Synthesis of Traditional Chinese Medicines-Derived Carbon Dots for Bioimaging and Therapeutics

Jing He; Jia Chen; Hongdeng Qiu

doi:10.7536/PC221024

Progress in Chemistry >

2023 , Vol. 35 >Issue 5: 655 - 682

DOI: https://doi.org/10.7536/PC221024

Review

Synthesis of Traditional Chinese Medicines-Derived Carbon Dots for Bioimaging and Therapeutics

Jing He ¹^,² ,
Jia Chen ^,¹^,²^,^* ,
Hongdeng Qiu ^,¹^,²^,^*

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¹ Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences,Lanzhou 730000, China
² University of Chinese Academy of Sciences,Beijing 101408, China

* Corresponding author e-mail: hdqiu@licp.cas.cn(Hongdeng Qiu);

jiachen@licp.cas.cn(Jia Chen)

Received date: 2022-11-01

Revised date: 2022-12-01

Online published: 2023-02-20

Supported by

CAS “Light of West China” Program(xbzg-zdsys-202008)

Youth Innovation Promotion Association CAS(2021420)

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Abstract

Carbon dots (CDs), with particle size less than 10 nm, are a new type of zero-dimensional photoluminescence nanomaterials. Due to the obvious advantages of adjustable fluorescence emission and excitation wavelength, light stability, low toxicity, good water solubility and biocompatibility, etc., CDs have been widely researched in recent years. As a treasure of ancient Chinese science, Traditional Chinese medicine (TCM) is rich in various active ingredients and plays a variety of pharmacodynamic effects, which has been used for thousands of years. TCM-CDs prepared with TCM as carbon source can create some special functions, and then may play a greater medicinal value. In this paper, the synthesis of TCM-CDs and its application in biological imaging and medical therapy are reviewed. Firstly, different synthetic methods of TCM-CDs (including hydrothermal, pyrolysis, solvothermal and microwave assisted method) are introduced in detail, and their advantages and disadvantages are compared. Subsequently, the latest research on TCM-CDs in biological imaging and medical treatment is comprehensively analyzed. This paper focuses on the application of imaging different types of cells in vitro and the distribution and uptake of TCM-CDs guided by imaging in vivo (mice, zebrafish, etc.). In addition, the intrinsic pharmacological activities of these TCM-CDs (including antibacterial, anti-inflammatory, hemostatic, antioxidant and anticancer, etc.) and their mechanisms are also discussed in order to improve and promote their clinical application. Finally, the importance of TCM-CDs research, the main problems and challenges in this fields and the future development direction are summarized and outlooked.

Contents

1 Introduction

2 Synthetic method of TCM-CDs

2.1 Hydrothermal method

2.2 Pyrolysis method

2.3 Solvothermal method

2.4 Microwave assisted method

3 Application of TCM-CDs in bioimaging

3.1 In vitro imaging

3.2 In vivo imaging

4 Application of TCM-CDs in therapeutics

4.1 Anti-bacterial

4.2 Anti-inflammatory

4.3 Hemostasis

4.4 Anti-oxidation

4.5 Anti-cancer

4.6 Other therapeutic effects

5 Conclusion and outlook

Key words： carbon dots; traditional chinese medicines; synthesis methods; bioimaging; therapeutics

Cite this article

Jing He , Jia Chen , Hongdeng Qiu . Synthesis of Traditional Chinese Medicines-Derived Carbon Dots for Bioimaging and Therapeutics[J]. Progress in Chemistry, 2023 , 35(5) : 655 -682 . DOI: 10.7536/PC221024

1 Introduction

As a new type of luminescent zero-dimensional Carbon nanomaterials, fluorescent Carbon dots (CDs) have attracted great attention of researchers since they were first discovered and reported by Scrivens et al. In 2004^[1]. The size of CDs is generally less than 10 nm, and the structure usually consists of an amorphous carbon center and an outer shell with abundant O/N/S functional groups or polymer chains. As shown in fig. 1, fluorescent CDs can be further divided into Graphene quantum dots (GQDs), Carbon quantum dots (CQDs), Carbon nanodots (CNDs) and Carbon polymer dots (CPDs) according to the differences in the microstructure and quantum confinement of Carbon nuclei^[2].

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图1 CDs的分类和相应结构的示意图^[2]

Fig.1 Schematic illustration of classifications and corresponding structures of CDs^[2]

Based on the above structural characteristics, CDs not only have the high Quantum Yield (QY) and tunable emission wavelength of traditional semiconductor Quantum dots,It also exhibits some excellent optical properties, including excellent photoluminescence (PL) properties, low biotoxicity, good biocompatibility, easy chemical modification and high chemical passivation, and photobleaching resistance (high photostability)^[3⇓~5]. At present, CDs have been widely used in optoelectronic devices, photocatalysis, medical therapy, chemical sensing, optical bioimaging, chromatographic stationary phases and other fields^[6,7]^[8,9]^[10]^[11,12]^[13,14]^[15⇓~17]. CDs have gradually become one of the research hotspots in many of the above fields, and are considered to be a potential alternative to semiconductor quantum dots^[18].

With the development of society, the production concept of green chemistry has gradually penetrated into the hearts of the people, and using "green" biomass as raw materials has gradually become a hot spot in production research. Especially in the synthesis of precursors, a large number of green precursors can be used as natural carbon sources for the synthesis of CDs, which has significant advantages such as abundant and renewable raw materials, no chemical pollution and environmental friendliness^[19]. Various green carbon precursors, including fruits, vegetables, and various foods and beverages, have been studied and applied to achieve material synthesis with good material properties, high cost-effectiveness, and economic and environmental protection^[20]. Among these green precursors, Traditional Chinese Medicines (TCM) have attracted wide attention because of their unique medicinal value.

In the long history of China for thousands of years, traditional Chinese medicine, which is rich in active ingredients and can play a variety of pharmacodynamic effects, has played an important role in China's medical care as a safe, economical and effective drug. Since the discovery of artemisinin by a team led by Chinese pharmacologist Tu Youyou, which pioneered a new way to treat malaria, traditional Chinese medicine has gained more and more attention^[21]. A large number of clinical practices have proved that traditional Chinese medicine has unique curative effect on many special diseases such as SARS and COVID-19^[22]. However, traditional Chinese medicinal materials are usually large in size, and their functions can not be effectively brought into play, so their medicinal value can not be fully reflected. Therefore, researchers have begun to try to prepare various nanoscale traditional Chinese medicine substances. Interestingly, as a unique class of drugs in the clinical application of traditional Chinese medicine, the synthesis method of traditional Chinese medicine charcoal is similar to the preparation process of CDs, and the diameter of the synthesized nanomaterials is less than 10 nm, so it is called traditional Chinese medicine CDs (TCM-CDs)^[23]. As early as 2013, Wu et al. Tried to use the rhizome of traditional Chinese medicine giant knotweed (Reynoutria japonica Houtt.) As a carbon source to prepare blue fluorescent CDs by hydrothermal method, which had high Photoluminescence (PL) QY, and was successfully applied to the highly sensitive detection of Reynoutria japonica Houtt. For the first time^[24]. In addition, traditional Chinese medicine CDs can also be used for the sensing analysis of metal ions such as Fe³⁺, Ag⁺, and Cu²⁺^[25]^[26]^[27]. For example, Lycium chinense Miller, known as the "master of Chinese health preservation", can be used as both a carbon source and a nitrogen source, and the green fluorescent CDs synthesized have a QY as high as 21.8%^[28]. Based on the highly selective PL quenching characteristics of CDs, fluorescence images with highly sensitive detection (detection limit of 83 nM) of Ag⁺ can be directly captured and analyzed using a smartphone (Figure 2). In addition to the detection of Fe³⁺, CDs from Lycium barbarum have been successfully used as fluorescent probes for multicolor imaging of human cervical cancer (HeLa) cells due to their low toxicity and excellent biocompatibility^[29]. Therefore, fluorescent CDs synthesized from such inexpensive and environmentally friendly traditional Chinese medicine sources have smaller particle sizes, which are expected to create greater medicinal value and unexpected surprises.

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图2 N-CDs的合成路线及其在Ag⁺传感和RGB颜色分析中的应用^[28]

Fig. 2 Schematic illustration of synthesis route for N-CDs and the application in Ag⁺ sensing and RGB color analysis^[28]

Up to now, there have been some reports on the synthetic precursors, optical properties and therapeutic applications of CDs in biosensing and hemostasis, but there has been no report on the main synthetic methods of CDs in traditional Chinese medicine and their comprehensive review in bioimaging and medical therapy^{[30⇓⇓~33]}. Therefore, on the basis of previous studies, we focus on the comparison and review of the advantages and disadvantages of different synthesis methods of traditional Chinese medicine CDs, and comprehensively investigate the latest progress of traditional Chinese medicine CDs in the rapidly developing field of biological imaging.In particular, this study focuses on applications in imaging different types of cells in vitro and imaging-guided distribution and uptake of CDs in vivo (mice and zebrafish, among others). In addition, the intrinsic pharmacological activities of these CDs (including antibacterial, anti-inflammatory, hemostatic, antioxidant, and anticancer, etc.) and their mechanisms of action are discussed to improve and promote their clinical applications. Finally, we describe the important issues and challenges in the synthesis of CDs from traditional Chinese medicine and their potential applications in bioimaging and medical therapy, and share our views on future development.

2 Synthesis of carbon dots in traditional Chinese medicine

From the viewpoint of green chemistry, the preparation of CDs from natural products using various synthetic methods has become a matter of concern. It has been shown that the precursor plays an important role in determining the optical properties and biological activity of CDs. As a treasure house of Chinese civilization, the scientific nature of traditional Chinese medicine comes from thousands of years of clinical application. Traditional Chinese medicine (TCM) is a class of medicinal substances produced by processing plants, animals, or other biomass. In view of the characteristics of rich carbon source, low cost, simple preparation, large-scale production and environmental friendliness, using traditional Chinese medicine as the precursor of CDs synthesis is an ideal choice and strategy for the preparation of functionalized CDs. Compared with CDs produced by chemical reagents, some CDs prepared from traditional Chinese medicines can be self-passivated during the synthesis process, because the carbonization and surface passivation occur simultaneously due to the large amount of carbides in traditional Chinese medicines, while heteroatom-containing compounds or long-chain compounds in traditional Chinese medicines can act as surface passivators^[34]. For a long time, Chinese researchers have conducted extensive research on the preparation of CDs from traditional Chinese medicine, and have made a series of significant progress in the quality control and application of CDs synthesis.

In terms of the synthesis methods of CDs, their controlled synthesis is still in the early stage of development, which is usually divided into two main categories: "top-down" and "bottom-up". The former can be achieved by physical or chemical resolution of carbon materials, while the latter involves pyrolysis or carbonization of small organic molecules. In the synthesis of CDs using the "top-down" approach, harsh experimental conditions (e.g., strong acid and arc discharge), tedious operating procedures, and expensive equipment are usually employed, greatly limiting their practical application. Therefore, considering the relatively simple preparation process and cost economic benefits, the "bottom-up" route with traditional Chinese medicine as the precursor is an effective way to obtain high-quality CDs. At present, the common "bottom-up" synthesis methods are hydrothermal method, pyrolysis method, solvothermal method and microwave-assisted method.

2.1 Hydrothermal method

Hydrothermal method is the most common method to prepare CDs of traditional Chinese medicine^[35]. Distilled water is used as a common solvent and a reaction medium, the raw materials are simultaneously added into a polytetrafluoroethylene-lined stainless steel autoclave, and the raw materials are subjected to hydrothermal reaction and heating for a period of time at a certain temperature (80-300 deg C) and under high pressure to prepare the target product CDs^[36]. In general, hydrothermally synthesized CDs do not require additional passivation on the surface, thereby maximizing safety and reducing toxicity^[37]. This method has attracted wide attention because it is simple, inexpensive, and allows one-pot synthesis of heterogeneous reactions.

CDs prepared from different traditional Chinese medicine materials show different luminescent properties due to their different particle size, passivation degree and surface groups. Chang et al. Have prepared fluorescent CDs extracted from ginger (Zingiber officinale Roscoe) and hydrothermally obtained at 300 ° C for cell imaging^[38]. At the same time, it has been proved that the synthesized CDs can effectively inhibit the growth of human hepatoma cells. Mewada et al. Used Trapa bispinosa Roxb. As a precursor, the pink extract was converted into a reddish-brown solution by hydrothermal heating at 90 ℃ for 2 H, and luminescent CDs with good water solubility and biocompatibility were obtained^[39]. They speculated that the strong green fluorescence of the CDs might be due to the recombination of electron-hole pairs of impurity atoms and oxygen-containing functional groups. Zhao et al. Prepared fluorescent N and S co-doped CDs by hydrothermal method using garlic (Allium sativum L.) as precursor^[40]. Unlike most previously reported CDs, the prepared CDs exhibited excellent free radical scavenging activity, which further extends their biological applications. Yu et al. Synthesized green CDs with QY as high as 50.78% by hydrothermal method at 200 ° C for 5 H using the traditional Chinese medicine Jinhua Bergamot (Citrus Bergamot) as precursor^[25].

It is experimentally proved that the hydrothermal temperature is usually the determining factor for the carbonization of the main components during the preparation of CDs^[41]. The effect of reaction temperature on the optical properties of CDs has been studied by many researchers. In the general model, the CDs consist of two key components, a carbon-graphite core and an amorphous carbon region^[42,43]. The increase of temperature leads to the growth of nuclei and the simultaneous reduction of amorphous regions, affecting the optical properties. However, it is difficult to form graphite nuclei at low temperatures, so polymer-like CDs are usually obtained. On the contrary, the higher reaction temperature will completely carbonize the functional groups on the amorphous surface. Therefore, the highest QY is reached in the intermediate temperature range where the core-shell structure coexists. Li et al. Synthesized three kinds of CDs (CDs-100, CDs-150 and CDs-180) by hydrothermal method at different temperatures (100, 150 and 180 ° C) for 6 H using Salvia miltiorrhiza Bunge as carbon source, and their average diameters were 16.94, 1.53 and 2.03 nm, respectively^[44]. They speculated that during the synthesis of CDs, polymers were formed first, followed by carbonization to form sp² hybrid carbon nuclei. As the temperature increases, the size of CDs starts to decrease and then increases slightly. This may be due to the fact that the higher temperature produces a large amount of polymer or carbon nuclei. After the temperature continues to rise, the degree of carbonization gradually increases, resulting in a slight increase in the size of carbon nuclei and CDs. After a series of structural changes, the characterization results showed that the surface of the prepared CDs contained a polymer similar to Salvia miltiorrhiza and abundant functional groups such as -OH, -COOH and C = O, which endowed CDs with high antioxidant capacity and multiple enzyme activities.

The reaction time also affects the relevant optical properties of the traditional Chinese medicine CDs^[45]. Although there are some differences in carbon source and reaction temperature, QY usually reaches a maximum value in a time related to reaction temperature, and then gradually decreases with the extension of reaction time. Based on Mentha haplocalyx Briq., Shen et al. Synthesized a yellow transparent solution by a relatively simple hydrothermal method, which showed bright blue fluorescence characteristics under ultraviolet lamp, and established a fluorescence quantitative method for rapid detection of Mentha haplocalyx Briq. Based on the fluorescence characteristics of CDs^[46]. They explored the effects of different hydrothermal temperatures and times on the synthesis of CDs. The experimental results show that when the temperature is lower than 120 ℃ and the carbonization time is 8 ~ 12 H, a large amount of polymer can be seen by electron microscopy, indicating that the carbonization is not complete at low temperature and CDs is not easy to form. When the temperature reached 180 ° C and the reaction time was 8 H, no polymer was found by electron microscopy, indicating that all compounds had been carbonized. It can be seen that the control of hydrothermal time and temperature is particularly critical for the quality of CDs synthesis. Raveendran et al. Also studied the CDs of Mentha haplocalyx^[47]. Without other modification or functionalization, they synthesized mint CDs by a simple hydrothermal method at 200 ℃ for 5 H. Its average diameter is about 4 nm, and its surface is rich in hydroxyl functional groups. The CDs can be used as a promising fluorescent sensor for highly selective and sensitive dual analyte detection of biologically relevant Fe³⁺ and ascorbic acid.

