A mineralized enzyme complex powder for disrupting biofilm and its preparation method and antibacterial application

Abstract

This invention discloses a mineralization enzyme composite powder for disrupting biofilms, its preparation method, and its antibacterial applications. The composite powder is a hybrid nanoparticle prepared by a one-step biomineralization process. The composite powder contains nucleases and bactericidal enzymes; the nucleases are one or more of deoxyribonucleases, ribonucleases, or non-specific nucleases in any proportion; the bactericidal enzymes are single or multiple enzymes, wherein the multi-enzyme cascade generates hydroxyl radicals. This invention achieves a synergistic effect of biofilm degradation and antibacterial activity by integrating multiple natural enzymes. It not only significantly improves the stability of nucleases but also enhances the cascade catalytic efficiency of the bactericidal enzymes, thereby effectively disrupting biofilms and increasing the generation efficiency of hydroxyl radicals. This invention also provides the antibacterial applications of the composite powder in vitro and in vivo, exhibiting good stability and catalytic performance, suitable for various anti-infection scenarios.

Description

Technical Field

[0001] This invention relates to new materials in the field of biomedicine, specifically a mineralizing enzyme composite powder for disrupting biological membranes, its preparation method, and its antibacterial applications. Background Technology

[0002] Bacterial infections and related diseases pose a persistent threat to global public health, seriously endangering human health. According to the World Health Organization, infectious diseases remain one of the leading causes of death, with bacterial infections accounting for a significant proportion, and the mortality rate from these infections is on the rise in some regions. Furthermore, over 80% of refractory infections are directly related to the formation of bacterial biofilms, which hinder antibiotic penetration and significantly weaken their efficacy. Biofilms are mainly composed of extracellular polymeric substances (EPS), including polysaccharides, proteins, lipids, and eDNA. Among these, eDNA, as the longest molecule in EPS, promotes initial bacterial attachment to the surface and induces bacterial aggregation. Simultaneously, through interactions with other EPS components, it forms a tight network structure, ultimately aggregated into a dense physical barrier, effectively protecting bacteria from attack by the host's immune system. Therefore, efficiently disrupting the biofilm structure, especially its key backbone component eDNA, has become a potential strategy for eradicating bacterial infections at their source.

[0003] Using nucleases to cleave eDNA is an effective strategy for clearing biofilms. Based on this strategy, natural enzymes such as deoxyribonuclease I can be used to specifically degrade eDNA, thereby disrupting the biofilm's skeletal structure and promoting the penetration and efficacy of other antibacterial components. However, DNase I has significant shortcomings in practical use: as a biological enzyme, it is easily inactivated by temperature changes, pH fluctuations, or the presence of other proteases, and its stability and duration of action are limited; furthermore, it lacks bactericidal ability and cannot completely eliminate released bacteria. Therefore, although DNase I can theoretically degrade eDNA, these shortcomings limit its practical application in the field of anti-biofilm treatment.

[0004] Therefore, there is an urgent need to develop a novel antibacterial material. This material aims to effectively enhance the stability of DNase I, enabling it to powerfully disrupt bacterial biofilms and further endow it with direct bactericidal activity, thereby synergistically achieving the root-cause elimination of drug-resistant bacteria. Summary of the Invention

[0005] To address the aforementioned issues, this invention provides a mineralized enzyme composite powder for disrupting biofilms. It enhances the stability of natural enzymes through a one-step biomineralization process and cleverly utilizes biomineralization to form nanowires. During the formation of nanowires, a cascade catalytic system of glucose oxidase and peroxidase is constructed, improving catalytic efficiency and the generation efficiency of hydroxyl radicals, ultimately achieving synergistic and efficient antibacterial action through multiple mechanisms.

[0006] A second objective of this invention is to provide a method for preparing a mineralizing enzyme composite powder for disrupting biological membranes.

[0007] A third objective of this invention is to provide an antibacterial application of a mineralizing enzyme composite powder for disrupting biofilms.

[0008] The technical solution adopted in this invention is a mineralization enzyme composite powder for disrupting biofilms. The composite powder is a hybrid nanoparticle prepared by one-step biomineralization and contains nuclease and bactericidal active enzyme. The nuclease is one or more of deoxyribonuclease, ribonuclease or nonspecific nuclease in any proportion; The bactericidal active enzyme is a single enzyme or multiple enzymes, wherein the multiple enzyme cascade generates hydroxyl radicals.

[0009] Furthermore, the nuclease is a natural deoxyribonuclease (DNase I), and the bactericidal active enzymes are glucose oxidase (GOx) and horseradish peroxidase (HRP). The DNase I enzyme activity concentration is 400-800 U / mL, the GOx mass concentration is 0.05-1 mg / mL, and the HRP mass concentration is 0.25-0.5 mg / mL.

