Penetrating photocatalytic nano-antibacterial agent, preparation method and application

Abstract

This invention provides a permeable photocatalytic nano-antibacterial agent, its preparation method, and its application. The agent uses graphitic carbon nitride nanosheets as the core, with a molecular weight of 10 encapsulated on their surface. 3 ~10 7 The hyaluronic acid nanoparticles form nanostructures with a particle size of 200-400 nm. The nano-graphite phase carbon nitride, with its sharp-edged, sheet-like structure, can physically disrupt drug-resistant bacterial biofilms. Hyaluronic acid enhances the stability of the nano-antibacterial agent in the physiological environment and is degraded by hyaluronidase within the drug-resistant bacterial biofilm, thus disrupting the biofilm's structural stability and promoting efficient penetration and accumulation of the antibacterial agent within the biofilm. Under laser irradiation, the nano-graphite phase carbon nitride catalyzes the generation of reactive oxygen species, synergistically achieving efficient biofilm removal. This nano-antibacterial agent possesses excellent solution stability, biosafety, and photocatalytic activity, making it suitable for treating diseases related to drug-resistant bacterial biofilm infections.

Description

Technical Field

[0001] This invention relates to the field of photocatalytic materials technology, specifically to a permeable photocatalytic nano-antibacterial agent, its preparation method, and its application. Background Technology

[0002] Bacterial infection refers to an acute infection, either local or systemic, caused by pathogenic or opportunistic pathogens invading the human body, growing and multiplying, and producing metabolites or secondary metabolites. Currently, antibiotics are the standard treatment for bacterial infections in clinical practice. However, the overuse of antibiotics promotes the development of bacterial resistance, even leading to the emergence of bacteria resistant to multiple antibiotics, commonly known as multidrug-resistant bacteria. According to statistics from the World Health Organization, 700,000 people die globally each year from drug-resistant bacterial infections, and this number is projected to rise to 10 million by 2050, significantly increasing the burden on public healthcare and finances.

[0003] During infection, scattered bacterial cells attach to the body or tissues, proliferate, and secrete polysaccharide matrix, lipid proteins, and fibrin to encapsulate them, forming a membrane-like structure known as a drug-resistant bacterial biofilm. Compared to planktonic bacteria, drug-resistant bacterial biofilms possess unique structures and microenvironments (such as hypoxia, nutrient deprivation, and low metabolism), reducing the resistant bacteria's sensitivity to the environment and resisting the host's immune system attack. This leads to a significant increase in antibiotic resistance within the biofilm. Therefore, traditional antibiotics are often ineffective in eliminating drug-resistant bacterial biofilms.

[0004] Photocatalytic antibacterial technology utilizes photocatalysts to generate electron-hole pairs upon light excitation. These pairs react with water and oxygen on the surface to produce highly oxidizing reactive oxygen species (ROS), such as superoxide radicals and hydroxyl radicals. These ROS destroy the cell membranes, proteins, and DNA of microorganisms, leading to their death and achieving highly efficient sterilization. Compared to traditional antibacterial methods, photocatalytic antibacterial technology offers advantages such as broad-spectrum efficiency, durability, safety, and low drug resistance. Currently commonly used photocatalytic materials mainly include titanium dioxide (TiO2), graphitic carbon nitride (g-C3N4, abbreviated as C3N4), bismuth oxide (Bi2O3), and silver-based composite photocatalytic materials. References can be made to Chinese patent application (CN110169979A) for a photocatalytic antibacterial film with good antibacterial effect, and Chinese patent (CN113842937B) for a visible light catalyst. These photocatalysts can efficiently utilize sunlight, expanding the practical application scope of photocatalytic antibacterial technology.

[0005] C3N4 is a carbon-based material that is readily available and inexpensive, and it also has excellent biocompatibility, which has attracted widespread attention. However, its poor water solubility means it cannot be stably preserved in physiological solutions for long periods, thus limiting its application and transformation in clinical drug-resistant bacterial biofilm infections. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a photocatalytic nano-antibacterial agent. This nano-antibacterial agent can improve the solution stability of photocatalytic drugs and destroy the biofilm structure by consuming hyaluronidase in the biofilm of drug-resistant bacteria, thereby improving its efficiency in treating diseases related to drug-resistant bacterial biofilm infections.

[0007] In a first aspect, the present invention provides a photocatalytic nano-antibacterial agent, the nano-antibacterial agent comprising nano-graphitic carbon nitride as a photocatalytic drug, wherein hyaluronic acid is coated on the surface of the nano-graphitic carbon nitride, the hyaluronic acid having a molecular weight range of 10. 3 -10 7 Da; wherein the nano-graphite phase carbon nitride has a sheet-like structure.