Xu et al. Synthesized CDs from Aloe vera by a simple and green method without further chemical modification^[48]. Due to its simple synthesis process and the use of aloe vera as a carbon source, which is inexpensive and environmentally friendly, it has great potential for cell imaging and drug delivery applications. To ensure the excellent performance of Aloe CDs, they optimized the time and temperature of the synthesis. The results showed that the fluorescence intensity increased with the reaction time up to 11 H, but decreased when the reaction time exceeded 11 H. At the same time, the fluorescence intensity increased with the increase of reaction temperature. Finally, 180 ° C was selected as the optimal temperature because the fluorescence intensity did not increase significantly when this temperature was exceeded.

In addition, CDs with down-conversion and up-conversion photoluminescence properties can also be synthesized in one step by hydrothermal method using cabbage (Brassica oleracea Linnaeus var. capitata Linnaeus) as natural carbon source^[49]. CDs with a QY of 16.5% exhibited excellent solubility and stability in aqueous media, excellent photobleaching resistance, and consistent PL in the biological pH range.

Atchudan et al. Successfully synthesized durable fluorescent N-CDs using Lily (Lilium brownii F. E. Brown var. Colchesteri Wils.) as a green raw material by an economical hydrothermal method (Fig. 3A)^[50]. Different from the traditional synthesis method of two reactants, Lily can be used as both carbon and nitrogen sources. The synthesized N-CDs have the advantages of excellent monodispersity, good biocompatibility, bright fluorescence and high QY fluorescence persistence, which can be used as a fluorescent probe without any chemical modification, and have potential applications in the selective and sensitive detection of Fe³⁺ and live cell imaging.

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图3 (A)荧光N-CDs的合成和应用示意图^[50];(B)CDs荧光探针制备及检测Cr(Ⅵ)的原理图^[51]

Fig. 3 (A) Schematic illustration for the synthesis and applications of fluorescent N-CDs^[50]; (B) Schematic of the preparation and detection of Cr(Ⅵ) using CDs fluorescent probe^[51]

Poria polysaccharide is the main effective component of Poria cocos (Schw.) Wolf, which is rich in carbon and oxygen. Therefore, it can be used as a potential raw material for the synthesis of CDs derived from traditional Chinese medicine. However, the water solubility of pachyman is poor, which greatly limits its application. For the first time, Huang et al. Synthesized CDs from pachyman and ethylenediamine by a novel, simple and green hydrothermal method^[51]. The synthesized CDs have abundant hydrophilic groups, such as hydroxyl, carboxyl, and amino moieties, which greatly improve the solubility of CDs in aqueous solution. The pachymaran precursor can also be used for the detection of Cr⁴⁺ after modification, broadening the application range of traditional Chinese medicine ingredient nanoparticles (Fig. 3B).

Perilla Frutescens (L.) Britt, as a traditional medicinal plant, can be used to treat symptoms of respiratory system and spleen meridian related problems. Chen et al. First used perilla as a carbon source to simply synthesize fluorescent CDs by hydrothermal method at 260 ℃ for 5 H^[26]. The CDs not only have low cytotoxicity, high photostability, good water solubility and biocompatibility, but also have a wide fluorescence response range to Ag⁺. Therefore, CDs have potential applications in live cell imaging and serve as an effective probe for an inexpensive, highly sensitive and selective sensor in environmental and biological scenarios.

The preparation of CDs by hydrothermal method is one of the most mature single-step synthesis technologies, which has the advantages of simple preparation steps, low experimental requirements, and easy to achieve high quantum yield of CDs^[52]. It is crucial to know the content of TCM raw materials when selecting TCM sources as precursors for CDs synthesis. After synthesis, the functional groups present in the starting materials remain on the CDs as dopants, enhancing the fluorescence of the CDs. In addition, the hydrothermal synthesis of CDs needs to fully consider and verify the reaction temperature and time. Since the synthesis of traditional Chinese medicine CDs involves dehydration and carbonization, the hydrothermal temperature should be higher than 100 ° C. The time required to complete the carbonation reaction can be decided based on the color change in the precursor solution. CDs solutions are usually yellow, orange, or brown. If no color change is observed, it means that the duration is too short for carbonization to occur. A black solution was obtained, which may indicate that the duration of CDs synthesis was too long and the carbon particles were no longer fluorescent.

2.2 Pyrolysis method

The steps of pyrolysis synthesis of CDs are similar to those of traditional Chinese medicine processing. The conventional method comprises the following steps of: grinding the traditional Chinese medicine into slag by a certain method, putting the slag into a quartz crucible, covering the quartz crucible, tightly wrapping the quartz crucible with tinfoil paper, and calcining the quartz crucible in a muffle furnace to form powder. The organic matter in the carbon source is gradually converted into CDs through high temperature heating, dehydration, degradation, carbonization and other processes in vacuum or inert atmosphere. High concentrations of acid or base are generally used for pyrolytic cleavage of carbon precursors into nanoparticles. Watermelon peel, sago residue, coffee residue, plant leaves and other biomass raw materials can be used as carbon sources for the preparation of CDs by pyrolysis. The properties of the obtained carbon dots can be adjusted by changing the pyrolysis conditions, such as pyrolysis temperature, pyrolysis time, and the pH value of the reaction system.

Litchi seed has the effects of regulating qi, relieving pain, dispelling cold and dissipating stagnation in traditional Chinese medicine. Xue et al. Obtained blue CDs with QY of 10. 6% and high biological activity (low cytotoxicity) by pyrolysis of litchi seeds at 300 ℃ for 2 H, and the average size was 1. 12 nm^[53]. Then it was used to detect methylene blue (MB) and fluorescence imaging of human hepatoma HepG2 cells. Then they used Peanut Shells, a classic Chinese herbal medicine with the function of astringing the lung and relieving cough, as the carbon source to synthesize N-doped fluorescent CDs by pyrolysis^[54]. Peanut shell CDs have good stability, light resistance, and high chemical stability, and have been successfully applied to multicolor imaging of living cells (fig. 4A). On this basis, Zhu et al. Studied the effect of pyrolysis temperature on the luminescence characteristics of CDs synthesized from various plant leaves (lotus leaves, pine needles, Chinese parasol leaves, and palm leaves) (Fig. 4 B)^[55]. The experimental results show that the optimal pyrolysis temperature is different when different carbon sources are used to synthesize CDs.

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图4 (A)从花生壳合成荧光CDs及其应用于多色活细胞成像的示意图^[54];(B)从植物叶片热解合成CDs,等离子体和微波辐射增强PL的示意图^[55];(C)榴莲皮CDs的合成与应用示意图^[56]

Fig. 4 (A)Schematic illustration of the synthesis of fluorescent C-dots from peanut shells and their application in multicolor living cell imaging^[54]; (B)Schematic synthesis of CDs from pyrolysis of plant leaves and the PL enhancement by plasma and microwave irradiation^[55]; (C)Schematic representation of synthesis and applications of Durian Peel-based CDs^[56]

Durian Peel has the effect of treating abdominal cold, deficiency of essence and blood, and premature graying of hair. Blue fluorescent CDs with an average diameter of about 10 nm were successfully synthesized from durian peel by pyrolysis at 250 ℃ for 5 H^[56]. The experimental results show that CDs can be used as an effective dopant to improve the performance of electrodes and supercapacitors due to its high specific surface area and the introduction of surface functional groups, which provide pseudocapacitive behavior.

The introduction of inert gas in the reaction can prevent CDs from being oxidized by air, so as to obtain stronger fluorescence, such as the preparation of strong blue CDs by heating Gynostemma pentaphyllum (Thunb.) Makino in nitrogen atmosphere^[57]. The synthesized CDs not only have good stability and low biological toxicity, but also can promote the expression of related gene mRNA.

To sum up, pyrolytic carbonization is a very classical method to prepare CDs of traditional Chinese medicine, which shows many advantages, including simplicity, convenience and low toxicity^[58]. The synthesis of traditional Chinese medicine CDs can usually be achieved in one step without too much equipment or complicated processes. However, due to its low QY and high equipment requirements, it is urgent to seek better solutions to adapt to future development.

2.3 Solvothermal method

According to the above, a large number of studies have shown that the preparation of traditional Chinese medicine CDs is usually hydrothermal or pyrolysis, and water is mainly used as the reaction solvent. By changing the type of reaction solvent, the PL properties of CDs can be effectively adjusted to achieve the multicolor emission of CDs^[59].

The solvothermal synthesis method is to mix the traditional Chinese medicine carbon source with one or more solvents in a stainless steel autoclave with Teflon lining. After a period of continuous heating, the original carbon source is converted into CDs under high temperature and pressure in air or inert atmosphere. Similar to the hydrothermal method, the solvothermal method has the advantages of environmental friendliness, low cost, convenient operation, and simple equipment. A variety of carbon precursors are available for the synthesis of CDs via solvothermal methods.

The choice of solvent is the key to determine the performance of traditional Chinese medicine CDs. Unlike hydrothermal synthesis, solvothermal methods involve a variety of solvents other than water. Wang et al. Prepared ethanol-Papaya CDs (E-CDs) and water-papaya CDs (W-CDs), respectively^[60]. The water base contains a large amount of carbohydrates and a small amount of water-soluble macromolecules, which are conducive to the production of W-CDs. In contrast, in 90% ethanol, more organic macromolecules resulted in larger size of E-CDs. The results showed that W-CDs had better fluorescence stability.

Sugar cane (Saccharum sinensis Roxb.) has also been used as a carbon source for the synthesis of traditional Chinese medicine CDs by the ethanol solvent method^[61]. The synthesis method has the significant advantages of low cost, environmental friendliness, short time consumption, simple procedure and the like. The average particle size of CDs is about 1 nm, and the CDs show good spherical symmetry and dispersion. CDs was also modified with copper ketone (CPZ) to eliminate the interference of other metals, and then the selective determination of Cu²⁺ in water was realized. In addition, Sim et al. Also carried out research on CDs of sugarcane^[62]. CDs was synthesized from sugarcane and urea using ethanol as solvent. It further forms a composite photocatalyst (CDs/g-C₃N₄) with graphitized carbon nitride (g-C₃N₄). It is observed that the higher the proportion of CDs, the stronger the photosensitization of CDs/g-C₃N₄, which overcomes the low charge separation efficiency of CDs/g-C₃N₄. Since sugarcane is used as a green precursor, the preparation of CDs/g-C₃N₄ is cost-effective and environmentally friendly.

The use of organic solvents in the synthesis of CDs promotes the carbonization process, resulting in large changes in the photophysical properties of carbon nanoparticles. As shown in Fig. 5, green CDs were prepared from Codonopsis pilosula extracted and processed by one-step solvent method at room temperature^[63]. Compared with the synthesis strategy of green CDs by hydrothermal or pyrolysis treatment and using biomass Chinese medicinal materials as precursors, this method does not require expensive heating equipment and long-term high-temperature heating process, and has significant advantages such as more convenient operation, lower energy consumption and more economical synthesis cost.The obtained Codonopsis pilosula-derived CDs (CP-CDs) have excellent fluorescence properties (QY up to 12.8%) and high photostability, and no passivation or functionalization treatment is required on the surface of the CP-CDs.

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图5 CP-CDs的制备工艺及应用的示意图^[63]

Fig.5 Schematic of the preparation procedure and application of CP-CDs^[63]

2.4 Microwave-assisted method

The conventional synthesis methods described above usually involve high temperature conditions and have the disadvantages of expensive heating equipment or high energy consumption, which is not in line with the core concept of green chemistry. With the increasing concern for environmental sustainability, it is desirable to develop greener methods to prepare CDs. The microwave-assisted method is a method for directly carbonizing a traditional Chinese medicine source into traditional Chinese medicine CDs under microwave radiation. The principle of microwave treatment is to realize the violent movement and mutual friction of drug molecules by microwave. Compared with typical synthesis methods, microwave treatment has obvious advantages. Due to its high efficiency, simple equipment and operation, the microwave-assisted method is a cost-effective method with a strong competitive advantage to produce large quantities of fluorescent CDs^[64]. The microwave-assisted method allows the synthesis of CDs from precursors using a household microwave oven or a scientific microwave oven under the uniform heating effect of microwave radiation. After absorption of microwave radiation by the precursor, heat is generated by dielectric heating^[65]. Unlike the hydrothermal method, microwave irradiation directly heats the target molecules instead of conduction and/or convection. It is easy to operate and can produce high-quality CDs of traditional Chinese medicine.

Microwave-assisted method can also effectively solve the problem of uncontrollable temperature and time. The method has the advantages of high processing speed, high precision, low pollution and the like. It is suitable for charcoal medicinal materials with light texture. In this method, the drug is placed in glassware and carbonized with high heat into a brown product on the outside and a black product that burns inside. Architha et al. Reported that Mentha canadensis Linnaeus was processed by this method, and blue fluorescent CDs (M-CDs) with a particle size of about 2. 43 nm were successfully synthesized^[66]. The microwave processing technology of M-CDs with uniform appearance and controllable quality is easy to operate, energy-saving and efficient, and can be popularized in production.

Silkworm pupa (Bombyx mori L.) is rich in protein and chitosan, so it has the potential to be used as a natural carbon source to synthesize self-carbonized and doped CDs. Feng et al. Used microwave-assisted synthesis to discuss this^[67]. The obtained CDs have QY up to 46% and achieve 5.72% N-doping. Their microwave-assisted method enabled the synthesis of uniform CDs with satisfactory repeatability due to its reduced time consumption and precise control of pressure and temperature. In addition, the CDs show potential for use in cellular imaging due to low toxicity and good imaging quality (fig. 6).

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图6 活蚕蛹中提取制备的SC-CDs及其蓝光荧光的示意图^[67]

Fig. 6 Schematic illustration for the preparation of SC-CDs derived from alive silkworm chrysalis and the presentation of blue photoluminescence^[67]

Isnaeni et al. Synthesized two CDs from ginger (Zingiber officinale Roscoe) and galangal (Alpinia officinarum) by microwave treatment for 5 – 40 min^[68]. The appearance of CDs as a brown solution indicates that they are completely dissolved in the aqueous solvent. The sample did not have any sedimentation after a few weeks, indicating that the CDs and the water solvent were a stable colloid, and the light emitted was mostly cyan or light green. It was observed that the color of CDs solution was gradually deepened when the microwave treatment time was prolonged, indicating that the amount of synthesized CDs was also gradually increased.

