Enzyme response antibacterial carbon dots as well as preparation method and application thereof

Abstract

The invention relates to an enzyme response antibacterial carbon dot and a preparation method and application thereof.The carbon dot takes a copper-doped carbon dot as a core and is covalently grafted with a hyaluronic acid shell layer through amidation reaction to form a composite nano material Cu-CDs (at) HA, specifically, soluble copper salt and folic acid are subjected to a hydrothermal reaction to obtain the copper-doped carbon dot, carboxyl is activated through EDC / NHS in a buffer system, and the carbon dot is prepared. And performing grafting reaction with hyaluronic acid to obtain a target product. The hyaluronidase-containing antibacterial composition can be specifically degraded and release antibacterial components under the action of hyaluronidase, shows good bacterial responsive bactericidal performance, has the minimum inhibitory concentrations of 8 g / mL and 16 g / mL for staphylococcus aureus and escherichia coli respectively, and has peroxidase-like activity and free radical scavenging capacity. The antibacterial carbon dots can be used for preparing intelligent wound dressings, antibacterial coatings and other biomedical materials, and have the advantages of responsive drug release, efficient antibacterial property and promotion of wound repair.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanofunctional materials and biomedical technology, and particularly relates to a copper-doped carbon dot hyaluronic acid grafted composite with enzyme-responsive antibacterial function, a preparation method thereof and application thereof in preparing antibacterial medical materials. BACKGROUND

[0002] As a new type of carbon-based nanomaterial, carbon dots (CDs) have attracted much attention in the field of biomedicine, especially in the development of new antibacterial materials, due to their excellent biocompatibility, low toxicity and easy functionalization. Among them, metal (such as silver, copper, zinc, etc.) doped carbon dots exhibit stronger synergistic antibacterial effect than single components by integrating the intrinsic properties of carbon nanomaterials and the antibacterial activity of metal ions, providing a new strategy to effectively overcome the problem of drug resistance of traditional antibiotics, and have become a research hotspot of nano-antibacterial materials.

[0003] However, existing antibacterial carbon dot materials, especially copper-doped carbon dots (Cu-CDs), still face several severe challenges in the process of moving towards practical biomedical applications, mainly in the following aspects:

[0004] 1. Insufficient antibacterial efficacy

[0005] A large number of reported carbon dot materials have a high minimum inhibitory concentration (MIC), which is not sufficient to take effect at low doses, limiting their practical application value.

[0006] For example, Wang et al. (Effect of ultra-trace Ag doping on the antibacterial performance of carbon quantum dots [J]. Journal of Environmental Chemical , 2022, 10: 107112.) prepared Ag, N-CQDs nanoclusters using citric acid, ascorbic acid, silver nitrate and ammonia as raw materials by hydrothermal method, and the minimum inhibitory concentration (MIC) of the nanoclusters against gram-negative bacteria Engineering and gram-positive bacteria E. coli was 250 µg / mL and 200 µg / mL, respectively. Similarly, Travlou et al. (S-and N-doped carbon quantum dots: Surface chemistry dependent antibacterial activity [J]. S. aureus , 2018, 135: 104-111.) reported that the MIC of sulfur-doped carbon dots against Escherichia coli was 128 μg / mL, which had limited antibacterial efficacy. Carbon

[0007] 2. Single mechanism and lack of intelligent responsiveness

[0008] Most of the antibacterial carbon dots have persistent and non-selective bactericidal effects, showing a "always-on" non-selective bactericidal mode. Wang et al. (Infection microenvironment-related antibacterial nanotherapeutic strategies [J]. Biomaterials , 2022, 280: 121249.) review points out that this non-selective antibacterial mode will indiscriminately attack normal cells in vivo applications, while killing pathogenic bacteria, it may damage normal tissue cells and destroy beneficial microbial communities, leading to potential toxicity and microecological imbalance, which is not conducive to the microenvironment balance and high-quality healing of wounds.

[0009] In addition, the complex infection microenvironment requires antibacterial materials to be able to dynamically respond and synergistically regulate. The ideal infection microenvironment-regulated antibacterial material should be able to both produce bactericidal reactive oxygen species (ROS) at the infection site through its peroxidase-like activity, and have antioxidant activity to scavenge excess free radicals to alleviate inflammation. However, Wang et al. (Superantioxidant and high antibacterial ability bi-functional xylitol / 2-hydroxypropyl-β-cyclodextrin carbon dots with hydroxyl-functionalized for rainbow trout preservation [J]. Food Research International , 2025, 203:115792.) points out that most antibacterial carbon dot materials only focus on ROS generation to enhance bactericidal performance, while carbon dot materials with "two-way regulation" of both high ROS generation ability and free radical scavenging (antioxidant) ability are still very rare, which makes it difficult for them to synergistically manage the complex infection oxidative stress environment.

[0010] 3. Stability and biological applicability need to be improved

[0011] Although Cu-CDs have good antibacterial performance, they are prone to aggregation in physiological environments and have poor stability, which may lead to premature release of metal ions in the body environment, bringing potential toxicity risks and reducing their long-term antibacterial efficiency, limiting their further biomedical applications.