[0008] As a preferred technical solution, the particle size of nano-graphite phase carbon nitride coated with hyaluronic acid is 200-400 nm.

[0009] As a preferred technical solution, the sheet-like structure with sharp edges has the same characteristics as shown in the accompanying drawings. Figure 3 The transmission electron microscope (TEM) images are essentially the same, and the photocatalytic nano-antibacterial agent has the same characteristics as those shown in the attached drawings. Figure 4 The transmission electron microscope images are essentially the same.

[0010] As a preferred technical solution, the sheet-like structure includes a thickness that gradually decreases from the center to the edge of the nano-graphitic carbon nitride, forming a wedge-shaped or blade-shaped profile with a tip width ≤10nm.

[0011] As a preferred technical solution, the hyaluronic acid is modified with disulfide bonds, and the modification ratio of disulfide bonds is 1-3 disulfide bonds per 100 hyaluronic acid disaccharide units.

[0012] As a preferred technical solution, the method for modifying the disulfide bonds includes dissolving hyaluronic acid in a buffer solution, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide to activate the carboxyl group for 10-30 min, then adding cystamine, wherein the molar ratio of cystamine to the disaccharide unit of hyaluronic acid is 0.1:1-0.5:1, and reacting at room temperature for 12-24 h; after the reaction is completed, adding ethanol to precipitate, centrifuging to collect the precipitate, washing and drying to obtain disulfide-modified hyaluronic acid. Secondly, the present invention provides a method for preparing the aforementioned photocatalytic nano-antibacterial agent, specifically including the following steps: S1. Disperse graphitic carbon nitride powder in 36-38% HCl, mix and carry out etching reaction at room temperature. After the reaction is completed, centrifuge and filter, and wash the precipitate with water until neutral. S2. Add the precipitate to a hyaluronic acid aqueous solution, mix well, centrifuge and filter to obtain the permeable photocatalytic nano-antibacterial agent; As a preferred technical solution, in step S1, the mass / volume ratio of graphite phase carbon nitride powder to HCl is (1-5):2; the rotation speed of the magnetic stirrer is 600-1200 rpm; and the acid etching reaction time is 0.5-4 h.

[0013] As a preferred technical solution, in step S2, a cell disruptor is used for ultrasonication, with an ultrasonic power of 100~300W; the ultrasonication time is 2-5 hours, with a 4-second interval between every 2 seconds of ultrasonication; the molecular weight of the hyaluronic acid is in the range of 10. 3 -10 7 Da; The concentration of hyaluronic acid aqueous solution is 5-40 mg / mL; Thirdly, the present invention provides the application of the aforementioned photocatalytic nano-antibacterial agent in the preparation of a treatment for biofilm infection of drug-resistant bacteria.

[0014] The beneficial effects of this invention are as follows: 1. The permeable photocatalytic nano-antibacterial agent of the present invention uses graphite phase carbon nitride powder as the reaction substrate, and obtains a first nano system through acid etching reaction and physical exfoliation reaction. Then, hyaluronic acid is coated on the surface of the first nano system to improve the solution stability of the photocatalytic drug. At the same time, hyaluronic acid can consume hyaluronidase in the biofilm of drug-resistant bacteria, reduce the compactness of the biofilm, and thus make it easier for the photocatalytic drug to penetrate into the biofilm.

[0015] When the permeable photocatalytic nano-antibacterial agent penetrates into the tissue infected by the biofilm of drug-resistant bacteria, the abundant hyaluronidase inside the infected tissue further degrades hyaluronic acid, exposing the sharp, sheet-like structure of the photocatalytic drug—nano-graphite phase carbon nitride. This structure can further disrupt the integrity of the biofilm structure, thereby forming pores of various shapes inside the biofilm, achieving high permeability and accumulation of the photocatalytic drug in the infected tissue.

[0016] 2. After the permeable photocatalytic nano-antibacterial agent of the present invention is enriched in the biofilm, the nano-graphite phase carbon nitride will catalyze the oxygen in the surrounding environment to undergo a photochemical reaction to generate reactive oxygen species when irradiated by a laser of a certain wavelength. This will cause irreversible damage to the biomolecules of drug-resistant bacteria, completely disintegrate the biofilm, and effectively prevent the emergence of new resistance in drug-resistant bacteria.

