Environment-responsive charge conversion carbon quantum dots, and preparation method and application thereof

Abstract

The application belongs to the technical field of carbon quantum dot materials, and particularly relates to an environment-responsive charge conversion carbon quantum dot as well as a preparation method and application thereof, the preparation method of the quantum dot comprises the following steps: (1) preparing quantum dots by using chlorogenic acid to obtain ChACDs; (2) modifying the ChA CDs by using an imidazole compound to obtain the environment-responsive charge conversion carbon quantum dot. The quantum dot has excellent biocompatibility and antibacterial activity, and also has a significant ability of treating skin infection and promoting wound healing.

Description

Technical Field

[0001] The present invention belongs to the technical field of carbon quantum dot materials, and specifically relates to an environmentally responsive charge-converting carbon quantum dot and a preparation method and application thereof. Background Art

[0002] Antibiotic resistance is a threat to human health. It occurs when antibiotics act on bacteria, causing mutations in certain genes within their genomes, resulting in reduced or complete loss of antibiotic sensitivity. These mutations can occur spontaneously or as a result of selective pressure from antibiotics. Antibiotic resistance caused by selective pressure from antibiotics is particularly threatening to human health (it takes 4-5 years for bacteria to develop resistance through genetic mutations, while the spread of resistance within a biofilm takes only 2-3 months). Specifically, bacteria exist in two common states: planktonic and biofilm-bound. During infection, bacteria secrete extracellular polymeric substances (EPS) to encapsulate bacterial communities, forming a dense three-dimensional structure called a biofilm. This structure serves as a protective barrier against the host immune system, environmental stressors, and antibiotics. While antibiotics effectively kill planktonic bacteria and those in the surface layer of biofilms, bacteria deeply embedded in the biofilm, due to its unique, dense structure, often evade antibiotic action and persist. If bacterial biofilms persist in patients' bodies for a long time, they will continuously release planktonic bacteria, triggering new infections and leading to chronic and persistent infections. Furthermore, mature biofilms can be hundreds to thousands of times more resistant to most antibiotics than individual planktonic bacteria, greatly increasing the difficulty of clinical treatment. Therefore, effective therapeutic strategies to combat established bacterial biofilms are of great significance.

[0003] In order to eradicate biofilms, a series of nanomaterials have been designed and their effectiveness has been confirmed in in vitro and in vivo experiments, including organic nanoparticles (such as liposomes, polymer nanoparticles, dendrimers, cyclodextrins, solid lipid nanoparticles, etc.) and inorganic nanoparticles (such as metal nanoparticles such as gold, silver, and copper, metal oxide nanoparticles such as zinc oxide and aluminum oxide, quantum dots, fullerenes, and organic-inorganic hybrid materials). Although metals such as gold and silver and metal oxide nanoparticles such as ferrous oxide, copper oxide, and zinc oxide have shown strong antibacterial activity, the release of metal ions caused by these materials in vivo can produce nonspecific biological toxicity, which greatly limits the application of nanomaterials in the antibacterial field.

[0004] Carbon quantum dots (CDs) are attracting increasing attention due to their economical and simple preparation, high water solubility, high degree of functionalization, and excellent biocompatibility. Furthermore, their small size allows them to penetrate various natural biological barriers in the body, including ion channels, the blood-brain barrier, and the glomerular filtration barrier. These characteristics of CDs are expected to overcome the limitations of nanomaterials in antimicrobial applications and demonstrate great potential in the biomedical field. Summary of the Invention

[0005] Based on this, the present invention provides an environmentally responsive charge-conversion carbon quantum dot, which is prepared by chlorogenic acid and then modified with an imidazole compound; it can selectively protonate the imidazole group in an acidic environment to achieve charge conversion, can quickly damage and penetrate the biofilm and attach to the bacteria colonized in the biofilm, thereby achieving the purpose of resisting drug-resistant bacteria.

[0006] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0007] On the one hand, the present invention provides a method for preparing environmentally responsive charge-converting carbon quantum dots, comprising: (1) preparing chlorogenic acid into quantum dots to obtain ChA CDs; and (2) modifying ChA CDs with an imidazole compound to obtain environmentally responsive charge-converting carbon quantum dots.

[0008] Preferably, the above-mentioned imidazole compound is n is an integer and 1≤n≤5.

[0009] More preferably, the imidazole compound is 1-(3-aminopropyl)imidazole.

[0010] Preferably, the mass / volume ratio of ChACDs and imidazole compounds is (3-6) mg / mL.

[0011] Preferably, the above preparation method satisfies one or more combinations of the following conditions: (a) in step (1), chlorogenic acid is prepared to obtain ChACDs by hydrothermal synthesis; (b) in step (2), the imidazole compound is modified to ChACDs by EDC / NHS method.

[0012] More preferably, in the above preparation method, step (1) comprises: placing the ChA aqueous solution in a high-pressure reactor, and then heating it at 220°C-240°C to obtain a ChACDs aqueous solution; step (2) comprises: (i) mixing EDC, NHS and 2-morpholineethanesulfonic acid to obtain a reactant; (ii) mixing the reactant with an imidazole compound, adjusting the pH to 7-9, and reacting to obtain environmentally responsive charge-conversion carbon quantum dots.

[0013] Another aspect of the present application provides an environmentally responsive charge conversion carbon quantum dot prepared by the preparation method of the present application.

[0014] Still another aspect of the present application provides any one of the following:

[0015] (i) a composition comprising the environmentally responsive charge conversion carbon quantum dot of the present application; the composition is used for antibacterial, inhibiting the growth of drug-resistant bacteria, treating skin pyogenic infection or promoting wound healing;

[0016] (ii) a preparation comprising the environmentally responsive charge conversion carbon quantum dot of the present application or the composition of (i); and a carrier.

[0017] Still another aspect of the present application provides the use of the environmentally responsive charge conversion carbon quantum dot of the present application in antibacterial.

[0018] Preferably, the above use comprises the use of the environmentally responsive charge conversion carbon quantum dot of the present application in antibiotic-resistant bacteria.

[0019] More preferably, the antibiotic-resistant bacteria comprises one or more of methicillin-resistant Staphylococcus aureus, penicillin-resistant Enterococcus, vancomycin-resistant Enterococcus, carbapenem-resistant Enterobacter, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa or pan-resistant Acinetobacter.

[0020] Preferably, the above use comprises the use of the environmentally responsive charge conversion carbon quantum dot of the present application in the preparation of a composition or a preparation for destroying the bacterial membrane system, reducing the metabolic level of bacteria or promoting the accumulation of bacterial ROS.

[0021] Preferably, the above use comprises the use of the environmentally responsive charge conversion carbon quantum dot of the present application in the preparation of a pharmaceutical composition or a pharmaceutical preparation for treating skin pyogenic infection and / or promoting wound healing.

