
==== Front
Regen Biomater
Regen Biomater
rb
Regenerative Biomaterials
2056-3418
2056-3426
Oxford University Press

10.1093/rb/rbae105
rbae105
Research Article
AcademicSubjects/MED00010
AcademicSubjects/SCI01410
Constructing a highly efficient multifunctional carbon quantum dot platform for the treatment of infectious wounds
Zhang Hangzhen Key Laboratory of Advance Technologies of Materials, Ministry of Education, College of Medicine and School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China

Bai Jiafan Key Laboratory of Advance Technologies of Materials, Ministry of Education, College of Medicine and School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China

Chen Xiangli Key Laboratory of Advance Technologies of Materials, Ministry of Education, College of Medicine and School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China

Wang Linyu Key Laboratory of Advance Technologies of Materials, Ministry of Education, College of Medicine and School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China

Peng Wenzhen Department of Biochemistry and Molecular Biology, College of Basic and Forensic Medicine, Sichuan University, Chengdu 610041, China

Zhao Yuancong Key Laboratory of Advance Technologies of Materials, Ministry of Education, College of Medicine and School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China

https://orcid.org/0000-0002-7034-3215
Weng Jie Key Laboratory of Advance Technologies of Materials, Ministry of Education, College of Medicine and School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China

Zhi Wei Key Laboratory of Advance Technologies of Materials, Ministry of Education, College of Medicine and School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China

Wang Jianxin Key Laboratory of Advance Technologies of Materials, Ministry of Education, College of Medicine and School of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, China

Zhang Kai National Engineering Research Center for Biomaterials, College of Biomedical Engineering, Sichuan University, Chengdu 610064, China

Zhang Xingdong National Engineering Research Center for Biomaterials, College of Biomedical Engineering, Sichuan University, Chengdu 610064, China

Correspondence address. E-mail: jwang@swjtu.edu.cn (J.W.)
2024
24 8 2024
24 8 2024
11 rbae10513 5 2024
13 7 2024
31 7 2024
05 9 2024
© The Author(s) 2024. Published by Oxford University Press.
2024
https://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Antibiotic resistance poses a huge threat to public health, which has increased the difficulty and transmission of disease treatment, as well as the burden and cost of medical institutions. In response to the current problems and challenges in inflammation control and treatment of bacterial infected wounds, inspired by antibacterial mechanisms based on active elements such as N, S, Cu and tannic acid (TA), a highly efficient multifunctional carbon quantum dot platform was proposed in this study and constructed through their special assembly in a solvothermal reaction system for the treatment of infected wounds. By introducing active elements such as N, S and Cu, this carbon quantum dot platform is endowed with antibacterial properties, while also achieving good angiogenesis promoting performance through the use of ion Cu. Meanwhile, the good antioxidant activity of TA (one of the precursors used) enables this platform to have better immunomodulatory performance in vivo. The research results on the treatment of bacterial infection models indicate that the multifunctional carbon quantum dots obtained can accelerate the healing of infected wounds by inhibiting bacterial infection, regulating immunoreaction, accelerating collagen deposition and promoting angiogenesis. This multifunctional carbon quantum dot platform shows good clinical application prospects in treating bacterial infected wounds. Additionally, the fluorescence characteristics of such carbon dots can be expected to realize visual therapy in the future.

Graphical abstract

multifunctional fluorescent carbon dots
antibacterial
immunomodulation
bacterial labeling
promoting angiogenesis
National Key Technologies R&D Program of China 2016YFC1102001 Natural Science Foundation of Sichuan Province 10.13039/501100018542 2022NSFSC0369
==== Body
pmcIntroduction

Bacterial infections have long been a major global public health challenge. The continued overuse and misuse of antibiotics has led to an increase in bacterial resistance. Therefore, the development of new antimicrobial agents has become more urgent [1, 2]. At present, researchers try to find a variety of methods to solve this issue, such as photothermal therapy, magnetic therapy, and the use of metals and metal-oxide nanoparticles [3–8]. However, these methods have some disadvantages in the treatment process that cannot be ignored. For example, photothermal therapy and magnetic therapy are limited in local treatment due to operational complexity and equipment requirements, while some metal and metal-oxide nanoparticles are both costly and toxic. Therefore, this needs to find a new strategy to develop a highly effective antibacterial agent with low toxicity, good biocompatibility and different from traditional small-molecule antibiotics. Carbon dots (CDs), a new type of nanomaterial, have attracted much attention in biomedical applications such as drug delivery [9], bioimaging [10] and antimicrobial agents [11] due to their unique physicochemical and optical properties [12]. They are one of the most widely studied antimicrobial materials with low drug resistance and good biocompatibility to date [13–15]. However, most reported antimicrobial CDs were endowed with antimicrobial function by means of coupling with certain antibacterial materials, such as antibiotics, peptides and quaternary ammonium salts, etc. or with the help of photodynamic power [16–19]. These methods have obvious drawbacks such as cumbersome preparation process, high cost and toxicity. Liu et al. [20] also reported a Cu-doped CD with nanoenzyme activity, which can achieve antibacterial effect through its enzymatic activity to generate reactive oxygen species. However, the related mechanism and system are complicated, making it difficult to be promoted.

Tannic acid (TA), a naturally derived polyphenolic compound, has good biocompatibility, antioxidant, anti-inflammatory and in vivo immunomodulatory properties, as well as a certain degree of antimicrobial activity, which facilitate tissue repair [21–23]. As far as we know, there have been no reports on the synthesis of antibacterial CDs with TA as a precursor yet. In this study, TA would be used as a precursor to explore the synthesis of antibacterial CDs which would be expected to promote tissue repair, and its mechanism was also discussed. In view of the inability of a single TA to synthesize fluorescent CDs, we would functionalize the surface of the CDs by introducing some materials containing N and S heteroatoms to prepare CDs with strong antibacterial and antioxidant activities [24, 25]. In addition, considering that TA contains a large number of phenolic hydroxyl structures, they can provide a large number of reaction sites for the chelation of active metal ions (Cu2+, Zn2+, Mg2+, etc.), which is conducive to the loading of active metal ions and endows CDs with higher biological activity and more functions. Therefore, we designed a highly efficient synergistic antibacterial system by introducing Cu2+, which has antibacterial and vascularizing functions, into the material system.[26, 27] Compared with other antibacterial materials, the fluorescent CDs are smaller in size and easier to contact bacteria through multivalent interactions, thus disrupting the stability and permeability of the bacterial membrane. Also, it would be easier for them to reach every corner of the bacteria through endocytosis, thus affecting the metabolic process inside the bacteria, as well as the synthesis of DNA and protein, and ultimately inhibiting the proliferation of the bacteria, thereby achieving efficient antibacterial activity. In addition, the introduction of Cu2+ would effectively promote vascularization and then wound healing. This simple, green and low-cost synthesis of CDs that have good biocompatibility, in vivo immune regulation and high antibacterial efficiency will open up new ideas for the regeneration and repair of infected tissue (Figure 1).