Subsequently, Li et al. Used Ginkgo biloba Linn. Fruit as the sole carbon source to synthesize two kinds of nitrogen-doped CDs (M-N-CDs/H-N-CDs) by microwave-assisted and hydrothermal methods, respectively^[69]. The synthesis time of microwave-assisted method (5 ~ 15 min) is much lower than that of hydrothermal method (8 ~ 16 H), and the particle size of microwave-assisted method (2. 82 nm) is smaller than that of hydrothermal method (3. 81 nm). However, as far as the luminescence characteristics are concerned, although both N-CDs show typical absorption and PL spectra, the fluorescence intensity of the hydrothermally synthesized H-N-CDs is much higher than that of the M-N-CDs synthesized under microwave irradiation. In addition, the QY and fluorescence lifetime of H-N-CDs are also larger than those of M-N-CDs. To sum up, microwave synthesis may be better than hydrothermal and pyrolysis methods in terms of preparation time and efficiency. Despite the above advantages of the microwave method, it is still rare in the synthetic application of traditional Chinese medicine CDs.

Similarly, Tejwan et al. Also synthesized a new type of spherical, water-soluble, green CDs with an average particle size of about 2 nm by a highly efficient microwave-assisted method using Panax ginseng water extract as raw material and seven consecutive cooling and heating cycles^[70]. The synthesized CDs have excitation-dependent fluorescence emission behavior, which can be used for the selective detection of Fe²⁺ by fluorescence quenching.

The microwave-assisted method combines microwave technology with chemical synthesis to provide efficient and uniform energy to the precursor solution, which is considered to be an important method to accelerate the formation of CDs in traditional Chinese medicine. Microwave-assisted technology can heat the reaction medium quickly and uniformly, shorten the reaction time, and greatly improve the yield and purity of the product^[71]. The main disadvantage is the uneven particle size distribution of CDs. Studies have shown that the size of CDs can be adjusted by post-treatment such as ultrasound, gel electrophoresis, column chromatography, centrifugation, dialysis and filtration^[72].

In general, among the above synthetic methods for the preparation of traditional Chinese medicine CDs, hydrothermal and pyrolysis methods are widely used because of their advantages of convenience, economy, easy operation and environmental friendliness. However, hydrothermal synthesis of traditional Chinese medicine CDs is generally considered to be a time-consuming process. Although the microwave-assisted method is not as frequently used as the hydrothermal method and pyrolysis method, its time-saving, low-cost, and easy-to-operate characteristics are very attractive for renewable green synthesis of traditional Chinese medicine CDs. A comparison of their advantages and disadvantages is summarized in Table 1.

表1 不同中药源及合成方法及条件制得的CDs的尺寸、荧光性质和荧光QY比较

Table 1 Comparison of size, fluorescence properties and fluorescence QY of CDs prepared from different TCM sources, synthesis methods and conditions

Source	Synthesis method	Reaction temperature ( ℃/W)	Reaction time (h)	Exciation (nm)	Emission (nm)	Average particle size (nm)	Quantum yield (%)	Surface modification	ref
Reynoutria japonica Houtt.	Hydrothermal	200	3	320	400	35	11.5	-	24
Citrus Bergamot	Hydrothermal	200	5	330	440	10	50.78	-	25
Perilla Frutescens(L.)Britt	Hydrothermal	260	5	360	450	2.8	9.01	-	26
Lycium chinense Miller	Hydrothermal	180	24	427	550	4.5	21.8	-	28
Lycium chinense Miller	Hydrothermal	200	5	350	430	3.3	17.2	NH₃·H₂O	29
Zingiber officinale Roscoe	Hydrothermal	300	2	325	400	4.3	13.4	-	38
Trapa bispinosa Roxb.	Hydrothermal	90	2	450	520	7.5	1.2	-	39
Allium sativum L.	Hydrothermal	200	3	360	442	10.7	17.5	-	40
Salvia miltiorrhiza Bunge	Hydrothermal	100~180	6	400	490	1.53~16.94	30~40	-	44
Mentha haplocalyx Briq.	Hydrothermal	200	5	360	450	7	7.64	-	46
Mentha haplocalyx Briq.	Hydrothermal	180	8	363	441	5	4.5	-	47
Aloe vera	Hydrothermal	180	11	441	503	5	10.3	-	48
Brassica oleracea Linnaeus var. capitata Linnaeus	Hydrothermal	140	5	345	432	4	12.5	-	49
Lilium brownii F. E. Brown var. Colchesteri Wils.	Hydrothermal	240	12	340	405	4	11	-	50
Poria cocos (Schw.) Wolf	Hydrothermal	200	5	376	450	4	4.8	-	51
Ginkgo biloba Linn.	Hydrothermal	160~200	8	420	520	3.81	3.33	-	69
Salvia miltiorrhiza Bunge	Hydrothermal	150	6	420	526	3.32	-	-	74
Charred Triplet	Hydrothermal	100	2	340	447	5.1	7.95	-	131
Litchi chinensis Sonn.	Pyrolysis	300	2	365	440	1.12	10.6	-	53
Peanut Shells	Pyrolysis	250	2	320	440	1.6	9.91	-	54
Durian Peel	Pyrolysis	250	5	368	480	10	11	-	56
Gynostemma pentaphyllum (Thunb.) Makino	Pyrolysis	400	4	320	400	2.49	5.7	-	57
Papaya	Solvothermal	200	5	370	450	3.4/10.8	18.98/18.39	ethanol	60
Saccharum sinensis Roxb.	Solvothermal	250	6	350	430	1	10.7	ethanol	61
Saccharum sinensis Roxb.	Solvothermal	120	8	360	460	5	-	Urea and ethanol	62
Codonopsis pilosula	Solvothermal	25	4	390	456	11.54	12.8	methanol	63
Mentha canadensis Linnaeus	Microwave	960	0.07~0.17	340	436	2.43	17	-	66
Bombyx mori L.	Microwave	210	0.75	350	440	19	46	-	67
Zingiberis rhizome and Alpinia officinarum	Microwave	450	0.08~0.67	-	-	10	-	-	68
Ginkgo biloba Linn.	Microwave	800	0.08~0.25	440	550	2.82	0.65	-	69
Panax ginseng	Microwave	700	0.5	380	500	2	8	AgNPs	70
Talinum paniculatum (Jacq.) Gaertn.	Microwave	700	0.5	380	470	2	-	Rutin	107

Based on the hydrothermal synthesis method, most researchers have carried out CDs synthesis between 180 ℃ and 200 ℃ within 2 H and 12 H. Since carbides are not activated at low temperatures, carbonization is better in the high temperature range. The carbonation reaction at lower temperature can be counteracted by prolonging the reaction time. The microwave-assisted method can greatly shorten the hydrothermal synthesis time. Most researchers carried out the reaction within 1 H, while others extended the synthesis time to a maximum of 2 H. The temperature of microwave-assisted synthesis is similar to that of traditional hydrothermal synthesis, and more temperatures are observed between 180 and 200 ℃. Hydrothermal and microwave methods are expected to continue to be used in the synthesis of traditional Chinese medicine CDs on a large scale because of their simple steps and low equipment cost. In order to realize the sustainability of the function of CDs, more traditional Chinese medicine sources should be explored as the carbon source of CDs, and traditional Chinese medicine raw materials rich in carbon and nitrogen compounds can be selected. Because CDs have the advantages of low cost and non-toxicity, the commercialization and scale of CDs should be seriously studied in the photoluminescence study of CDs in traditional Chinese medicine.

The possibility of synthesizing such bright fluorescent nanoparticles with simple methods and inexpensive precursors is the reason for the wide interest of CDs. However, the multiple carbonization reactions that drive the formation of luminescent CDs are largely uncontrolled, and the complex process mechanisms that occur during the formation of CDs seem difficult to explain. Moreover, the chemical reactions leading to the formation of carbon nanoparticles upon hydrothermal, microwave treatment, or thermal degradation synthesis of CDs are quite different from those generally occurring in organic chemistry. Therefore, the study of mechanism and process based on CDs is the basis of true CDs nanotechnology.

3 Application of traditional Chinese medicine carbon dots in biological imaging

Based on the advantages of traditional Chinese medicine CDs, such as good photoluminescence properties, water dispersibility, narrow particle size distribution and low cytotoxicity, researchers have successfully applied traditional Chinese medicine CDs to cell imaging in vitro and in vivo imaging^[54,73].

3.1 In vitro imaging

In recent years, traditional Chinese medicine CDs have greatly promoted the development of bioimaging-supported nanomedicine to provide efficient biomedical applications, including the effectiveness of molecular imaging, the improvement of image characteristics of traditional fluorescent agents, and the effective treatment of tumors guided by fluorescence imaging. Various reports have successfully developed multicolor TCM fluorescent CDs with excellent properties such as high fluorescence, excellent photostability, easy functionalization, and good biocompatibility, which have been widely used in a variety of biomedical applications from biology to medicine.

3.1.1 Cell imaging

Cancer affects human health and consumes millions of lives every year. Therefore, advanced methods for early cancer diagnosis are increasingly important for preventing tumor development. Specifically functionalized CDs can penetrate a variety of cancer cells and probe them more efficiently by FL imaging. The special recognition mechanism mainly depends on the interaction between the functional CDs and the cancer cell surface group. In cellular imaging, specific labeling of cancer cells remains a significant challenge in cellular imaging. Therefore, improving the specificity and targeting ability of fluorescent CDs is more important for tracking cancer cells.

Orange (Citrus junos Tanada) contains important chemical components such as hesperidin, citric acid, malic acid, succinic acid, sugars and vitamins. In 2012, Sahu et al. Attempted to synthesize CDs with high quantum yield (QY = 26%) from orange juice, and first applied CDs from traditional Chinese medicine to the in vitro cellular uptake experiment of human osteosarcoma (MG-63) cells^[74]. The agglomeration of green fluorescent CDs in MG-63 cells was clearly shown in the image under 488 nm excitation, and a clear nuclear region was observed. There was also no decrease in fluorescence intensity after prolonged excitation. The results show that CDs obtained from orange juice can be used as a potential alternative to organic dyes or semiconductor quantum dots for bioimaging.

Peppermint leaf is an important medicinal plant with antioxidant, anti-inflammatory, antimicrobial, and anticancer activities. Because positively charged nanoparticles have stronger adhesion to negatively charged cell membranes and higher transfection efficiency, researchers used peppermint leaves as precursors and passivated them with polyethyleneimine to synthesize peppermint leaf CDs (P-CDs) to increase cell uptake^[75]. Therefore, uptake studies of human breast cancer (MCF-7) cells were further developed in P-CDs. Enhanced uptake of P-CDs was found within 3 H after incubation. The small size enables P-CDs to be successfully taken up into cells, thereby providing fluorescence properties. Initially without P-CDs fluorescence, only LysoTracker red red fluorescence is produced. When the incubation time was extended to 3 H, bright green fluorescence was observed. In addition, enhanced cellular uptake is greatly affected in the cancer environment, resulting in high-contrast cellular labeling. Therefore, synthetic P-CDs can serve as an effective alternative to multifunctional imaging probes.

Atchudan et al. Studied their performance as fluorescent cell markers by incubating N-CDs synthesized from peach (Prunus persica) as a carbon source with human breast cancer (MDA-MB-231) cells for 4 H^[76]. The experimental images observed that MDA-MB-231 cells incubated with N-CDs were clearly illuminated under the microscope. Whereas MDA-MB-231 cells incubated without N-CD showed no fluorescence emission. The results showed that N-CDs could be rapidly absorbed by cells and successfully used as a cell bioimaging probe. In addition, they prepared N-CDs-4 from Prunus mume as a precursor in a similar way^[77]. The synthesized N-CDs-4 was internalized into MDA-MB-231 cells, which showed bright blue and green fluorescence under UV excitation, so the CDs could also be used as a good cell imaging agent.

Cytotoxic effects during cell imaging play a key role in determining the suitability of traditional Chinese medicine CDs as fluorescent imaging agents. Turmeric (Curcuma Longa) is a chemically active polyphenolic drug, which can show anticancer, anti-inflammatory, antioxidant and antibacterial behavioral effects in medical diagnosis and treatment. Mazrad et al. Synthesized multicolor turmeric CDs modified with polyethylene glycol, and first examined the cytotoxicity of turmeric CDs in human oral epidermoid carcinoma (KB) cells and canine renal epithelial continuous cells (MDCK) by MTT assay^[78]. The results showed that the cell activity of CD was close to 100% at high concentration. Inspired by this, they then reported that a series of CDs derived from mango (Mangifera indica L.) with various chemical components (such as phenols, antioxidants, and antibacterial agents) could synthesize multicolor (blue, green, and yellow) bioimaging probes (FCP-B, FCP-G, and FCP-Y) by regulating carbonization conditions, and the CDs were stably used for in vitro imaging of human lung adenocarcinoma (A549) cells (Fig. 7)^[79]. All CDs emitted strong fluorescence at the corresponding excitation wavelength, and the same effect was observed after 4 H of incubation. Thus, the confocal imaging results indicate that CDs are strong candidates for cellular imaging for diagnosis.

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图7 用FCP-B、FCP-G和FCP-Y处理的A549细胞在4 h内观察的共聚焦显微镜图像^[79]

Fig. 7 Confocal microscope images of A549 cells treated with FCP-B, FCP-G and FCP-Y, observed during a 4 h incubation period^[79]

Zhao et al. Also performed related cell imaging studies on synthetic garlic CDs^[40]. According to the experimental results, A549 cells labeled with garlic CDs showed bright blue, green and red fluorescence under ultraviolet, blue and green light excitation, respectively. Garlic CDs were mainly distributed in the cell membrane and cytoplasm, and the nucleus only had weak fluorescence. In addition, Sachdev et al. Used Hoechst 33342 dye as a nuclear marker to track the cellular distribution of coriander (Coriandrum sativum Linn.) CDs in A549 cells and human embryonic lung epithelial (L-132) cells^[80]. Green fluorescence around the nucleus, and a large number of coriander CDs distributed in the cytoplasm. However, colocalization of blue (Hoechst 33342) and green fluorescence (coriander CDs) was not observed, which also emphasizes the fact that this CDs cannot penetrate the nuclear interior.

Chai et al. Prepared nitrogen-doped fluorescent CDs (N-CDs) by hydrothermal method using Dendranthema morifolium as precursor^[81]. HeLa cells treated with N-CDs showed bright blue and green fluorescence under excitation at 405 and 488 nm under a fluorescence microscope. The results show that N-CDs have good biocompatibility and can be used for bioimaging. In addition, the CDs also realized the highly sensitive detection of hydroxybenzaldehyde (PHBA), one of the main components of Gastrodia elata, and was successfully applied to the detection of PHBA in blood and urine samples.

Raveendran et al. Synthesized fluorescent CDs (M-CDs) of mint origin by a green method^[82]. Considering the strong and stable photoluminescence of M-CDs, they tried to apply it to HeLa cell fluorescence imaging. The hydrophilicity and the presence of functional groups on the surface of M-CDs enable them to easily internalize them into cells by endocytosis, showing multicolor fluorescence images at different excitation wavelengths, and these results ensure the successful application of M-CDs as effective probes for imaging.

Li et al. Synthesized CDs by hydrothermal method using Brassica campestris (Brassica compestris L. var. purpurea Bailey) as carbon precursor and labeled HeLa cells^[83]. The results showed that CDs easily penetrated the cell membrane and labeled the cytoplasm, and a small amount of CDs entered the nucleus. The multicolor emission of CDs provides a wide wavelength region for observation, which makes CDs an ideal cell imaging agent.

Feng et al. Prepared fluorescent CDs (SC-CDs) with high QY using silkworm pupa (Bombyx mori L.) as natural carbon source^[67]. After co-incubation of SC-CDs with HeLa cells for 6 H, bright fluorescence images were obtained at 340, 495, and 550 nm excitation. Cell images showed that SC-CDs had good biocompatibility and weak interaction with intracellular proteins.