[0022] The present application has at least the following advantages:

[0023] (1) The environmentally responsive charge conversion carbon quantum dot provided by the present application has excellent biocompatibility, no obvious toxic side effects are shown by toxicity test, and has significant antibacterial activity.

[0024] (2) The environmentally responsive charge conversion carbon quantum dot provided by the present application has excellent antibacterial activity against antibiotic-resistant bacteria, such as MRSA (methicillin-resistant Staphylococcus aureus) and PAE (penicillin-resistant Enterococcus), and the inhibition rate can reach 99%.

[0025] (3) The environment-responsive charge conversion carbon quantum dots provided by the present application have significant abilities of treating skin infection and promoting wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Physical and chemical characterization of ChACDs and CDs-API; wherein, a is the TEM image and DLS particle size distribution of ChACDs; b is the TEM image and DLS particle size distribution of CDs-API (inset: high-resolution TEM image); c is the fluorescence emission spectra of ChACDs and CDs-API at 400-600 nm under 370 nm excitation light (inset: the color of aqueous solution of ChACDs (left) and CDs-API (right) under sunlight and ultraviolet light);

[0027] Figure 2 Structural characterization of ChACDs and CDs-API; wherein, a is the full element scanning XPS spectrum of ChACDs; b is the C element scanning XPS spectrum of ChACDs; c is the full element scanning XPS spectrum of CDs-API; d is the Fourier transform infrared (FTIR) spectrum of ChACDs and CDs-API;

[0028] Figure 3 Zeta potential of ChACDs and CDs-API under different pH environments;

[0029] Figure 4 Glutathione-like enzyme activity verification of CDs-API; wherein, a is the schematic diagram of the principle of DTNB colorimetric method for detecting GSH; b is the concentration-dependent and time-dependent catalytic activity;

[0030] Figure 5 In vitro antibacterial performance evaluation of CDs-API; wherein, a is the plate colony map after different treatments; b is the growth condition statistics of bacteria in liquid medium after MRSA treatment; c is the growth condition statistics of bacteria in liquid medium after PAE treatment; (**p<0.01, ***p<0.001);

[0031] Figure 6 MIC evaluation of CDs-API: the growth conditions of MRSA and PAE in agar plates and liquid medium under different concentrations of CDs-API treatment; wherein, a is the growth conditions of MRSA and PAE in agar plates under different concentrations of CDs-API treatment; b is the growth conditions of MRSA in liquid medium under different concentrations of CDs-API treatment; c is the growth conditions of PAE in liquid medium under different concentrations of CDs-API treatment; (*p<0.05, **p<0.01, ***p<0.001);

[0032] Figure 7 The growth of Escherichia coli, Klebsiella pneumoniae, and extended-spectrum β-lactamase-producing Klebsiella pneumoniae on LB agar plates under different treatments;

[0033] Figure 8 The ability of CDs-API to destroy mature biofilms of MRSA (a) and PAE (b) was evaluated by crystal violet method (**p<0.01);

[0034] Figure 9 SEM images of MRSA and PAE after treatment with CDs-API and control PBS (scale bar: 1 μm);

[0035] Figure 10 is the PI fluorescence value of MRSA and PAE after treatment with CDs-API and control PBS;

[0036] Figure 11 is the diSC3(5) fluorescence value of MRSA and PAE after treatment with CDs-API and control PBS;

[0037] Figure 12 ATP levels of MRSA and PAE bacterial suspension after different treatments (***p<0.001);

[0038] Figure 13 is the absorbance of ABDA at 378 nm after MRSA and PAE were treated with ChACDs and CDs-API at different times;

[0039] Figure 14 MTT toxicity evaluation of HUVEC after treatment with a series of concentrations of CDs-API;

[0040] Figure 15 The weight changes of mice during treatment; a is the MRSA infection group, b is the PAE infection group;

[0041] Figure 16 H&E staining of the main organs (heart, liver, spleen, lung, and kidney) of mice in each group; a is the MRSA infection group; b is the PAE infection group; (Scale bar: 100 μm);

[0042] Figure 17 This is the timeline of the mouse skin suppurative infection experiment;

[0043] Figure 18 The in vivo antibacterial properties of CDs-API treated with MRSA; a shows the changes in the infection site of mice during treatment; b shows the bacterial growth in the infection site of mice in different treatment groups after treatment; c shows H&E staining of the skin at the infection site after treatment in different treatment groups; (Scale bar: 100 μm);

[0044] Figure 19 The in vivo antibacterial properties of PAE-treated CDs-API; a is the changes in the infection site of mice during treatment; b is the bacterial growth in the infection site of mice after treatment in different treatment groups; c is the H&E staining of the skin at the infection site after treatment in different treatment groups; (Scale bar: 100 μm). DETAILED DESCRIPTION

[0045] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0046] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context clearly has a different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0047] An embodiment of the present invention provides a method for preparing environmentally responsive charge-conversion carbon quantum dots, comprising: (1) preparing chlorogenic acid (ChA) into quantum dots to obtain ChACDs; and (2) modifying ChACDs with an imidazole compound to obtain environmentally responsive charge-conversion carbon quantum dots (abbreviated as CDs-API, the same below).

[0048] It should be noted that the present invention achieves acid-responsive charge conversion by modifying the surface of ChACDs with imidazole groups and selectively protonating the imidazole groups in low pH environments. This means that the quantum dots can selectively protonate the imidazole groups in acidic environments to achieve charge conversion, rapidly penetrating biofilms and attaching to bacteria colonized within them. The enzyme-like activity of the CDs-API can effectively consume the high levels of reduced glutathione in biofilms, inducing the accumulation of reactive oxygen species (ROS), degrading the extracellular polymer matrix, disrupting bacterial DNA and protein structure, and disrupting the bacterial redox balance to kill the bacteria.

[0049] It should also be noted that chlorogenic acid (ChA), also known as coffee tannin or coffee tannic acid, is a naturally occurring polyphenolic compound primarily formed by the ester condensation of caffeic acid and quinic acid. Found in high concentrations in honeysuckle, eucommia bark, and coffee beans, it is known as "plant gold" and exhibits multiple biological activities, including antiviral, antibacterial, and free radical scavenging. ChACDs are highly soluble in water and possess significant glutathionease-like activity. By consuming GSH, they disrupt the cellular redox balance, inducing oxidative stress and cell death. This property opens up the possibility of using CDs in the antibacterial field. However, these low-toxic CDs maintain a negative charge in aqueous solution. Compared to positively charged nanomaterials, while this avoids damage to normal tissue cells to a certain extent and has higher biocompatibility, their antibacterial effect is also somewhat suppressed. Therefore, the present invention further modifies the surface of ChACDs with imidazole compounds to produce environmentally responsive charge-conversion carbon quantum dots, enhancing their antibacterial effect.