Figure 1. Schematic diagram of the synthesis of antibacterial CDs and their treatment in bacterial infections of wounds.

Results

Morphological and structural characterization of carbon dots

The morphologies of CDs, Cu-CDs1 and Cu-CDs2 were characterized using transmission electron microscopy (TEM), as shown in Figure 2A–F. It can be seen that CDs, Cu-CDs1 and Cu-CDs2 were well-dispersed with average particle sizes of about 4.59, 4.58 and 4.62 nm, respectively. High-resolution TEM (HRTEM) images show that both CDs, Cu-CDs1 and Cu-CDs2 had well-resolved lattice fringes with crystal plane spacing of 0.28, 0.23 and 0.2 nm, corresponding to the (020) and (100) crystal plane of graphitic carbon, respectively. X-ray diffraction (XRD) spectrum of CDs (Figure 2I) exhibits a strong diffraction peak around 24°, which is due to the typical vibration of graphitic carbon, indicating high crystallinity of CDs, thus being in good agreement with the TEM results. The Raman spectrum (Figure 2J) showed the degree of CDs defects and graphitization, both CDs and Cu-CDs2 showed two characteristic peaks at 1342 and 1554 cm−1, while Cu-CDs1 indicated two characteristic peaks at 1368 and 1593 cm−1, these peaks correspond to the disordered structure or defects (D band) and sp2 graphitic structure (G band) of the carbon material, respectively, showing that the carbon atoms are hybridized by disordered and sp2 hybridization [28, 29]. The intensity ratios of the D and G bands (ID/IG) were 0.85, 0.76 and 0.73 for CDs, Cu-CDs1 and Cu-CDs2, respectively, indicating that both the proportion of indeterminate carbon in the structure and the degree of graphitization increased from CDs to Cu-CDs2. Moreover, the zeta potentials of CDs, Cu-CDs1 and Cu-CDs2 were −25, −19.23 and −6.41 mV (Figure 2G), respectively, indicating that they had good dispersion properties and the surface potential of the material increased due to the doping of copper [30].

Figure 2. TEM and HRTEM images (inset) of (A) CDs, (C) Cu-CDs1 and (E) Cu-CDs2. Particle size distribution diagrams of (B) CDs, (D) Cu-CDs1 and (F) Cu-CDs2. (I) XRD spectra of CDs. (J) Raman spectra of CDs and Cu-CDs2. (L) Raman spectra of Cu-CDs1. (G) Zeta potential of CDs, Cu-CDs1 and Cu-CDs2. (H) FTIR spectra of CDs, Cu-CDs1 and Cu-CDs2. (K) The full XPS spectra of CDs, Cu-CDs1 and Cu-CDs2. (M) High-resolution XPS spectra of C 1s and O 1s of CDs and Cu-CDs2.

The compositions of the CDs and Cu-doped CDs were investigated using Fourier transform infrared (FTIR) spectra and X-ray photoelectron spectroscopy (XPS). As shown in Figure 2H, the FTIR spectra show that CDs, Cu-CDs1 and Cu-CDs2 possessed abundant polar functional groups, such as O-H/N-H (3500 − 3100 cm−1), C=O (1650 − 1600 cm−1), which ensured their good water solubility. Meanwhile, C=C (1530 − 1450 cm−1) and C-O (1200 − 1100 cm−1) were detected on the surface of CDs, while C-N (1410 − 1380 cm−1) was found on the surface of CDs, Cu-CDs1 and Cu-CDs2, indicating the formation of polyaromatic structures in the CDs during the synthesis process. Furthermore, some typical peaks of CDs were observed at 1190, 1063, 999 and 577 cm−1, which were attributed to the stretching vibration of C-S, S=O and C-C=S groups [31], respectively, indicating the successful doping of S on the surface of CDs. While the stretching vibration intensity of S=O at 999 cm−1 decreased and C=O at 1620 cm−1 increased for Cu-CDs1 and Cu-CDs, indicating that the phenolic hydroxyl group of TA underwent redox reactions with Cu2+ upon doping with copper ions [32], and two new strong absorption peak at 1090 and 622 cm−1 of Cu-CDs2 were attributed to the stretching vibration of N-Cu-N and Cu-O [33, 34], respectively, indicating that copper was incorporated during the synthesis of Cu doped CDs, resulting in successful formation of Cu coordination compound.

To further determine the element composition and valence state of the CDs, XPS were performed. As shown in Figure 2K, the full XPS spectra revealed that the surfaces of CDs, Cu-CDs1 and Cu-CDs2 contained five typical peaks of C 1s (284.2 eV), N 1s (400.9 eV), O 1s (532.1 eV), S 2s (228.1 eV) and S 2p (163.4 eV). It was further verified that N and S were involved in the formation process of CDs, which have affected their structure and properties, such as enhanced fluorescent properties and antibacterial activity. In particular, Cu-CDs1 and Cu-CDs2 also contained Cu 2p (932 eV) typical peaks, indicating the successful doping of Cu on the surface of Cu-CDs1 but with a low content of 0.13% (Supplementary Table S1). Therefore, in this study, the Cu-CDs2 with 5.65% Cu content was prepared by increasing the proportion of Cu2+ in the reactions system. In the high-resolution XPS spectrum (Figure 2M and Supplementary Figures S1 and S2), it can be seen that the C 1 s spectra can be deconvoluted into C-C/C=C (284.6 eV), C-N (285.5 eV), C-O (286.2 eV), C=O (287.2 eV) and COOH (288.8 eV). The N 1s spectra can be deconvoluted into pyrrole nitrogen (399.8 eV), graphite nitrogen (401.6 eV), and N-O (406.8 eV). The O 1s spectra can be deconvoluted into C=O (531.8 eV) and C-O (533 eV) [35, 36]. The S 2p spectra mainly consists of two peaks centered at 164 and 167.6 eV, which indicates the presence of sulfur in two forms, the former peak can be deconvoluted into S 2p3/2 (163.8 eV) and S 2p1/2 (165 eV), while the latter peak can be deconvoluted into peaks located at 168.1, 168.8 and 169.9 eV for C-SOX (X = 2, 3, 4) [37]. The peaks at 932.8 and 934.9 eV in the Cu 2p spectra were corresponded to the Cu 2p3/2 feature of Cu+ and Cu2+, and the peaks at 952.4 and 954.5 eV were attributed to the Cu 2p1/2 feature of Cu+ and Cu2+ [20, 38]. The results further validated the successful introduction of N, S and Cu, which provided the structural basis for the antibacterial activity of N, S and Cu active elements and Cu2+ promoted vascularization. In addition, the increase of the COOH content in the C 1s spectra and C=O in the O 1s spectra (Supplementary Table S2) further demonstrated that the coordination and redox reactions between Cu2+ and the phenolic hydroxyl group of TA increased CDs’ oxidation degree, which is consistent with the analysis results of FTIR.