Wan et al. Also explored the cell imaging of CDs synthesized from Abelmoschus esculentus (Linn.) Moench^[84]. After HeLa cells were incubated with CDs for 3 H, bright blue fluorescence was observed in the cytoplasm, while a small amount of CDs could penetrate into the nucleus and be located in the surrounding area of the nucleus. In addition, the fluorescence signal of the cells dropped significantly after further incubation with the addition of 2,4,6-trinitrophenol (THF). Based on this, the CDs can be used as an effective fluorescent probe for the highly sensitive detection of THF.

To demonstrate the potential application of the synthesized Lycium barbarum CDs as bioimaging probes, in vitro cellular uptake experiments were performed^[29]. HeLa cells were labeled with the prepared Lycium barbarum CDs, and bright blue, blue-green, and green luminescence were observed in living cells at different wavelengths, respectively. When Fe³⁺ was added to the cells labeled with CDs, the fluorescence intensity of the cells decreased significantly. The results indicate that Lycium barbarum CDs are membrane-permeable and can be used for cellular multicolor imaging and visualization of Fe³⁺ in living cells.

Recently, Liu et al. Synthesized blue fluorescent CDs from Alisma plantago-aquatica Linn., and successfully applied them to bioimaging of HeLa cells^[85]. The CDs material has good cell permeability and strong fluorescence intensity, and can be developed as a bioluminescent dye in the future.

Two kinds of CDs (M-N-CDs/H-N-CDs) synthesized from Ginkgo biloba Linn. Were studied by cell imaging^[69]. Bright blue and green fluorescence images in HeLa cells and human esophageal squamous carcinoma (KYSE-410) cells were clearly observed at 405 and 488 nm by confocal laser scanning microscopy (CLSM). The fluorescence in the nucleus was found to be weak, indicating that the CDs mainly entered the cytoplasm. Therefore, the CDs is a promising bioimaging agent and has a certain potential for practical application. Similarly, Chinese wax gourd (Benincasa hispida (Thunb.) Cogn.) CDs synthesized as carbon source were co-incubated with HepG2 cells^[86]. It was observed that all human hepatoma (HepG2) cells exhibited bright blue fluorescence under UV excitation, and the CDs were also mainly internalized to the cytoplasmic region around the nucleus.

Ma et al. Used fluorescent CDs synthesized by purple leaf plum (Prunus cerasifera Ehrhart f. atropurpurea (Jacq.) Rehd.) fruits to incubate with HepG2 cells, and the cells treated with CDs showed bright blue and green fluorescence under the excitation of 405 and 488 nm^[87]. The results showed that the CDs could penetrate the cell membrane and diffuse into the cytoplasm and nucleus.

Litchi is a qi-regulating drug, which has the effects of promoting qi circulation, dissipating stagnation, dispelling cold and relieving pain in traditional Chinese medicine treatment. Xue et al. First attempted to develop a method for the preparation of green fluorescent CDs using litchi seeds as precursors^[53]. The CDs were incubated with HepG2 cells, and bright blue fluorescence was observed in the intracellular region when excited at 405 nm. Subsequently, they developed a near-infrared fluorescent CDs (NB-CDs) using the exocarp of litchi as the carbon source. Because NB-CDs have near-infrared fluorescence emission, low toxicity, the ability to target cancer cells and high solubility, they can be used for near-infrared fluorescence imaging targeting cancer cells^[88]. The fluorescence intensity of human hepatoma (Bel-7404) cells treated with NB-CDs was significantly higher than that of human normal liver (HL-7702) cells, indicating that NB-CDs could enhance the uptake and effective release of chlorin E6 (Ce6) by cancer cells. In addition, the experimental results show that tumor cells can absorb NB-CDs and participate in the photochemical reaction of oxygen in biological tissues under the excitation of specific wavelength light to produce singlet oxygen or free radicals, which leads to the decrease of Ce6 concentration and fluorescence intensity.

Ginsenoside Re is a kind of tetracyclic triterpenoid derivative, which has been found as a variety of active ingredients in Panax medicinal plants, and has attracted much attention because of its anticancer effect. Yao et al. Used CDs (Re-CDs) synthesized from ginsenoside Re to image human malignant melanoma (A375) cells to study apoptosis and necrosis^[89]. As shown in Fig. 8, with the increase of the concentration of Re-CDs, the apoptosis and damage were obviously enhanced. Further experiments confirmed that the anticancer mechanism of Re-CDs was to inhibit cancer cell proliferation and induce apoptosis through reactive oxygen species (ROS) -mediated pathway or other pathways.

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图8 Hoechst/PI双染色法对细胞凋亡和坏死的高含量成像分析^[89]

Fig. 8 High-content imaging analysis of cell apoptosis and necrosis by hoechst/PI double-staining method^[89]

In addition, in vitro imaging studies have been performed on normal human and animal cells. Alam et al. Observed that purified cabbage (Brassica oleracea Linnaeus var. capitata Linnaeus) CDs exhibited low cytotoxicity against human epidermal keratinocyte (HaCaT) cells at high concentrations^[49]. Subsequently, during in vitro imaging, the cells treated with CDs showed three different colors of blue, green, and red under the confocal microscope. The CDs have the advantages of green synthesis, high biocompatibility, low cytotoxicity, good optical properties and cell imaging effect, and have broad application prospects in the biomedical field.

Atchudan et al. Synthesized highly durable fluorescent N-doped CDs (FN-CDs) using West Indian gooseberry (Phyllanthus acidus (L.) Skeels) as carbon source by one-step hydrothermal method^[90]. They used CLSM to further explore the feasibility of FN-CDs for cell imaging. Rat liver epithelial (Clone9) cells treated with FN-CDs showed bright illumination under laser irradiation and maintained good cell morphology. Cells emit multicolor fluorescence in blue, green, and red at different excitation wavelengths of 405, 488, and 555 nm laser pulses, respectively. This multicolor emission shows the advantages of FN-CDs (the ability to provide a broader wavelength observation area), which indicates the great potential of FN-CDs as cell imaging agents.

There is no doubt that traditional Chinese medicine CDs have shown excellent performance in cell bioimaging applications, which is beneficial to enhance researchers' knowledge and understanding of cells. However, each specific mechanism of the interaction of TCM CDs with targets in cells, cytotoxicity, and optimal conditions for bioimaging processes need to be further studied in depth and carefully to make significant contributions to biology and biomedicine.

3.1.2 Bacterial imaging

Thota et al. Reported the synthesis of CDs using Cymbopogon citratus (D. C.) Stapf as a carbon source via a simple hydrothermal route, and obtained a high fluorescence quantum yield (QY = 23.3%)^[91]. They successfully applied it to multicolor live cell (Saccharomyces cerevisiae BY4742) imaging, demonstrating the excellent biocompatibility of the prepared CDs.

Bhatt et al. Used Ocimum sanctum Linn. CDs as a bio-friendly fluorescent probe for cell imaging of B. subtilis and E. coli bacteria (Fig. 9)^[92]. Internalized cells were excited at 405, 488, and 561 nm and emitted blue, yellow, and red fluorescence, respectively. The well-dispersed fluorescence images of the bacterial cells indicated that the CDs had successfully penetrated the cell membrane. In addition, the reaction time required for CDs to adhere to bacterial cells to form bacteria-CDs conjugates was quite low, indicating that CDs can easily and rapidly penetrate into bacterial cells.

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**图9 在蓝、黄、红三色区(A)E. Coli和(B)B.Subtilis存在下的细菌生物成像^[92]**

Fig. 9 Bioimaging of bacteria in presence of CDs in blue, yellow and red region (A) E. Coli and (B) B.Subtilis^[92]

Durrani et al. Obtained a Ca, N, S-doped green fluorescent CDs (Mis-mPD-CDs) by hydrothermal reaction of m-phenylenediamine with Salvadora persica, an ancient plant^[93]. They applied CDs to sense and image Congo red (CR) in living cells (A5 49, Candida albicans (C. albicans), E. coli, and S. aureus) (Figure 10). The results showed that CDs could rapidly penetrate into all cells within 2 min and showed green fluorescence, and mainly accumulated in the nucleolus of A549 cells. In addition, CDs can also label bacterial cells rapidly and have a long retention time. This is the first report of using CDs to rapidly detect CR in living cells, which provides a novel and efficient platform for detecting intracellular CR and expands the application of CDs as a dye biosensor.

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**图10 基于Mis-mPD-CDs的细胞内CR成像与传感:A549、C. albicans、E. coli和S. aureus的CLSM图像,用不同浓度的CR处理,然后与Mis-mPD-CDs孵育30 min^[93]**

Fig. 10 Imaging and sensing of intracellular CR based on Mis-mPD-CDs. CLSM images of A549, C. albicans, E. coli, and S. aureus, and treated with CR of different concentrations and then incubated with Mis-mPD-CDs for 30 min^[93]

Lin et al. Prepared a variety of traditional Chinese medicine source CDs (including onion, ginger and garlic)^[94]. Among them, onion (Allium cepa Linn.) CDs exhibited antibacterial activity against E. coli and S. aureus bacteria. They then evaluated the cell viability of P. fragilis after exposure to onion CDs using green SYTO and red PI fluorescence staining. After 4 H of co-incubation, it was observed by cell images that the number of viable cells decreased and the number of dead cells increased with the increase of the concentration of onion CDs. This indicated that the cell membrane integrity was damaged, the viability of P. fragilis was reduced, and the cell morphology was changed after treatment with onion CDs. Therefore, the synthesized onion CDs have the potential for bacteriostatic application.

Bacterial bioimaging based on traditional Chinese medicine CDs has been successfully used for fluorescence imaging. However, these studies mainly focused on the imaging of several bacteria, such as Staphylococcus aureus and Escherichia coli, and other bacterial types with different effects need to be further studied. Traditional Chinese medicine CDs with specific affinity for each bacterium should be developed for effective application. And carefully consider the cytotoxicity of CDs against various types of bacteria for further utilization. In addition, the fluorescence interference of culture medium on CDs of traditional Chinese medicine and the real-time monitoring of bacteria need to be further studied.

At present, the research on in vitro imaging of traditional Chinese medicine CDs is mainly focused on human health or common cells such as cancer cells and bacteria, while the imaging of other important organs (such as stem cells, neurons, nuclei, mitochondria and lysosomes) needs to be further explored. These potential applications in in vitro imaging reflect that traditional Chinese medicine CDs are expected to create new achievements in biomedical applications and can be used as effective nanocarriers for manufacturing drug delivery systems for cancer cells. But current CDs-based probes still have shortcomings in some aspects, and more efforts should be taken in developing multifunctional CDs with better characteristics, such as excellent targeting ability, ease of surface function, low cytotoxicity, high QY, and stability of in vitro imaging. For example, the fluorescence emission of CDs shows a red shift by further increasing the excitation wavelength, while the fluorescence intensity is usually quite low. Similarly, the targeted properties and applications of TCM CDs in in vivo imaging and clinical diagnosis should be studied in detail.

3.2 In vivo imaging

Based on in vitro studies, many efforts have contributed to in vivo biomedical applications based on fluorescent CDs, and Yang et al. First reported CDs that achieved in vivo imaging of mice by three injection routes^[95]. Subsequently, more and more studies have been carefully conducted to explore the in vivo application of CDs in animal models.

TCM CDs have been proven to be successfully used for fluorescence imaging of cells, taking advantage of their visible excitation and emission wavelengths, single-spot-level fluorescence brightness, and high photostability. TCM CDs have been evaluated for optical bioimaging applications, including their cellular uptake and fluorescence brightness in cellular environments, and their in vivo imaging in mouse models^[96]. In this section, we summarize the biodistribution, clearance, and uptake of TCM CDs in mice and other animals (e.g., zebrafish, Paramecium).

3.2.1 Mouse imaging

Although CDs play a crucial role in current biomedical research, they may have chronic toxicity, low stability, and potentially undesirable accumulation in organs/tissues. Therefore, the biodistribution and in vivo uptake of CDs must be evaluated.

In addition to in vitro imaging, mango CDs have also been tried for in vivo mouse imaging studies^[79]. Based on the different synthesis conditions, there are some differences in the size of the prepared multicolor traditional Chinese medicine CDs. It was observed that the distribution of CDs with different colors in the body was not exactly the same, and CDs with small particles were easier to be cleared from the body than those with large particles (Fig. 11).

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图11 杧果CDs的小鼠体内成像图。尾静脉注射5 mg·kg^-1体重后FCP-B、FCP-G和FCP-Y在BALB/c裸鼠体内的生物分布。FCP-B(a,b)、FCP-G(c,d)和FCP-Y(e,f)的体内分布及相应强度^[79]

Fig. 11 In vivo biodistribution of FCP-B, FCP-G and FCP-Y in balb/c nude mice after tail vein injection of 5 mg·kg^-1 of body weight. The in vivo biodistribution and corresponding intensities of FCP-B (a, b), FCP-G (c, d) and FCP-Y (e, f) respectively

In biosensing and biophotonics, red/near-infrared fluorescent CDs (R-CDs) deserve more attention due to their high tissue penetration, low photodamage, and negligible autofluorescence interference. However, the synthesis and purification of R-CDs are time-consuming, which hinders their large-scale industrial production and application. Liu et al. Used Taxus chinensis (Pilger) Rehd., the raw material for the production of anticancer drug paclitaxel, as the reaction precursor to obtain deep red fluorescent CDs with extremely narrow half-peak width (FWHM ≈ 20 nm) and high quantum yield (QY = 59%) by solvothermal method (Fig. 12)^[97]. Through a series of detailed characterizations, it is found that CDs has a unique polymer characteristic composed of a carbon core and a polymer chain shell, and a single photoluminescence center is controlled by a π-conjugated system formed by N heterocyclic and aromatic rings, which explains the reasons for the excellent characteristics of CDs, such as narrow full width at half maximum, deep red luminescence and high QY. Through cell imaging and in vivo imaging analysis, it was observed that intravenously injected CDs rapidly entered the whole body of mice through blood circulation and were rapidly excreted from the body through two metabolic pathways, renal system and hepatobiliary system. Therefore, CDs can be used as excellent deep red fluorescent probes for one- and two-photon bioimaging, with good biocompatibility and low cytotoxicity, and with minimal post-retention time. Therefore, the synthesized CDs have broad application prospects in the fields of drug delivery, photothermal therapy, and optoelectronic conversion.

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图12 CDs的单光子和双光子生物成像。(A)在不同时间点静脉注射CDs的仰卧裸鼠的体内成像(Ⅰ:胸部区域,Ⅱ:肝脏区域,Ⅲ:小肠区域,Ⅳ:大肠区域,Ⅴ:膀胱区域);(B)在不同时间点静脉注射CDs的裸鼠的实时离体成像^[97]

Fig. 12 One-photon and two-photon bioimaging of CDs. (A) In vivo imaging of supine nude mice with intravenous injection of CDs at different time points (Ⅰ: thoracic region, Ⅱ: area of liver, Ⅲ: area of small intestine, Ⅳ: area of large intestine, Ⅴ: bladder region). (B)Real-time ex vivo imaging of nude mice with intravenous injection of CDs at different time points

The near-infrared fluorescence emission characteristics of near-infrared fluorescent CDs (NB-CDs) synthesized with litchi as carbon source are also suitable for continuous imaging of tumors in vivo^[88]. As shown in Fig. 13, NBCD-PEG-Ce6-Tf was subcutaneously injected into the tumor area of mice, followed by real-time imaging. When the NBCD-PEG-Ce6-Tf solution was injected into the tumor region of mice, the fluorescence signal was immediately detected from the tumor, and the near-infrared fluorescence signal in the tumor was detectable for up to 120 H. Therefore, these results indicate that NBCD-PEG-Ce6-Tf can be used as a near-infrared fluorescence imaging probe for mouse tumors and can be used for fluorescence image-guided tumor PDT.

