A peptide-like biomimetic carbon dot, its preparation method and application

Abstract

This invention provides a peptide-like biomimetic carbon dot, its preparation method, and its application, belonging to the field of nanobiomaterials and antibacterial technology. Using carbon dots as a matrix, antibacterial peptide molecules are grafted onto the carbon dot matrix and doped with N and Fe heteroatoms. These molecules bind to the carbon dot matrix via amide bonds or electrostatic interactions. The molecular weight of the antibacterial peptide molecules is 1000 Da to 5000 Da; the doping amount of N heteroatoms is 8 at% to 18 at%, and the doping amount of Fe heteroatoms is 1 at% to 5 at%. The loading amount of the antibacterial peptide molecules is 5 wt% to 25 wt%. This achieves a dual synergistic function of targeted anti-biofilm activity and cell selectivity. The three functions work together to effectively remove bacterial biofilms while maintaining both anti-biofilm activity and cell selectivity, avoiding damage to normal host cells at effective antibacterial concentrations.

Description

Technical Field

[0001] This invention relates to the fields of nanobiomaterials and antibacterial technology, and more specifically, to a peptide-like biomimetic carbon dot, its preparation method, and its application. Background Technology

[0002] Bacterial biofilms are microbial communities formed by bacteria and their secreted extracellular polymeric matrix (EPS). They exhibit strong drug resistance and resilience. Statistics show that approximately 80% of clinical infections are related to biofilms, such as infections associated with medical catheters, wound infections, and artificial joint infections. Traditional antibiotics struggle to penetrate the dense EPS barrier, failing to effectively kill bacteria within the biofilm. Furthermore, long-term use can induce drug resistance in bacteria, leading to recurrent infections and posing a significant challenge to clinical treatment.

[0003] Polypeptides, as a type of non-natural peptide-like polymers, have a cationic amphiphilic structure similar to antimicrobial peptides (AMPs). They can bind to bacterial membranes through electrostatic interactions, disrupting membrane integrity. They also have advantages such as high stability, low immunogenicity, and low susceptibility to inducing drug resistance, showing good application potential in the field of antibacterial agents.

[0004] Carbon dots (CDs) are a class of carbon-based nanomaterials with a size of less than 10 nm. They have excellent biocompatibility, fluorescence properties, easy functionalization and low cytotoxicity, and can be used as carriers to achieve targeted delivery and fluorescence tracing of antibacterial molecules. By combining the biomimetic antibacterial properties of peptides with the structural advantages of carbon dots, peptide-like biomimetic carbon dots can be constructed, which is expected to achieve a synergistic improvement in targeted anti-biofilm and biocompatibility.

[0005] Currently, a search reveals that some studies have combined peptides with carbon dots for antibacterial purposes, but existing technologies still have many shortcomings: First, most peptide-carbon dot composite materials only focus on antibacterial effects and lack targeting of biofilms, making it difficult to achieve precise removal of biofilms; Second, some materials cannot simultaneously achieve both anti-biofilm activity and cell selectivity, and are prone to damaging normal host cells at effective antibacterial concentrations. In addition, existing anti-biofilm nanomaterials mostly rely on a single antibacterial mechanism (such as simple membrane disruption or ROS oxidation), which has limited antibacterial effect and is easily tolerated by bacteria. For example, patent CN115671264A discloses a cascaded nanozyme, which can target and catalyze the destruction of biofilms, but it relies on the synergy between glucose oxidase and nanozyme, resulting in a complex structure and poor stability. Patent CN108483426B discloses low-toxicity carbon dots based on peptides, but it lacks anti-biofilm targeting and has weak antibacterial activity. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of existing peptide-carbon dot composite materials, such as poor targeting and high cytotoxicity, as well as the single antibacterial mechanism and poor stability of existing anti-biofilm nanomaterials. The invention provides a peptide-based biomimetic carbon dot that is easy and controllable to prepare, has stable peptide loading, uniform heteroatom doping, and has both targeted anti-biofilm and cell selective functions, along with its preparation method and applications.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A biomimetic carbon dot-like peptide is characterized by using carbon dots as a matrix, grafting antibacterial peptide molecules onto the carbon dot matrix and doping them with N and Fe heteroatoms.

[0008] The antimicrobial peptide molecule is terminally modified with at least one of amino or thiol groups, and binds to the carbon dot matrix through amide bonds or electrostatic interactions. The molecular weight of the antimicrobial peptide molecule is 1000 Da to 5000 Da.