Optical properties of carbon dots

The UV-vis absorption spectra of CDs, Cu-CDs1 and Cu-CDs2 are shown in Figure 3A–C. Which exhibit an absorption peaks at 267 and 270 nm, respectively, corresponding to the π–π* transitions of aromatic sp2 domains in the carbon cores, while the absorption peak at 363 nm for Cu-CDs2 should be due to the n−π* transitions caused by edge transition of CDs. The edge band of CDs has been reported many times and refers to the edge atoms in the crystalline carbon nuclei, especially in some blue fluorescent CDs where this feature is often present [39]. As shown in Figure 3A, when CDs was dissolved in water, it appeared light yellow and exhibit bright green fluorescence under UV irradiation (λ = 365 nm). When a small amount of Cu2+ ions were doped in the raw material system, the prepared Cu-CDs1 showed a brownish yellow color when dissolved in water (Figure 3B), and they emitted dull green fluorescence under UV irradiation (λ = 365 nm), however, their fluorescence performance was relatively poor. Considering this, this study further doped more Cu2+ and allowed them to participate in the system reaction and prepared Cu-CDs2. As shown in Figure 3C, Cu-CDs2 showed a transparent and colorless state when dissolved in water, which was in sharp contrast to the color of the initial CDs solution (light yellow). Moreover, it exhibited blue fluorescence under 365 nm UV excitationn, and its fluorescence performance was improved compared to Cu-CDs1. Therefore, from the changes in solution color and fluorescence color, it is further verified that Cu2+ participated in the reaction in this system.

Figure 3. Absorption spectra, PL excitation and PL emission spectra (photographs of CDs under daylight (left) and UV light (excited at 365 nm) (right)) of (A) CDs, (B) Cu-CDs1 and (C) Cu-CDs2. PL emission spectra of (D) CDs, (E) Cu-CDs1 and (F) Cu-CDs2 under excitation of different wavelengths of light.

To further investigate the fluorescence properties of the prepared CDs, the analysis of fluorescence spectra were performed in this study. As shown in Figure 3D–F, the maximum emission peaks of CDs, Cu-CDs1 and Cu-CDs2 were at 495, 455 and 488 nm with quantum yields of 10%, 1% and 12%, respectively. We further tested the variation on the fluorescence properties of CDs, Cu-CDs1 and Cu-CDs2 at the same excitation wavelength. As shown in Supplementary Figure S3, the fluorescence emission wavelength was blue-shifted from CDs to Cu-CDs2, and the fluorescence intensity showed a decrease followed by an increase. The results indicated that the fluorescence performance of Cu-CDs1 was poor when doped with a small amount of Cu2+. While the Cu2+ content was increased, we obtained Cu-CDs2 with good fluorescence performance, which will provide the possibility for CDs and Cu-CDs2 to be used in fields such as fluorescent labeling. We further investigated the effects of different pH levels, NaCl concentration and hydrogen peroxide concentration environments, and laser radiation time on the fluorescence intensity of CDs and Cu-CDs2. Supplementary Figure S4 shows that the fluorescence intensity of CDs slightly decreased with increasing pH, but the relative change was not significant, while the fluorescence intensity of Cu-CDs2 remained basically stable with changing pH value. Even when CDs and Cu-CDs2 were in a medium environment of 2 mol/l NaCl, 0.1 mmol/l H2O2 or exposed to an Xe lamp for 1 h, their fluorescence intensity still remained stable. The results indicate that CDs and Cu-CDs2 have good fluorescence stability, which provides a basis for their application in complex biological environments.

In vitro antimicrobial test

The antibacterial effect of CDs against E. coli (Gram-negative) and S. aureus (Gram-positive) was evaluated using the plate counting method. Figure 4A and B show that the number of colonies decreased obviously on the plates with increasing concentrations of CDs, corresponding to an improvement of antibacterial performance. When the bacteria were treated with 0.1 mg/ml of CDs, there were only a small number of E. coli and S. aureus on the plates, and the antibacterial rates of CDs against E. coli and S. aureus reached 99.03% and 99.79%, respectively. Gao et al. [12] synthesized CDs by using ampicillin as raw material. When the concentration of CDs were 0.7 mg/l and under visible light irradiation, they showed excellent antibacterial capacity only against S. aureus, however, the antibacterial capacity was not obvious in the dark. In contrast, the CDs prepared in this study have the advantages of low cost, short preparation time, excellent antimicrobial capacity against both S. aureus and E. coli, and easy operation without the use of photodynamic power.

Figure 4. (A) Plate count images of E. coli and S. aureus after co-culture with different concentrations of CDs, AMP, Cu-CDs1 and Cu-CDs2. (B) Antibacterial rate 3D plots of CDs with different concentrations against E. coli and S. aureus. Antibacterial rate of CDs, Cu-CDs1 and Cu-CDs2 with different concentrations against (C) E. coli and (D) S. aureus. (E) SEM images of E. coli and S. aureus after treatment with CDs, Cu-CDs1 and Cu-CDs2 (The arrows point to perforation, collapse and outflow of bacterial contents, respectively). (F) Fluorescence microscopy images of E. coli and S. aureus incubated with CDs and Cu-CDs2, respectively (fluorescence images were captured under the excitation of 488 and 405 nm for CDs and Cu-CDs2, respectively) (* indicates significant differences, *P < 0.05, **P < 0.01, ***P < 0.001).

The antimicrobial properties of CDs, Cu-CDs1 and Cu-CDs2 have been also compared. As shown in Figure 4C and D, compared to CDs, the antibacterial properties of Cu-CDs1 increased slightly due to less copper doping, however, Cu-CDs2 had a significantly enhanced antibacterial effect was its antibacterial rates against E. coli and S. aureus were 99.86% and 100%, respectively. When its concentration was only 0.08 mg/ml, as shown in Figure 4A, even at such concentration, a large number of E. coli and S. aureus were still present after treatment with the ampicillin sodium antibiotic (AMP), while the number of bacteria decreased substantially after treatment with CDs and Cu-CDs1, and they almost disappeared after treatment with Cu-CDs2. These results showed that the antimicrobial CDs prepared in this study had superior antimicrobial effect compared with traditional antibiotic AMP, and that the antimicrobial properties of CDs could be greatly improved due to copper doping and the antimicrobial effect increased significantly with increasing the copper content. The antibacterial properties of Cu-doped CDs were also reported [40], these Cu-doped CDs (with a copper content of 3.95%) at a concentration of 0.156 mg/ml exhibited significant antibacterial effect against S. aureus, however, didn’t work against E. coli. In contrast, the Cu-doped CDs prepared in this study have excellent antibacterial performance against both E. coli and S. aureus.