[0050] It should also be noted that the environmentally responsive charge-converting carbon quantum dots (CDs-API) in the present invention maintain a negative charge under normal physiological conditions (pH = 5.5), reducing nonspecific effects on normal cells and tissues, and accumulating at positively charged infection sites through electrostatic attraction; after contacting the acidic environment of the biofilm, the surface functional group imidazole group is protonated, quickly reversing the surface charge of the CDs-API to positive, and the CDs-API gradually adsorbs on the negatively charged bacteria to exert the material's enzyme-like activity and antibacterial activity to kill bacteria with a fixed value in the biofilm and destroy the biofilm. After verification by the present invention, it was found that CDs-API has excellent bactericidal activity, can effectively inhibit the growth of drug-resistant bacteria, and also has a certain destructive effect on mature biofilms.

[0051] In some specific examples, the above imidazole compound can be n is an integer and 1≤n≤5.

[0052] It should be noted that the imidazole compound in the present invention can be preferably The compound shown, wherein n can be 1, 2, 3, 4 or 5; wherein, n is more preferably 3, that is, the imidazole compound is 1-(3-aminopropyl)imidazole, and the environmentally responsive charge-conversion carbon quantum dots prepared by the imidazole compound have the best performance.

[0053] In some specific examples, the mass / volume of ChACDs and imidazole compound is (3-6) mg / mL.

[0054] It should be noted that the imidazole compound (1-(3-aminopropyl)imidazole) is in liquid form at room temperature, so "mass / volume" refers to the ratio of the mass of ChACDs to the volume of the imidazole compound; the ratio is (3-6) mg / mL, such as 3 mg / mL, 4 mg / mL or 5 mg / mL; in addition, it should be understood that "mg / mL" can be uniformly converted according to international standard units and does not specifically include only "mg / mL" here.

[0055] In some specific examples, the above preparation method satisfies one or more combinations of the following conditions:

[0056] (a) In step (1), chlorogenic acid (ChA) is prepared by a hydrothermal synthesis method to obtain ChACDs; specifically, the present invention uses ChA as a precursor substance and can synthesize ChACDs by a traditional "top-down" method - a hydrothermal method; it should be understood that the hydrothermal method in the present invention is carried out in a manner known in the art;

[0057] (b) In step (2), the imidazole compound is modified to ChA CDs by the EDC / NHS method. Specifically, in the present invention, the imidazole compound can be modified to ChA CDs by various methods, such as the EDC / NHS method. The EDC / NHS method is performed in a manner known in the art.

[0058] In some specific examples, in the above preparation method,

[0059] Step (1) comprises: placing a ChA aqueous solution in a high-pressure reactor, and then heating it at 220° C.-240° C. to obtain a ChACDs aqueous solution; specifically, the method of synthesizing ChACDs by the hydrothermal method in the present invention can be to heat the high-pressure reactor in an oven, and the oven temperature can be 220° C.-240° C., such as 225° C., 230° C., or 235° C.;

[0060] Step (2) comprises: (i) mixing EDC, NHS and 2-morpholineethanesulfonic acid to obtain a reactant; (ii) mixing the reactant with an imidazole compound, adjusting the pH to 7-9, and reacting to obtain environmentally responsive charge conversion carbon quantum dots.

[0061] An embodiment of the present invention further provides an environmentally responsive charge-converting carbon quantum dot prepared by the preparation method of the present invention.

[0062] The present invention also provides any one of the following substances:

[0063] (i) A composition comprising the environmentally responsive charge-converting carbon quantum dots of the present invention; the composition is used for antibacterial purposes, inhibiting the growth of drug-resistant bacteria, treating suppurative skin infections, or promoting wound healing;

[0064] (ii) a formulation comprising the environmentally responsive charge-conversion carbon quantum dots of the present invention or the composition of (i); and a carrier.

[0065] It should be noted that the above-mentioned composition refers to the environmentally responsive charge-conversion carbon quantum dots in the present invention prepared in combination with other effective active compounds; for example, the antibacterial composition may be composed of environmentally responsive charge-conversion carbon quantum dots and one or more compounds with antibacterial activity (such as antibiotics); for example, the composition for inhibiting the growth of drug-resistant bacteria may be composed of environmentally responsive charge-conversion carbon quantum dots and a compound with the activity of inhibiting the growth of drug-resistant bacteria; similarly, the composition of drugs for treating suppurative skin infections or the composition of drugs for promoting wound healing is composed of environmentally responsive charge-conversion carbon quantum dots and a drug composition that can treat suppurative skin infections or a compound that promotes wound healing; in addition, it should be understood that the purpose of the composition of the compound can be to enhance the therapeutic effect or to combine different therapeutic effects, and the specific selection can be based on clinical practice.

[0066] It should also be noted that the preparation can be the environmentally responsive charge-conversion carbon quantum dots in the present invention or the above-mentioned composition with the addition of a carrier to prepare different preparations, such as sprays, injections, powders, tablets, capsules, ointments or patches, etc.; of course, it should be understood that the choice of carrier can be based on different dosage forms according to the types known in the art; in addition, in specific products, antibacterial preparations can be in the form of sprays, ointments or patches, etc.; pharmaceutical preparations for inhibiting the growth of antibiotic-resistant bacteria can be tablets, powders or capsules, etc.; pharmaceutical preparations for treating skin purulent infections or promoting wound healing can be powders, capsules, injections or patches, etc.

[0067] An embodiment of the present invention further provides an application of the environment-responsive charge-conversion carbon quantum dots in the present invention in antibacterial applications.

[0068] In some specific examples, the above applications include: application of the environmentally responsive charge-converting carbon quantum dots of the present invention in antibiotic-resistant bacteria.

[0069] In some specific examples, the antibiotic-resistant bacteria include one or more of methicillin-resistant Staphylococcus aureus, penicillin-resistant Enterococci, vancomycin-resistant Enterococci, carbapenem-resistant Enterobacteriaceae, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, or pan-resistant Acinetobacter.

[0070] It should be noted that the antibiotic-resistant bacteria in the present invention are well known to the present invention, such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococci, carbapenem-resistant Enterobacteriaceae, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa or pan-resistant Acinetobacter (usually resistant to antibiotics such as aminoglycosides, cephalosporins, penicillins, tetracyclines, sulfonamides, etc.).

[0071] In some specific examples, the above applications include: the use of the environmentally responsive charge-conversion carbon quantum dots of the present invention in the preparation of compositions or preparations, which are used to destroy bacterial membrane systems, reduce bacterial metabolic levels, or promote bacterial ROS accumulation.

[0072] It should be noted that when evaluating the antibacterial properties of the materials in vitro, it was observed that CDs-API showed a more significant antibacterial effect than ChACDs; however, the two materials showed similar ROS generation capabilities. The antibacterial mechanism of CDs-API may include the following aspects: (1) Cell membrane damage: CDs-API can damage the bacterial cell membrane, causing changes in membrane permeability and membrane potential; (2) Decreased metabolic level: CDs-API reduces the metabolic level of bacteria, which may be an important factor leading to bacterial death; (3) GSH consumption and ROS accumulation: CDs-API can exert GSH-like enzyme activity, consume reduced GSH in bacteria, lead to ROS accumulation, and then damage the bacterial DNA and protein structure in the extracellular matrix.