[0009] The doping concentration of N heteroatoms is 8 at% to 18 at%, and the doping concentration of Fe heteroatoms is 1 at% to 5 at%. Specifically, an N doping concentration of 8 to 18 at% significantly increases the surface positive charge density of carbon dots, enhancing their electrostatic adsorption capacity for negatively charged bacteria. Simultaneously, N atom incorporation into the carbon framework can form various configurations such as graphitic nitrogen and pyridine nitrogen, effectively regulating the band structure of the carbon dots and improving fluorescence quantum yield. Controlling the doping concentration within this range avoids carbon structural defects and destruction of luminescent centers caused by excessive N doping. 1 to 5 at% doping concentrations... At an at% Fe doping level, uniformly distributed catalytic active centers are formed in the carbon framework, enabling efficient catalysis of Fenton-like reactions in infected microenvironments, generating high concentrations of hydroxyl radicals. This doping range ensures sufficient catalytic activity while avoiding non-specific oxidative damage, increased biotoxicity, and Fe ion leakage risks caused by excessive Fe doping. Within this specific ratio range, N doping and Fe doping form an optimal synergistic effect: the enhanced bacterial adsorption capacity of N doping allows the Fe-catalyzed hydroxyl radicals to act at closer distances, improving the utilization rate of reactive oxygen species; the Fe-doped catalytic reaction can further oxidize and destroy the structure of the adsorbed bacteria, forming a "capture-kill" closed loop.

[0010] The loading of antimicrobial peptide molecules is 5wt%~25wt%, which ensures sufficient density of antimicrobial active groups to achieve efficient sterilization, while avoiding problems such as material aggregation, decreased biocompatibility and increased cost caused by excessive loading.

[0011] Another objective is the method for preparing the aforementioned peptide-like biomimetic carbon dots, characterized by comprising the following steps: The carbon source, antimicrobial peptide, nitrogen source, and iron source were dissolved in deionized water, and the pH was adjusted to maintain the stability of the amphiphilic structure of the peptide, thus obtaining a precursor mixture.

[0012] The precursor mixture was subjected to a carbonization reaction, followed by post-processing, to obtain peptide-like biomimetic carbon dots.

[0013] In this embodiment of the invention, the antimicrobial peptide is at least one of poly(N-allylglycine) derivatives and polylysine peptides, used to target and recognize bacterial membranes and extracellular polymers of biomembranes and disrupt membrane integrity.

[0014] The N source is at least one of ammonia, urea, and ethylenediamine. The N source is used for heteroatom doping and adjusting the pH of the solution to promote the interaction between peptides and carbon sources.

[0015] The Fe source is at least one of ferric chloride, ferrous sulfate, and ferric nitrate, ensuring that the Fe ions have Fenton-like activity and catalyze the generation of hydroxyl radicals. The two work synergistically to optimize the targeted anti-biofilm performance.

[0016] In this embodiment of the invention, the particle size of the peptide-like biomimetic carbon dots is 3nm~10nm, the Zeta potential is +15mV~+40mV, and the quantum yield is 5%~30%. The carbon source is at least one of citric acid, urea, and glucose, used to improve the fluorescence quantum yield and heteroatom doping uniformity of the carbon dots. The carbon source forms a carbon core with a conjugated structure through a hydrothermal carbonization reaction, providing reaction sites for peptide molecule grafting and heteroatom doping. Furthermore, citric acid contains abundant carboxyl groups, which can undergo amidation reaction with the amino groups of peptide molecules during carbonization, enhancing the binding stability of peptides and carbon dots. Urea decomposes during carbonization to provide a nitrogen source, achieving in-situ N doping and simplifying the preparation process. Glucose, as a natural biomass carbon source, has excellent biocompatibility and renewability. The carbon dots formed after carbonization are rich in hydroxyl groups on their surface, facilitating subsequent functionalization modification.

[0017] The antimicrobial peptide molecules are terminally modified with at least one of amino or thiol groups, and bind to the carbon dot matrix via amide bonds or electrostatic interactions. The molecular weight of the antimicrobial peptide molecules is 1000 Da to 5000 Da. The terminal modification with amino or thiol active functional groups enables them to undergo specific chemical reactions with active groups such as carboxyl and epoxy groups on the carbon dot surface, forming stable covalent bonds. This significantly improves the grafting stability and anti-desorption ability of the peptides on the carbon dot surface, ensuring the structural integrity of the material in complex physiological environments. Peptide chains below 1000 Da are too short, making it difficult to effectively insert into and disrupt the bacterial membrane bilayer structure, resulting in insufficient antimicrobial activity. Peptide chains above 5000 Da are too long, with large steric hindrance, limiting grafting density, and potentially posing an immunogenicity risk. Peptides within this molecular weight range possess both good membrane insertion ability and appropriate spatial conformation, enabling the formation of a high-density active layer on the carbon dot surface, achieving efficient membrane-breaking and sterilization.

[0018] In this embodiment of the invention, the mass ratio of carbon source: antimicrobial peptide: N source: Fe source is 1~2:0.2~0.8:0.5~1.5:0.05~0.2; the pH is adjusted to 6.5~8.0; the carbonization reaction temperature is 160℃~200℃, and the carbonization reaction time is 8h~12h.