[0012] These studies show that it is crucial to develop new carbon dots with lower MIC and higher antibacterial efficacy.

[0013] Hyaluronic acid (HA) is a natural polysaccharide with good biocompatibility, biodegradability and moisturizing properties, and is widely used in biological medical materials such as wound dressings. Especially noteworthy is that in the wound microenvironment infected by Staphylococcus aureus, Streptococcus pyogenes and other pathogenic bacteria, these bacteria will specifically overexpress a specific enzyme, hyaluronidase, which can specifically degrade hyaluronic acid. This pathological characteristic provides an ideal and natural trigger mechanism for designing enzyme-responsive intelligent antibacterial materials.

[0014] Currently, there have been studies using HA as an enzyme-responsive carrier to encapsulate traditional antibiotics, such as Lee et al. (Light-stimulated carbon dot hydrogel: Targeting and clearing infectious bacteria in vivo [J]. ACS Applied Bio Materials , 2022, 5(2): 761-770.) encapsulated conventional antibiotics in HA hydrogel, achieving enzyme-controlled release, but these systems usually rely on external drugs and have single functions.

[0015] Integrating the enzyme-responsive properties of HA with Cu-CDs, which have inherent high enzyme-like activity and antibacterial activity, through chemical bonding, to construct a multifunctional nanoplatform that integrates "intelligent targeted release, high-efficiency synergistic antibacterial, and oxidative stress microenvironment bidirectional regulation" has so far been a research blank. Developing a new type of nanomaterial that can respond to the infected microenvironment, precisely release antibacterial components, and have multiple biological activities is of great significance and value for improving the treatment efficiency of infectious wounds, reducing side effects, and promoting tissue regeneration. SUMMARY

[0016] The purpose of the present application is to provide an enzyme-responsive antibacterial carbon dot that responds to hyaluronidase degradation to release copper ions in a wound infection environment, exerting broad-spectrum antibacterial action with antioxidant and enzyme-like activity, solving the problem of strong bacterial drug resistance and inaccurate release mechanism of existing antibacterial materials.

[0017] To achieve the above-mentioned purpose of the application, the present application first provides a preparation method of an enzyme-responsive antibacterial carbon dot, which comprises the following steps:

[0018] Specifically, the preparation method of the enzyme-responsive antibacterial carbon dot comprises the following steps: 1) using folic acid as a carbon source and a soluble copper salt as a copper source, subjecting an aqueous solution containing the copper source and the folic acid to a hydrothermal reaction at 180-220°C, and subjecting the reaction liquid to purification treatment to obtain copper-doped carbon dots in which copper is doped in the form of Cu(II) and Cu(I) in the carbon dots; 2) in a buffer solution system having a pH value of 5.5-6.7, subjecting the copper-doped carbon dots to carboxyl activation using a condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and an activating agent N-hydroxysuccinimide, then adding an aqueous hyaluronic acid solution, adjusting the pH value to neutral, and performing amidation reaction to form stable amide bonds by covalently grafting hyaluronic acid (HA) to the surface of the Cu-CDs, and subjecting the reactants to purification treatment to obtain enzyme-responsive antibacterial carbon dots grafted with hyaluronic acid.

[0019] Further, the mass ratio of the copper source to the folic acid in the step 1) is preferably 1:(1-5).

[0020] Further, the time of the hydrothermal reaction in the step 1) is preferably 6-12 h.

[0021] Further, the purification treatment of the reaction liquid in the step 1) includes freeze-drying the Cu-CDs powder after suction filtration, high-speed centrifugation, and small-molecular-weight dialysis bag purification of the reaction liquid.

[0022] Further, the high-speed centrifugation is preferably at a speed of 8000-10000 r / min for 10-20 min.

[0023] Further, the small-molecular-weight dialysis bag purification is preferably performed using a dialysis bag having a molecular weight cut-off of 500-1500 Da.

[0024] Further, the buffer solution system in the step 2) preferably uses a MES buffer solution having a concentration of 0.05-0.2 M.

[0025] Further, the mass ratio of the copper-doped carbon dots to the hyaluronic acid in the step 2) is preferably 1:(1.5-2.5).

[0026] Further, the reaction time of the carboxyl activation is preferably 0.5-2 h, and the reaction time of the amidation reaction is preferably 10-14 h.

[0027] Further, the purification treatment of the reactants in the step 2) is freeze-drying the final product Cu-CDs@HA carbon dot complex after dialysis purification of the reactants using a large-molecular-weight dialysis bag.

[0028] Further, the large-molecular-weight dialysis bag purification is preferably performed using a dialysis bag having a molecular weight cut-off of 8000-12000 Da.

[0029] Further, the application also provides an enzyme-responsive antibacterial carbon dot prepared by the above preparation method, which takes copper-doped carbon dot antibacterial active ingredient as a core, copper element is doped in the carbon dot in the form of Cu(II) and Cu(I), the core surface is rich in amide bond, and the hyaluronic acid molecule is connected to form a core-shell structure nanocomposite with a particle size of 2-10 nm, the copper-doped carbon dot antibacterial active ingredient is released by responding to the hyaluronidase enzyme hydrolysis, and has peroxidase-like activity and free radical scavenging capacity.