[0073] In some specific examples, the above applications include: use of the environmentally responsive charge-conversion carbon quantum dots of the present invention in the preparation of pharmaceutical compositions or pharmaceutical preparations, which are used to treat suppurative skin infections and / or promote wound healing.

[0074] It should be noted that, in the present invention, animal experiments have shown that CDs-API can effectively treat suppurative skin infections in mice and has a certain promoting effect on wound healing; CDs-API has low cytotoxicity and good biocompatibility in mice.

[0075] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0076] 1. Preparation of Quantum Dot CDs-API

[0077] Example 1

[0078] (1) Take 150 mg of ChA (chlorogenic acid) into a 50 mL centrifuge tube, add 30 mL of ultrapure water, dissolve thoroughly, and then transfer into a 50 mL PTFE-lined high-pressure reaction kettle. Preheat the high-temperature oven to 230°C, and when the temperature is stable, place the high-pressure reaction kettle containing the ChA aqueous solution into the oven, and heat for 2 h. After the reaction kettle is naturally cooled to room temperature, remove the liner, filter the solution through a microporous filter membrane (0.02 μm) to remove impurities, and then place the obtained brown transparent solution into a dialysis bag (1 kDa) for dialysis purification with ultrapure water for 8 h to obtain a ChACDs aqueous solution (which can be freeze-dried according to the needs of the experiment).

[0079] (2) Take 50 mg of EDC (1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride) and 25 mg of NHS (N-hydroxysuccinimide) into a 10 mL centrifuge tube, add 1 mL of MES (2-morpholinoethanesulfonic acid) (pH = 5.5) to dissolve thoroughly. In a dark environment, take 2 mL of the above-synthesized ChACDs aqueous solution into the centrifuge tube, and incubate at 37°C for 1 h. Then immediately add 1 mL of API (1-(3-aminopropyl)imidazole), and adjust the pH of the solution to 8 with PBS. The reaction solution is incubated at 4°C overnight. After the reaction, the solution is transferred into a dialysis bag (molecular weight cut-off 3 kDa), and then dialyzed with ultrapure water for 24 h (in this process, the ultrapure water is replaced every 2 h to ensure complete removal of small molecular impurities). After dialysis, the light brown yellow solution is taken out from the dialysis bag, and freeze-dried to obtain a stable solid light yellow powder, which is the product CDs-API-1 (environment-responsive charge conversion carbon quantum dots), and stored at 4°C in the dark for future use.

[0080] Example 2

[0081] The difference between Example 2 and Example 1 is that the imidazole compound is different. The imidazole compound in Example 2 is 1-(3-aminoethyl)imidazole. The other steps are the same as in Example 1, and quantum dots CDs-API-2 are prepared.

[0082] Example 3

[0083] The difference between Example 3 and Example 1 is that the imidazole compound is different. The imidazole compound in Example 3 is 1-(3-aminobutyl)imidazole. The other steps are the same as in Example 1, and quantum dots CDs-API-3 are prepared.

[0084] Comparative Example 1

[0085] The difference between Comparative Example 1 and Example 1 is that the carbon dot precursor is different. The carbon dot precursor used in Comparative Example 1 is citric acid urea. The other steps are the same as in Example 1, and quantum dots CDs-API-4 are prepared.

[0086] 2. Quantum Dot CDs-API Characterization

[0087] In the following experiments, CDs-API refers only to the quantum dots in Example 1, and does not include the quantum dots in Example 2 and Example 3. The quantum dots in Example 2 are only CDs-API-2, the quantum dots in Example 3 are only CDs-API-3, and the quantum dots in Comparative Example 1 are only CDs-API-4.

[0088] (1) Transmission electron microscopy test

[0089] The shape and size of the materials were observed by transmission electron microscopy (TEM). The results showed that ChACDs and CDs-API were monodispersed, spherical with similar diameters and uniform size. High-resolution TEM images ( Figure 1 b (upper right) shows that the lattice distance of CDs-API is 0.22 nm, and the crystal structure is consistent with graphene, revealing the typical graphite-like structure formed during the synthesis process.

[0090] (2) Dynamic light scattering test

[0091] The particle size of the nanomaterials was analyzed by dynamic light scattering (DLS), and the average size of ChACDs was 2.35 nm, and the average size of CDs-API was 2.38 nm ( Figure 1 (a inset, 1b inset). After modification of API, the shape and size of CDs did not change significantly.

[0092] (3) Fluorescence test

[0093] In addition, the synthesized ChACDs and CDs-API can emit dazzling blue fluorescence under 365nm excitation light, and after modification with API, the fluorescence of ChACDs solution is significantly enhanced ( Figure 1 c). This suggests that the presence of surface functional groups may affect the fluorescence properties of carbon quantum dots. Different functional groups can alter the surface properties of carbon quantum dots, thereby affecting their fluorescence quantum yield. Specific functional groups can also act as electron donors or acceptors, participating in the energy transfer process and thus regulating the photoluminescence behavior of carbon quantum dots. By introducing specific functional groups into the surface of carbon quantum dots, not only can the solubility of carbon quantum dots be altered, but also their fluorescence properties can be modulated. This may be of great significance for the development of fluorescent materials and biomarkers based on carbon quantum dots.

[0094] (4) X-ray test

[0095] The elemental composition, functional group composition and surface structure of ChA CDs and CDs-API were further characterized. The elemental composition of ChACDs and CDs-API was analyzed by X-ray photoelectron spectroscopy (XPS). The results showed that ChACDs were mainly composed of C and O elements ( Figure 2 a). In the high-resolution XPS spectrum of the carbon 1s orbital, three characteristic peaks were observed. These peaks correspond to different carbon atomic environments, suggesting the presence of different functional groups on the surface of carbon quantum dots, located at 284.8, 286, and 289 eV, respectively ( Figure 2 b), carbon-carbon bonds, carbon-oxygen bonds, and carbon-oxygen double bonds, respectively. This indicates that the surface functional groups of ChACDs are mainly composed of carbon atoms and abundant hydroxyl, carbonyl, and carboxyl groups.

[0096] (5) XPS test

[0097] Next, the charge conversion group API was modified onto the surface of ChACDs by NHS / EDC method, and XPS confirmed the successful introduction of N element ( Figure 2 c); In order to analyze the functional group composition of ChACDs and CDs-API and study the changes during the CDs-API reaction, the Fourier transform infrared spectroscopy (FTIR) of ChACDs and CDs-API was further measured ( Figure 2 d) Similar characteristic absorption peaks appeared in the two groups of samples. The presence of these characteristic peaks indicates that ChACDs and CDs-API have similar functional group compositions, such as stretching vibrations of carbon-hydrogen bonds and carbon-oxygen bonds. In the absorption spectrum of CDs-API, new characteristic absorption peaks can be observed, such as 1545 cm -1 The nitrogen-hydrogen bond at 1448 cm -1 The carbon-nitrogen bonds at the two positions are both amide bonds, which proves that the API is connected to the ChACDs surface through amide bonds, that is, the charge conversion group is successfully modified onto the ChACDs surface.