[0019] In this embodiment of the invention, the post-processing involves centrifugation followed by filtration of the supernatant through a 0.1μm~0.22μm filter membrane, dialysis through a dialysis bag, and then vacuum freeze-drying at -60℃~-70℃ and 0MPa~0.01MPa.

[0020] In this embodiment of the invention, the centrifugation speed is 8000rpm~12000rpm and the centrifugation time is 10min~15min; the molecular weight cutoff of the dialysis bag is 500Da~1000Da and the dialysis time is 24h~48h, and the deionized water is replaced every 8h~12h to remove unreacted impurities and improve the purity and biocompatibility of the material.

[0021] The aforementioned peptide-like biomimetic carbon dots were post-modified to obtain peptide-like biomimetic carbon dots with amide bond reinforcement.

[0022] In this embodiment of the invention, the specific post-modification step is as follows: the peptide-like biomimetic carbon dots are dispersed in 0.01 mol / L to 0.05 mol / L phosphate buffer solution with pH 7.2 to 7.4, and a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is added as a crosslinking agent. The mixture is stirred at room temperature for 2 to 4 hours, then centrifuged, dialyzed, and freeze-dried to obtain amide bond-reinforced peptide-like biomimetic carbon dots. The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) to N-hydroxysuccinimide (NHS) is 1:1 to 1.2.

[0023] The peptide-like biomimetic carbon dots have a particle size of 3nm~10nm, a zeta potential of +15mV~+40mV, and a quantum yield of 5%~30%. Controlling the particle size within the 3nm~10nm range ensures excellent cell penetration and biomembrane permeability of the carbon dots while avoiding rapid renal clearance due to excessively small size or capture by the reticuloendothelial system due to excessively large size. The zeta potential controlled at +15mV~+40mV ensures strong electrostatic adsorption to negatively charged bacterial membranes while avoiding non-specific cytotoxicity and serum protein adsorption caused by excessively high positive charges. The 5%~30% fluorescence quantum yield endows the material with good fluorescence tracing ability, enabling real-time visual monitoring of the antibacterial process and providing timely feedback for clinical treatment.

[0024] In this embodiment of the invention, the antimicrobial peptide is at least one of poly(N-allylglycine) derivatives and polylysine peptides, used to target and recognize bacterial membranes and extracellular polymers of biofilms and disrupt membrane integrity. Furthermore, the antimicrobial peptide molecules inherit the cationic amphiphilic structural characteristics of natural antimicrobial peptides, and can specifically recognize abundant anionic components (such as teichoic acid, lipopolysaccharide, extracellular DNA, etc.) in bacterial membranes and biofilm EPS, disrupting their integrity by inserting into the membrane bilayer structure, thus having the advantage of physical bactericidal action and being less likely to induce bacterial resistance.

[0025] Amide bond linkage provides a highly stable chemical bond, suitable for applications requiring long-term stability; electrostatic bonding is simple and rapid, enabling peptide self-assembly modification under mild conditions while preserving the peptide's natural conformation and biological activity; both bonding methods can be flexibly selected according to application requirements, expanding the material's applicability.

[0026] In this embodiment of the invention, N doping is used to enhance the positive charge density and fluorescence performance of the carbon dot surface, while Fe doping is used to endow the carbon dot with Fenton-like activity, catalyzing the generation of hydroxyl radicals. This enables the carbon dot to catalyze the decomposition of hydrogen peroxide in a weakly acidic infection microenvironment to generate highly oxidizing hydroxyl radicals (·OH). Through the synergistic effect of N doping and Fe doping, the carbon dot's targeting adhesion ability and catalytic bactericidal performance are optimized, achieving efficient removal of biofilms.

[0027] It should be noted that this step is for scenarios that require further improvement in the stability of peptide loading. The appropriate step can be selected based on the actual scenario. Specifically, for biomimetic carbon dots that are initially bound mainly by electrostatic interactions, the post-modification step transforms the unstable electrostatic binding into stable covalent amide bond linkages, significantly improving the grafting stability of peptide molecules on the carbon dot surface. This prevents peptide desorption in complex physiological environments (high salt, high protein, wide pH range) and ensures the structural integrity and functional durability of the material.