Next, to explore the mechanism of the antimicrobial effect of CDs. We used scanning electron microscopy (SEM) to observe the morphological changes of bacteria and the disruption of bacterial membranes before and after treatment with antimicrobial CDs. As shown in Figure 4E, the E. coli and S. aureus without CDs treatment remained typically spherical or rod shape, and the surface was intact and smooth. However, the bacteria were damaged to different degrees after treatment with CDs, Cu-CDs1 and Cu-CDs2. In contrast, Cu-CDs2 caused greater damage to both E. coli and S. aureus, as reflected by deformation, perforation and collapse of the bacterial membrane, and even the phenomenon of partial bacterial content flowing out. Particularly, form SEM images, it can be observed that the surface deformation of S. aureus treated with CDs was less pronounced than E. coli. This kind of antimicrobial mechanism of CDs against S. aureus might be primarily attributed to the endocytosis of bacterial membranes by CDs, which in turn led to great damage to the DNA or proteins of S. aureus and ultimately achieved highly efficient antimicrobial activity.

Bacterial labeling imaging

We further explored the potential of antimicrobial CDs in bacterial labeling. E. coli and S. aureus were incubated with CDs and Cu-CDs for a period of time, respectively, and then observed under fluorescence microscopy. As shown in Figure 4F, E. coli and S. aureus treated without CDs and Cu-CDs2 did not show any fluorescence signal under fluorescence microscope, while the bacteria treated with CDs and Cu-CDs2 showed green and blue fluorescence with wide distribution, respectively. The results showed that CDs and Cu-CDs2 had good labeling imaging effects on both E. coli and S. aureus, indicating their potential as fluorescent probes for bacterial imaging. Furthermore, the results of bacterial labeling experiments showed that the antimicrobial CDs could bind freely to bacteria and distribute evenly within the bacteria, further verifying the mechanism of endocytosis of CDs by bacterial membranes, which is consistent with the SEM results.

Characterization of biological properties of carbon dots

Antioxidation properties

Antioxidant CDs can improve the wound healing process by regulating the excessive production of reactive oxygen species [41–43]. In this study, the antioxidant properties of CDs were evaluated by 1,1-2-phenyl-2-bitter base hydrazyl (DPPH) radical scavenging method which is based on a phenomenon that after scavenging free radicals, the color of DPPH will change from purple to yellow [44, 45]. As shown in Figure 5A, when the concentration of CDs was in the range of 0–17 μg/ml, the color of the solution gradually changed from purple to light yellow as the concentration of CDs increased, corresponding to the weakening of the UV absorption peak at 517 nm in Figure 5B, and the scavenging rate of DPPH radical was subsequently increased. When the concentration of CDs was only 17 μg/ml, the scavenging rate of DPPH reached 94.73% (Figure 5D). This is due to the phenolic hydroxyl groups on the surface of CDs can be oxidized into quinones, which can capture free radicals and then endow CDs with excellent antioxidant properties [46]. We further compared the antioxidant properties of CDs, Cu-CDs1 and Cu-CDs2. With the doping of Cu2+ in CDs, the color of the DPPH solution incubated with Cu-CDs1and Cu-CDs2 showed a deepening phenomenon (Figure 5C). This is attributed to the reactions between the phenolic hydroxyl groups of TA and copper ions, which consumed a portion of the phenolic hydroxyl groups and thus diminished the antioxidant performance, but still maintained a certain antioxidant activity, which lays a good foundation for their in vivo immunomodulatory capacity.

Figure 5. (A) Photos and (B) UV-vis spectrum of DPPH after co-incubation with different concentrations of CDs. (C) Photos of DPPH after co-incubation with CDs, Cu-CDs1 and Cu-CDs2 at a concentration of 200 μg/ml. (D) Scavenging rate of DPPH for different concentrations of CDs, Cu-CDs1 and Cu-CDs2. (E) Photos of hemolysis for different concentrations of CDs (top) and CDs, Cu-CDs1 and Cu-CDs2 at a concentration of 200 μg/ml (bottom). (F) Hemolysis rates for different concentrations of CDs, Cu-CDs1 and Cu-CDs2. (G) Fluorescent images of AO/PI stained L929 cells after 3 days of co-culture with CDs (I is the blank group, II is CDs group, III is Cu-CDs1 group and IV is Cu-CDs2 group). (H) Cytotoxicity for different concentrations of CDs, Cu-CDs1 and Cu-CDs2 on L929 cells (* indicates significant differences, *P < 0.05, **P < 0.01, ***P < 0.001).

Hemocompatibility

Good hemocompatibility is an important indicator for biomaterials [47, 48]. According to F756-2000 standard from the American Society for Testing and Materials (ASTM), the hemolysis rate of medical devices must be less than 5% [49], for that, we evaluated the hemocompatibility of CDs, Cu-CDs1 and Cu-CDs2. As shown in Figure 5E and F, compared with the positive control (PC), there was no significant hemolysis in the supernatant after treatment with CDs in the concentration range of 0–200 μg/ml. Moreover, after treatment with Cu-CDs1 and Cu-CDs2, the supernatant also did not show significant hemolysis. Even when the concentrations of CDs, Cu-CDs1 and Cu-CDs2 reached 200 μg/ml, their hemolysis rate was still less than 5%, reaching the standard of medical device use. The results showed that in the concentration range of 0–200 μg/ml, CDs, Cu-CDs1 and Cu-CDs2 had good blood compatibility.

Cytotoxicity assay

The cytotoxicity of CDs, Cu-CDs1 and Cu-CDs2 on L929 cells was assessed through MTT method. As shown in Figure 5G and H, compared with the blank group, the survival rate of L929 cells decreased slightly with increasing material concentration and doped Cu2+ content. When the concentration of CDs, Cu-CDs1 and Cu-CDs2 reached 100 μg/ml, the RGR value remained above 84%. According to the standard, if the RGR value is greater than 75%, it is evaluated as noncytotoxic and can be used as medical materials. In addition, it can be seen that the cell morphology of both the material and the blank group was intact and evenly distributed. The results indicate that CDs, Cu-CDs1 and Cu-CDs2 all exhibit good cytocompatibility, demonstrating that the antimicrobial materials prepared in this study can meet the basic requirements of biomedical materials, which creates the basic conditions for subsequent in vivo therapeutic experiments.