[0098] (VI) CDs-API acid response charge conversion ability test

[0099] The Zeta potential of ChACDs and CDs-API in a neutral environment (pH = 5.5) and an acidic environment (pH = 7.5) was measured and analyzed by dynamic light scattering (DLS); specifically, 1 mg / mL ChACDs and CDs-API PBS solutions were prepared, and the Zeta potential of the nanomaterials was measured at pH = 5.5, 25°C and pH = 7.5, 25°C, respectively.

[0100] The results are as follows Figure 3As shown in the figure, under a neutral pH of 7.5, both ChACDs and CDs-API are negatively charged, which to some extent avoids damage to healthy cells and tissues and enhances the biosafety of the material. In the acidic environment of bacterial biofilms (pH ≤ 5.5), the negative charge of ChACDs decreases. This may be attributed to its abundant surface groups: when the pH decreases, the H+ concentration in the solution increases and binds to the carboxyl groups on the surface of ChACDs, causing the carboxyl groups to change from -COO- in a neutral environment to -COOH, while the hydroxyl groups remain in the form of -OH. At this time, neither the carboxyl groups (-COOH) nor the hydroxyl groups (-OH) carry a negative charge. Therefore, as the pH decreases, the negative charge on the surface of ChACDs also decreases to a certain extent. CDs-API, however, rapidly reverses its charge to a positive charge (pKa < 6.8) due to the protonation of the imidazole groups on its surface. This charge reversal not only facilitates the aggregation of the material around bacteria in the biofilm but also gives the positively charged ChACDs an enhanced bactericidal effect.

[0101] (VII) CDs-API glutathionease activity test

[0102] The glutathione peroxidase catalytic activity of CDs-API was detected using a glutathione peroxidase detection kit (DTNB method). The new substance TNB generated by the reaction of DTNB and reduced GSH has a characteristic absorption peak at 412 nm, and its absorbance is proportional to the GSH content (see Figure 4 a); specifically, the CDs-API was first diluted to 1 mg / mL with prepared PBS, DTNB solution (100 μM) and GSH solutions of different concentrations (1 mM, 2 mM, 3 mM, 4 mM) were added to the diluted solution, and the mixture was incubated at room temperature. The absorbance of the solution at 412 nm was detected using a microplate reader at different incubation times (0 min, 10 min, 20 min, 30 min, 40 min).

[0103] The results are as follows Figure 4 As shown in b, time-dependent and GSH concentration-dependent absorbance changes were observed at 412 nm ( Figure 4 b), indicating that GSH is effectively scavenged by the CDs-API. Reduced GSH plays an important role in maintaining bacterial redox balance. GSH depletion can promote ROS accumulation in the biofilm environment. This type of GSH enzyme activity provides the material with effective bactericidal potential.

[0104] 3. In vitro antibacterial test of quantum dot CDs-API

[0105] In the following experiments, CDs-API refers only to the quantum dots in Example 1, excluding the quantum dots in Example 2 and Example 3. The quantum dots in Example 2 are only CDs-API-2, and the quantum dots in Example 3 are only CDs-API-2.

[0106] 5 μL of frozen glycerol culture of MRSA (methicillin-resistant Staphylococcus aureus) and PAE (penicillin-resistant enterococci) was inoculated into a screw-cap tube containing 5 mL of LB liquid medium. The inoculated medium was placed in a constant temperature shaker and cultured at 37°C and 200 rpm. During the bacterial growth process, the absorbance value (OD) of the bacterial solution at a wavelength of 600 nm was regularly measured using a biological spectrophotometer. 600 When the absorbance reaches 0.5, the amount of bacteria is about 10 8 CFU / mL, at this time the bacteria are in the logarithmic growth phase.

[0107] (1) In vitro antibacterial activity test

[0108] Take appropriate amount of MRSA and PAE strains and inoculate them into a screw cap tube containing LB liquid medium, place it in a constant temperature shaker, and culture it at 37°C, 220 rpm for 3 hours, at which time the bacteria are in the logarithmic growth phase. Dilute the bacterial solution with PBS (0.01M, pH 7.4) to a bacterial volume of about 10 6 CFU / mL; ChACDs, CDs-API and CDs-API-4 were prepared into 1 mg / mL solutions with PBS; 100 μL of PBS, ChACDs, CDs-API and CDs-API-4 solutions were placed in a 96-well plate respectively, 100 μL of the above bacterial suspension was added to each well and mixed, and incubated at room temperature for 20 minutes; after 20 minutes, 100 μL of the mixed sample was inoculated on the LB agar plate, and the sample was evenly spread on the surface of the agar plate using a spreader to ensure that the bacteria were evenly distributed and allowed to stand for 10 minutes; the plate was placed upside down and incubated in a constant temperature incubator at 37°C for 18 hours; after 18 hours, the plate was taken out and photographed to record the bacterial growth on each agar plate and the number of colonies was counted to evaluate the effect of the antibacterial agent. In addition, 5 μL of the mixed system was inoculated into 5 mL of LB liquid medium in each well and cultured overnight on a constant temperature shaker at 37°C and 200 rpm. The optical density (OD) of the bacterial solution at 600 nm was then measured using a spectrophotometer. 600 ).

[0109] The results showed that both ChACDs and CDs-API had significant antibacterial effects against MRSA and PAE, and at the same concentration, the antibacterial effect of CDs-API was superior ( Figure 5a), while the blank control results of CDs-API-4 and PBS were similar, with colonies growing all over the plates, indicating that there was no antibacterial effect; at the same time, the OD of LB liquid culture medium under different treatments was measured using a spectrophotometer. 600 , the bacterial growth was evaluated by statistical analysis of the changes in absorbance values, and ChACDs and CDs-API obtained the same results as the plate experiment ( Figure 5 b and Figure 5 c); Similarly, CDs-API-4 and PBS blank controls showed dark purple and had higher absorbance, which was consistent with the results of the plate experiment.

[0110] (2) Minimum inhibitory concentration determination

[0111] MRSA and PAE strains were cultured in LB liquid medium and grown in a 37°C constant temperature shaker to the logarithmic phase. The bacterial solution was diluted with PBS (0.01M, pH 7.4) to a bacterial volume of about 10 6 CFU / mL.

[0112] ChACDs and CDs-API were prepared with PBS to different concentrations (final concentrations were 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1 mg / mL, respectively). 100 μL of PBS, ChACDs, and CDs-API solutions were placed in a 96-well plate, and 100 μL of the above bacterial suspension was added to each well, mixed, and incubated at room temperature for 20 minutes. After 20 minutes, the same operation as the in vitro antibacterial activity experiment was adopted to observe the growth of each group of bacteria in LB liquid culture medium and LB agar plates.