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图13 用NBCD-PEG-Ce6-Tf在不同时间点对小鼠肿瘤进行实时近红外荧光成像^[88]

Fig. 13 Real-time NIR fluorescence images of the mice tumor with NB-CDs at different time points^[88]

In order to study the distribution and transport of Panax notoginseng CDs (Pn N-CDs) in vivo, Zheng et al. used BALB/C mice as an animal model system. By observing the fluorescence intensity of the chest of BALB/C mice injected with Pn N-CDs (Fig. 14), it was found that clear strong fluorescence signals were gradually observed in the chest and abdomen of mice after 3 hours, and the fluorescence intensity gradually increased after 9 hours^[98]. With the passage of time, the fluorescence gradually decreased and finally disappeared 12 H after injection. They speculated that Pn N-CDs injected into mice could flow through the blood to the whole body, then gradually be absorbed by the stomach, and eventually be excreted through the large intestine and bladder. Interestingly, it was also observed that a small amount of fluorescence signal was captured in the mouse brain, indicating that the synthesized CDs could cross the BBB, which may be due to the strong affinity of the very small and multifunctional Pn N-CDs with the BBB endothelial cell membrane. This study is the first to demonstrate that CDs derived from traditional Chinese medicine can cross the blood-brain barrier, thus providing a potential strategy for real-time tracking and diagnosis of multiple brain diseases.

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图14 BALB/c小鼠不同时间间隔注射Pn N-CDs后的体内成像^[98]

Fig. 14 In vivo imaging of male BALB/c mice after injection of Pn N-CDs at different time intervals^[98]

In addition, some scholars have prepared pea CDs (p-CDs) from pea (Pisum sativum Linn.) By a simple hydrothermal method^[99]. P-CDs can specifically bind to Cryptococcus neoformans pathogenic fungi, showing the potential of staining Cryptococcus neoformans. Furthermore, they used Cryptococcus neoformans to infect mice and P-CDs to track the location of the fungus, demonstrating the bioimaging promise of P-CDs.

In a word, the surprising results of traditional Chinese medicine CDs in vivo imaging in mice provide a lot of useful information for researchers. In addition, the study of the application of traditional Chinese medicine CDs in targeting and drug delivery can monitor the therapeutic effect and other clinical applications. Meanwhile, performing its surface modification to reduce nonspecific adsorption onto CDs in vivo and ensuring rapid extraction and transport of CDs to targeted tissues to reduce the potential cytotoxicity of CDs when used as cancer imaging agents in diagnostics deserves further investigation.

3.2.2 Zebrafish imaging

Zebrafish has become a popular model for the practical research of CDs because of its easy genetic screening and expression, rapid embryonic development and optical in vivo imaging. Fish at early stages of development are very sensitive to toxic substances, so it is more accurate and appropriate to use embryos to assess toxicity, transport and biocompatibility. Crinis Carbonisatus, as a traditional Chinese medicine, has the effect of astringing and stopping bleeding. It is recorded in Yi Xue Zhong Zhong Shen Xi Lu that its properties of removing blood stasis and generating new blood are similar to those of Panax notoginseng, so it is good at treating hematemesis and epistaxis. Zhang et al. Innovatively reported a kind of carbonized human hair CDs (HCDs) and used it for fluorescence imaging of zebrafish embryos in vivo^[100]. Intense fluorescence was observed at 12 hpf (hour post fertilization) after 8 H of incubation with HCDs. With the extension of incubation time, the fluorescence intensity remained unchanged during incubation, indicating that HCDs penetrated into the embryo and had no significant effect on embryonic development, reaching saturation after 8 H. After 72 hpf, the embryo hatched into a normally developing larva, and the fluorescence was still bright. The fluorescence intensity began to decrease on the 5th day, and was mainly concentrated in the abdomen and tail of zebrafish larvae. After 15 days of larval culture, the fluorescence was completely reduced. HCDs may be removed from the larva by the digestive system (fluorescence in the abdomen and intestine) or quenched by certain metabolites or substances in zebrafish. The photostability and decay process of HCDs as a bioprobe in zebrafish suggest that the fluorescence imaging is dose-dependent and time-dependent. The results show that HCDs decay slowly in the embryo and are photostable in vivo.

Wei et al. Synthesized CDs for zebrafish imaging analysis using Gynostemma pentaphyllum as carbon source (Fig. 15)^[57]. It was observed that the embryo showed strong fluorescence after incubation with CDs for 22 H, indicating that CDs could enter the embryo through the chorion. In addition, the fluorescence signal accumulated in the digestive system (gastrointestinal tract) of zebrafish after 5 days of incubation, which may indicate that CDs tend to enter zebrafish through the mouth. Based on the striking properties of toothbrush-tree-based CDs in cells in vitro, CDs were also bioimaged in zebrafish in vivo^[93]. Similar to the results of in vitro cell imaging, the in vivo zebrafish experiment of CDs also obtained satisfactory cell imaging.

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图15 CDs在斑马鱼体内的光致发光衰减。0.4 mg/mL HCDs和SCDs溶液在不同时间点暴露2 d后斑马鱼胚胎的荧光图像^[100]

Fig. 15 The photoluminescence decay of CDs in zebrafish. The fluorescent microscopic images of bright field and fluorescent field of zebrafish embryos after exposure to 0.4 mg/mL HCDs and SCDs solutions for 2 days at different time points^[100]

In addition to mouse and zebrafish, Paramecium (P. caudatum), as a representative protozoan, is often used in the study of protozoan model systems. Zheng et al. Used CLSM to study the applicability of CDs synthesized from Panax notoginseng (Pn N-CDs) in protozoa imaging^[98]. It was observed that the whole cell membrane of Paramecium showed a uniform fluorescence distribution after 1 H of incubation. At the same time, uniform Pn N-CDs were also attached to the nuclear membrane of the nucleus. The ability of Pn N-CDs to enter Paramecium cells and successfully attach and label the cell membrane and nuclear envelope may be attributed to the specific recognition function of this CD on the phospholipid bilayer of the cell membrane/membrane structure. In addition, they attempted plant imaging. Similar to Paramecium, a clear boundary between onion epidermal cells was observed, which may imply that Pn N-CDs are attached to the cell membrane or in the periplasm of the cell. These results also indicate that Pn N-CDs have the function of specific recognition of cell membrane. Plant imaging expands the application of traditional Chinese medicine CDs in biological imaging and provides a new perspective for plant labeling research.

The above studies suggest that TCM CDs have great application prospects in bioimaging of animal models, which is expected to support efficient medical diagnosis and treatment. The development of traditional Chinese medicine CDs with good biocompatibility and efficient clearance ability is more critical to provide excellent fluorescent probes for tumor tracking, drug delivery, disease monitoring and further biomedical applications. In order to eliminate the nonspecific absorption of CDs, it may be necessary to modify the surface of CDs. Therefore, the specific imaging of in vivo targets should be considered in the future development of traditional Chinese medicine CDs.

Since fluorescence is the main characteristic of traditional Chinese medicine CDs in medical imaging, it is necessary to obtain high QY traditional Chinese medicine CDs in order to obtain the best biological imaging performance. Can be achieved, for example, by heteroatom doping^[101]. Although studies on the toxicity of traditional Chinese medicine CDs have confirmed their biocompatibility in vivo and in vitro, the long-term accumulation of CDs in targeted tissues or organs may cause irritation, inflammation, nephrotoxicity, hepatotoxicity, and immune system problems. It is still a challenge for scientists to study the possibility of the existence of CDs in vivo and the ability of CDs to be released in vitro. It is necessary to ensure that the CDs of traditional Chinese medicine have sufficient photostability to analyze the stability of fluorescence signals for further application in vivo.

In conclusion, CDs have now emerged as a new type of fluorescent nanomaterials, and their application in biomedicine based on fluorescence imaging seems to be promising for further clinical applications, and the relevant characteristics of traditional Chinese medicine CDs in bioimaging applications have been summarized in Table 2. Although these CDs image-based biomedicine have been reported in the initial stages, they will have great potential in clinical medicine if their possibilities are successfully evaluated in clinical trials. For further development of CDs, CDs with bright fluorescence emission in the red/near-infrared spectral region need to be synthesized to penetrate tissues more effectively. More research is needed to use the surface functionalization of CDs for controllable coupling with bioactive substances to achieve specific targeting in cell and in vivo imaging and related biomedical applications. Although CDs imaging-based nanomedicine has promising potential for further clinical medicine, some key aspects need more attention before transferring to the clinical setting.

表2 中药CDs在生物成像中的应用

Table 2 The application of TCM-CDs in bioimaging

Source	FL color	Applied Ex/Em (nm)	Application	Biotarget	ref
Citrus junos Tanada	blue, green	405, 488/-	Imaging of cells	MG-63	74
Mentha haplocalyx Briq	blue, green, red	360, 470, 530/447, 525, 593	Imaging of cells	MCF-7	75
Prunus persica	blue	-	Imaging of cells	MDA-MB-231	76
Prunus mume	blue, green	365/-	Imaging of cells	MDA-MB-231	77
Curcuma Longa	blue, yellow, red	405, 488, 543/-	Imaging of cells	KB	78
Mangifera indica L.	blue, green, yellow	488, 488, 513/505, 530, 560	Imaging of cells	A549	79
Allium sativum L.	blue, green, yellow	385, 480,550/-	Imaging of cells	A549	40
Coriandrum sativum Linn.	green	470/525	Imaging of cells	L-132	80
Dendranthema morifolium	blue, green	405, 488/-	Imaging of cells	HeLa	81
Mentha haplocalyx Briq.	blue, yellow, red	380, 480, 590/-	Imaging of cells	HeLa	82
Brassica compestris L.var.purpurea Bailey	blue, yellow, red	405, 488, 559/-	Imaging of cells	HeLa	83
Bombyx mori L.	blue, yellow, red	340, 495, 550/-	Imaging of cells	HeLa	67
Abelmoschus esculentus (Linn.) Moench	blue	340/410	Imaging of cells	HeLa	84
Lycium chinense Miller	blue, glaucous, green	400, 415, 485/-	Imaging of cells	HeLa	29
Alisma plantago-aquatica Linn.	blue, yellow, red	340, 460, 520/-	Imaging of cells	HeLa	85
Ginkgo biloba Linn.	blue, green	405, 488/-	Imaging of cells	HeLa, KYSE-410	69
Benincasa hispida (Thunb.) Cogn.	blue	365/-	Imaging of cells	HepG2	86
Prunus cerasifera Ehrhart f. atropurpurea (Jacq.) Rehd.	blue, green	405, 488/-	Imaging of cells	HepG2	87
Litchi chinensis Sonn	blue	405/-	Imaging of cells	HepG2	88
Ginsenoside Re	blue to red	360-530/-	Imaging of cells	A375	89
Brassica oleracea Linnaeus var. capitata Linnaeus	blue, green, red	405, 488, 543/-	Imaging of cells	HaCaT	49
Phyllanthus acidus (L.) Skeels	blue, green, red	405, 488, 555/-	Imaging of cells	Clone9	90
Cymbopogon citratus (D. C.) Stapf	blue, yellow, red	-	Imaging of bacteria	BY4742	91
Ocimum sanctum Linn.	blue, yellow, red	405, 488, 561/-	Imaging of bacteria	B. subtilis, E. coli	92
Salvadora persica	green	488/550	Imaging of bacteria	C. albicans,E. coli, S. aureus	93
Allium cepa Linn.	green	-	Imaging of bacteria	E.coli, S.aureus	94
Mangifera indica L.	blue to yellow	-	In vivo imaging	mice	79
Taxus chinensis (Pilger) Rehd.	red	640/705	In vivo imaging of tumor	mice	97
Litchi chinensis Sonn.	blue to red	-	In vivo imaging of tumor	mice	88
Panax notoginseng	blue to red	-	In vivo imaging of tumor	BALB/c mice	98
Pisum sativum Linn.	blue	-	In vivo imaging	mice	99
Crinis Carbonisatus	blue, green, red	385, 480, 550/-	In vivo imaging	Zebrafish	100
Gynostemma pentaphyllum (Thunb.) Makino	blue, green, red	-	In vivo imaging	Zebrafish	57
Salvadora persica	green	488/550	In vivo imaging	Zebrafish	93
Panax notoginseng	blue, yellow, red	405, 488, 543/-	In vivo imaging	P. caudatum	98

4 Application of traditional Chinese medicine carbon dot in medical treatment

Traditional Chinese medicine has shown more unique and satisfactory efficacy and low toxicity than commercial drugs. The material basis of traditional Chinese medicine for disease prevention and treatment comes from bioactive parts or active chemical components. Therefore, medical researchers usually focus their attention on finding components with various biological activities in TCMs. However, the absorption, metabolism and excretion of drugs by organisms are complex processes, so the pharmacological effects of drugs should not be attributed entirely to the unique chemical composition of the drug, but also to the physical characteristics of the drug. This has inspired researchers that changing the physical state may be an effective research method for new drugs of traditional Chinese medicine.

Nanomedicine has become a highly active research field, and the emergence of CDs has contributed new forces to the development of nanomedicine. The key to CDs of traditional Chinese medicine is to process and manufacture high and new technologies with specific functions on the basis of inheriting and developing the advantages and characteristics of traditional Chinese medicine. Existing CDs can only be used to treat diseases by loading pharmacophores or as drug carriers, and require expensive chemical materials and complex modification processes. Excitingly, TCMs as precursors can overcome these limitations through their specific efficacy, which naturally attracted the attention of researchers. The medical therapeutic effects of CDs and their specific functional mechanisms are mainly reflected in the following aspects.

4.1 Antibacterial action

Infections caused by fungi, bacteria, parasites, or viruses cause many serious diseases. The treatment of diseases caused by these bacterial infections will become more challenging due to bacterial multidrug resistance (MDR). Traditional antibiotics/antibacterial drugs can not meet the urgent need to effectively prevent and treat a wide range of bacterial infections. It is imperative to find and develop alternative antibacterial technologies to effectively combat MDR, prevent and treat diseases and their adverse side effects. CDs, as a potential fluorescent nanomaterial, have been used to identify and inactivate different types of bacterial species in various photosensitizers (PS)^[102,103]. CDs, which exhibit powerful photodynamic effects due to their optical properties, have been used to kill bacteria and cancer cells under visible light irradiation^[104].

The currently known antibacterial mechanism of CDs is shown in Fig. 16^[105]. The positively charged CDs on the surface interact electrostatically with the negatively charged bacteria, facilitating the internalization of CDs and killing the bacteria. This CDs behavior is particularly critical for the success of photodynamic antimicrobial chemotherapy (PACT). Bacteria can also be killed by breaking down the bacterial cell wall, resulting in leakage of cytoplasmic material, which may cause secondary side effects. In addition, the higher temperature caused by the release of ROS by photothermal therapy (PTT) effect or PACT effect can directly damage bacterial DNA and protein, so it is widely used. CDs are also valued for their ability to generate highly reactive ROS. CDs present light absorption in the visible and near infrared spectrum to destroy bacteria through the photoinduced production of ROS by the classical mechanism.

