good stability and process excellent both CAT and SOD abilities. Unlike the previous report that the function can change to highly oxidant, these nanoparticles hold high anti-oxidation abilities even under high temperature or low pH condi-tions, which are conducive to its storage, transport, and long-term therapy. Both in vitro and in vivo studies demonstrated the high ROS scavenging ability and excellent biocompatibility of CuZn-CDs. We also proved that intramyocardial injection of CuZn-CDs in myocardial I/R mice could significantly decrease the infarct area of the heart. Our study provides a novel therapeutic agent against heart I/R injury and other ROS related disease. We expect that this study may pave the way for the development of enzyme-mimicking nanomaterials for biomedical and clinical use.

Supporting InformationSupporting Information is available from the Wiley Online Library or from the author.

AcknowledgementsS.X., T.Z., and X.W. contributed equally to this work. This work was financially supported by National Key Research and Development Program of China (NO. 2019YFA0708700), National Natural Science Foundation of China (Grant No. 81900395, 51672309, 91849209), National Key Research and Development Program of China (NO. 2019YFA0708700), Fundamental Research Funds for Central Universities (18CX07009A), and Shandong Provincial Natural Science Foundation of China (Grant No. ZR2017BH066). The authors also acknowledge the Young Taishan scholars program of Shandong province (Grant No. tsqn20182027). The authors thank Dr. Cuiyun Liu, Dr. Lifan He, Prof. Wencheng Zhang for their help in animal experiments. The authors also thank Prof. Ye Tao, Dr. Can Yu, and Dr. Lirong Zheng from Beijing Synchrotron Radiation Facility, Institute of High Energy Physics, Chinese Academy of Sciences for their support in measurements and data analysis. All animal experiments were carried out in accordance with NIH Guide for the Care and Use of Laboratory Animals (eighth edition, 2011). All experimental protocols involving animal use were approved by Institutional Animal Care and Use Committee of Qingdao University Medical College (No. 20181201c5715190131009).

Conflict of InterestThe authors declare no conflict of interest.

Data Availability StatementThe data that support the findings of this study are available from the corresponding author upon reasonable request.

Keywordsantioxidation, biological activity, carbon dots, copper zinc dopants, photocatalysis

Received: April 15, 2021Revised: May 8, 2021

Published online:

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