[0017] 3. The permeable photocatalytic nano-antibacterial agent solution of the present invention has better stability, which is conducive to long-distance transportation and long-term storage of drugs. Moreover, it can directly act on the treatment of drug-resistant bacterial biofilm infections without complicated processing. It is simple to operate, has significant efficacy, and has value for further promotion. Attached Figure Description

[0018] Figure 1 This is a particle size and potential diagram of C3N4 and C3N4@HA in Embodiment 1 of the present invention.

[0019] Figure 2 This is a particle size distribution of C3N4@HA with different HA concentrations according to the present invention.

[0020] Figure 3 This is a transmission electron microscope image of C3N4 in Embodiment 1 of the present invention.

[0021] Figure 4 This is a transmission electron microscope image of C3N4@HA in Embodiment 1 of the present invention.

[0022] Figure 5 This is a comparison chart of the stability of C3N4@HA prepared with HA of different molecular weights in Example 1 of this invention.

[0023] Figure 6 This is a diagram showing the photocatalytic performance of C3N4@HA according to the present invention.

[0024] Figure 7 This is a comparison diagram of the permeation effects of C3N4 and C3N4@HA in biomembranes according to the present invention.

[0025] Figure 8 This is a scanning electron microscope image of the biofilm removal effect of C3N4 and C3N4@HA under light conditions according to the present invention.

[0026] Figure 9 This invention describes the therapeutic effect of C3N4@HA on drug-resistant bacterial biofilm infections.

[0027] Figure 10 This describes the changes in hyaluronidase content in tissues infected with drug-resistant bacterial biofilms after treatment with C3N4@HA. Detailed Implementation

[0028] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, the specific embodiments of this application will be described in detail below with reference to the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of this application and not intended to limit it. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0029] The terms “comprising” and “having”, and any variations thereof, used in this application are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not limited to the steps or units listed, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to such process, method, product, or apparatus.

[0030] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly or implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0032] This invention provides a permeable photocatalytic nano-antibacterial agent, which uses hyaluronic acid to functionalize the surface of nano-graphitic carbon nitride, thereby achieving efficient permeation of photocatalytic drugs into drug-resistant bacterial biofilms and light-mediated removal of drug-resistant bacterial biofilms, thus achieving efficient treatment of drug-resistant bacterial biofilm infections.

[0033] Combination Figure 1 In an exemplary embodiment of the present invention, a permeable photocatalytic nano-antibacterial agent is provided. This nano-antibacterial agent comprises a photocatalytic drug—nano-graphite phase carbon nitride—with hyaluronic acid coated on the surface of the nano-graphite phase carbon nitride. The hyaluronic acid has a molecular weight range of 10. 3 -10 7 Da; the concentration of the hyaluronic acid aqueous solution is 5-40 mg / mL; wherein, the nano-graphite phase carbon nitride has a sheet-like structure with sharp edges.

[0034] Preferably, the particle size of the nano-graphite phase carbon nitride coated with hyaluronic acid is 200-400 nm.

[0035] According to a second aspect of the present invention, a method for preparing the aforementioned permeable photocatalytic nano-antibacterial agent is provided, specifically comprising the following steps: S1. Disperse graphitic carbon nitride powder in 36-38% HCl, mix thoroughly with a magnetic stirrer at room temperature, and carry out the etching reaction. After the reaction is completed, centrifuge and filter to discard the supernatant. Wash the precipitate with deionized water until neutral. S2. Add the precipitate, which has been washed to neutral, to a hyaluronic acid aqueous solution, and then perform ultrasonication, centrifugation, and filtration to obtain the permeable photocatalytic nano-antibacterial agent; Preferably, in step S1, the mass / volume ratio of graphite phase carbon nitride powder to HCl is (1-5):2; the rotation speed of the magnetic stirrer is 600-1200 rpm; and the acid etching reaction time is 0.5-4 h.

[0036] It should be noted that graphitic carbon nitride, as a graphite-like layered material, is bonded to its layers by relatively weak van der Waals forces. The etching reaction refers to the process where, under the action of a strong acid, hydrogen ions embed themselves between the layers of carbon nitride, weakening the interlayer forces through protonation and causing the layered structure to peel off, forming a nanoscale sheet-like structure.

[0037] Preferably, in step S2, a cell disruptor is used for ultrasonication; the ultrasonic temperature is 4°C; the ultrasonic power is 100~300W; the ultrasonic time is 2-5 hours, with a 4-second interval between every 2 seconds of ultrasonication; and the molecular weight of the hyaluronic acid is in the range of 10. 3 -10 7 Da; The concentration of hyaluronic acid aqueous solution is 5-40 mg / mL; In a third aspect of the present invention, an application of the aforementioned permeable photocatalytic nano-antibacterial agent is provided in the preparation of a treatment for biofilm infections caused by drug-resistant bacteria.