[0028] More specifically as follows: S1: Preparation of precursor mixture: Disperse carbon source, antimicrobial peptide, N source, and Fe source in deionized water at a mass ratio of (1~2):(0.2~0.8):(0.5~1.5):(0.05~0.2), stir for 10-30 minutes until completely dissolved, adjust the pH to 6.5-8.0 to maintain the stability of the amphiphilic structure of the peptide, and obtain the precursor mixture. Specifically, this ratio range can ensure the formation and growth of carbon dots, and also achieve effective grafting of peptides and uniform doping of heteroatoms. Adjusting the pH of the precursor solution to a neutral to slightly alkaline range of 6.5-8.0 maintains the stability of the amphiphilic conformation of the antimicrobial peptide and prevents it from being over-protonated under acidic conditions or hydrolyzed and inactivated under alkaline conditions. This pH range also promotes the dehydration and carbonization reaction of the carbon source, which is beneficial to the formation and growth of carbon dots. S2: Co-carbonization reaction: The precursor mixture is transferred to a polytetrafluoroethylene reactor and placed in an oven at 160℃~200℃ for 8h~12h. After natural cooling to room temperature, a crude product is obtained. The reaction time of 8h~12h ensures the full formation and growth of carbon points, while avoiding agglomeration caused by over-reaction. S3: Purification: The crude product is centrifuged, and the supernatant is filtered sequentially through a 0.22μm filter membrane, dialyzed through a dialysis bag, and freeze-dried to obtain peptide-like biomimetic carbon dot powder. Specifically, centrifugation at 8000rpm~12000rpm removes large particle precipitates; filtration through a 0.22μm filter membrane removes small insoluble matter; a 500Da~1000Da dialysis bag selectively removes unreacted small molecule precursors, salt ions, and oligomers, while retaining peptide-like biomimetic carbon dots with appropriate molecular weight; dialysis for 24h~48h with water changes every 8h~12h ensures thorough removal of impurities and improves material purity; freeze-drying preserves the structure and activity of the material, facilitating long-term storage and use.

[0029] The carbon source mentioned in step S1 is a mixture of citric acid and urea, with a mass ratio of citric acid to urea of ​​1:(0.8~1.2). This mixture is used to improve the fluorescence quantum yield and heteroatom doping uniformity of the carbon dots. Specifically, citric acid and urea are mixed in a ratio of 1:(0.8~1.2) to form a synergistic effect. Citric acid provides abundant carboxyl groups during carbonization, which react with the amino groups of peptide molecules to enhance grafting stability. Urea decomposes at high temperature to provide a nitrogen source, enabling in-situ N doping. The two work together to form a carbon core with high fluorescence quantum yield, and the fluorescence performance is better than that of carbon dots prepared with a single carbon source.

[0030] Secondly, urea, as a nitrogen source, is uniformly dispersed in the precursor solution, achieving uniform molecular-level doping of N elements during carbonization, thus avoiding the problem of uneven doping that may be caused by external nitrogen sources. Uniform N doping ensures the uniformity of surface charge distribution of carbon dots, which is conducive to the formation of stable surface potential and controllable bacterial adsorption capacity.

[0031] The N source is at least one of ammonia, urea, and ethylenediamine. The N source is used for heteroatom doping and adjusting the pH of the solution to promote the interaction between peptides and carbon sources.

[0032] The Fe source is at least one of ferric chloride, ferrous sulfate, and ferric nitrate, ensuring uniform dissolution of Fe ions and enhancing Fenton-like activity. Specifically, the selected N source (ammonia, urea, ethylenediamine) serves as both a dopant and a pH adjuster: it decomposes at high temperatures to provide N atoms for incorporation into the carbon framework; simultaneously, it provides an alkaline environment in solution, adjusting the pH of the precursor solution to a suitable range (6.5~8.0), simplifying the preparation process and avoiding the introduction of additional impurities by adding external acid-base adjusters.

[0033] The Fe source is selected from water-soluble iron salts such as ferric chloride, ferrous sulfate, and ferric nitrate to ensure that Fe ions are uniformly dispersed in the precursor solution in an ionic state, avoiding agglomeration and precipitation. During the carbonization process, these iron salts undergo coordination-carbonization reactions with the carbon source, and Fe ions are reduced and stably incorporated into the carbon skeleton to form uniformly distributed catalytic active centers, ensuring the uniformity and stability of Fenton-like activity.

[0034] In step S3, the centrifugation speed is 8000rpm~12000rpm, and the centrifugation time is 10min~15min. The molecular weight cutoff of the dialysis bag is 500Da~1000Da, the dialysis time is 24h~48h, and the deionized water is replaced every 8h~12h to remove unreacted impurities and improve the purity and biocompatibility of the material. Specifically, centrifugation at 8000rpm~12000rpm for 10min~15min can effectively remove large carbon aggregates, unreacted raw material precipitates and insoluble matter formed during the reaction without settling the target product (peptide-like biomimetic carbon dots, particle size 3nm~10nm), thereby improving the purity and dispersibility of the product.

[0035] By using dialysis bags with a molecular weight cutoff of 500 Da to 1000 Da, salt ions with a molecular weight below 500 Da, small molecule precursors (such as unreacted citric acid, urea, iron salts, etc.) and byproducts can diffuse freely during use; while peptide-like biomimetic carbon dots with a molecular weight above 1000 Da are retained. This cutoff range is exactly between the target product and impurities, achieving efficient separation.