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图16 CDs的一般杀菌作用机制。(a)CDs和细菌细胞壁之间最初静电相互作用。(b)CDs内化,在细菌膜中插入,以及细胞质物质泄漏的不可逆破坏。(c)CDs促进细菌光动力灭活,产生ROS和DNA损伤^[105]

Fig. 16 General bactericidal mechanisms of action of CDs. (A) Schematic representation of the initial electrostatic interaction between CDs and the bacterial cell wall. (B) CDs internalization, intercalation in the bacterial membrane, and irreversible disruption with a leak of cytoplasmatic material. (C) CD-promoted bacterial photodynamic inactivation with ROS production and DNA damage^[105]

Yoon et al. Used mushroom (Agaricus bisporus (lang.) Sing) as a raw material to prepare CDs (MCDs) with high blue fluorescence under the excitation of 360 nm UV light, and the emission at 456 nm was the most prominent^[106]. Under LED visible light illumination, MCDs can produce ROS (such as OH · and E. coli), which can directly adhere to the surface of E. Coli and induce cell membrane damage. Scanning electron microscopy (SEM) images clearly showed the morphology of E. coli cells before and after the addition of MCDS (Figure 17). The bacteria without MCDs showed clear and smooth cell surface under visible light with well-defined bacterial morphology. The bacterial surface was partially damaged when treated with MCDs without light irradiation. By contrast, bacterial cells treated with MCDs and visible light were highly damaged. During cell degradation, photoexcited MCDs release ROS such as singlet oxygen, which causes cytosolic and cellular destruction. Therefore, CDs can be considered to inhibit the growth of bacteria.

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图17 大肠杆菌的SEM图像:(A~C)光照射不含MCDs,(D~F)MCDs在黑暗条件下处理,以及(G~I)MCDs在可见光照射下处理12 h^[106]

Fig. 17 SEM images of E. coli: (A~C) light irradiation without MCDs, (D~F) MCDs treated under dark conditions, and (G~I) MCDs treated with visible light irradiation for 12 h^[106]

Drugs can bind to the functional groups on the surface of CDs through covalent bonds, and achieve drug delivery through CDs to exert pharmacodynamic effects. Tejwan et al. Studied CDs loaded with rutin prepared from aqueous root extract of traditional Chinese medicine red ginseng (Talinum paniculatum (Jacq.) Gaertn.) As a drug delivery system, which showed excellent free radical scavenging activity and dose-dependent antibacterial effect against Gram-negative E. coli and Gram-positive S. aureus bacteria^[107]. They observed that CDs and RUT alone did not significantly inhibit bacterial growth, whereas the CDs-RUT nanohybrid showed a dose-dependent synergistic effect in inhibiting bacterial growth. In addition, a dose-dependent DPPH radical and H₂O₂ scavenging activity was observed for CDs. Therefore, the synthesized CDs can not only enhance the antioxidant defense mechanism of cells, but also enhance the pharmacological effects of natural bioactive hydrophobic antioxidant molecules by increasing their hydrophilicity. Similar to bacterial growth inhibition, the CDs-RUT nanohybrid significantly increased ROS levels by 4 to 5 times in both bacteria compared to the control. On the other hand, ROS induction by free drug RUT was minimal. Therefore, the CDs can improve the pharmacological effects of hydrophobic antibacterial molecules by increasing their hydrophilicity.

In addition, Lin et al. Prepared four fluorescent CDs with various Chinese medicinal materials (onion, ginger, garlic) and other natural products (fish) as carbon sources^[94]. Among them, onion CDs (O-CDs) exhibited the best antibacterial activity against Pseudomonas fragilis, which was stable over a wide pH range. As shown in Fig. 18, onion CD also exhibited antibacterial activity against representative Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of onion CDs against P. Fragilis were 2 mg·mL^-1 and 4 mg·mL^-1, respectively.

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图18 (a)洋葱CDs对细菌细胞壁和细胞膜完整性影响的示意图;洋葱CDs对脆弱假单胞菌胞外(b)AKP和(c)ATP释放的影响^[94]

Fig. 18 (a) Schematic illustration of the effect of the onion carbon dots on the integrity of the bacterial cell walls and membranes. Effect of onion carbon dots on the release of extracellular (b) AKP and (c) ATP content in Pseudomonas fragi^[94]

4.2 Anti-inflammatory effect

Inflammation is a dynamic process including injury, anti-injury and repair, which is a response of the body to the stimulation of various injury factors. All factors that can cause tissue and cell damage can cause inflammation. There are many kinds of inflammatory factors, including physical factors, chemical factors, biological factors, tissue necrosis and allergy. Traditional Chinese medicine CDs have also attracted extensive research interest in biomedical treatment of inflammation because of their unique advantages such as good biocompatibility, photostability and inherent targeting of functional groups.

Abnormal ROS production is one of the important mediators of inflammation. Therefore, the development of nanocatalytic drugs to catalyze ROS scavenging reactions in pathological areas has broad application prospects in anti-inflammatory therapy. As shown in Fig. 19, the synthesized TCM CDs is an antioxidant multienzyme^[108]. It can directly scavenge excessive ROS, including H₂O₂,

O 2 · -

, OH ·, etc., and effectively reduce the inflammatory response of liver tissue through its inherent passive targeting, which has a high application prospect in the anti-inflammatory treatment of nanocatalytic drugs.

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图19 用于清除ROS和抗炎治疗的CDs示意图^[108]

Fig. 19 Schematic illustration of CDs for ROS depletion and anti-inflammation therapy^[108]

Arthritis generally refers to inflammatory diseases occurring in human joints and surrounding tissues. Aurantii fructus ymulturus (AFI) is a medicinal and edible plant, and AFI Carbonisatas (AFIC), the carbonized processed drug of AFI, has long been used to treat inflammatory and metabolic diseases. However, the material basis and mechanism of action of AFIC are still unclear. On the basis of previous studies, Wang et al. Produced a new type of substance, Fructus Aurantii Immaturus CDs (AFIC-CDs), after carbonizing AFI by pyrolysis, and explored the anti-hyperuricemia and anti-gouty arthritis effects of AFIC-CDs^[109]. Studies have shown that AFIC-CDs effectively reduce the inflammatory response induced by monosodium urate (MSU) crystals by inhibiting the production of inflammatory cytokines (IL-1β and TNF-α), and play an important role in the pathological process of acute gouty arthritis. Subsequently, they synthesized a new type of CDs derived from Mulberry Silk worm Cocoon (MSC-CDs) by pyrolysis method to study the anti-inflammatory properties^[110]. Mulberry cocoons have been used for the treatment of inflammatory diseases for hundreds of years. However, after many years of research, there is little information about its anti-inflammatory components and underlying mechanisms. In this study, they innovatively used three classical experimental models of inflammation to evaluate the anti-inflammatory bioactivity of MSC-CDs. The results showed that MSC-CDs had significant anti-inflammatory activity, which may be related to the inhibition of the expression of inflammatory cytokines IL-6 and TNF-α, and provided a basis for further study of the potential effective material basis of MSC-CDs.

Psoriasis is a chronic inflammatory skin disease. CDs have many advantages, which provide hope for the development of new nanomedicines for the treatment of difficult diseases. Zhang et al. Established a green and simple calcination method to prepare novel non-toxic PCC-CDs based on Phellodendri Cortex (PCC), which has obvious anti-psoriatic effect^[111]. Using a mouse model of psoriasiform skin, they demonstrated for the first time that the prepared PCC-CDs had significant anti-psoriatic activity to improve the appearance, psoriasis area, and histopathological morphology of the back skin and right ear of IMQ-induced mice. The underlying mechanism of its antipsoriatic effect may be related to the inhibition of macrophage M1 polarization and the relative promotion of M2 polarization. These results suggest that PCC-CDs may be used as a candidate anti-psoriatic drug in the clinical treatment of psoriasis, which not only provides a basis for further broadening the biological application of CDs, but also provides a potential hope for the application of nano-drugs in the treatment of difficult diseases.

Acute lung injury (ALI) refers to the damage of alveolar epithelial cells and pulmonary capillary endothelial cells caused by non-cardiac diseases. Lipopolysaccharide (LPS) is the key cause of ALI. Under the stimulation of LPS, neutrophils rapidly invade lung tissue, resulting in the activation of related inflammatory cells and the release of a series of inflammatory factors. MPO, MDA, SOD and GSH are important indicators reflecting the level of oxidative stress in lung tissue. Armeniacae Semen Amarum Carbonisata (AsAC) is a charcoal processed product of bitter almonds, which has long been used as an anti-inflammatory agent. According to the changes of histological observation and cytokine data, ASAC-CDs synthesized by Zhao et al. Using bitter almond by one-step simple pyrolysis method can effectively inhibit the expression level of inflammatory factors (IL-6, IL-1β and TNF-α), showing satisfactory anti-inflammatory effect^[112]. To a certain extent, it can reduce the increase of neutrophils in the blood, reduce the chemotaxis of neutrophils to inflammatory sites, thereby reducing the release of inflammatory mediators and inhibiting LPS-induced lung tissue injury and deterioration. The results showed that ASAC-CDs could reduce the content of MDA and MPO in rat lung tissue on the one hand, and on the other hand, it could reduce the damage of inflammation to the lung by improving the effect of antioxidants. On the other hand, ASAC-CDs can increase the activity of SOD and the content of GSH in rat lung tissue to a certain extent, indicating that ASAC-CDs also has good antioxidant activity (Fig. 20).

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图20 ASAC-CDs改善LPS诱导的大鼠急性肺损伤(ALI)的宏观图像:(A)生理盐水组;(B)示范组;(C)阳性对照组;(D)大剂量ASAC-CDs组;(E)中剂量ASAC-CDs组;低剂量ASAC-CDs组^[112]

Fig. 20 Macroscopic images of ASAC-CDs ameliorating an LPS-induced acute lung injury (ALI) in rats. (A) Normal saline group; (B) model group; (C) positive control group; (D) high-dose ASAC-CDs group; (E) medium-dose ASAC-CDs group; and (F) low-dose ASAC-CDs group^[112]

Lonicerae japonicae Flos (LJF) is commonly used to treat fever, furuncles, carbuncles, and tumors. Carbonized LJF (LJFC) is the first choice for detoxification and relief of dysentery and its related symptoms. Wu et al. Explored the effect on LPS-induced fever and hypothermia model in rats based on synthetic LJFC-derived CDs (LJFC-CDs)^[113]. The results showed that LJFC-CDs could significantly reduce the inflammatory response induced by LPS, which was manifested by the expression of TNF-α, IL-1β, IL-6 and the recovery of normal body temperature. Therefore, LJFC-CDs may have some anti-inflammatory effects and have the potential to reduce symptoms such as fever and hypothermia caused by inflammation.

Taken together, these findings are helpful to understand the anti-inflammatory mechanisms of TCM CDs as an effective nanocatalytic drug to control ROS-induced oxidative damage and inhibit the expression of anti-inflammatory factors. However, there are still some challenges to directly identify the reaction intermediates and clarify the relationship between TCM CDs and catalytic activity and complex surface functionalization.

4.3 Hemostasis

In traditional Chinese medicine, a charcoal drug extracted from charcoal is also considered to be a CDs extracted from natural products. Traditional Chinese medicine charcoal has a long history and rich clinical accumulation in the treatment of hemorrhagic diseases^[114]. In particular, its prominent hemostatic effect has been valued by physicians of past Dynasties, and after a large number of clinical practices, the great theory of "carbonized hemostasis" has gradually been formed. "Charred hemostasis", as its name implies, refers to traditional Chinese medicines with different carbon sources, such as carbonized hair, Schizonepeta tenuifolia, Phellodendron, thistle and so on, which have hemostatic effect after being processed into charcoal^[115]^[116]^[111]^[117]. Through the study of CDs in carbonized drugs such as Schizonepeta tenuifolia, Cirsium japonicum and carbonized Pollen Typhae, it was found that CDs existed in most of the carbonized drugs and had the characteristics of low toxicity, good water solubility and biocompatibility^[118]^[117]^[119]. In addition, all extracted pure CDs showed good hemostatic effect. From the Qin and Han Dynasties to the present, after more than 2000 years, traditional Chinese medicine CDs is still active in the treatment of various hemorrhagic diseases, the fundamental reason is that traditional Chinese medicine CDs based on charcoal medicine is safe and has exact hemostatic effect.

The coagulation system protects the pressurized circulatory system by producing and preventing thrombi, a complex process known as hemostasis. Routine coagulation parameters include activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB). Among them, the PT value is related to the overall efficiency of the extrinsic coagulation pathway, while the APTT is related to the intrinsic coagulation pathway. The common coagulation pathway and the activity that promotes the conversion of FIB to fibrin in plasma are related to TT and FIB levels.

CDs in charcoal drugs can increase the solubility of glycosides in water by affecting the glycosidic acid. Luo et al. Studied the effect of new water-soluble CDs on baicalin, the main component of Scutellaria baicalensis Georgi, and found that pure CDs could significantly improve the solubility of baicalin in water^[120]. The results showed that the oral bioavailability of baicalin CDs was 1.7 times higher than that of pure baicalin. In addition, baicalin and wogonoside in Scutellaria baicalensis Georgi are carbonized into carbonized baicalein and wogonoside, which are easily absorbed and have strong hemostatic effect. In the process of high temperature carbonization, CDs is one of the key substances to stop bleeding, which can be directly used in the treatment of bleeding symptoms due to blood heat. At the same time, it can promote the absorption of glycosides and indirectly enhance the hemostatic effect.

In addition, the hemostatic effect of Cirsii Japonici (CJ) is also very obvious, but the active ingredients and related mechanisms of the hemostatic effect of CJ are still unclear. Therefore, Wang et al. Synthesized the novel CDs of Cirsium japonicum DC. (CJ-CDs) and evaluated its pharmacodynamics in vivo and coagulation parameters in rats^[117]. The results showed that CJ-CDs had a significant inhibitory effect on bleeding caused by tail amputation or liver injury in mice, and the hemostatic effect may be related to the common coagulation pathway FIB system.

Phellodendri Cortex Carbonisatus (PCC) has been used to treat many diseases (such as anemia, enteritis, diarrhea, etc.) for more than 1000 years in the ancient prescription "Fifty-two Wards". However, the basic material composition and mechanism of biological transformation after charcoal burning are still unclear. Therefore, scholars discovered the existence of a new substance Phellodendron CDs (PCC-CDs) from the aqueous extract of Phellodendron amurense, and studied the hemostatic activity of PCC-CDs by tail-clipping bleeding and liver injury models in mice^[121]. The results showed that the mice treated with PCC-CD showed satisfactory hemostatic effect (comparable to hemostatic agents), and it was found for the first time that the hemostatic mechanism was through the activation of the FIB system to exert hemostatic effect. Synthetic PCC-CDs have excellent stability, are suitable for long-term storage, and can be used as complementary and alternative therapeutic drugs for acute traumatic hemorrhage.

Pollen Typhae (Ptollen Typhae Carbonisata, PTC) is a calcined herbal medicine that has been used for thousands of years as a hemostatic agent to promote hemostasis. Yan et al. Discovered and isolated novel water-soluble CDs (PTC-CDs) from the aqueous extract of PTC^[119]. Pharmacodynamic studies showed that PTC-CDs had the ability to inhibit bleeding in tail-clipping and liver injury models in mice. In addition, these effects may be related to the intrinsic anticoagulant activity and activation of the fibrinogen system as assessed by blood coagulation parameters. These results not only shed new light on the materials-based research of drugs, but also provide new insights into the potential biomedical and healthcare applications of CDs and lay a solid foundation for future drug discovery.