[0038]

Example 1

[0039] 5mg of hyaluronic acid (molecular weight: 10) 3 Add Da to 1 mL of deionized water to obtain an aqueous solution of HA.

[0040] The precipitate, washed until neutral, was resuspended in an aqueous HA solution. The cell was subjected to an exfoliation reaction in a cell sonicator at 4°C with an ultrasonic power of 100W for 2 hours (2 seconds per sonication, with a 4-second interval). The mixture was then centrifuged and filtered to obtain a C3N4@HA nano suspension.

[0041]

Example 2

[0042] 40mg of hyaluronic acid (molecular weight: 10) 4 Add Da to 1 mL of deionized water to obtain an aqueous solution of HA.

[0043] The precipitate, washed until neutral, was resuspended in an aqueous HA solution. The cell fragmentation reaction was then performed in a cell sonicator at 4°C and 300W for 5 hours (2 seconds of sonication followed by a 4-second interval). The fragments were centrifuged and filtered to obtain C3N4@HA-10. 4 Nano suspension.

[0044]

Example 3

[0045] 30mg of hyaluronic acid (molecular weight: 10) 7 Add Da to 2 mL of deionized water to obtain an aqueous solution of HA.

[0046] The precipitate, washed until neutral, was resuspended in an aqueous HA solution. The cell fragmentation reaction was then performed in a cell sonicator at 4°C and 200W for 4 hours (2 seconds of sonication followed by a 4-second interval). The fragments were centrifuged and filtered to obtain C3N4@HA-10. 7 Nano suspension.

[0047]

Example 4

[0048] 40mg of hyaluronic acid (molecular weight: 10) 6 Add Da to 2 mL of deionized water to obtain an aqueous solution of HA.

[0049] The precipitate, washed until neutral, was resuspended in an aqueous HA solution. The cell fragmentation reaction was then performed in a cell sonicator at 4°C and 300W for 3 hours (2 seconds of sonication followed by a 4-second interval). The fragments were centrifuged and filtered to obtain C3N4@HA-10. 6 Nano suspension.

[0050]

Example 5

[0051] 5 mg of disulfide-modified hyaluronic acid (molecular weight: 10) 4 Da (with a disulfide bond modification ratio of 1.8 disulfide bonds per 100 disaccharide units) was added to 1 mL of deionized water to obtain a disulfide bond modified hyaluronic acid aqueous solution.

[0052] The precipitate, washed until neutral, was resuspended in a disulfide-modified hyaluronic acid aqueous solution. The precipitate was then subjected to a cell sonication reaction in a cell sonicator at 4°C and 100W for 2 hours (2 seconds of sonication followed by a 4-second interval). The resulting product was centrifuged and filtered to obtain C3N4@HA-10. 4 -SS nano suspension.

[0053]

Example 6

[0054] 20 mg of disulfide-modified hyaluronic acid (molecular weight: 10) 4 Da (with a disulfide bond modification ratio of 2.2 disulfide bonds per 100 disaccharide units) was added to 1 mL of deionized water to obtain a disulfide bond modified hyaluronic acid aqueous solution.

[0055] The precipitate, washed until neutral, was resuspended in a disulfide-modified hyaluronic acid aqueous solution. The precipitate was then subjected to a cell sonication reaction in a cell sonicator at 4°C and 200W for 3 hours (2 seconds of sonication followed by a 4-second interval). The resulting product was centrifuged and filtered to obtain C3N4@HA-10. 4 -SS nano suspension.

[0056]

Example 7

[0057] 30 mg of disulfide-modified hyaluronic acid (molecular weight: 10) 4Da (with a disulfide bond modification ratio of 2.5 disulfide bonds per 100 disaccharide units) was added to 2 mL of deionized water to obtain a disulfide bond modified hyaluronic acid aqueous solution.

[0058] The precipitate, washed until neutral, was resuspended in a disulfide-modified hyaluronic acid aqueous solution. The precipitate was then subjected to a cell sonication reaction in a cell sonicator at 4°C and 300W for 4 hours (2 seconds of sonication followed by a 4-second interval). The resulting product was centrifuged and filtered to obtain C3N4@HA-10. 4 -SS nano suspension.