[0030] The application also provides application of the enzyme-responsive antibacterial carbon dot as a bacterial-responsive antibacterial material in preparation of medical materials for preventing and / or treating bacterial infection.

[0031] Specifically, the bacterial infection is an infection related to overexpression of hyaluronidase.

[0032] The application further provides an antibacterial composition for treatment of infectious wounds, which comprises the enzyme-responsive antibacterial carbon dot and a pharmaceutically or materially acceptable carrier. The antibacterial composition of the application sufficiently integrates the intelligent antibacterial properties of the enzyme-responsive antibacterial carbon dot and the structural properties of the carrier, realizes efficient response to specific enzymes in the wound infection process, and improves the bioavailability and action persistence of the drug through the adaptation of the carrier, thereby providing a novel solution with targeting, safety and compliance for clinical treatment of infectious wounds.

[0033] Specifically, the antibacterial composition of the application can include, but is not limited to, specific forms such as gel, film or fiber in physical state, which are all conventional drug or medical material dosage forms in the art, and the enzyme-responsive antibacterial carbon dot and the suitable high molecular material are prepared into the corresponding forms by physical mixing, cross-linking or spinning and other known processes, the preparation method belongs to the existing mature technology, and the skilled person in the art can routinely select and implement according to the actual application requirements. Among them, the gel state is a semi-solid or solid preparation formed by compounding the enzyme-responsive antibacterial carbon dot and a biocompatible polymer matrix, which has a continuous three-dimensional network structure and adjustable rheological properties, can adapt to different forms of wound surface, maintain a moist microenvironment of the wound surface, and realize controllable release of the drug; the film state is a uniform planar thin layer structure prepared by casting, drying or electrospinning process by uniformly dispersing the enzyme-responsive antibacterial carbon dot in the film-forming material, which is beneficial to covering the wound surface, has a certain air permeability to allow gas exchange, and blocks external microorganisms; and the fiber state is a filament, short fiber or non-woven fabric form prepared by wet spinning, melt spinning or electrospinning by integrating the enzyme-responsive antibacterial carbon dot into a spinning solution, which has a high specific surface area and porosity, and good mechanical adaptability, and can further form a flexible network substrate, which is beneficial to cell adhesion and tissue fluid exchange.

[0034] More specifically, based on the different physical states and clinical application requirements described above, the antibacterial composition of the present application can also include, but is not limited to, different product forms configured as wound dressings, antibacterial sprays or antibacterial coatings, etc. These product forms are all common dosage form application methods in the medical field, and their processing methods such as cutting and packaging of dressings, filling and atomization of sprays, coating and curing of coatings, etc. also belong to the prior art. When configured as a wound dressing, the composition with a gel, film or fiber state is compounded with a substrate such as non-woven fabric, foam, hydrogel, etc., and further processed into a medical product with a specific size and shape that can be directly applied to the wound surface; when configured as an antibacterial spray, the composition is formulated into a suspension or solution dosage form together with suitable solvents, propellants and film-forming aids, and fine droplets are formed by pressure or mechanical atomization device to uniformly cover the irregular or large area wound surface to form a thin film or gel layer with antibacterial function; when configured as an antibacterial coating, the composition is firmly attached to the surface of other medical devices or materials in the form of a thin film through physical adsorption, chemical bonding or blending into a film, so as to have long-term, stable and intelligent antibacterial performance.

[0035] Compared with the prior art, the enzyme-responsive antibacterial carbon dots of the present application have the following beneficial effects:

[0036] 1. High-efficiency catalytic sterilization: The present application realizes a leap in antibacterial efficiency through the unique "copper-doped carbon core-hyaluronic acid shell" structural design. The simultaneous presence of copper elements Cu(II) and Cu(I) in Cu-CDs@HA makes it exhibit a minimum inhibitory concentration (MIC) significantly lower than most existing carbon-based nanomaterials against common gram-positive bacteria (such as Staphylococcus aureus Staphylococcus aureus ) and gram-negative bacteria (such as Escherichia coli Escherichia coli ). Specifically, the MIC value against Staphylococcus aureus can be below 8 µm / mL, and the MIC value against Escherichia coli can be below 16 µm / mL, which is several times higher than the carbon dot materials based on conventional precursors such as citric acid commonly reported in the literature (whose MIC value is usually higher than 25-50 µm / mL). This ultra-high antibacterial efficiency means that lower drug doses can be used in clinical applications to achieve effective infection control, not only reducing material costs, but also greatly reducing the risk of potential systemic side effects.