Cirsium Setosum Carbonisata (CSC) has the effects of cooling blood, relieving pain and clearing heat in traditional Chinese medicine, and can treat sores, carbuncles, swelling and pain. CSC-CDs based on silymarin charcoal synthesis showed moderate hemostatic activity in mouse models of tail-clipping hemorrhage and liver injury. Coagulation parameters in mice were evaluated, and it was observed that the PT value was decreased and the FIB value was increased in mice treated with CSC-CDs, indicating that the hemostatic effect of CSC-CDs may be related to the stimulation of extrinsic coagulation activity and the activation of the FIB system^[122].

Schizonepetae Herba Carbonisata (SHC) has been used in traditional Chinese medicine to treat hemorrhagic diseases for more than 1000 years. However, there are few studies on its hemostatic components and mechanisms^[123]. In this study, Zhang et al. Prepared SHC by a modified pyrolysis method using Schizonepeta tenuifolia (SH) as the sole precursor, and developed novel water-soluble CDs in SHC aqueous extract for the first time^[116]. Pharmacodynamic experiments showed that SHC-CDs had obvious inhibitory effect on bleeding in mouse tail-clipping bleeding and liver injury models. In addition, these effects may be related to exogenous coagulant activity and activation of the FIB system, based on the results of evaluation of coagulation parameters in mice. This study is an initial evaluation of the hemostatic activity of SHC-CDs, and further studies are needed to elucidate the mechanisms underlying these effects. Therefore, they developed a new mouse tail-cutting model and needle toxin model to evaluate the hemostatic biological activity of SHC-CDs^[118]. The results showed that SHC-CDs inhibited the hemorrhagic activity of Trypanosoma acuminatum venom by increasing PLT. Therefore, SSC-CDs not only have the potential effect of anti-bleeding caused by needle toxin, but also have a good application prospect in diseases caused by PLT reduction.

Pollen Typhae Carbonisata (PTC) is also a charred herbal medicine, which has been widely used in traditional Chinese medicine for thousands of years in the treatment of hemorrhagic diseases. Yan et al. Discovered and isolated new water-soluble CDs (PTC-CDs) from the aqueous solution of PTC, which exert hemostatic effects by stimulating the intrinsic coagulation pathway and activating the FIB system^[119]. In addition, the significant antitumor effect of PTC-CDs was also revealed by pharmacological experiments. The degree of increase in APTT, FIB, and PLT was significantly reduced in rats treated with PTC-CDs.

Juncus effuses (Juncus effusus L.) is a perennial herb of Juncaceae, which has diuretic, cooling and sedative effects. Its charcoal-processed material, Junci Medulla Carbonisata (JMC), has been used in traditional Chinese medicine for nearly 900 years to treat bleeding disorders, but its potential bioactive components and mechanism of action have not been identified despite its long history of use. Cheng et al. First discovered the novel CDs (JMC-CDs) in JMC, and explored the hemostatic mechanism of JMC-CDs by measuring the coagulation parameters of rats^[124]. The results showed that JMC-CDs had excellent hemostatic effect through the extrinsic coagulation pathway and the activation of the FIB system.

Egg yolk oil (EYO) is obtained by refining gallus gallus fimderus brisson. It has been used in traditional medicine in China for more than a thousand years. EYO has analgesic, antioxidant, and anti-aging properties, and is now widely used to treat a variety of burns, as well as acute and chronic eczema in clinics. Zhao et al. Isolated a new substance from EYO and obtained EYO-CDs by pyrolysis^[125]. The experimental results suggest that the main hemostatic mechanism of EYO-CDs is to stimulate the activation of the intrinsic coagulation system and the FIB system.

The above studies show that CDs are widely used in the treatment of hemostasis based on the characteristics of charcoal drugs. Traditional Chinese medicine will produce a large amount of carbon after carbonization at high temperature. Its hemostatic effect may be due to the enhanced astringency of CDs produced by carbonization of traditional Chinese medicine, and the enhanced adsorption and astringency of drugs. There are many loose holes in its structure, which can cause physical adsorption to accelerate hemostasis. In addition, because of the special structure of the carbon surface, it can activate plasma coagulation factors and split platelets, and release platelet factors to promote coagulation. At the same time, it releases substances to enhance the tension of smooth muscle and cause vasoconstriction, which is beneficial to hemostasis. Some scholars believe that high temperature carbonization is the key to the material basis of hemostatic efficacy of traditional Chinese medicine CDs. In recent years, scholars have introduced the characterization technology of nanoscience into charcoal drugs to study the material basis of the hemostatic effect of charcoal drugs. The results showed that CDs existed in many charred drugs, and the structural characteristics, physicochemical properties and biological activities of CDs in different charred drugs were different. These TCM CDs are derived from a variety of natural products with different biological activities, which are worthy of further study.

4.4 Antioxidation

Oxidative stress refers to the imbalance between oxidative and antioxidant systems in the body, and the tendency of oxidation to produce a large number of ROS intermediates. ROS is a general term for oxygen-containing substances, including

O 2 · -

, OH ·, H₂O₂, O₃, and ¹O₂, which are related to intracellular redox balance and enzyme antioxidation. Normal levels of ROS play a decisive role in signal regulation and homeostasis of cells, but excessive ROS accumulation may lead to oxidative damage, inflammation, and various diseases, even cancer. Based on this, Wei et al carried out an in vitro (HeLa cells) and in vivo (zebrafish) study on the oxidative stress resistance of synthetic Gynostemma pentaphyllum fluorescent CDs induced by H₂O₂^[57]. The results showed that the content of ROS in the H₂O₂ induced group was significantly increased, indicating that exogenous H₂O₂ changed the normal metabolism and caused oxidative stress in vivo and in vitro. However, the content of ROS in the group added by CDs decreased significantly, indicating that fluorescent CDs had an effect on the generation of ROS. They then evaluated oxidative stress-related genes in zebrafish and observed that CDs could protect zebrafish from oxidative stress by increasing ROS-related enzymes, thus reducing ROS content through compensatory mechanisms. Therefore, Gynostemma pentaphyllum CDs can be used as an antioxidant to effectively reduce ROS damage in HeLa cells and zebrafish.

Salvia miltiorrhiza Bunge is a common plant of Salvia in traditional Chinese medicine. Because of the presence of phenolic hydroxyl groups or o-diphenolic hydroxyl groups on its surface, some studies have pointed out that salvianolic acid, as the main active ingredient of Salvia miltiorrhiza, has a strong antioxidant effect^[126]. Therefore, Salvia miltiorrhiza has been highly valued by researchers and widely studied as an antioxidant. Inspired by this, Li et al. Synthesized multifunctional antioxidant CDs by one-step hydrothermal method using Salvia miltiorrhiza as carbon source^[44]. Compared with the natural extract of Salvia miltiorrhiza, the prepared CDs had better antioxidant activity and stronger ability to scavenge ROS. CDs can mimic the antioxidant defense process of plant cells, as shown in Fig. 21. CDs can catalyze the generation of H₂O₂ from

O 2 · -

due to its unique superoxide dismutase (SOD) activity, and then further convert H₂O₂ to OH · by Fenton reaction. CDs can reduce OH · to H₂O because of its strong scavenging ability. In vitro, CDs have excellent ability to scavenge intracellular ROS and protect cells from oxidative stress. Because these CDs have antioxidant properties, they can scavenge ROS and thus alleviate the abiotic stress of plants, so they have a wide range of applications in the field of botany. Subsequently, they synthesized a Salvia miltiorrhiza derived CDs^[127]. Based on their abundant surface functional groups (-OH and -COOH), CDs hydroponics promoted Ca²⁺ signaling and plant environmental adaptation under salinity and nutrient deficiency conditions, which in turn triggered ROS-independent Ca²⁺ activation in plant roots. Therefore, the CDs can be used for crop improvement as both a Ca²⁺ signal amplifier and a ROS scavenger.

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图21 CDs减轻盐胁迫下莴苣氧化损伤的机理示意图^[44]

Fig. 21 Schematic of the Mechanism of CDs Alleviating the Oxidative Damage of Italian Lettuce under Salt Stress^[44]

4.5 Anticancer effect

Cancer, namely malignant tumor, has become one of the most important problems that endanger human health, and the effective prevention and treatment of cancer has become the main direction of global researchers. Traditional Chinese medicine has a good and unique anti-tumor effect. At present, more than 100 kinds of traditional Chinese medicines have been proved to have anti-tumor effects in clinic, and some of them can also be used in combination with radiotherapy and chemotherapy to reduce toxicity and enhance efficacy. In addition, biocompatible CDs have great potential as clinical biomedical applications. The combination of traditional Chinese medicine and CDs is expected to effectively improve biocompatibility and degradability, reduce anti-cancer side effects, increase tumor accumulation and improve drug efficacy to overcome many limitations of traditional methods.

Ginger (Zingiberis rhizoma) has been used as a traditional medicine for a long time and contains rich anticancer active components. Curcumin, a hydrophobic polyphenol, is a major component of ginger, which has antibacterial, anticancer, hepatoprotective and other biological activities. Curcumin can inhibit the growth of human hepatoma cells (HepG2) and change the surface morphology of cells, trigger pro-apoptotic factors to promote apoptosis, but has low toxicity to other cells. 6-Gingerol, another abundant component of ginger, also has pharmacological activity against tumor cell metastasis and tumor cell invasion. In addition, 6-gingerol can also inhibit the invasion of HepG2 cells by regulating the organization of matrix metallopeptidase 9 (MMP-9) and metallopeptidase inhibitor 1 (TIMP1). Inspired by this, Li et al. Prepared novel CDs (G-CDs) based on ginger for anticancer studies^[38]. The results showed that G-CDs could up-regulate the expression of p53 gene in cancer cells and induce the level of intracellular ROS, which had a high inhibitory effect on the growth of HepG2 cells, but had a low toxicity to normal breast epithelial cells (MCF-10a) and mouse hepatocytes (FL83B). At the same time, in vivo studies have found that G-CDs have significant anti-hepatoma activity, which can accumulate in the tumor site through the enhanced osmotic retention effect (EPR) of solid tumors, and then inhibit the growth of tumors. In addition, G-CDs are excreted in urine within 1 H and are not stored in large quantities in the body, thus avoiding potential biological toxicity.

Yao et al. Synthesized CDs of ginsenoside Re source (Re-CDs) with a diameter of about 4.6 nm^[89]. Compared with the ginsenoside Re raw material, the synthesized Re-CDs exhibited low toxicity to normal cells and higher efficiency in inhibiting cancer cell proliferation. In addition, researchers have found that the anticancer activity of Re-CDs is related to the generation of a large amount of ROS and CASPASE-3-related apoptosis.

Kiwifruit (Actinidia deliciosa) is highly concerned and loved by people because of its medicinal value in the treatment of hepatitis, edema, gastric cancer and breast cancer. Arul et al. Prepared nitrogen-doped CDs (N-CDs) by a simple hydrothermal method using kiwifruit fruit extract as carbon precursor and NH₃·H₂O as nitrogen source^[128]. The synthesized N-CDs has blue fluorescence, the average particle size is 3. 59 nm, and the graphitization property with the interlayer spacing of about 0. 21 nm. N-CDs were found to display some level of anticancer activity when tested against mouse fibroblast (L-929) cells and human breast cancer (MCF7) cells. In addition, N-CDs also showed significant catalytic activity for the reduction of rhodamine B in the presence of NaBH₄.

Even though traditional Chinese medicine CDs have made some progress in anti-cancer and tumor bioimaging, up to now, there are few reports on the application of traditional Chinese medicine CDs in tumor bioimaging and therapy. The reason may be attributed to the critical conditioning factor of red CDs required for bioimaging. Therefore, the synthesis of red-emitting traditional Chinese medicine CDs in the near-infrared band is essential for their application in anticancer therapy and tumor bioimaging. In addition, new therapeutic methods combining traditional Chinese medicine CDs with methods such as photodynamic and photothermal therapy are expected to improve the efficacy of cancer treatment.

4.6 Other therapeutic effects

(1) Glycyrrhizae Radix et Rhizoma (GRR) is one of the ten core traditional Chinese medicines commonly used in the treatment of gastroesophageal reflux disease, which has been used in the treatment of digestive system diseases for hundreds of years. Liu et al. Synthesized licorice CDs (GRR-CDs) using GRR as a precursor by an environmentally friendly one-step pyrolysis process^[129]. The results confirmed that GRR-CDs could reduce the oxidative damage of gastric mucosa and tissues caused by alcohol, and restore the expression of malondialdehyde, superoxide dismutase and nitric oxide in serum and tissues of mice, indicating that GRR-CDs had obvious anti-ulcer activity, which provided a new idea for the study of the effective substance basis of GRR.

(2) Analgesia In addition to its anticancer effect, ginger has been used as a painkiller for thousands of years and can be used to treat pain for various reasons. Zhang et al. Prepared a novel eco-friendly ginger CDs (ZR-CDs) from ZR by direct pyrolysis^[130]. They confirmed that ZR-CDs had obvious analgesic effect by classical hot plate method, tail immersion method and acetic acid writhing method, and proved for the first time that the analgesic effect of ZR-CDs was mediated by regulating the systemic mechanism of opioids in brain tissue and serotonin level in serum.

(3) Hypoglycemic Charred Triplet (JSX) is a charcoal traditional Chinese medicine that promotes appetite and digestion, and is widely used in the treatment of dyspepsia in China. Studies have shown that there is a relationship between the rate of carbohydrate degradation during digestion and the regulation of postprandial blood glucose and insulin levels. Sun et al. Developed JSX-derived CDs (JSX-CDs) with an average diameter of 4.4 ~ 6.4 nm by pyrolysis^[131]. Due to the abundance of surface groups, JSX-CDs have obvious solubility and bioactivity, and do not require further modification or external surface passivators. The results of pharmacodynamic studies showed that JSX-CDs had good hypoglycemic effect and safe hypoglycemic activity, which was a new hypoglycemic drug with broad prospects.

(4) Ziziphus jujuba Mill. Is a commonly used qi-tonifying drug in clinic, which can tonify the middle and benefit qi, enhance muscle strength, and treat anemia by promoting bone marrow hematopoiesis. Recently, Xu et al. Successfully prepared J-CDs with excellent anemia treatment effect based on jujube (Fig. 22)^[132]. The CDs have excitation-dependent properties, with excitation at 405, 488, and 561 nm resulting in bright blue, green, and red fluorescence emission, respectively, and the synthesized J-CDs can be used for cell imaging over a wide range of detectable wavelengths. It is able to stimulate the self-renewal of erythroid progenitor cells in vitro and in vivo, specifically enhancing erythroid cell proliferation by regulating the hypoxia response pathway and up-regulating the phosphorylation level of STAT5. Therefore, they have great potential for use as therapy-related anemia cancer drugs.

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图22 J-CDs的治疗贫血功能示意图^[132]

Fig. 22 Schematic of the function of J-CDs^[132]

(5) Scutellariae Radix Carbonisata (SRC) is a traditional Chinese medicine obtained by calcining Scutellariae Radix for the treatment of allergic diseases. However, the function and mechanism of action of the carbonized part of SRC have not been elucidated so far. Kong et al. Isolated novel water-soluble Scutellaria CDs (SRC-CDs) of 2 – 9 nm from the aqueous extract of SRC^[133]. They conducted anti-inflammatory studies of SRC-CDs at the cellular level. The results showed that the anti-inflammatory effect was closely related to the stabilization of mast cell activation, which may be related to the reduction of mast cell activation, the inhibition of RBL-2H3 cell degranulation, and the reduction of histamine and inflammatory factors. Therefore, SRC-CDs have a positive effect on anaphylaxis. The demonstration of the antiallergic activity and related mechanism of SRC-CDs in this paper fills the gap in this research field and lays a foundation for the future research and development of new drugs. SRC-CDs may become a potential drug for the treatment of allergic diseases.