[0059] In Examples 5-7 of this invention, hyaluronic acid, after being modified by disulfide bonds, is encapsulated on the surface of graphitic carbon nitride to form a core-shell structured nano-antibacterial agent with glutathione responsiveness. The disulfide bonds introduced into the hyaluronic acid molecular chain remain stable in a normal physiological environment, ensuring good colloidal stability of the nano-antibacterial agent during storage and transportation, while also prolonging its duration of action. When the nano-antibacterial agent penetrates into tissue infected by drug-resistant bacterial biofilms, the highly expressed (approximately 2-4 times that of healthy tissue) glutathione (GSH) in the infected microenvironment can specifically reduce and cleave the disulfide bonds, triggering rapid degradation of the hyaluronic acid layer; the nano-antibacterial agent maintains a relatively intact structure before reaching the infected tissue, thereby enabling targeted degradation and site-specific release.

[0060] It is worth noting that the modification of disulfide bonds can be achieved by chemically bonding them to hyaluronic acid, although in some embodiments, loading them onto C3N4 has a similar effect. In contrast, the disulfide bonds on the surface loaded onto carbon nitride are more easily attacked by the ROS generated on the C3N4 surface, and the concentration of disulfide bonds will slowly decrease before the "nanoknife effect" takes effect on the biofilm surface, resulting in a weakening of the targeted release effect.

[0061] The materials presented in this invention will now be discussed in detail, taking into account specific test results.

[0062] Particle size and potential testing C3N4 and C3N4@HA prepared according to the method in Example 1 were diluted with deionized water to form a suspension with a concentration of 10 μg / mL of nano-graphitic carbon nitride, and then their potential and particle size were measured at 25 °C.

[0063] like Figure 1 As shown, the particle sizes of C3N4 and C3N4@HA are 180.3±7.4nm and 203.4±5.6nm, respectively; the potential of C3N4 is -11.4±2.1mV, and the potential of C3N4@HA is -28.4±2.1mV.

[0064] The potential and particle size results of C3N4 and C3N4@HA show that the particle size of C3N4@HA is increased compared to g-C3N4. This is because HA forms a hydration layer on the outer surface of g-C3N4. In addition, since the surface of HA-modified g-C3N4 has a large number of ionized carboxyl groups carrying a large number of negative charges, the surface potential of C3N4@HA is lower than that of C3N4, which indicates that it has successfully modified HA.

[0065] C3N4@HA-10 prepared according to the methods in Examples 2, 3 and 4 4 C3N4@HA-10 6 and C3N4@HA-10 7 The carbon nitride particles were diluted with deionized water to a concentration of 10 μg / mL in the graphite phase, and then their particle size was measured at 25 °C.

[0066] The results are as follows Figure 2 As shown, C3N4@HA-10 4 C3N4@HA-10 6 and C3N4@HA-10 7 The particle sizes were 243.1±9.2 nm, 269.6±8.9 nm and 303.1±10.1 nm, respectively.

[0067] As can be seen from the above, the particle size of the first nanosystem of the present invention after loading antibacterial drugs on the surface is between 200-400 nm.

[0068] Morphology test C3N4 and C3N4@HA prepared according to the method in Example 1 were dropped onto the surface of a copper mesh, dried, and then their morphology was measured under a transmission electron microscope.

[0069] Transmission electron microscopy results as follows Figure 3 , 4 As shown in the figure, it is clear that C3N4 exhibits a two-dimensional sheet-like structure with surface wrinkles. Figure 3 This facilitates the disruption of biofilm integrity based on the "nanoknife effect"; while C3N4@HA has a similar surface morphology to g-C3N4 ( Figure 4 This indicates that the encapsulation of HA did not significantly alter the shape and structure of C3N4.

[0070] Stability test C3N4 and C3N4@HA-10 prepared by the method in Example 2 were used. 4 And select HA (molecular weight: 10) 2 10 8 Da), and C3N4@HA-10 were prepared separately according to the method in Example 1. 2and C3N4@HA-10 8 The C3N4 concentration was diluted to the same concentration (20 μg / mL) with phosphate-buffered saline (PBS), and its particle size distribution was measured at the initial stage and after 48 h to evaluate its stability.

[0071] Experimental results are as follows Figure 5 As shown, unmodified C3N4 readily aggregates in PBS solution, with the average particle size increasing by 210% after 48 hours, indicating poor stability. Using HA of different molecular weights (molecular weight: 10... 2 10 4 and 10 8 Modify C3N4@HA-10 2 C3N4@HA-10 4 and C3N4@HA-10 8 After storage in PBS solution for 48 hours, C3N4@HA-10 2 and C3N4@HA-10 8 The particle size increased by about 150%; in contrast, C3N4@HA-10 4 The particle size remained essentially unchanged, indicating a molecular weight of 10. 4 HA can improve the solution stability of photocatalytic drugs.