[0037] 2、Multiple synergistic antibacterial mechanism: The Cu-CDs@HA of the application innovatively integrates two seemingly contradictory ROS regulation mechanisms, efficient peroxidase-like activity and strong free radical scavenging ability (antioxidant property), to achieve the intelligent function of "one material with double effects". The Cu-CDs@HA not only has the antibacterial property of copper ions, but also exhibits excellent peroxidase-like activity, can catalyze endogenous hydrogen peroxide (H2O2) to produce a large number of highly toxic hydroxyl radicals (·OH) in the wound microenvironment, cause fatal oxidative damage to bacteria through chemical kinetic therapy, form a strong synergy with the antibacterial effect of copper ions, greatly improve the bactericidal efficiency and effectively avoid the drug resistance path of traditional antibiotics; at the same time, it also shows high removal rate in DPPH and ABTS free radical scavenging experiments, and this excellent antioxidant capacity enables it to timely remove excess harmful free radicals at the infection site after completing the sterilization task, thereby effectively relieving the oxidative stress and excessive inflammatory response of the tissue. The time sequence regulation ability of "killing bacteria first and then anti-inflammatory" of the application creates a good microenvironment for cell proliferation and tissue regeneration, breaks the limitation of traditional antibacterial materials "only breaking but not building", and shows excellent potential for promoting wound healing.

[0038] 3、Intelligent response and targeted sterilization: The application skillfully uses the excess expression of hyaluronidase in the infected microenvironment as a specific biological trigger switch. In healthy tissues, the intact HA shell effectively shields the internal Cu-CDs core, maximally reducing its toxicity to normal cells; and once it reaches the bacterial infection site, the hyaluronidase secreted by the pathogenic bacteria will specifically degrade the HA shell, releasing the highly active Cu-CDs antibacterial core on demand and quickly. This "intelligent switch" mechanism realizes the precise targeted delivery and controlled release of drugs, significantly improves the selectivity of treatment, realizes the upgrade from "general sterilization" to "precise sniping", and effectively safeguards the safety of normal tissue cells and the stability of skin microecology while eradicating infection.

[0039] At the same time, the enzyme-responsive antibacterial carbon dots also have excellent biocompatibility and healing potential, and the hyaluronic acid shell is a natural component of the human body, which has excellent biocompatibility and moisturizing properties, and can provide a good moist environment for wound healing. As an intelligent nanomaterial, the enzyme-responsive antibacterial carbon dots can be easily integrated into various medical dressing substrates to construct new composite antibacterial materials, which have wide application prospects in the field of biomedicine, especially in the management of chronic infected wounds. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 is a transmission electron microscope and particle size statistical graph of the enzyme-responsive antibacterial carbon dots.

[0041] Figure 2 is a Fourier transform infrared spectrum of the enzyme-responsive antibacterial carbon dots.

[0042] Figure 3 are the chemical structure characterization of enzyme-responsive antibacterial carbon dots, wherein A is an X-ray photoelectron spectrogram; B is a high-resolution X-ray photoelectron spectrogram of Cu element; C is an X-ray diffraction pattern.

[0043] Figure 4 are the antibacterial performance diagrams of enzyme-responsive antibacterial carbon dots; wherein A is the minimum inhibitory concentration of Staphylococcus aureus and Escherichia coli; B is the SEM image of bacteria after treatment.

[0044] Figure 5 are the antioxidant properties of enzyme-responsive antibacterial carbon dots, wherein A is the DPPH method free radical scavenging performance; B is the ABTS method free radical scavenging performance.

[0045] Figure 6 are the enzyme responsiveness of enzyme-responsive antibacterial carbon dots, wherein A is the crystal violet staining diagram of Cu-CDs@HA treated Escherichia coli biofilm; B is the crystal violet staining diagram of Cu-CDs@HA treated Escherichia coli biofilm after adding hyaluronidase; C is the statistical diagram of biomass tested by enzyme label instrument. Embodiment

[0046] The embodiments of the present application will be described in detail below with reference to the accompanying drawings and specific examples. It should be noted that the examples are for illustrative purposes only, and are intended to provide a thorough understanding of the technical solutions of the present application and to guide those skilled in the art to implement and apply the present application. It should be understood that these descriptions do not constitute any limitation on the scope of protection of the present application.

[0047] Unless otherwise explicitly stated, the production processes, experiments, detection or analysis methods involved in the embodiments of the present application are all considered to be conventional methods known to those skilled in the art, which only need to be implemented in accordance with conventional conditions or relevant product instructions, and the steps and names involved are all generally clear and unambiguous in the art.

[0048] The instruments, equipment, raw materials, reagents or samples used in the examples do not have any special restrictions on the source, and are all conventional products that can be purchased through normal commercial channels or prepared by known methods, and the source does not have a substantial impact on the implementation results of the present application.

[0049] The scientific and technical terms used in the present application, unless otherwise explicitly defined, all have the meanings generally understood by those skilled in the art in the technical field described in the present application. If there is a conflict, the definition in the specification shall prevail.

[0050] The terms "comprise", "contain", "include", and the like as used herein are to be construed in an open, non- limiting sense, i.e. meaning "comprising, containing, including, and the like". The term "and / or" includes any and all combinations of one or more of the associated listed items. The number terms such as "one", "a", and the like do not exclude a plurality; "plurality" or "a plurality" means two or more.

[0051] The terms "preferably", "more preferably", "most preferably" and the like as used herein are to be construed as describing a particularly preferred aspect, but not necessarily a necessity of the application. The terms "about" and "substantially" as used herein are to be construed as leaving some leeway in the meaning to account for natural variations in the described parameters.