In vivo antibacterial and wound healing efficacy

After demonstrating that CDs, Cu-CDs1 and Cu-CDs2 have excellent antibacterial properties, we further investigated their therapeutic ability after bacterial infection on skin wounds of SD rats (Ethical approval number: KS2019052). Firstly, we established a model of total skin defect induced with S. aureus, and Figure 6A showed the wound healing process in SD rats, compared with the uninfected control group (Control), the suppuration and inflammation were visible on the wound after modeling (0 day) in each infected group, and the wound area increased compared with the initial size, indicating the successful modeling in this experiment. Subsequently, the wounds treated with CDs, Cu-CDs1 and Cu-CDs2 started to crust and the size of wound was obviously reduced, compared with the infected control group (Control-inf), especially when the wound was treated with Cu-CDs2, its healing was maximally accelerated (Figure 6B). On the 15th day of treatment, both the control group (healing rate of 76.5%) and the control-inf group (healing rate of 65.7%) had some unhealed area. This is because the inflammatory secretions were not controlled in time, the healing speed was slower than that of the material group. CDs have good antimicrobial properties and can effectively inhibit bacteria at the wound site, thus resulting in an increased healing rate (82.9%) compared with the control group, while the wounds treated with Cu-CDs1 showed a higher healing rate (85.1%) on the 15th day of treatment. Compared with the CDs group, the effect of promoting repair for doped CDs is not obvious when they have a lower copper content. However, with increasing Cu2+ content, Cu-CDs2 exhibited stronger antibacterial effect and better tissue repair ability. A healing rate of 99.6% was seen on day 15 for Cu-CDs2, showing that Cu-CDs2 significantly accelerated wound healing (Figure 6C). Although photodynamic therapy and antimicrobial gold nanoparticles were also reported to treat bacterially infections and have achieved certain therapeutic effects, the preparation and treatment processes were complex [50]. In contrast, our materials are simpler and less expensive in preparation and our treatment method can achieve good curative effect without relying on any photodynamic power, thus providing a simple and effective way for treatment and tissue repair of infected wounds.

Figure 6. (A) Representative photographs of S. aureus infected wounds after treatment with different materials on days 0, 1, 5, 10 and 15. (B) Wound area and (C) wound healing rate after different treatments. (D) Gram staining, (E) H&E staining and (F) Masson staining images of wound tissue in the control group, control-inf group, CDs group, Cu-CDs1 group and Cu-CDs2 group (* indicates significant differences, *P < 0.05, **P < 0.01, ***P < 0.001).

Histological and biochemical evaluation of skin infected wound healing

S. aureus is a Gram-positive bacterium that stains blue-purple in the Gram staining, hence, we can use Gram staining to evaluate bacterial infection of traumatic tissue and the antibacterial effect of materials on S. aureus. As seen in Figure 6D, after being infected by S. aureus, bacteria could be observed in all groups, among which a lot of bacteria were found in the blank infection group. In addition, because the wound was exposed, some bacteria were also detected in the blank uninfected group. After material treatment, bacteria were effectively suppressed, demonstrating that the as-prepared CDs have excellent in vivo antibacterial properties, with Cu CDs2 being the most prominent. To further assess the tissue regeneration of the wound, we performed H&E and Masson staining of the traumatic tissue. As shown in Figure 6E and F, compared with the control and control-inf groups, the materials groups can reduce inflammatory cell infiltration, promote epidermal tissue continuity, and enhance collagen deposition. In particular, after treatment with Cu-CDs2, the epidermis was greatly restored on Day 10, and collagen was arranged in a regular and oriented manner. On Day 15, the infiltration of inflammatory cells has subsided significantly, and the whole skin layer has basically healed and resembles the surrounding normal tissue, and a large amount of collagen deposition and more regenerative attachment formation can be seen, which effectively improved the tissue matrix. The results showed that bacterial invasion greatly hindered wound healing, however, it can be seen that the introduction of the CDs prepared in this study with excellent in vivo and in vitro antibacterial properties not only promoted wound healing by inhibiting bacteria but also accelerated wound repair and regeneration due to the regulatory effect of Cu2+ on promoting vascularization.

To assess inflammation and angiogenesis of traumatic tissue, we performed immunohistochemical staining analysis of TNF-α and CD31. As shown in Figure 7A and B, positive expression of inflammatory factors was observed in all groups. However, the materials group could reduce the expression of inflammatory factors to some extent, which is attributed to the good antioxidant activity of CDs. This endows them with better in vivo immunomodulatory properties, resulting in effective inhibition of traumatic inflammatory. Meanwhile, more neovascularization was formed in the wound after treatment with the CDs, especially Cu-CDs2 by virtue of its excellent promoting vascularization effect of Cu2+. The results suggest that CDs can play an active role in vivo immune modulation and angiogenesis, thereby maximizing the acceleration of wound healing.

Figure 7. (A) TNF-α immunostaining and (B) CD31 immunostaining (The arrows point to new blood vessels) images of wound tissue in the control group, control-inf group, CDs group, Cu-CDs1 group and Cu-CDs2 group. (C) The relative expression of iNOS mRNA in tissues by real-time quantitative PCR. (D) The relative expression of Arginase-1 mRNA in tissues by real-time quantitative PCR (* indicates significant differences, *P < 0.05, **P < 0.01, ***P < 0.001).

There are two phenotypes of macrophages, namely M1 type or M2 type, where M1 type macrophages mainly secrete TNF-α pro-inflammatory factors, which have pro-inflammatory effect and exert host immune function, while M2 macrophages mainly secrete anti-inflammatory factors including IL-4, which have functions of promoting extracellular matrix reconstruction and inflammatory intervention [51]. To quantitatively detect the in vivo immune regulatory effect of CDs, we conducted real-time fluorescence quantitative analysis of the expression levels of two phenotypes (M1 or M2) of macrophages. Because they each have respective marker proteins such as iNOS and ARG-1, the ability of immune regulation of CDs can be analyzed by detecting the mRNA expression levels of iNOS and ARG-1. As shown in Figure 7C, when we used the control group as a reference, it can be seen that the mRNA expression level of M1-type marker protein (iNOS) was significantly higher than that in the control-inf group, indicating that M1 macrophages significantly increased and the wounds were still in a high inflammatory phase. However, after treatment with the CDs, Cu-CDs1 and Cu-CDs2, the mRNA expression level of iNOS was significantly reduced, which was due to the excellent antimicrobial properties of the CDs that can inhibit bacterial growth and the good antioxidant activity that can effectively stimulate in vivo immune modulation, resulting in a relative reduction of tissue inflammation. In addition, Figure 7D shows the mRNA expression level of M2 type marker protein (ARG-1) was also significantly higher in the control-inf group which was undergoing a high inflammatory phase, indicating that M2 macrophages significantly increased and actively engaged in inflammatory intervention to maintain immune homeostasis. While the mRNA expression levels of ARG-1 in the materials groups were significantly lower than the control and control-inf groups, which might be due to the fact that the materials groups were in the low inflammatory zone or had passed the inflammatory phase, resulting in relatively fewer M2 type macrophages. The results further validated that CDs, Cu-CDs1 and Cu-CDs2 can reduce tissue inflammation and promote wound repair through in vivo immune modulation.