[0072] Photocatalytic performance test The photocatalytic production of reactive oxygen species (ROS) from C3N4@HA was detected using a SOSG reactive oxygen species fluorescent probe kit. 5 mL of g-C3N4 and C3N4@HA prepared according to the method in Example 1 were thoroughly mixed with 100 μL of SOSG (5 μM) reagent. The mixtures were then irradiated with a 440 nm laser for 3 min. The release behavior of ROS was subsequently detected using a fluorescence spectroscopy system, with an excitation wavelength of 504 nm and a generation wavelength of 525 nm.

[0073] Experimental results are as follows Figure 6 As shown in the figure, it is clear that the g-C3N4 group showed no obvious fluorescence signal under the condition of no laser irradiation; however, after 3 minutes of irradiation with a 440nm laser, both the g-C3N4 and C3N4@HA groups showed obvious fluorescence signals, and the fluorescence intensities of the two were similar, indicating that the antibacterial agent of HA-modified nano-graphitic carbon nitride maintained the photocatalytic performance of C3N4 well.

[0074] Evaluation of osmotic behavior within biofilms The permeation behavior of C3N4@HA prepared according to the method in Example 1 within the biofilm was observed using laser confocal scanning electron microscopy. Methicillin-resistant Staphylococcus aureus (MRSA) bacterial suspension (10... 9A mixture of CFU / mL trypsin soybean broth (TSB) and 24-well plates was added at a ratio of 1:100 and incubated at 37°C for 12 h. Unattached bacteria were gently washed three times with sterile PBS to harvest the MRSA biofilm. Then, PBS, Cy5.5-labeled C3N4, and Cy5.5-labeled C3N4@HA (C3N4 concentration 5 μg / mL) prepared according to Example 1 were added to the wells, and the plates were incubated at 37°C for 0.5 h. The fluorescence signal distribution within the biofilm was then observed using a laser confocal scanning electron microscope.

[0075] The results are as follows Figure 7 As shown, with PBS as the negative control group, the Cy5.5-labeled g-C3N4 group exhibited a strong fluorescence signal. The intensity of the fluorescence signal decreased with increasing biomembrane depth, indicating that the sheet-like structure of C3N4 can effectively disrupt the integrity of the biomembrane and promote the penetration of photocatalytic drugs within the biomembrane. Compared with the Cy5.5-labeled C3N4 group, the Cy5.5-labeled C3N4@HA group still showed a strong fluorescence signal at 50 μm. This can be attributed to the degradation of HA by hyaluronidase within the biomembrane, while simultaneously exposing the sharp edges of C3N4. The combined effect of these two factors significantly enhances the drug's penetration and accumulation within the biomembrane.

[0076] Anti-biofilm efficacy test The biofilm scavenging ability of C3N4@HA prepared according to the method in Example 1 was observed using scanning electron microscopy. MRSA bacterial culture (10... 9 The MRSA biofilm was then harvested by mixing CFU / mL with TSB medium at a ratio of 1:100 and added to a 24-well plate, and incubated at 37°C for 12 h. Bacteria not attached to the TSB medium were gently washed three times with sterile PBS. PBS, HA, C3N4, and C3N4@HA (C3N4 concentration 5 μg / mL) prepared according to Example 1 were then added to the wells. After incubation at 37°C for 1 h, the plates were irradiated with blue light for 30 min. Subsequently, all samples were fixed in 2.5% glutaraldehyde solution for 12 h, then dehydrated using a gradient of ethanol (30, 50, 70, 80, 90, and 100%, v / v), vacuum dried, and the biofilm removal effect of different treatment groups was observed using scanning electron microscopy.

[0077] The results are as follows Figure 8As shown, compared with the PBS group, HA can be degraded by hyaluronidase in the biofilm, leading to the destruction of the biofilm's integrity, but it has almost no anti-biofilm effect. C3N4 can also destroy the structural integrity of the biofilm through its sharp, sheet-like structure. Subsequently, under light conditions, a photocatalytic reaction occurs, generating a large amount of reactive oxygen species, thereby killing drug-resistant bacteria, with a biofilm clearance rate of 30%. Compared with C3N4, C3N4@HA can enhance the drug enrichment effect by consuming hyaluronidase inside the biofilm, and then dissolve most of the biofilm (>85%) under light-mediated ablation. This indicates that HA-modified photocatalytic drugs can significantly enhance their permeation behavior inside the biofilm, thereby achieving the photocatalytic and efficient biofilm disintegration effect under light conditions.