[0036] Dialysis for 24-48 hours ensures thorough removal of impurities, and replacing deionized water every 8-12 hours maintains the concentration gradient inside and outside the membrane, accelerating the diffusion process. Thorough dialysis removes small molecule residues that may cause cytotoxicity, significantly improving the biocompatibility and clinical safety of the material.

[0037] Furthermore, the EDC / NHS molar ratio of 1:1 to 1.2 ensures the high efficiency of carboxyl activation: EDC activates carboxyl groups to form unstable O-acyl isourea intermediates, which are then converted into semi-stable NHS esters by NHS, thus improving the efficiency of the amidation reaction; the reaction time of 2 to 4 hours ensures sufficient cross-linking while avoiding material aggregation caused by over-reaction; and the buffer system with a pH of 7.2 to 7.4 maintains the optimal active pH range for EDC / NHS.

[0038] After being incubated for 72 hours in simulated physiological conditions (PBS buffer, cell culture medium, serum), the peptide-like biomimetic carbon dots with reinforced peptides showed a detachment rate of <5%, which was significantly better than the unreinforced group (detachment rate >20%), ensuring the reliability of long-term antibacterial application.

[0039] An application of a peptide-like biomimetic carbon dot, wherein the peptide-like biomimetic carbon dot is used to prepare anti-biofilm antibacterial agents, antibacterial coatings on the surface of biomedical materials, or for targeted treatment of clinical bacterial infections.

[0040] Specifically, this type of peptide biomimetic carbon dots includes, but is not limited to: preparing cleaning solutions or coating materials for removing biofilms from the surface of medical devices; preparing antibacterial dressings or gel formulations for treating refractory wounds such as chronic wound infections and diabetic foot ulcers; preparing targeted delivery systems for treating in vivo bacterial infections such as respiratory tract infections and urinary tract infections; and preparing surface-modified coatings for implantable medical devices (such as catheters, artificial joints, and heart valves) to prevent biofilm-related infections.

[0041] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention achieves a dual-function synergy of targeted antimicrobial action and cell selectivity by grafting antimicrobial peptide molecules onto a carbon dot matrix and doping it with N and Fe heteroatoms. On the one hand, the cationic amphiphilic structure of the antimicrobial peptide molecules can target and recognize bacterial membranes and extracellular polymers of biofilms, thereby disrupting membrane integrity. N doping enhances the positive charge density on the carbon dot surface, further improving the targeting and enrichment ability. Fe doping imparts Fenton-like activity, which can catalyze the generation of hydroxyl radicals, forming a dual antimicrobial effect with the membrane disruption mechanism of the peptides. This effectively removes bacterial biofilms, and the antimicrobial mechanism does not rely on a single action pathway, making it less likely to induce drug resistance in bacteria. On the other hand, the carbon dots themselves possess excellent biocompatibility and low cytotoxicity. Combined with the structural differences between bacterial membranes and host cell membranes, precise cell-selective recognition is achieved, avoiding damage to normal host cells and meeting the biosafety requirements for clinical applications.

[0042] 2. This invention limits the key structural parameters and component ratios of peptide-like biomimetic carbon dots to ensure stable material performance that can be controlled as needed. The carbon dopants are doped with N and Fe atoms, which synergistically optimize the targeted anti-biofilm performance, improving both the fluorescence characteristics and positive charge density of the carbon dots and ensuring the efficient performance of Fenton-like activity. The peptide molecules are stably grafted through amide bonds or electrostatic interactions, resulting in a low detachment rate. After the material is placed in a physiological environment, it exhibits excellent dispersibility and functional stability.

[0043] 3. This invention employs a co-carbonization method as the primary preparation process. Carbon source, antimicrobial peptide, N source, and Fe source are mixed in a precise mass ratio, and carbonization, peptide grafting, and heteroatom doping are completed in a single step. This simplifies the cumbersome reaction steps of existing post-modification methods and effectively avoids problems such as peptide structure destruction and uneven heteroatom doping that easily occur in co-carbonization. During the preparation process, the peptide loading and N / Fe heteroatom doping amount can be precisely controlled by adjusting the precursor ratio. The parameters for purification steps such as centrifugation, filtration, and dialysis are clearly defined, resulting in good process repeatability. Furthermore, the selected carbon, N, and Fe sources are all widely available and inexpensive conventional reagents, requiring no complex equipment or special raw materials.

[0044] 4. The peptide-like biomimetic carbon dots of the present invention can be widely used in the preparation of antimicrobial agents against biofilms, the construction of antimicrobial coatings on the surface of biomedical materials, and the targeted treatment of clinical bacterial infections. They are suitable for various infection scenarios caused by Gram-positive bacteria and Gram-negative bacteria (such as Staphylococcus aureus and Escherichia coli), as well as the antimicrobial modification of various biomedical materials such as medical catheters, artificial joints, and wound dressings. Attached Figure Description

[0045] Figure 1 This is a transmission electron microscope (TEM) image of the peptide-like biomimetic carbon dots obtained in Example 1 of the present invention.