[0052] The present application relates to the description of numerical parameters (such as amount, concentration, temperature, time, etc.), which should be understood to naturally exist within a reasonable deviation range caused by measuring instruments, operation errors, statistical fluctuations, etc. The deviation range should be within the limits acceptable by those skilled in the art according to common sense. Embodiment

[0053] Embodiment 1

[0054] 0.5 g of copper sulfate pentahydrate (CuSO4·5H2O) and 1 g of folic acid were weighed into 100 mL of deionized water, stirred at room temperature for 30 min in the dark, and then ultrasonically treated for 30 min to obtain a uniformly dispersed precursor solution.

[0055] The precursor solution was transferred to a polytetrafluoroethylene-lined high-pressure reaction kettle, sealed, heated to 220°C, and then hydrothermally reacted for 8 h. After the reaction was completed, the reaction liquid was naturally cooled to room temperature to obtain a dark brown reaction liquid.

[0056] The reaction liquid was filtered using a 0.45 μm microporous filter membrane to remove possible larger particles or carbonized impurities, the filtrate was collected and transferred to a high-speed centrifuge tube, centrifuged at 8500 r / min for 15 min, the supernatant was taken, and a dialysis bag with a molecular weight cutoff of 1000 Da was filled with the supernatant, and dialysis was performed in ultrapure water for 7 days, and the ultrapure water was replaced every 4 h to completely remove unreacted Cu 2+ ions, folic acid residues, and small molecule byproducts.

[0057] The dialysate was transferred to a clean freeze-drying bottle, pre-frozen in a -80°C ultra-low temperature refrigerator for 24 h, and then transferred to a freeze-drying machine for vacuum freeze-drying for 48 h to obtain a fluffy black-brown Cu-CDs solid powder, which was stored at 4°C in the dark.

[0058] Embodiment 2

[0059] Take 50.0 mg of Cu-CDs powder prepared in Example 1, disperse in 10 mL of 0.1 M MES (2-(N-morpholino)ethanesulfonic acid) buffer solution with pH value of 5.5, and ultrasonic treatment for 30 min to form a uniform and stable black Cu-CDs dispersion.

[0060] Add 50.0 mg of EDC·HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and 25.0 mg of NHS (N-hydroxysuccinimide) into the above dispersion in sequence, and carry out a carboxyl activation reaction under room temperature and light shielding for 1 h with magnetic stirring to activate the carboxyl groups on the surface of Cu-CDs to form an active ester intermediate, which is convenient for subsequent grafting.

[0061] Take 100.0 mg of hyaluronic acid (HA) with a molecular weight of about 1×10 6 Da, and dissolve in 10 mL of deionized water to obtain a HA solution.

[0062] Slowly add the HA solution drop by drop into the activated Cu-CDs dispersion under stirring, and after the addition is completed, adjust the pH value of the mixture to 7.0 with 0.1 M NaOH solution, and carry out stirring reaction under room temperature and light shielding for 12 h to make the amidation grafting reaction fully proceed and realize the grafting and coating of HA on Cu-CDs.

[0063] After the reaction, the mixture is loaded into a dialysis bag with a molecular weight cut-off of 10,000 Da, and dialyzed in ultrapure water for 48 h, and the water is changed every 6-8 h during the dialysis to completely remove the unreacted EDC, NHS, by-products (such as isourea), and free hyaluronic acid which is not grafted.

[0064] Collect the dialysate, and vacuum freeze-dry according to the method of Example 1 to prepare the enzyme-responsive antibacterial carbon dots Cu-CDs@HA brownish flocculent solid.

[0065] Figure 1 The transmission electron microscope (TEM) photo of the enzyme-responsive antibacterial carbon dots Cu-CDs@HA shows that the prepared Cu-CDs@HA is approximately spherical, well dispersed, and has no obvious agglomeration, and the average particle size D is 2.36±0.80 nm.

[0066] Figure 2 The Fourier transform infrared spectrum (FTIR) of Cu-CDs and Cu-CDs@HA is given, and compared with Cu-CDs, it can be seen that the Cu-CDs@HA has characteristic absorption peaks of amide I band and amide II band near 1650 cm -1 and 1550 cm -1 , which confirms the successful formation of amide bond (−CONH−) and proves that HA has been grafted onto the surface of Cu-CDs.

[0067] Further,Figure 3 The characteristic peaks of C, O, N, Cu elements are clearly shown in the X-ray photoelectron spectroscopy (XPS) survey spectrum of (A), while the binding energy position in the high-resolution Cu 2p spectrum of (B) confirms that Cu element is successfully doped in carbon dots in the form of Cu(II) and Cu(I). Cu(II) can catalyze hydrogen peroxide (H2O2) to generate highly toxic hydroxyl radicals (·OH) through Fenton-like reaction, and Cu(I) can also participate in similar reactions and usually has higher catalytic efficiency. Although single Cu(II) or Cu(I) also has certain antibacterial properties, the coexistence of Cu(II) and Cu(I) creates a dynamic and sustainable "ROS factory". The synergistic effect of the multi-valence copper ions in the present application can produce stronger and more persistent oxidative stress than single Cu 2+ valence, thereby greatly improving the antibacterial efficiency of the material.