[0113] like Figure 6 The results showed that as the concentration of CDs-API increased, the number of MRSA and PAE colonies decreased and the growth inhibition became more pronounced, indicating that the antibacterial activity of CDs-API is concentration-dependent. At a concentration of 1 mg / mL, CDs-API achieved an inhibition rate of 99% against both MRSA and PAE (Table 1).

[0114] Table 1 Inhibitory rate of different concentrations of CDs-API against MRSA and PAE

[0115]

[0116] In addition, the antibacterial rates of CDs-API-2 and CDs-API-3 at a concentration of 1 mg / mL were tested according to the above method, and the results are shown in Table 2 below.

[0117] Table 2 Inhibitory rate of CDs-API-2 and CDs-API-3 against MRSA and PAE

[0118]

[0119] (3) Broad-spectrum antibacterial activity test

[0120] Three strains, Escherichia coli (E. coli), Klebsiella pneumoniae (K. pneumoniae), and ESBLs-producing K. pneumoniae, were selected as models for evaluating the in vitro broad-spectrum antibacterial ability of CDs-API. Monoclonal colonies were picked from agar plates and inoculated into screw-capped tubes containing LB liquid culture medium. The inoculated screw-capped tubes were placed in a constant temperature shaker and incubated at 37°C and 200 rpm for 3 hours until the bacteria grew to the logarithmic phase (OD 600 =0.5); 100 μL of PBS (0.01 M, pH 7.4), ChACDs, and CDs-API (final concentration 1 mg / mL) were each added to a 96-well plate. 100 μL of the pre-prepared bacterial solution was added to each well containing the above solutions. The 96-well plate was gently shaken to ensure uniform mixing. Incubate at room temperature for 20 minutes to allow the materials to fully function. 10 μL of the mixture was aspirated from each well and evenly spread onto an LB agar plate. The plate was inverted and incubated overnight in a 37°C incubator. The next day, the plate was removed and the resulting colonies were photographed.

[0121] The results are as follows Figure 7 As shown in the results, both ChACDs and CDs-API had inhibitory effects on the growth of these bacteria, but the antibacterial ability of ChACDs was weak and unstable at the same concentration, while CDs-API had a better and more stable broad-spectrum antibacterial ability.

[0122] (IV) Destructive ability of CDs-API on mature biofilms

[0123] Crystal violet (CV) dye was used to detect the degree of destruction of bacterial biofilm. First, a sterile 96-well plate was prepared to collect MRSA and PAE bacterial cultures in the logarithmic growth phase, ensuring that the bacterial count was approximately 10 8CFU / mL; then, 200 μL of each of the above two bacterial solutions was taken and added to the corresponding wells of a pre-prepared sterile 96-well plate; the inoculated 96-well plate was placed in a 37°C constant temperature incubator for 36 hours to obtain a mature biofilm; then the bacterial suspension in the well was carefully aspirated, and the remaining light yellow paste (PAE is yellow-green) was the bacterial biofilm. The remaining suspended bacteria and culture medium were gently rinsed with PBS; then PBS, ChA CDs, and CDs-API solution were added to the biofilm (the final concentration of ChA CDs and CDs-API was 1 mg / mL), gently shaken to mix the liquid, and placed in a 37°C constant temperature incubator for 3 hours; after taking out, the supernatant was gently aspirated, and 200 μL of 0.1% CV dye was added to each well at room temperature and incubated in a dark environment for 15 minutes to ensure that the biofilm was fully stained. Then, it was washed three times with PBS to completely remove unbound CV dye. Next, 200 μL of 95% ethanol was added to each well to dissolve and elute the CV dye immobilized on the biofilm. Finally, the absorbance of the eluate from each well was measured at 590 nm using a multifunctional microplate reader to assess the biofilm content in each group.

[0124] The results are as follows Figure 8 As shown in the results, compared with the PBS control group, ChACDs only had a certain destructive effect on mature biofilms, while the CDs-API treatment group had a more excellent destructive effect on the mature biofilms of MRSA and PAE.

[0125] (5) Bacterial morphology observation

[0126] The surface morphology of bacteria under different treatments was observed using scanning electron microscopy (SEM). PAE and MRSA (10 8 CFU / mL) and incubated with PBS (0.01 M, pH 7.4), ChACDs (1 mg / mL), or CDs-API (1 mg / mL) for 20 minutes. The bacterial pellet was then obtained by low-speed centrifugation (5000 rpm for 3 minutes). The supernatant was gently aspirated, and the cells were washed 1-2 times with PBS to remove excess culture medium. The collected bacterial pellet was resuspended in 2.5% glutaraldehyde solution and fixed at 4°C for 24 hours. The cells were then dehydrated in different concentrations of ethanol (50%, 70%, 80%, 90%, and 100%) at room temperature for 10 minutes, vacuum-dried, sprayed with gold, and observed and photographed using a SEM.

[0127] The results showed that the bacteria in the PBS control group were normal in shape, MRSA was nearly spherical, and PAE was rod-shaped. The bacterial surface was smooth and the structure was intact ( Figure 9After treatment with CDs-API, the surfaces of the two bacteria were deformed, with the cell walls and membranes shrinking, collapsing, and even rupturing. Cells became stacked and adhered, and even cellular contents leaked out (red arrows). This suggests that CDs-API's antibacterial ability may be achieved by destroying the bacterial cell walls or membranes.

[0128] (6) Verification of bacterial membrane system damage

[0129] The fluorescent dye propidium iodide (PI) usually cannot penetrate the intact cell membrane. However, when the cell membrane is damaged, PI can enter the cell and bind to DNA, emitting red fluorescence. This characteristic is used to use PI as an indicator of changes in bacterial membrane permeability. Prepare MRSA and PAE bacterial suspension (10 6 CFU / mL), 500 μL of each were added with 100 μL of PBS and 100 μL of CDs-API solution (final concentration was 1 mg / mL), respectively, and incubated at 37°C for 1 h and 2 h. 50 μM PI fluorescent probe solution was added, and the fluorescence intensity at an excitation wavelength of 535 nm was measured.

[0130] Compared with the PBS control group, the fluorescence intensity of MRSA and PAE bacterial suspensions incubated with CDs-API was significantly enhanced, and after 2 h of incubation, the fluorescence intensity of the bacterial suspension was further enhanced ( Figure 10 ), indicating that CDs-API damaged the bacterial cell membrane, causing changes in membrane permeability. PI entered the bacteria, bound to DNA in the nucleoid structure, and released red fluorescence. The longer the incubation time, the more severe the cell membrane damage caused by CDs-API, the more PI entered, and the stronger the fluorescence intensity. This result demonstrates that CDs-API significantly damages the bacterial cell membrane, causing changes in membrane permeability, and that this damage is time-dependent.