In general, traditional Chinese medicine CDs, as an emerging nanomaterial, has been widely used in the medical field due to its remarkable therapeutic effects, such as good photoluminescence properties, good chemical stability and low toxicity, water dispersibility and biocompatibility. However, it is worth noting that more and more attention has been paid to the study of the biological activity of CDs, which is expected to reveal its various pharmacological and active effects. However, the therapeutic mechanism of traditional Chinese medicine CDs has not been studied in depth, and scientists need to continue to explore. In addition, as nanomaterials, it is another important challenge to clarify the metabolic process of traditional Chinese medicine CDs in vivo.

5 Conclusion and prospect

In the past decade, CDs, as a new type of fluorescent carbon nanomaterials, have made many major breakthroughs from their basic optical properties to their potential applications, which have attracted much attention from researchers. As a new branch of CDs, traditional Chinese medicine CDs have been deeply used in the fields of disease treatment and bioimaging. Potential therapeutic effects and fluorescence characteristics are meaningful markers to distinguish TCM CDs from other CDs. The medicinal value of traditional Chinese medicine may make traditional Chinese medicine CDs contain medicinal substances without drug loading, which can effectively avoid harmful effects. In addition, the application of traditional Chinese medicine CDs in bioimaging has laid a solid foundation for therapeutic diagnosis, which is expected to make a major breakthrough in the near future. Although great progress has been made in traditional Chinese medicine CDs in a short period of time, there are still many problems to be solved.

(1) The luminescence mechanism of CDs. The photoluminescence of CDs is one of the most convincing features. However, the luminescence mechanism of traditional Chinese medicine CDs is still unclear, usually due to the difficulty in determining the structure of the synthesized traditional Chinese medicine CDs, resulting in the lack of a universally applicable luminescence mechanism, which restricts the structural modification of traditional Chinese medicine CDs and the improvement of luminescence properties to meet clinical needs. Therefore, the structure and composition of CDs based on the synthesis of traditional Chinese medicine sources and the related luminescence mechanism need to be analyzed.

(2) In vivo toxicity evaluation of traditional Chinese medicine CDs. Compared with traditional QDs, CDs show low toxicity, but their potential effects on humans are still unclear, and there is still widespread concern about the risks of CDs in therapeutic use. Recently, it has been reported that the small molecular products produced by the photodegradation of CDs can cause certain toxicity^[134]. On the other hand, some CDs have novel cytotoxicity, such as ROS generation toxicity, dose-dependent toxicity, etc. Even though various related studies have been conducted in different cells and animals, and the excellent biocompatibility of traditional Chinese medicine CDs has been reported, the in vivo toxicity of traditional Chinese medicine CDs still needs to be systematically evaluated for clinical diagnosis and treatment. Therefore, the risk of traditional Chinese medicine CDs in clinical treatment still needs further attention and research. Therefore, higher requirements are put forward for the synthesis of traditional Chinese medicine CDs in practical application in the future.

(3) Development and application of traditional Chinese medicine CDs based on infrared/near-infrared emission. More and more attention has been paid to the application of traditional Chinese medicine CDs in biological imaging. It should be noted that most of the reported traditional Chinese medicine CDs show blue or green emission overlapping with the background biofluorescence in organisms, and thus, are somewhat limited in terms of bioimaging. While IR/NIR emitting CDs have little damage to biological matrices, deep tissue penetration and minimal autofluorescence background of biological samples. In addition, red CDs have long absorption and emission wavelengths, which can overcome the interference caused by autofluorescence in biological systems and effectively improve the signal-to-noise ratio of fluorescence detection. Therefore, stable and efficient synthesis of IR/NIR emitting traditional Chinese medicine CDs is essential for bioimaging applications. This important challenge has far-reaching potential value for traditional Chinese medicine CDs in bioimaging and clinical diagnosis and treatment.

(4) Application of traditional Chinese medicine CDs in other subcellular biological imaging. At present, the research on traditional Chinese medicine CDs in vitro cell imaging is mainly focused on cancer cells and bacteria, and other important organs need to be further explored. For example, in neuronal cells, traditional Chinese medicine CDs with spontaneous penetration ability and special functional modifications based on unique neural membrane lipids and proteins have developed influential methods for clinical diagnosis and treatment in the central nervous system. Staining of the nucleus is essential to reveal its morphology and its role in cellular metabolism, growth, differentiation, and heredity. Due to their small nanosize, traditional Chinese medicine CDs as newly developed nanomaterials can infiltrate into the nucleus to bind DNA and RNA. In addition, as one of the important subcellular organelles, mitochondria are the power body that produces ATP and metabolic centers in the cell. Dysfunction of mitochondria leads to many diseases, including inflammation, neurodegenerative diseases, diabetes, and cardiac dysfunction. Therefore, tracking the state and behavior of mitochondria is essential for regulating cellular dyes and mitochondrial diseases. Another important subcellular organelle, as the lysosome of the digestive system in the cell, plays a crucial role in cell signal transduction, endocytosis, and autophagy. Fluorescent CDs lysosomal imaging can be used to understand the relationship between abnormal lysosomes and related diseases. These studies will provide a new way to develop fluorescent probes based on CDs for subcellular labeling. But there is still a long way to go to develop effective CDs probes for fluorescence imaging of subcellular organelles and analysis of dynamic changes in living cells.

(5) The function of attenuation and synergism of the combination of traditional Chinese medicine and CDs. Most traditional Chinese medicines have certain toxic and side effects. Therefore, in view of the introduction of nanomaterial CDs, it is expected to improve the therapeutic effect of traditional Chinese medicine, improve biocompatibility, reduce the dosage, save limited important resources, and reduce the toxic and side effects of traditional Chinese medicine. Based on the changes of the main active ingredients and chemical properties in the processing process, the preparation of toxic traditional Chinese medicine sources into traditional Chinese medicine CDs is also expected to achieve the purpose of reducing toxicity and enhancing efficacy, which may greatly promote the promotion and application of this kind of traditional Chinese medicine in clinic.

(6) Realization of therapeutic diagnosis. Due to the characteristics of traditional Chinese medicine, the composition of prescriptions is complex, and the mechanism of action of traditional Chinese medicine is usually difficult to explain. Because of the therapeutic effect, the research on the integration of diagnosis and treatment of traditional Chinese medicine CDs seems to be endless. According to existing research, bioimaging and treatment effects are independent of each other. In addition, the metabolism of traditional Chinese medicine CDs in vivo needs to be further analyzed. Compared with classical medicine, the circulation of traditional Chinese medicine CDs in living body and organs is not clear, and the interaction with living molecules is complex, which leads to the limitation of CDs in clinical application. Moreover, the study of bioimaging is still in its very infancy. The combination of bioimaging and therapy will provide unlimited possibilities for achieving diagnostic therapy, which also becomes a great challenge.

All in all, even though there are still some controversies and challenges, fluorescent CDs synthesized based on traditional Chinese medicine sources, as a novel nanomaterial, have many opportunities and bright future in bioimaging and medical treatment.

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Outlines

模态框（Modal）标题

Abstract

Cite this article

1 Introduction

图1 CDs的分类和相应结构的示意图[2]

图2 N-CDs的合成路线及其在Ag+传感和RGB颜色分析中的应用[28]

2 Synthesis of carbon dots in traditional Chinese medicine

2.1 Hydrothermal method

图3 (A)荧光N-CDs的合成和应用示意图[50];(B)CDs荧光探针制备及检测Cr(Ⅵ)的原理图[51]

2.2 Pyrolysis method

图4 (A)从花生壳合成荧光CDs及其应用于多色活细胞成像的示意图[54];(B)从植物叶片热解合成CDs,等离子体和微波辐射增强PL的示意图[55];(C)榴莲皮CDs的合成与应用示意图[56]

2.3 Solvothermal method

图5 CP-CDs的制备工艺及应用的示意图[63]

2.4 Microwave-assisted method

图6 活蚕蛹中提取制备的SC-CDs及其蓝光荧光的示意图[67]

表1 不同中药源及合成方法及条件制得的CDs的尺寸、荧光性质和荧光QY比较

3 Application of traditional Chinese medicine carbon dots in biological imaging

3.1 In vitro imaging

3.1.1 Cell imaging

图7 用FCP-B、FCP-G和FCP-Y处理的A549细胞在4 h内观察的共聚焦显微镜图像[79]

图8 Hoechst/PI双染色法对细胞凋亡和坏死的高含量成像分析[89]

3.1.2 Bacterial imaging

图9 在蓝、黄、红三色区(A)E. Coli和(B)B.Subtilis存在下的细菌生物成像[92]

图10 基于Mis-mPD-CDs的细胞内CR成像与传感:A549、C. albicans、E. coli和S. aureus的CLSM图像,用不同浓度的CR处理,然后与Mis-mPD-CDs孵育30 min[93]

3.2 In vivo imaging

3.2.1 Mouse imaging

图11 杧果CDs的小鼠体内成像图。尾静脉注射5 mg·kg-1体重后FCP-B、FCP-G和FCP-Y在BALB/c裸鼠体内的生物分布。FCP-B(a,b)、FCP-G(c,d)和FCP-Y(e,f)的体内分布及相应强度[79]

图12 CDs的单光子和双光子生物成像。(A)在不同时间点静脉注射CDs的仰卧裸鼠的体内成像(Ⅰ:胸部区域,Ⅱ:肝脏区域,Ⅲ:小肠区域,Ⅳ:大肠区域,Ⅴ:膀胱区域);(B)在不同时间点静脉注射CDs的裸鼠的实时离体成像[97]

图13 用NBCD-PEG-Ce6-Tf在不同时间点对小鼠肿瘤进行实时近红外荧光成像[88]

图14 BALB/c小鼠不同时间间隔注射Pn N-CDs后的体内成像[98]

3.2.2 Zebrafish imaging

图15 CDs在斑马鱼体内的光致发光衰减。0.4 mg/mL HCDs和SCDs溶液在不同时间点暴露2 d后斑马鱼胚胎的荧光图像[100]

表2 中药CDs在生物成像中的应用

4 Application of traditional Chinese medicine carbon dot in medical treatment

4.1 Antibacterial action

图16 CDs的一般杀菌作用机制。(a)CDs和细菌细胞壁之间最初静电相互作用。(b)CDs内化,在细菌膜中插入,以及细胞质物质泄漏的不可逆破坏。(c)CDs促进细菌光动力灭活,产生ROS和DNA损伤[105]

图17 大肠杆菌的SEM图像:(A~C)光照射不含MCDs,(D~F)MCDs在黑暗条件下处理,以及(G~I)MCDs在可见光照射下处理12 h[106]

图18 (a)洋葱CDs对细菌细胞壁和细胞膜完整性影响的示意图;洋葱CDs对脆弱假单胞菌胞外(b)AKP和(c)ATP释放的影响[94]

4.2 Anti-inflammatory effect

图19 用于清除ROS和抗炎治疗的CDs示意图[108]

图20 ASAC-CDs改善LPS诱导的大鼠急性肺损伤(ALI)的宏观图像:(A)生理盐水组;(B)示范组;(C)阳性对照组;(D)大剂量ASAC-CDs组;(E)中剂量ASAC-CDs组;低剂量ASAC-CDs组[112]

4.3 Hemostasis

4.4 Antioxidation

图21 CDs减轻盐胁迫下莴苣氧化损伤的机理示意图[44]

4.5 Anticancer effect

4.6 Other therapeutic effects

图22 J-CDs的治疗贫血功能示意图[132]

5 Conclusion and prospect

References

图1 CDs的分类和相应结构的示意图^[2]

图2 N-CDs的合成路线及其在Ag⁺传感和RGB颜色分析中的应用^[28]

图3 (A)荧光N-CDs的合成和应用示意图^[50];(B)CDs荧光探针制备及检测Cr(Ⅵ)的原理图^[51]

图4 (A)从花生壳合成荧光CDs及其应用于多色活细胞成像的示意图^[54];(B)从植物叶片热解合成CDs,等离子体和微波辐射增强PL的示意图^[55];(C)榴莲皮CDs的合成与应用示意图^[56]

图5 CP-CDs的制备工艺及应用的示意图^[63]

图6 活蚕蛹中提取制备的SC-CDs及其蓝光荧光的示意图^[67]

图7 用FCP-B、FCP-G和FCP-Y处理的A549细胞在4 h内观察的共聚焦显微镜图像^[79]

图8 Hoechst/PI双染色法对细胞凋亡和坏死的高含量成像分析^[89]

**图9 在蓝、黄、红三色区(A)E. Coli和(B)B.Subtilis存在下的细菌生物成像^[92]**

**图10 基于Mis-mPD-CDs的细胞内CR成像与传感:A549、C. albicans、E. coli和S. aureus的CLSM图像,用不同浓度的CR处理,然后与Mis-mPD-CDs孵育30 min^[93]**

图11 杧果CDs的小鼠体内成像图。尾静脉注射5 mg·kg^-1体重后FCP-B、FCP-G和FCP-Y在BALB/c裸鼠体内的生物分布。FCP-B(a,b)、FCP-G(c,d)和FCP-Y(e,f)的体内分布及相应强度^[79]

图12 CDs的单光子和双光子生物成像。(A)在不同时间点静脉注射CDs的仰卧裸鼠的体内成像(Ⅰ:胸部区域,Ⅱ:肝脏区域,Ⅲ:小肠区域,Ⅳ:大肠区域,Ⅴ:膀胱区域);(B)在不同时间点静脉注射CDs的裸鼠的实时离体成像^[97]

图13 用NBCD-PEG-Ce6-Tf在不同时间点对小鼠肿瘤进行实时近红外荧光成像^[88]

图14 BALB/c小鼠不同时间间隔注射Pn N-CDs后的体内成像^[98]

图15 CDs在斑马鱼体内的光致发光衰减。0.4 mg/mL HCDs和SCDs溶液在不同时间点暴露2 d后斑马鱼胚胎的荧光图像^[100]

图16 CDs的一般杀菌作用机制。(a)CDs和细菌细胞壁之间最初静电相互作用。(b)CDs内化,在细菌膜中插入,以及细胞质物质泄漏的不可逆破坏。(c)CDs促进细菌光动力灭活,产生ROS和DNA损伤^[105]

图17 大肠杆菌的SEM图像:(A~C)光照射不含MCDs,(D~F)MCDs在黑暗条件下处理,以及(G~I)MCDs在可见光照射下处理12 h^[106]

图18 (a)洋葱CDs对细菌细胞壁和细胞膜完整性影响的示意图;洋葱CDs对脆弱假单胞菌胞外(b)AKP和(c)ATP释放的影响^[94]

图19 用于清除ROS和抗炎治疗的CDs示意图^[108]

图20 ASAC-CDs改善LPS诱导的大鼠急性肺损伤(ALI)的宏观图像:(A)生理盐水组;(B)示范组;(C)阳性对照组;(D)大剂量ASAC-CDs组;(E)中剂量ASAC-CDs组;低剂量ASAC-CDs组^[112]

图21 CDs减轻盐胁迫下莴苣氧化损伤的机理示意图^[44]

图22 J-CDs的治疗贫血功能示意图^[132]