[0068] Figure 3 The X-ray diffraction (XRD) spectrum of (C) shows that Cu-CDs has a broad diffraction peak at 24°, which is attributed to the amorphous graphite structure (002 crystal plane) of the carbon core, while the sharp crystalline diffraction peak attributed to copper species in the spectrum confirms that copper is successfully doped into carbon dots in the form of crystals. After subsequent grafting with hyaluronic acid (HA) to form Cu-CDs@HA, the intensity of these copper characteristic peaks decreases significantly, which indicates that the successful coating of HA reduces the crystallinity or exposure ratio of copper species on the surface of the material, inhibits its rapid oxidation and realizes the long-acting release of copper ions, which is the key structural basis for the material to exhibit excellent long-acting antibacterial performance.

[0069] Example 3

[0070] The in vitro antibacterial activity of the enzyme-responsive antibacterial carbon dots Cu-CDs@HA prepared in Example 2 against Gram-positive bacteria Staphylococcus aureus ( S. aureus ) and Gram-negative bacteria Escherichia coli ( E. coli ) was tested by microbroth dilution method, and the morphology of dead bacteria was observed.

[0071] The minimum inhibitory concentration (MIC) determination results (corresponding Figure 4 A) show that Cu-CDs@HA exhibits significant antibacterial effect on both test strains. Specifically, the MIC value of Cu-CDs@HA against Staphylococcus aureus is 8 µg / mL, and the MIC value against Escherichia coli is 16 µg / mL, which exhibits stronger antibacterial activity against Gram-positive bacteria, which is speculated to be due to the thick and loose peptidoglycan cell wall structure of Gram-positive bacteria, which is more susceptible to the contact and destruction of the cell membrane by the active copper components in the material.

[0072] Figure 4The bacterial morphology scanning electron microscopy (SEM) observation of B further confirmed its antibacterial mechanism. Compared with the intact normal bacteria, the bacteria treated by Cu-CDs@HA appeared serious morphological damage, and the bacterial surface shrinkage, collapse, cell wall / membrane rupture and even disintegration could be clearly observed, resulting in a large amount of intracellular content leakage. This consistent morphological evidence is consistent with the antibacterial mechanism of Cu-CDs@HA by destroying the cell membrane integrity, triggering the content leakage, and thus leading to bacterial death.

[0073] Example 4

[0074] The free radical scavenging ability (antioxidant property) of the enzyme-responsive antibacterial carbon dots Cu-CDs@HA prepared in Example 2 was evaluated by DPPH and ABTS free radical scavenging experiments. Specifically, different concentrations of Cu-CDs and Cu-CDs@HA solutions were mixed with DPPH or ABTS free radical working solution, respectively, and reacted for a certain time at room temperature in the dark. Then, the absorbance was measured at 517 nm (DPPH) or 734 nm (ABTS) wavelength using an enzyme marker, and vitamin C was used as a positive control. The antioxidant activity was evaluated by calculating the free radical scavenging rate.

[0075] Figure 5 At a concentration of 1 mg / mL, Cu-CDs@HA had a DPPH free radical scavenging rate of up to 71.5%, and an ABTS free radical scavenging rate of up to 99%. This excellent antioxidant property helps to kill bacteria while removing excess harmful free radicals at the infection site, relieving oxidative stress and creating a more conducive microenvironment for wound healing. The DPPH scavenging rate of Cu-CDs@HA after grafting HA decreased slightly, mainly because HA, as a macromolecular polymer, was coated on the surface of carbon dots, producing steric hindrance, which hindered the contact of DPPH free radicals with the active sites of carbon dots. The difference between the two for ABTS was not significant, and even the effect of Cu-CDs@HA was slightly better, because ABTS was a water-soluble free radical, and the excellent hydrophilicity of HA helped the material to disperse in the aqueous phase, maintaining the contact area with the free radicals.

[0076] Example 5

[0077] To evaluate the catalytic performance of the enzyme-responsive antibacterial carbon dots prepared in Example 2, TMB (3,3',5,5'-tetramethylbenzidine) was used as a chromogenic substrate for peroxidase (POD) activity kinetic test.

[0078] In the reaction system containing different concentrations of H2O2, Cu-CDs@HA was quantitatively added, and the absorbance change of oxidized TMB at 652 nm was monitored. The results showed that Cu-CDs@HA could efficiently catalyze the oxidation of TMB in the presence of H2O2, and the absorbance value of the reaction solution rapidly increased within 5 min.

[0079] The Michaelis-Menten equation was used to fit the reaction rate, and the Michaelis constant (Km) of Cu-CDs@HA for the substrate H2O2 was calculated to be 0.087 mM, and the maximum reaction rate (V max ) was 2.315 µM / s, which was significantly lower than that of various reported nanoscale enzymes, indicating that it had higher affinity for the substrate H2O2 and more prominent peroxidase-like catalytic efficiency.

[0080] Based on this property, Cu-CDs@HA can utilize low concentrations of endogenous H2O2 in the wound microenvironment to efficiently generate highly toxic hydroxyl radicals (·OH) through chemical kinetics, thereby achieving its high antibacterial effect.