[0131] The charge-dependent aggregation release characteristics of diSC3(5) were used as an indicator of bacterial membrane potential changes to monitor the potential changes of the bacterial cell membrane phospholipid bilayer. 6 800 μL of each 4 μM diSC3(5) solution (800 μL of each 4 μM diSC3(5) solution) and 60 μL of KCl (100 mM) were added and incubated at room temperature for 1 hour. 100 μL of PBS and 100 μL of LCDs-API solution (final concentration of 1 mg / mL) were added to the samples, respectively. The samples were incubated at room temperature for 1 hour and 2 hours. The fluorescence intensity was measured at an excitation wavelength of 620 nm.

[0132] The results showed that the fluorescence intensity of diSC3(5) in the MRSA and PAE bacterial suspensions in the CDs-API group was significantly enhanced, and the fluorescence intensity after 2 h of co-incubation was greater than that after 1 h ( Figure 11 ), indicating that CDs-API can effectively damage the bacterial cell membrane and change the membrane potential of the bacterial cell membrane. With the increase of incubation time, the membrane damage is aggravated and the membrane potential change increases, causing more diSC3(5) to be released into the solution.

[0133] The above two experiments prove that CDs-API can effectively destroy the bacterial cell membrane structure, causing a series of membrane damages such as changes in bacterial membrane permeability and membrane potential.

[0134] (VII) Study on bacterial metabolism level

[0135] First, prepare MRSA and PAE bacterial suspensions in the logarithmic growth phase and dilute them with PBS to a bacterial volume of approximately 10 6 CFU / mL, mix the above two bacterial solutions with PBS, 1mg / mL ChACDs, and 1mg / mL CDs-API respectively, and incubate at room temperature for 30 minutes to allow the materials to fully exert their effects. After incubation, collect the bacteria by centrifugation (5000rpm, 3min). Move to ice, use bacterial lysis solution to lyse the collected bacteria, and centrifuge again (12000g, 5min, 4℃) to collect the supernatant. Prepare a sterile 96-well plate, add 100μL ATP working solution to each well, let it stand at room temperature for 5 minutes, then add 100μCDs-API solution (1mg / mL) and mix gently, and finally use a multifunctional enzyme reader to detect the chemiluminescence value of each well.

[0136] The results showed that compared with the PBS control group, the ATP content in the CDs-API treatment group decreased significantly ( Figure 12 ), indicating that bacterial metabolic activity has decreased or even stagnated. This decline in metabolic levels can be considered a sign of bacterial death. It can be concluded that CDs-API can significantly affect bacterial metabolic levels, causing a decrease or even cessation of metabolic activity, which may be one of the causes of bacterial death.

[0137] (8) ROS signal detection

[0138] The singlet oxygen fluorescence probe ABDA (9,10-anthracenediyl-bis(methylene)dicarboxylic acid) was used as an indicator to monitor the decomposition of ABDA by the decrease in absorbance at 378 nm, thereby determining the content of ROS in the system. 8 CFU / mLMRSA, 10 8 CFU / mL PAE, 10 8CFU / mLMRSA+1mg / mL ChACDs, 10 8 CFU / mL PAE+1mg / mL ChA CDs; ②10 8 CFU / mL MRSA, 10 8 CFU / mL PAE, 10 8 CFU / mLMRSA+1mg / mL CDs-API, 10 8 CFU / mLPAE+1 mg / mL CDs-API were mixed and incubated at room temperature, and the absorbance of the mixed system at 378 nm at different reaction times (10 min, 20 min, 30 min, 40 min) was detected using a microplate reader.

[0139] like Figure 13 The results showed that the absorbance of ABDA did not decrease significantly when incubated with bacteria, indicating that bacteria do not produce a large amount of ROS during normal growth; however, when ChACDs and CDs-API were incubated with bacteria, the absorbance decreased significantly, and the absorbance continued to decrease with the increase of incubation time, proving that both ChACDs and CDs-API can promote the accumulation of bacterial ROS, and ROS continues to increase with time.

[0140] (IX) In vitro biocompatibility of CDs-API

[0141] Human umbilical vein endothelial cells (HUVEC) were selected as the research object for in vitro cytotoxicity study. First, HUVEC cells were cultured at 5×10 3 Cells were seeded at a density of 100 cells / well in a 96-well plate and incubated with a series of concentrations of CDs-API (1 mg / mL, 3 mg / mL, 5 mg / mL, 7 mg / mL, and 10 mg / mL) for 48 hours. Cell viability was then determined using the MTT assay. 10 μL of methyl thiazolyltetrazolium (MTT, 0.5 mg / mL, pH 7.4) was added to each well and incubated for another 4 hours. The culture was terminated, the supernatant gently aspirated, and 100 μL of DMSO was added to each well. The cells were shaken for 10 minutes to fully dissolve viable mitochondria and reduce MTT to blue-purple crystalline formazan. Finally, the absorbance of each well at 490 nm was measured using a multi-functional microplate reader and the results were recorded.

[0142] The results are as follows Figure 14 Even at a high concentration of 10 mg / mL, the cell survival rate was almost unaffected, indicating that CDs-API had almost no cytotoxicity in vitro and could be used in in vivo anti-infection treatment experiments.

[0143] 4. In vivo testing of quantum dots

[0144] In the following experiments, CDs-API refers only to the quantum dots in Example 1, excluding the quantum dots in Example 2 and Example 3. The quantum dots in Example 2 are only CDs-API-2, and the quantum dots in Example 3 are only CDs-API-2.

[0145] The animals used in this experiment have been approved by the Experimental Animal Welfare Ethics Review Committee of Chongqing Medical University. Eighteen 6-week-old female BALB / c mice were selected and subjected to a one-week adaptive feeding for skin suppurative infection modeling. First, the healthy mice were randomly divided into two groups: MRSA infection group and PAE infection group, and then the two groups were divided into three subgroups (n=3), namely: PBS blank control group, ChACDs treatment group and CDs-API treatment group. The back of the mouse was depilated, and a circle with a diameter of about 9 mm was marked at a fixed position with a punch. Under ether gas anesthesia, surgical scissors were used to cut off the marked circular skin area on the back of the mouse, and 100 μL MRSA bacterial solution and PAE bacterial solution (10 8 CFU / mL) was applied to the open wound site after the skin was cut. After 24 hours of cage rearing, obvious pus was observed in the wound site, proving that the skin suppurative infection model was successfully established.

[0146] (1) Biocompatibility of CDs-API in vivo

[0147] After successfully establishing a mouse back wound infection model, the mice were treated with CDs and CDs-API according to different groups, and the changes in mouse weight were continuously recorded. After the treatment, the mouse organs were surgically removed and H&E staining was performed to assess the toxicity of the materials on the mouse organs. The results showed that during the 12-day treatment, the mice's weight did not decrease significantly and maintained a steady increase over time ( Figure 15 ); HE staining results of major organs showed that compared with the PBS control group, the morphological structures of various organs in the CDs and CDs-API treatment groups were normal, with no obvious toxic damage or histological changes ( Figure 16 These results demonstrate that CDs-API has no significant toxicity to mouse organs, has high biocompatibility, does not cause damage to the body when treating bacterial infections, and is highly safe.