[0046] Figure 2 The images show the effect of peptide-like biomimetic carbon dots obtained in Examples 1-3 of this invention on the removal of Staphylococcus aureus biofilm.

[0047] Figure 3 The images show the toxicity test results of the peptide-like biomimetic carbon dots obtained in Examples 1-3 of this invention on 3T3 cells.

[0048] Figure 4 The figure shows the carbon dot stability test results of the peptide-like biomimetic obtained in Example 1 of the present invention. Detailed Implementation

[0049] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0050] Example 1: Preparation of a peptide-like biomimetic carbon dot: (1) Preparation of precursor mixture: Take 1.0 g citric acid (carbon source), 0.5 g poly(N-allyl glycine) (antimicrobial peptide, molecular weight 3000 Da), 1.0 g urea (N source), and 0.1 g ferric chloride (Fe source), disperse them in 50 mL deionized water, stir at 180 rpm for 20 min until completely dissolved, adjust the pH to 7.0 with ammonia water to obtain the precursor mixture; (2) Co-carbonization reaction: The precursor mixture was transferred to a polytetrafluoroethylene reactor, placed in an oven at 180°C for 10 h, and then naturally cooled to room temperature to obtain the crude product. (3) Purification: The crude product was centrifuged at 10,000 rpm for 12 min. The supernatant was filtered through a 0.22 μm filter membrane. The filtrate was then transferred to a dialysis bag with a molecular weight cutoff of 800 Da. Dialysis was performed for 36 h, with deionized water replaced every 12 h. After freeze-drying, peptide-like biomimetic carbon dot powder was obtained. (4) Post-modification: The powder obtained in step (3) was dispersed in 0.03 mol / L phosphate buffer with pH=7.3, and EDC and NHS (molar ratio 1:1.1) were added. The mixture was stirred at room temperature for 3 h, centrifuged at 10000 rpm for 12 min, and the supernatant was dialyzed for 24 h. After freeze-drying, the modified peptide-like biomimetic carbon dots were obtained.

[0051] The peptide-like biomimetic carbon dots obtained in this embodiment have a particle size of 5~8 nm, a Zeta potential of +25~+35 mV, a quantum yield of 15~25%, a peptide loading of 12~18 wt%, an N doping amount of 12~15 at%, and an Fe doping amount of 2~4 at.

[0052] Example 2: Preparation of a peptide-like biomimetic carbon dot: (1) Preparation of precursor mixture: Take 1.0g glucose (carbon source), 0.2g polylysine peptide (antimicrobial peptide, molecular weight 1000Da), 0.5g ethylenediamine (N source), and 0.05g ferrous sulfate (Fe source), disperse them in 40mL deionized water, stir at 150rpm for 10min until completely dissolved, adjust the pH to 6.5 with hydrochloric acid to obtain precursor mixture; (2) Co-carbonization reaction: The precursor mixture was transferred to a polytetrafluoroethylene reactor, placed in an oven at 160°C for 12 hours, and then naturally cooled to room temperature to obtain the crude product. (3) Purification treatment: The crude product was centrifuged at 8000 rpm for 15 min, the supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was transferred to a dialysis bag with a molecular weight cutoff of 500 Da. Dialysis was performed for 24 h, and the deionized water was replaced every 8 h. After freeze drying, peptide-like biomimetic carbon dot powder was obtained.

[0053] The peptide-like biomimetic carbon dots obtained in this embodiment have a particle size of 3nm~6nm, a Zeta potential of +15~+25 mV, a quantum yield of 5%~15%, a peptide loading of 5wt%~10wt%, an N doping amount of 8at%~12at, and an Fe doping amount of 1at%~2at.

[0054] Example 3: Preparation of a peptide-like biomimetic carbon dot: (1) Preparation of precursor mixture: Take 1.0g urea (carbon source), 0.8g poly(N-allyl glycine) derivative (antimicrobial peptide, molecular weight 5000Da), 1.5g ammonia (N source), and 0.2g ferric nitrate (Fe source), disperse them in 60mL deionized water, stir at 200rpm for 30min until completely dissolved, adjust the pH to 8.0 with ammonia to obtain precursor mixture; (2) Co-carbonization reaction: The precursor mixture was transferred to a polytetrafluoroethylene reactor, placed in an oven at 200°C for 8 hours, and then naturally cooled to room temperature to obtain the crude product. (3) Purification: The crude product was centrifuged at 12000 rpm for 10 min, the supernatant was filtered through a 0.22 μm filter membrane, and the filtrate was transferred to a dialysis bag with a molecular weight cutoff of 1000 Da. Dialysis was performed for 48 h, and the deionized water was replaced every 12 h. After freeze drying, peptide-like biomimetic carbon dot powder was obtained. (4) Post-modification: The powder obtained in step (3) was dispersed in 0.05 mol / L phosphate buffer with pH=7.4, and EDC and NHS (molar ratio 1:1) were added. The mixture was stirred at room temperature for 4 h, centrifuged at 12000 rpm for 10 min, and the supernatant was dialyzed for 24 h. After freeze drying, the modified peptide-like biomimetic carbon dots were obtained.