To further evaluate the biosafety in vivo of the materials, the heart, liver, spleen, lung and kidney of each group of SD rats were removed and analyzed using H&E staining on Day 15 postoperatively. As can be seen in Supplementary Figure S5, there were no obvious histopathological abnormalities such as damage, necrosis or inflammation in any of the groups, indicating that there were no obvious toxic side effects on the organs of SD rats in each treatment group. Therefore, the CDs, Cu-CDs1 and Cu-CDs2 have good biosafety in vivo, which makes them possess great potential as an antimicrobial and repair material for skin wounds.

Discussion

Although there have been sporadic reports on the preparation of antimicrobial CDs using different strategies, there are fewer reports on the hybrid application of CDs in both antibacterial and promoting tissue repair. For example, Miao et al. [52] used a method by coupling CDs with antimicrobial materials to obtain antimicrobial CDs, Wang et al. [11] and Gao et al. [12] synthesized them using materials with stronger antimicrobial properties. The antimicrobial CDs prepared by Yan et al. [53] need to be irradiated with blue light to be endowed with antibacterial activity, while the ones prepared by Otis et al. [54] showed the antibacterial effect only against Gram-negative bacteria. Additionally, these methods have some drawbacks of cumbersome preparation process, low chemical stability, harsh antibacterial conditions and operation methods and poor broad-spectrum antibacterial activity. Moreover, the above CDs lack the ability to scavenge free radicals induced through inflammatory reactions, thus, it is difficult to achieve optimal tissue repair. In view of this, we designed the CDs with antioxidant and immunomodulatory properties by introducing TA, S and Cu2+ with antimicrobial properties. The reasons for choosing S and Cu2+ in terms of antibacterial properties are as follows: (i) The sulfur containing structure in the CD can react with some proteins and lipopolysaccharide on the bacterial membrane, causing damage to the integrity and stability of the cell membrane [55]. (ii) The introduction of Cu2+ ions into the system can enhance the destruction of the permeability of the cell membrane and cause the imbalance of substances inside and outside the cell, as well as the leakage of cytoplasm, which in turn causes the inactivity of enzymes and interferes with internal metabolic processes such as DNA replication, transcription and repair within the bacteria [56, 57], to achieve an enhanced antibacterial activity.

In addition to antibacterial properties, angiogenesis is another important link in wound healing [58, 59]. The pro-angiogenic function of Cu2+ has been reported, which can participate in the regulation of vascular endothelial growth factor, stimulate the proliferation and differentiation of endothelial cells as well as collagen synthesis and deposition, ultimately promoting angiogenesis [60, 61]. Additionally, the use of TA (precursors) in this study can effectively scavenge free radicals and protect wounds from oxidative stress with its antioxidant activity. On the other hand, it can induce in vivo immunomodulation to decrease the expression of TNF- and promote the conversion of macrophages from M1 type to M2 type, thereby reducing tissue inflammation. Meanwhile, its immunomodulatory properties can also accelerate the regeneration of new blood vessels in wound tissue [62, 63]. The use of Cu2+ and TA created a synergistic pro-angiogenic effect.

Therefore, under the synergistic action of TA, S and Cu2+, not only the double efficient antibacterial effect of CDs against Gram-negative and Gram-positive bacteria but also enhanced in vivo immunomodulation and angiogenesis could be realized. At the same time, the potential of the antibacterial CDs for bacterial fluorescent labeling provides a great possibility for realizing visual antibacterial and therapeutic. All these will provide huge guarantees for multifunctional CDs in the treatment of infectious wounds.

Conclusion

TA- and thioacetamide-based antibacterial CDs doped with N, S and Cu2+ were successfully synthesized by a simple one-step hydrothermal method. The obtained antibacterial CDs can fluorescently label bacteria and inhibit the proliferation of bacteria. Additionally, the use of tannin (one of precursors used) and ions N, S and Cu allow CDs to achieve stronger functions in antibacterial, immunoregulation and promoting vascularization. Compared with traditional dressing, such antibacterial CDs show better application prospects in treatment of infected trauma. Furthermore, the fluorescence characteristics of such CDs can be expected to realize visual therapy.

Supplementary Material

rbae105_Supplementary_Data

Funding

This work was supported by The National Key Technologies R&D Program of China (grant number 2016YFC1102001) and the Natural Science Foundation of Sichuan Province (grant number 2022NSFSC0369).

Supplementary data

Supplementary data are available at Regenerative Biomaterials online.

Conflicts of interest statement. The authors declare no conflict of interest.
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References