[0078] Efficacy test of treatment for drug-resistant bacterial wound infections To induce neutropenia in mice, cyclophosphamide (150 mg / kg) was injected intraperitoneally for three consecutive days before establishing the wound infection model. Mice were anesthetized with 4% chloral hydrate (40 mg / kg), their backs were shaved and prepared, and after alcohol disinfection, a circular wound approximately 5 mm in diameter was created. 50 μL of MRSA (10 mg / kg) was then instilled into the wound. 7 A wound infection model was constructed using a bacterial suspension containing CFU / mL. The bacteria were then treated with PBS, HA, C3N4, and C3N4@HA (C3N4 concentration 20 μg / mL) prepared according to the method in Example 1, and subjected to blue light (10 mW / cm²). 2 Irradiate for 30 minutes, and monitor the wound healing of mice after treatment.

[0079] Animal experiment results such as Figure 9 As shown, the infected wounds of mice in the PBS and HA groups healed slowly, with residual wound area reaching over 75% after 14 days of treatment. After treatment with C3N4 mediated by light, the healing rate of infected wounds was significantly better than that of the PBS and HA groups. This is because photocatalytic therapy can kill some drug-resistant bacteria, thereby promoting the healing of infected wounds. Compared with other groups, mice treated with C3N4@HA+ light showed the best wound healing effect, with a wound healing rate of >80% at the treatment endpoint. This is because C3N4@HA can enhance the penetration and enrichment effect of photocatalytic drugs by consuming hyaluronidase in the biofilm. Subsequently, under light conditions, photocatalytic therapy can rapidly kill pathogens and efficiently eliminate drug-resistant bacterial biofilm infections.

[0080] Hyaluronidase content test in infected tissue Mice with MRSA wound infection were treated with PBS, HA, C3N4, and C3N4@HA (C3N4 concentration of 20 μg / mL) prepared according to the method in Example 1, and then subjected to blue light (10 mW / cm²). 2Irradiation for 30 minutes. On the second day after treatment, infected tissue around the wound was collected, and the content of HAase (hyaluronidase) in the infected tissue was monitored using an ELISA kit to evaluate the ability of C3N4@HA to consume HAase in the infected tissue.

[0081] ELISA experimental results are as follows Figure 10 As shown, on the second day after treatment, the HAase content in the infected tissue of mice in the C3N4@HA + light-treated group was 4.1 ng / mg tissue, which was much lower than the HAase content in the PBS, HA and C3N4 + light-treated groups. This indicates that C3N4@HA effectively reduces the HAase content in the infected tissue by consuming it through surface-modified HA, thereby promoting the repair and healing of the infected tissue.

[0082] Performance testing of disulfide bond-modified nano-antibacterial agents Take the C3N4@HA-10 prepared in Example 5 4 -SS nano-suspension was diluted with PBS buffer to a graphitic carbon nitride concentration of 20 μg / mL and divided into two groups: the experimental group was treated with glutathione to a final concentration of 10 mM, while the control group was treated with an equal volume of PBS buffer. Both groups were incubated at 37°C. Particle size changes were measured at 0, 2, 4, 8, and 12 hours. The results showed that the particle size in the control group remained essentially unchanged within 12 hours; in the experimental group, the particle size began to decrease 2 hours after the addition of glutathione, decreased by approximately 30% after 4 hours, and decreased by approximately 50% after 8 hours, indicating that the introduction of disulfide bonds endowed the nano-antibacterial agent with good glutathione-responsive degradation ability.

[0083] The effect of reactive oxygen species on disulfide bond degradation behavior To investigate the effect of reactive oxygen species generated during photocatalysis on disulfide bond stability, C3N4@HA-10 prepared in Example 5 was used. 4 -SS nanospray suspension, diluted with PBS buffer to a carbon nitride concentration of 20 μg / mL in the nanographite phase, was divided into two groups: the experimental group was irradiated with a 440 nm laser (power density 10 mW / cm²). 2 The samples were irradiated for 30 minutes, while the control group was stored in the dark. After irradiation, the content of free thiol groups in both groups was determined using Ellman's reagent to evaluate the degree of disulfide bond breakage. Simultaneously, glutathione (final concentration 10 mM) was added to both groups, and the samples were incubated for another 4 hours before particle size changes were measured.