[0081] Example 6

[0082] To verify the intelligent enzyme response characteristics of Cu-CDs@HA, it was co-incubated with hyaluronidase (HAase) and PBS buffer (as a control), respectively, and the amount of Cu ion release in the supernatant was dynamically monitored. Figure 6 The results of A and B clearly showed that in the presence of HAase, Cu ions were rapidly and massively released within a certain time. From the statistical results of C, it was found that the release rate and cumulative release amount were significantly higher than those of the PBS control group. This result directly confirmed that Cu-CDs@HA could be specifically degraded by hyaluronidase overexpressed in the infected microenvironment, thereby intelligently releasing its antibacterial active components.

[0083] Figure 6 Compared with A, when hyaluronidase was introduced (to simulate the high enzyme microenvironment of the bacterial infection site), the bactericidal effect had a qualitative leap. With the concentration reaching 135 µg / mL, the enzyme response effect was significantly amplified. The enzyme-free group still had about 20% of the biofilm residue, while the experimental group treated with hyaluronidase had a sharp decrease in residual biomass to less than 5%. This significant difference strongly proved that Cu-CDs@HA had excellent enzyme responsiveness. The mechanism was that the high expression of hyaluronidase in bacterial biofilms or infected microenvironments could specifically degrade the HA shell on the material surface, which triggered the explosive release of the internal antibacterial active center (Cu-CDs) like "detonating a fuse", thereby achieving efficient penetration and removal of deep bacterial biofilms.

[0084] The enzyme-responsive antibacterial carbon dots Cu-CDs@HA prepared in Example 2 of this invention were compared with antibacterial materials reported in the literature. The specific comparison results are as follows:

[0085] Compare with Example 1

[0086] Lu et al. (Water-solvable carbon dots derived from curcumin and citric acid with enhanced broad-spectrum antibacterial and antibiofilm activity [J]. Materials Today Communications (2021, 26: 102000.) Highly water-soluble carbon dots were prepared by hydrothermal method using citric acid and curcumin as precursors. These carbon dots, at a concentration of 375 µg / mL, were effective against Gram-negative bacteria. E. coli and P. aeruginosa Its sterilization efficiency is up to 100%, effective against Gram-positive bacteria. S. aureus and B. subtilis Its bactericidal efficiency reaches 80%, and the specific mechanism is to destroy the bacterial cell membrane.

[0087] Although the carbon dot material has good broad-spectrum antibacterial and anti-biofilm activities, it lacks an enzyme-responsive release mechanism, which prevents it from achieving intelligent targeted drug release at the site of infection. At the same time, it has not been reported to have peroxidase-like activity or free radical scavenging ability, which prevents it from achieving dual regulation of bactericidal and anti-inflammatory effects, thus limiting its synergistic therapeutic potential in complex infectious microenvironments.

[0088] Compare with Example 2

[0089] Dou et al. (Multi-functional fluorescent carbon dots with antibacterial and gene delivery properties [J]. RSC Advances (2015, 5(58): 46817–46822.) Carbon dots were prepared by hydrothermal method using glucose and polyethyleneimine (PEI) as precursors, and quaternized with benzyl bromide at room temperature to obtain multifunctional fluorescent carbon dots with a particle size of about 3.5 nm. Regarding antibacterial properties, they exhibited activity against Gram-positive bacteria. S. aureus The MICs were 16 µg / mL (qCDl, linear PEI quaternization) and 32 µg / mL (qCDb, branched PEI quaternization); for Gram-negative bacteria E. coli The MICs were 16 µg / mL (qCDl) and 64 µg / mL (qCDb), and the mechanism involved membrane disruption.

[0090] In comparison, the MIC of Cu-CDs@HA against S. aureus was reduced to 8 µg / mL, against E. coli was 16 µg / mL, overcoming the limitations of high MIC, non-targeted release and lack of ROS regulation in the literature.

[0091] Example 3

[0092] Wang et al. (Effect of ultra-trace Ag doping on the antibacterial performance of carbon quantum dots [J]. Journal of Environmental Chemical Engineering , 2022, 10: 107112.) prepared Ag,N-CQDs nanoclusters using citric acid, ascorbic acid, silver nitrate and ammonia as raw materials by hydrothermal method, and the MIC against gram-negative bacteria E. coli and gram-positive bacteria S. aureus was 250 µg / mL and 200 µg / mL, respectively. SEM observation showed that the bacterial morphology was destroyed, and the mechanism was attributed to the strong adsorption of polar chemical groups and the membrane perforation effect of Ag atoms, but lacked the evaluation of light / enzyme trigger dependence and free radical scavenging ability.

[0093] In comparison, the MIC of Cu-CDs@HA against E. coli and S. aureus was reduced to 16 µg / mL and 8 µg / mL, respectively, with an ABTS free radical scavenging rate of up to 99%, and a 93.7% decrease in biofilm biomass through hyaluronidase response, overcoming the limitations of high MIC, non-targeted release and potential silver toxicity in the literature, significantly improving low-dose high-efficiency antibacterial and biocompatibility.