1 Yu M , LiP, HuangR, XuC, ZhangS, WangY, GongX, XingX. Antibacterial and antibiofilm mechanisms of carbon dots: a review. J Mater Chem B 2023;11 :734–54.36602120
2 Chai S , XieY, YangL. Antibacterial applications of elemental nanomaterials. Curr Opin Solid St M 2022;26 :101043.
3 Cao D , DingJ. Recent advances in regenerative biomaterials. Regen Biomater 2022;9 :rbac098.36518879
4 Liu Y , WangG, LuoH, ZhaoB, LiaoM, DaiQ, LiM, LiQ, CaoX. Phosphoserine enhanced Cu-doped bioactive glass dynamic dual-network hydrogel for craniofacial bone defect repair. Regen Biomater 2023;10 :rbad054.37287897
5 Hong X , TianG, ZhuY, RenT. Exogeneous metal ions as therapeutic agents in cardiovascular disease and their delivery strategies. Regen Biomater 2024;11 :rbad103.38173776
6 Qi X , HuangY, YouS, XiangY, CaiE, MaoR, PanW, TongX, DongW, YeF, ShenJ. Engineering robust Ag-decorated polydopamine nano-photothermal platforms to combat bacterial infection and prompt wound healing. Adv Sci (Weinh) 2022;9 :e2106015.35191211
7 Ma X , WangL, WangP, LiuZ, HaoJ, WuJ, ChuG, HuangM, MairL, HuangC, XuT, YingT, TangX, ChenY, CaiX, ZhengY. An electromagnetically actuated magneto-nanozyme mediated synergistic therapy for destruction and eradication of biofilm. Chem Eng J 2022;431 :133971.
8 Sun C , WangX, DaiJ, JuY. Metal and metal oxide nanomaterials for fighting planktonic bacteria and biofilms: a review emphasizing on mechanistic aspects. Int J Mol Sci 2022;23 :11348.36232647
9 Li S , SuW, WuH, YuanT, YuanC, LiuJ, DengG, GaoX, ChenZ, BaoY, YuanF, ZhouS, TanH, LiY, LiX, FanL, ZhuJ, ChenAT, LiuF, ZhouY, LiM, ZhaiX, ZhouJ. Targeted tumour theranostics in mice via carbon quantum dots structurally mimicking large amino acids. Nat Biomed Eng 2020;4 :704–16.32231314
10 Li R , WeiF, WuX, ZhouP, ChenQ, CenY, XuG, ChengX, ZhangA, HuQ. PEI modified orange emissive carbon dots with excitation-independent fluorescence emission for cellular imaging and siRNA delivery. Carbon 2021;177 :403–11.
11 Zhao D , LiX, XuM, JiaoY, LiuH, XiaoX, ZhaoH. Preparations of antibacterial yellow-green-fluorescent carbon dots and carbon dots-lysozyme complex and their applications in bacterial imaging and bacteria/biofilm inhibition/clearance. Int J Biol Macromol 2023;231 :123303.36657551
12 Xu Q , LiC, MengX, DuoX, FengY. Polyethylenimine-modified graphene quantum dots promote endothelial cell proliferation. Regen Biomater 2024;11 :rbae013.38525325
13 Wu L , YangY, HuangL, ZhongY, ChenY, GaoY, LinL, LeiY, LiuA. Levofloxacin-based carbon dots to enhance antibacterial activities and combat antibiotic resistance. Carbon 2022;186 :452–64.
14 Hajipour MJ , FrommKM, AshkarranAA, Jimenez de AberasturiD, de LarramendiIR, RojoT, SerpooshanV, ParakWJ, MahmoudiM. Antibacterial properties of nanoparticles. Trends Biotechnol 2012;30 :499–511.22884769
15 Wang H , SongZ, GuJ, LiS, WuY, HanH. Nitrogen-doped carbon quantum dots for preventing biofilm formation and eradicating drug-resistant bacteria infection. ACS Biomater Sci Eng 2019;5 :4739–49.33448817
16 Gao Z , YangD, WanY, YangY. One-step synthesis of carbon dots for selective bacterial inactivation and bacterial differentiation. Anal Bioanal Chem 2020;412 :871–80.31901958
17 Mazumdar A , HaddadY, MilosavljevicV, MichalkovaH, GuranR, BhowmickS, MoulickA. Peptide-carbon quantum dots conjugate, derived from human retinoic acid receptor responder protein 2, against antibiotic-resistant gram positive and gram negative pathogenic bacteria. Nanomaterials 2020;10 :325.32075033
18 Jijie R , BarrasA, BouckaertJ, DumitrascuN, SzuneritsS, BoukherroubR. Enhanced antibacterial activity of carbon dots functionalized with ampicillin combined with visible light triggered photodynamic effects. Colloids Surf B Biointerfaces 2018;170 :347–54.29940501
19 Romero MP , AlvesF, StringasciMD, BuzzaHH, CiolH, InadaNM, BagnatoVS. One-pot microwave-assisted synthesis of carbon dots and in vivo and in vitro antimicrobial photodynamic applications. Front Microbiol 2021;12 :662149.34234756
20 Liu M , HuangL, XuX, WeiX, YangX, LiX, WangB, XuY, LiL, YangZ. Copper doped carbon dots for addressing bacterial biofilm formation, wound infection, and tooth staining. ACS Nano 2022;16 :9479–97.35713471
21 Wang Y , LuoM, LiT, XieC, LiS, LeiB. Multi-layer-structured bioactive glass nanopowder for multistage-stimulated hemostasis and wound repair. Bioact Mater 2023;25 :319–32.36844363
22 Guo Z , XieW, LuJ, GuoX, XuJ, XuW, ChiY, TakuyaN, WuH, ZhaoL. Tannic acid-based metal phenolic networks for bio-applications: a review. J Mater Chem B 2021;9 :4098–110.33913461
23 Wu C , ZhouZ, YouX, GuoY, ChenP, LiH, TongX. Tannic acid-loaded hydrogel coating endues polypropylene mesh with hemostatic and anti-inflammatory capacity for facilitating pelvic floor repair. Regen Biomater 2022;9 :rbac074.36267152
24 Chen B , LiuM, LiC, HuangC. Fluorescent carbon dots functionalization. Adv Colloid Interface Sci 2019;270 :165–90.31265929
25 Varghese M , BalachandranM. Antibacterial efficiency of carbon dots against gram-positive and gram-negative bacteria: a review. J Environ Chem Eng 2021;9 :106821.
26 Li J , ZhaiD, LvF, YuQ, MaH, YinJ, YiZ, LiuM, ChangJ, WuC. Preparation of copper-containing bioactive glass/eggshell membrane nanocomposites for improving angiogenesis, antibacterial activity and wound healing. Acta Biomater 2016;36 :254–66.26965395
27 Yu Q , HanY, WangX, QinC, ZhaiD, YiZ, ChangJ, XiaoY, WuC. Copper silicate hollow microspheres-incorporated scaffolds for chemo-photothermal therapy of melanoma and tissue healing. ACS Nano 2018;12 :2695–707.29518321
28 Ding H , JiY, WeiJ, GaoQ, ZhouZ, XiongH. Facile synthesis of red-emitting carbon dots from pulp-free lemon juice for bioimaging. J Mater Chem B 2017;5 :5272–7.32264113
29 Yuan F , YuanT, SuiL, WangZ, XiZ, LiY, LiX, FanL, TanZ, ChenA, JinM, YangS. Engineering triangular carbon quantum dots with unprecedented narrow bandwidth emission for multicolored LEDs. Nat Commun 2018;9 :2249.29884873
30 Peng S , WangC, XieJ, SunS. Synthesis and stabilization of monodisperse Fe nanoparticles. J Am Chem Soc 2006;128 :10676–7.16910651
31 Yang X , GuoY, LiangS, HouS, ChuT, MaJ, ChenX, ZhouJ, SunR. Preparation of sulfur-doped carbon quantum dots from lignin as a sensor to detect Sudan I in an acidic environment. J Mater Chem B 2020;8 :10788–96.33156321