[0055] The peptide-like biomimetic carbon dots obtained in this embodiment have a particle size of 7nm~10nm, a Zeta potential of +30mV~+40mV, a quantum yield of 20%~30%, a peptide loading of 20wt%~25wt%, an N doping amount of 15at%~18at, and an Fe doping amount of 4at%~5at.

[0056] Results Analysis 1. Anti-biofilm performance test: Test method: The crystal violet staining method was used to evaluate the removal effect of the samples on Staphylococcus aureus biofilm. The mature biofilm formed by pre-culturing for 24 hours was co-incubated with the peptide-like biomimetic carbon dots (50 μg / mL) obtained in Examples 1 to 3 for 4 hours. After washing with phosphate buffer to remove airborne bacteria, the biofilm was fixed with methanol, stained with 0.1% crystal violet, dissolved in glacial acetic acid, and the absorbance was measured to calculate the biofilm removal rate. Test results: such as Figure 2 As shown, compared with the dense biofilm formed in the control group (without added materials), the biofilm structure was significantly disrupted after treatment with samples from Examples 1 to 3, demonstrating a significant removal effect. Quantitative analysis showed that the peptide-like biomimetic carbon dots obtained in Examples 1 to 3 all achieved a removal rate of over 99% for Staphylococcus aureus biofilm. Among them, Example 1 (optimized ratio group) showed the most outstanding removal effect, with a biofilm removal rate of over 99.5%, indicating that the peptide-like biomimetic carbon dots described in this invention have excellent removal ability for mature biofilms.

[0057] Effect analysis: The results confirm that the present invention can effectively penetrate and destroy the extracellular polymeric substance (EPS) matrix of biofilm by combining the targeted membrane-breaking effect of antimicrobial peptides with the synergistic effect of Fe doping-induced Fenton-like catalytic oxidation, thereby killing internal bacteria and achieving a highly efficient anti-biofilm effect.

[0058] 2. Cell selectivity (biocompatibility) test: Test methods: The MTT assay was used to evaluate the in vitro cytotoxicity of the samples against mouse fibroblasts (3T3). Different concentrations of the samples from Examples 1-3 were co-incubated with 3T3 cells for 24 h, followed by incubation for another 4 h with MTT reagent. After dissolving the formazan crystals with dimethyl sulfoxide, the absorbance was measured, and the relative cell viability was calculated. The cytotoxicity at the effective antibacterial concentration (50 μg / mL) was the primary focus.

[0059] Test results: such as Figure 3 As shown, at an effective antibacterial concentration of 50 μg / mL, the survival rate of 3T3 cells treated in Examples 1 to 3 all reached over 90%. Among them, Example 1 showed the lowest cytotoxicity and a cell survival rate of over 95%, which was not significantly different from the control group (no material added, cell survival rate set at 100%).

[0060] Effect Analysis: The results show that the peptide-like biomimetic carbon dots described in this invention have good biocompatibility with normal mammalian cells at effective antibacterial concentrations, exhibiting excellent cell selectivity. This is due to the precise control of the material particle size (3nm~10nm), the optimized regulation of the surface charge (Zeta potential +15mV~+40mV), and the biomimetic structural design of the peptide molecules, which enable them to effectively distinguish between bacteria and host cells, avoiding the defects of high non-specific cytotoxicity of traditional antibacterial materials.

[0061] 3. Physiological stability test: Test method: The peptide-like biomimetic carbon dots obtained in Example 1 were dispersed in phosphate buffer (PBS, pH 7.4, simulating physiological environment) and incubated in a constant temperature incubator at 37°C. Samples were taken at 1 day, 7 days, 14 days and 28 days. Dynamic light scattering (DLS) technology was used to determine the particle size and zeta potential changes of the material to evaluate its storage stability and structural integrity under physiological conditions.

[0062] Test results: such as Figure 4 As shown, during the 28-day continuous monitoring period, the particle size and zeta potential of the peptide-like biomimetic carbon dots obtained in Example 1 remained highly stable. Specifically, the particle size variation was ≤10% (initial particle size was about 5.2 nm, and after 28 days it was about 5.6 nm), and the zeta potential variation was ≤5 mV (initial potential was about +28.5 mV, and after 28 days it was about +26.5 mV).