[0084] Experimental results showed that after 30 minutes of laser irradiation, the content of free thiol groups increased by approximately 15% compared to the control group, indicating that the reactive oxygen species generated by photocatalysis could partially oxidize and break disulfide bonds. Further addition of glutathione resulted in a significantly greater reduction in particle size in the laser-irradiated group (approximately 65%) than in the control group (approximately 40%), suggesting that the pre-crack effect of reactive oxygen species on disulfide bonds could enhance the efficiency of subsequent responsive degradation of glutathione. These results indicate that during photocatalysis, the reactive oxygen species generated by photocatalysis first cause partial oxidative damage to the disulfide bonds, lowering the disulfide bond breaking barrier. Subsequently, the highly expressed glutathione in the infected tissue further reduces and breaks the disulfide bonds, accelerating the disintegration of the hyaluronic acid layer. This allows the disulfide-modified nano-antibacterial agent to more efficiently expose the sharp edges of graphitic carbon nitride during photocatalytic therapy, thereby enhancing its penetration and accumulation within the biomembrane.

[0085] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A permeable photocatalytic nano-antibacterial agent, characterized in that, The nano-antibacterial agent comprises nano-graphite phase carbon nitride as a photocatalytic drug, with hyaluronic acid coated on the surface of the nano-graphite phase carbon nitride. The hyaluronic acid has a molecular weight range of 10. 3 -10 7 Da; wherein the nano-graphite phase carbon nitride has a sheet-like structure.

2. The photocatalytic nano-antibacterial agent according to claim 1, characterized in that, The particle size of the nano-graphite phase carbon nitride coated with hyaluronic acid is 200-400 nm.

3. The photocatalytic nano-antibacterial agent according to claim 1, characterized in that, The nano-graphite phase carbon nitride has a sheet-like structure with sharp edges, and the sheet-like structure with sharp edges has a transmission electron microscope (TEM) image that is substantially the same as that in Figure 3 of the accompanying drawings. The photocatalytic nano-antibacterial agent has a TEM image that is substantially the same as that in Figure 4 of the accompanying drawings.

4. The photocatalytic nano-antibacterial agent according to claim 3, characterized in that, The sheet-like structure comprises a thickness that gradually decreases from the center to the edge of the nano-graphitic carbon nitride, forming a wedge-shaped or blade-shaped profile with a tip width ≤10nm.

5. The photocatalytic nano-antibacterial agent according to claim 1, characterized in that, The hyaluronic acid is modified with disulfide bonds, and the modification ratio is 1-3 disulfide bonds per 100 hyaluronic acid disaccharide units.

6. The photocatalytic nano-antibacterial agent according to claim 5, characterized in that, The method for modifying the disulfide bonds includes: dissolving hyaluronic acid in a buffer solution, adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide to activate the carboxyl group for 10-30 min, then adding cystamine, wherein the molar ratio of cystamine to the disaccharide unit of hyaluronic acid is 0.1:1-0.5:1, and reacting at room temperature for 12-24 h; after the reaction is completed, adding ethanol to precipitate, centrifuging to collect the precipitate, washing and drying to obtain disulfide-modified hyaluronic acid.

7. A method for preparing the photocatalytic nano-antibacterial agent according to claim 1, characterized in that, Includes the following steps: S1. Disperse graphitic carbon nitride powder in 36-38% HCl, mix and carry out etching reaction at room temperature. After the reaction is completed, centrifuge and filter, and wash the precipitate with water until neutral. S2. Add the precipitate to a hyaluronic acid aqueous solution, mix well, centrifuge and filter to obtain the photocatalytic nano antibacterial agent.

8. The preparation method according to claim 7, characterized in that, In step S1, the mass / volume ratio of graphite phase carbon nitride powder to HCl is (1-5):2, the rotation speed of the magnetic stirrer is 600-1200 rpm, and the acid etching reaction time is 0.5-4 h.

9. The preparation method according to claim 7, characterized in that, In step S2, a cell disruptor is used for sonication, with an ultrasonic power of 100-300W and a sonication time of 2-5 hours, with a 4-second interval between every 2 seconds of sonication; the molecular weight of the hyaluronic acid is in the range of 10. 3 -10 7 Da, the concentration of hyaluronic acid aqueous solution is 5-40 mg / mL.

10. The use of the photocatalytic nano-antibacterial agent according to claim 1 in the preparation of a medicament for treating biofilm infections caused by drug-resistant bacteria.

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