32 Salzer R. Peter R. Griffiths, James A. de Haseth: Fourier transform infrared spectrometry (2nd edn.). Anal Bioanal Chem 2008;391 :2379–80.
33 Wu W , ZhanL, FanW, SongJ, LiX, LiZ, WangR, ZhangJ, ZhengJ, WuM, ZengH. Cu-N dopants boost electron transfer and photooxidation reactions of carbon dots. Angew Chem Int Ed Engl 2015;54 :6540–4.25881689
34 Mu Z , HuaJ, FengS, YangY. A ratiometric fluorescence and light scattering sensing platform based on Cu-doped carbon dots for tryptophan and Fe(III). Spectrochim Acta A Mol Biomol Spectrosc 2019;219 :248–56.31048254
35 Ding H , WeiJ, ZhangP, ZhouZ, GaoQ, XiongH. Solvent-controlled synthesis of highly luminescent carbon dots with a wide color gamut and narrowed emission peak widths. Small 2018;14 :e1800612.29709104
36 Miao X , QuD, YangD, NieB, ZhaoY, FanH, SunZ. Synthesis of carbon dots with multiple color emission by controlled graphitization and surface functionalization. Adv Mater 2018;30 :1704740.
37 Ding H , WeiJ, XiongH. Nitrogen and sulfur co-doped carbon dots with strong blue luminescence. Nanoscale 2014;6 :13817–23.25297983
38 Padhan S , RoutTK, NairUG. N-doped and Cu,N-doped carbon dots as corrosion inhibitor for mild steel corrosion in acid medium. Colloids Surface A 2022;653 :129905.
39 Sharma A , GadlyT, GuptaA, BallalA, GhoshSK, KumbhakarM. Origin of excitation dependent fluorescence in carbon nanodots. J Phys Chem Lett 2016;7 :3695–702.27588560
40 Qing W , ChenK, YangY, WangY, LiuX. Cu2+-doped carbon dots as fluorescence probe for specific recognition of Cr (VI) and its antimicrobial activity. Microchem J 2020;152 :104262.
41 Zhu Y , ZhangJ, SongJ, YangJ, XuT, PanC, ZhangL. One-step synthesis of an antibacterial and pro-healing wound dressing that can treat wound infections. J Mater Chem B 2017;5 :8451–8.32264512
42 Garcia CD , BallesterAM, AlienaVA, CarabenRA, LlorisJM. Use of cyanoacrylate adhesives in general surgery. Surg Today 2015;45 :939–56.25344231
43 Archana D , SinghBK, DuttaJ, DuttaPK. Chitosan-PVP-nano silver oxide wound dressing: in vitro and in vivo evaluation. Int J Biol Macromol 2015;73 :49–57.25450048
44 Pedro AC , PanizOG, FernandesIAA, BortoliniDG, RubioFTV, HaminiukCWI, MacielGM, MagalhaesWLE. The importance of antioxidant biomaterials in human health and technological innovation: a review. Antioxidants 2022;11 :1644.36139717
45 Parham S , KharaziAZ, Bakhsheshi-RadHR, NurH, IsmailAF, SharifS, RamaKrishnaS, BertoF. Antioxidant, antimicrobial and antiviral properties of herbal materials. Antioxidants 2020;9 :1309.33371338
46 Andzi Barhé T , Feuya TchouyaGR. Comparative study of the anti-oxidant activity of the total polyphenols extracted from hibiscus sabdariffa L., Glycine max L. Merr., yellow tea and red wine through reaction with DPPH free radicals. Arabian J Chem 2016;9 :1–8.
47 Wei L , LiQ, ChenY, ZhangJ, MiY, DongF, LeiC, GuoZ. Enhanced antioxidant and antifungal activity of chitosan derivatives bearing 6-O-imidazole-based quaternary ammonium salts. Carbohydr Polym 2019;206 :493–503.30553350
48 Garlea A , MelnigV, PopaMI. Nanostructured chitosan-surfactant matrices as polyphenols nanocapsules template with zero order release kinetics. J Mater Sci Mater Med 2010;21 :1211–23.20033260
49 Iacopini P , CamangiF, StefaniA, SebastianiL. Antiradical potential of ancient Italian apple varieties of malus×domestica borkh. in a peroxynitrite-induced oxidative process. J Food Compos Anal 2010;23 :518–24.
50 Qiu L , WangC, LanM, GuoQ, DuX, ZhouS, CuiP, HongT, JiangP, WangJ, XiaJ. Antibacterial photodynamic gold nanoparticles for skin infection. ACS Appl Bio Mater 2021;4 :3124–32.
51 Wang N , LiangH, ZenK. Molecular mechanisms that influence the macrophage m1-m2 polarization balance. Front Immunol 2014;5 :614.25506346
52 Miao W , ZouW, ZhaoQ, WangY, ChenX, WuS, LiuZ, XuT. Coupling room-temperature phosphorescence carbon dots onto active layer for highly efficient photodynamic antibacterial chemotherapy and enhanced membrane properties. J Membr Sci 2021;639 :119754.
53 Yan H , LiP, WenF, XuQ, GuoQ, SuW. Green synthesis of carbon quantum dots from plant turmeric holds promise as novel photosensitizer for in vitro photodynamic antimicrobial activity. J Mater Res Technol 2023;22 :17–34.
54 Otis G , BhattacharyaS, MalkaO, KolushevaS, BolelP, PorgadorA, JelinekR. Selective labeling and growth inhibition of Pseudomonas aeruginosa by aminoguanidine carbon dots. ACS Infect Dis 2019;5 :292–302.30589261
55 Travlou NA , GiannakoudakisDA, AlgarraM, LabellaAM, Rodríguez-CastellónE, BandoszTJ. S- and N-doped carbon quantum dots: surface chemistry dependent antibacterial activity. Carbon 2018;135 :104–11.
56 Xi J , WeiG, AnL, XuZ, XuZ, FanL, GaoL. Copper/carbon hybrid nanozyme: tuning catalytic activity by the copper state for antibacterial therapy. Nano Lett 2019;20 :800–54.31808346
57 Chatterjee AK , ChakrabortyR, BasuT. Mechanism of antibacterial activity of copper nanoparticles. Nanotechnology 2014;25 :135101.24584282
58 Nosrati H , AramidehKR, NosratiA, KhodaeiM, BanitalebiDM, AshrafiDK, SanamiS, AlizadehZ. Nanocomposite scaffolds for accelerating chronic wound healing by enhancing angiogenesis. J Nanobiotechnol 2021;19 :1.
59 Sheng L , ZhangZ, ZhangY, WangE, MaB, XuQ, MaL, ZhangM, PeiG, ChangJ. A novel “hot spring”-mimetic hydrogel with excellent angiogenic properties for chronic wound healing. Biomaterials 2021;264 :120414.32980635
60 Chen J , HeJ, YangY, QiaoL, HuJ, ZhangJ, GuoB. Antibacterial adhesive self-healing hydrogels to promote diabetic wound healing. Acta Biomater 2022;146 :119–30.35483628
61 Wu C , ZhouY, XuM, HanP, ChenL, ChangJ, XiaoY. Copper-containing mesoporous bioactive glass scaffolds with multifunctional properties of angiogenesis capacity, osteostimulation and antibacterial activity. Biomaterials 2013;34 :422–33.23083929
62 Yang Y , GuoZ, ChenW, WangX, CaoM, HanX, ZhangK, TengB, CaoJ, WuW, CaoP, HuangC, QiuZ. M2 macrophage-derived exosomes promote angiogenesis and growth of pancreatic ductal adenocarcinoma by targeting E2F2. Mol Ther 2021;29 :1226–38.33221435
63 Spiller KL , NassiriS, WitherelCE, AnfangRR, NgJ, NakazawaKR, YuT, VunjakNG. Sequential delivery of immunomodulatory cytokines to facilitate the M1-to-M2 transition of macrophages and enhance vascularization of bone scaffolds. Biomaterials 2015;37 :194–207.25453950