[0148] (II) CDs-API in vivo anti-infection treatment trial

[0149] PBS (0.01M, pH 7.4), ChACDs (1 mg / mL) and CDs-API (1 mg / mL) were administered directly into the wound of each mouse at a dose of 50 μL. During the entire treatment period, different treatment drugs were administered at a fixed time every day, and the changes in wound size were recorded (see Figure 17 ). The body weight of the mice was recorded every other day; on the 12th day, the mice were sacrificed, the pus at the infection site was collected, diluted with PBS and inoculated into LB liquid culture medium and LB agar plates, incubated in a constant temperature incubator at 37°C for 18 hours, and the growth of the colonies was recorded by photographing. In addition, the back wound tissue and surrounding skin of the mice were obtained surgically, and the main organs (heart, liver, spleen, lungs, and kidneys) were removed by dissection. The tissues and organs were fixed with 4% paraformaldehyde solution for 48 hours. The fixed tissues were dehydrated and embedded in paraffin to prepare tissue sections with a thickness of 4-6 μm. The prepared tissue sections were stained with hematoxylin and eosin (H&E) and placed on a slice panoramic scanning system for photography to evaluate the structure and pathological conditions of the tissues.

[0150] On the 6th day of treatment, it was observed that the wounds of the PBS control group mice in the MRSA and PAE infection groups still had obvious edema and ulceration, while the wounds of the CDs and CDs-API treatment groups were significantly reduced and gradually healed with scabs ( Figure 18 a, 18b, 19a, 19b); At this stage, pus was collected from the wounds of mice in each group and inoculated into LB liquid culture medium. After incubation for 18 h, it was observed that the bacterial suspension in the PBS control group was obviously turbid, while the suspensions in the ChACDs group and CDs-API group were clear and transparent ( Figure 18 b, Figure 19 b), indicating that bacterial growth had been suppressed by day 6 of treatment. With continued treatment, the wounds of mice in the PBS control group repeatedly ruptured, demonstrating that bacteria were still proliferating in the wound site. In contrast, the wounds of mice in the ChACDs and CDs-API treatment groups gradually scabbed and healed. By the end of treatment, the wounds of mice in the CDs-API group were almost completely healed, healing faster than those in the ChACDs treatment group. This suggests that both ChACDs and CDs-API have certain antibacterial properties in vivo, with CDs-API being more effective than ChACDs.

[0151] After 12 days of treatment, the mice were sacrificed and the bacterial suspension from the infected wounds was obtained and cultured in LB solid medium. After incubation for 18 hours, the colonies on the solid medium were counted to assess the residual amount of bacteria in the infected area. Figure 18As shown in b and 19b, compared with the control group, there was almost no single colony formed in the bacterial solution smear of the wound infection site of the mice in the ChACDs and CDs-API treatment groups, indicating that the number of bacteria in the wound site was negligible, which was consistent with the results of the in vitro antibacterial ability test, further proving the antibacterial ability of the material. At the same time, tissues from the wound infection site were collected for H&E staining to assess the degree of infection of the wound. It can be seen that there is still a large amount of inflammatory cell infiltration in the wound site of the mice in the PBS control group (red arrow), and the inflammatory cell infiltration in the infected tissue of the ChACDs and CDs-API treatment groups was significantly reduced ( Figure 18 c, 19c), demonstrating that bacterial infection was fully inhibited.

[0152] These results demonstrate that both ChACDs and CDs-API inhibit the growth of MRSA and PAE in vivo, killing bacteria and reducing inflammation, thereby promoting skin recovery at the wound site in mice. In contrast, CDs-API demonstrated superior therapeutic efficacy by achieving effective treatment and killing bacteria at the site of infection in a shorter time.

[0153] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A method for preparing environmentally responsive charge-converting carbon quantum dots, characterized in that: include: (1) Chlorogenic acid was made into quantum dots to obtain ChA CDs; (2) ChA CDs were modified with imidazole compounds to obtain environmentally responsive charge-converting carbon quantum dots; In step (1), chlorogenic acid is prepared to obtain ChA CDs by a hydrothermal synthesis method, comprising: placing a chlorogenic acid ChA aqueous solution in a high-pressure reactor, and then heating it at 220° C.-240° C. to obtain a ChA CDs aqueous solution; In step (2), the imidazole compound is modified to ChA CDs by the EDC / NHS method, comprising: (i) mixing 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS) and 2-morpholinoethanesulfonic acid to obtain a reactant; (ii) mixing the reactant with the imidazole compound, adjusting the pH to 7-9, and reacting to obtain environmentally responsive charge-converting carbon quantum dots; Imidazole compounds are , n is an integer and 1≤n≤5.

2. The preparation method according to claim 1, characterized in that The imidazole compound is 1-(3-aminopropyl)imidazole.

3. The preparation method according to claim 1 or 2, characterized in that The mass / volume of ChA CDs and imidazole compounds was (3-6) mg / mL.

4. Environmentally responsive charge-converting carbon quantum dots prepared by the preparation method according to any one of claims 1 to 3.

5. A composition, characterized in that The composition comprises the environmentally responsive charge-conversion carbon quantum dots described in claim 4; the composition is used for antibacterial, inhibiting the growth of drug-resistant bacteria, treating skin suppurative infections or promoting wound healing.

6. A preparation, characterized in that The preparation comprises the environmentally responsive charge-conversion carbon quantum dots according to claim 4 or the composition according to claim 5; and a carrier.

7. Use of the environmentally responsive charge-converting carbon quantum dots according to claim 4 in antibacterial applications.

8. The use according to claim 7, characterized in that Applications include: Application of environmentally responsive charge-converting carbon quantum dots in antibiotic-resistant bacteria.

9. The use according to claim 8, characterized in that Antibiotic-resistant bacteria include one or more of methicillin-resistant Staphylococcus aureus, penicillin-resistant enterococci, vancomycin-resistant enterococci, carbapenem-resistant Enterobacteriaceae, carbapenem-resistant Acinetobacter baumannii, carbapenem-resistant Pseudomonas aeruginosa, or pan-resistant Acinetobacter.

10. Use of the environmentally responsive charge-conversion carbon quantum dots according to claim 4 in the preparation of a composition or preparation, wherein the composition or preparation is used to destroy the bacterial membrane system, reduce the bacterial metabolic level, or promote bacterial ROS accumulation.

11. Use of the environmentally responsive charge-conversion carbon quantum dots according to claim 4 in the preparation of a pharmaceutical composition or pharmaceutical preparation, wherein the pharmaceutical composition or pharmaceutical preparation is used to treat suppurative skin infections and / or promote wound healing.

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