[0094] Example 4

[0095] Qu et al. (Positively Charged Carbon Dots with Antibacterial and Antioxidant Dual Activities for Promoting Infected Wound Healing [J]. ACS Applied Materials & Interfaces , 2023, 15(15): 18608-18619.) prepared positively charged CDs using p-phenylenediamine and polyethyleneimine by one-step solvothermal method, which showed good biocompatibility and exhibited good antibacterial activity against S. aureusIt exhibits excellent antibacterial effects in a concentration-dependent manner, with bacterial survival decreasing with increasing concentration (0–100 μg / mL) and time, the mechanism being electrostatic interaction that disrupts the membrane; in terms of antioxidant activity, it can effectively scavenge O2. - • It neutralizes DPPH free radicals, promoting the relief of oxidative stress.

[0096] Although the material has both antibacterial and antioxidant functions, it lacks an enzyme-responsive release mechanism, which prevents it from achieving intelligent and targeted drug release at the site of infection. At the same time, there are no reports of it possessing peroxidase-like activity or anti-biofilm properties, which limits its ability to provide precise and synergistic treatment in complex infection microenvironments.

[0097] Compare with Example 5

[0098] Wang et al. (Metal-free nitrogen-doped carbon nanodots as an artificial nanozyme for enhanced antibacterial activity [J]. Journal of Cleaner Production (2023, 411: 137337.) reported a metal-free nitrogen-doped carbon nanodots (N-CNDs) prepared from tea polyphenols and ethylenediamine via a one-step hydrothermal method. This material exhibited oxidase-like activity under illumination, with a Km of 0.421 mM and a Vo. max The value is 1.59 µM / s. Regarding antibacterial properties, it is effective against bacteria in the absence of light. E. coli and S. aureus The inactivation rates were 59.72% and 37.15%, respectively; under light, these rates increased to 97.91% and 80.02%. For Gram-positive bacteria... S. aureus MIC 90 The concentration was 375 µg / mL, showing broad-spectrum light-dependent antibacterial activity, but data on free radical scavenging and enzyme response characteristics were lacking.

[0099] In contrast, the present invention Cu-CDs@HA not only targets S. aureus and E. coli The MICs of this study are significantly better than those in the literature, with an ABTS clearance rate of 99% and a peroxidase-like Km of only 0.087 mM (a reduction of 79.3%). Furthermore, it achieves a 93.7% reduction in biofilm biomass through HA enzyme response, overcoming the limitations of light-dependent and non-selective release in the literature and significantly improving targeting and biocompatibility.

[0100] The above embodiments of the present application do not describe all the details and do not limit the present application to the above described embodiments. Various changes, modifications, substitutions and variations of these embodiments, which are made without departing from the principles and spirit of the present application, should be included in the scope of protection of the present application.

Claims

1. A method for preparing an enzyme-responsive antibacterial carbon dot, characterized by The method comprises: 1) using folate as a carbon source and a soluble copper salt as a copper source, subjecting an aqueous solution containing the copper source and the folate to a hydrothermal reaction at 180-220 DEG C, and purifying the reaction solution to obtain copper-doped carbon dots in which copper is doped in the form of Cu(II) and Cu(I); 2) in a buffer solution system with a pH value of 5.5-6.7, subjecting the copper-doped carbon dots to carboxyl activation by using 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, adding an aqueous hyaluronic acid solution, adjusting the pH value to neutral, and performing an amidation reaction, and purifying the reaction product to obtain enzyme-responsive antibacterial carbon dots grafted with hyaluronic acid.

2. The method of claim 1 wherein The mass ratio of the copper source to the folate is 1:(1-5).

3. The method of claim 1 wherein The buffer solution system uses a MES buffer solution with a concentration of 0.05-0.2 M.

4. The method of claim 1 wherein The mass ratio of the copper-doped carbon dots to the hyaluronic acid is 1:(1.5-2.5).

5. Enzyme-responsive antibacterial carbon dots prepared by the method of any one of claims 1-4, wherein the carbon dots have a particle size of 2-10 nm, the antibacterial active ingredient of the copper-doped carbon dots is used as a core, copper is doped in the form of Cu(II) and Cu(I) in the carbon dots, the amide bond on the surface of the core is used to connect hyaluronic acid molecules to form a core-shell structure, and the copper-doped carbon dots antibacterial active ingredient with peroxidase-like activity and free radical scavenging capacity is released by responding to the enzyme hydrolysis of hyaluronidase.

6. Use of the enzyme-responsive antibacterial carbon dots of claim 5 in the preparation of medical materials for preventing and / or treating bacterial infections.

7. Use according to claim 6, characterized in that The bacterial infection is an infection associated with overexpression of hyaluronidase.

8. An antibacterial composition comprising the enzyme-responsive antibacterial carbon dots of claim 5 and a pharmaceutically or materially acceptable carrier.

9. The antibacterial composition of claim 8, wherein the antibacterial composition is in the form of a gel, a film or a fiber.

10. The antibacterial composition of claim 8, wherein the antibacterial composition is configured as a wound dressing, an antibacterial spray or an antibacterial coating.

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