[0063] Effect Analysis: These results demonstrate that the peptide-like biomimetic carbon dots described in this invention exhibit excellent colloidal and structural stability under physiological conditions (pH 7.4, 37℃). The antibacterial peptide molecules are firmly grafted onto the carbon dot surface through chemical bonding during the co-carbonization process, without significant desorption or degradation. No obvious aggregation or sedimentation was observed in the material. This excellent stability provides crucial assurance for its long-term circulation and sustained antibacterial activity in vivo, and also lays a solid foundation for the long-term storage of the formulation.

[0064] 4. Material morphology characterization Characterization method: The microstructure of the peptide-like biomimetic carbon dots obtained in Example 1 was observed using transmission electron microscopy (TEM); Characterization results: such as Figure 1 As shown, the peptide-like biomimetic carbon dots obtained in Example 1 exhibit a uniform spherical structure with good dispersibility and no obvious aggregation. The particle size distribution statistics show that the material size is mainly distributed in the range of 5nm to 8nm, which is consistent with the dynamic light scattering test results and meets the particle size range (3nm to 10nm) described in the claims. Performance analysis: Uniform nanoscale size and good dispersibility are the key foundations for the material to efficiently penetrate biological membranes, achieve intracellular delivery, and ensure batch-to-batch reproducibility. They also provide an ideal structural platform for subsequent functional modification and application.

[0065] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A peptide-like biomimetic carbon dot, characterized in that, Using carbon dots as a matrix, the carbon dot matrix is ​​grafted with antibacterial peptide molecules and doped with N and Fe heteroatoms; The antimicrobial peptide molecule is terminally modified with at least one of amino or thiol groups, and binds to the carbon dot matrix through amide bonds or electrostatic interactions. The molecular weight of the antimicrobial peptide molecule is 1000 Da to 5000 Da. The doping amount of N heteroatoms is 8at%~18at, and the doping amount of Fe heteroatoms is 1at%~5at; the loading amount of antimicrobial peptide molecules is 5wt%~25wt%.

2. The peptide-like biomimetic carbon dot according to claim 1, characterized in that, The peptide-like biomimetic carbon dots have a particle size of 3nm~10nm, a zeta potential of +15mV~+40mV, and a quantum yield of 5%~30%.

3. The peptide-like biomimetic carbon dot according to claim 1, characterized in that, The antimicrobial peptide molecule is at least one of poly(N-allylglycine) derivatives and polylysine peptides.

4. The method for preparing peptide-like biomimetic carbon dots according to claim 1, characterized in that, Includes the following steps: The carbon source, antimicrobial peptide, nitrogen source, and iron source were dissolved in deionized water, and the pH was adjusted to maintain the stability of the amphiphilic structure of the peptide to obtain a precursor mixture. The precursor mixture is subjected to a carbonization reaction. During the reaction, carbon dots doped with N and Fe are formed, and antibacterial peptides are grafted onto the surface of the carbon dots. After post-treatment, peptide-like biomimetic carbon dots are obtained.

5. The method for preparing peptide-like biomimetic carbon dots according to claim 4, characterized in that, The antimicrobial peptide is at least one of poly(N-allylglycine) derivatives and polylysine peptides; The N source is at least one of ammonia, urea, and ethylenediamine; The Fe source is at least one of ferric chloride, ferrous sulfate, and ferric nitrate; the carbon source is at least one of citric acid, urea, and glucose.

6. The method for preparing peptide-like biomimetic carbon dots according to claim 4, characterized in that, The carbon source is at least one of citric acid, urea, and glucose.

7. The method for preparing peptide-like biomimetic carbon dots according to claim 4, characterized in that, The mass ratio of carbon source: antimicrobial peptide: N source: Fe source is 1~2:0.2~0.8:0.5~1.5:0.05~0.2; adjust the pH to 6.5~8.0; the carbonization reaction temperature is 160℃~200℃, and the carbonization reaction time is 8h~12h.

8. The method for preparing peptide-like biomimetic carbon dots according to claim 4, characterized in that, Also includes: The aforementioned peptide-like biomimetic carbon dots were post-modified to obtain amide bond-strengthened peptide-like biomimetic carbon dots.

9. The method for preparing peptide-like biomimetic carbon dots according to claim 8, characterized in that, The specific steps of the post-modification are as follows: the peptide-like biomimetic carbon dots are dispersed in 0.01mol / L~0.05mol / L phosphate buffer solution with pH 7.2~7.4, and a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is added as a crosslinking agent. The mixture is stirred at room temperature for 2h~4h, centrifuged again, dialyzed, and freeze-dried to obtain amide bond-reinforced peptide-like biomimetic carbon dots. The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:1~1.

2.

10. The application of the peptide-like biomimetic carbon dots according to claim 1 in the preparation of anti-biofilm antibacterial agents, antibacterial coatings on the surface of biomedical materials, or in targeted therapy for clinical bacterial infections.

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