[0010] The DNase I in the hybrid nanoparticles can effectively disrupt biofilms under alkaline conditions or in complex biological environments, exposing bacteria. GOx consumes glucose to create an acidic environment, while the generated hydrogen peroxide (H2O2) serves as a substrate for HRP, continuing the reaction to generate hydroxyl radicals for synergistic bactericidal action. By integrating multiple natural enzymes, the biofilm degradability and antibacterial synergistic effect are achieved, which not only significantly improves the stability of DNase I but also enhances the cascade catalytic efficiency of GOx and HRP, thereby effectively disrupting biofilms and increasing the generation efficiency of hydroxyl radicals.

[0011] Furthermore, the composite powder is effective against Escherichia coli (E. coli). E. coli The clearance rate reached 94.78%, which is effective against drug-resistant bacteria. MRSA The clearance rate reached 97.45%, and the wound healing time was shortened to 7 days.

[0012] A method for preparing a mineralizing enzyme composite powder for disrupting biofilms includes the following steps: S1, DNase I, GOx and HRP are mixed and dissolved in phosphate buffer in a certain proportion to obtain an enzyme solution. The activity concentration of DNase I in the enzyme solution is 400-800 U / mL, the mass concentration of GOx is 0.05-1 mg / mL, and the mass concentration of HRP is 0.25-0.5 mg / mL. S2. The enzyme solution obtained in S1 is mixed with the metal salt solution at a volume ratio of 94-98:2-6. The mixture is thoroughly mixed and reacted in a constant temperature metal bath at 20-25℃ for 12-48 hours. During the synthesis process, inorganic components grow around the enzyme to form enzyme-based hybrid nanoparticles, which improve the stability and catalytic efficiency of the enzyme.

[0013] Furthermore, it also includes: washing and collecting the three-dimensional flower-like structure after allowing it to stand, and resuspending the resulting precipitate in phosphate buffer.

[0014] Furthermore, the concentration of the phosphate buffer solution is 0.5-2M.

[0015] Furthermore, the metal salt solution is a 100-200 mM CuSO4 solution.

[0016] The application of a mineralizing enzyme composite powder for disrupting biofilms in antibacterial products, said antibacterial products being wound dressings, wound topical medications, or prosthetic graft loading medications.

[0017] Furthermore, the glucose (Glu) concentration in the application environment is 1mM-10mM.

[0018] Furthermore, the antimicrobial product is used to treat or prevent infections associated with bacterial biofilms, including implant infections, catheter infections, or wound infections, and exhibits good stability and catalytic properties.

[0019] The beneficial effects of this invention are: This invention utilizes biomineralization technology to immobilize DNase I enzyme, significantly enhancing its catalytic stability and thus strengthening its shearing properties against bacterial biofilms. This synergistic catalytic system promotes the GOx-HRP cascade reaction, increasing the efficiency of hydroxyl radical generation by 7.2 times, ultimately achieving a multi-mechanism synergistic antibacterial effect. Experimental verification shows that this system... E. coli and drug-resistant bacteria MRSA The clearance rates reached 94.78% and 97.45%, and healing was achieved within 7 days in a diabetic wound infection model. This provides a new method for addressing biofilm-related infections and can be widely applied to scenarios such as biofilm infections in prosthesis transplants and catheters.

[0020] This invention utilizes a one-step biomineralization method to react DNase I, GOx, and HRP in Cu. 2+ Mineralization occurs in the presence of copper, which is essential for aerobic organisms and a key cofactor in various enzymatic processes, including energy production, metabolism, and signal transduction pathways. It not only possesses high biocompatibility but also releases Cu from DGH-YB. 2+ It can promote wound healing and bone regeneration. The compound DGH-YB was obtained, which significantly improved the stability of DNase I, giving DGH-YB higher catalytic activity and better stability compared to the free enzyme. DGH-YB can not only effectively scavenge biofilms, but also, in the presence of Glu, inhibit Gram-positive bacteria (…). MRSA ) and Gram-negative bacteria ( E. coli Both exhibit excellent bactericidal effects. Validated in vivo using a diabetic mouse biofilm wound infection model and a rat bone defect biofilm infection model, DGH-YB also demonstrates good activity. The absence of metal complexes enhances biosafety. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is the synthetic route for compound DGH-YB in the embodiments of the present invention.

[0023] Figure 2 This is a SEM image of DGH-YB from an embodiment of the present invention.

[0024] Figure 3 This is the energy dispersive X-ray spectrum of DGH-YB according to an embodiment of the present invention.

[0025] Figure 4 This invention relates to an embodiment of the invention that uses confocal microscopy to observe the distribution of enzymes labeled with various fluorescent dyes in DGH-YB.

[0026] Figure 5 This is the zeta point characterization of DGH-YB in the embodiment of the present invention.

[0027] Figure 6 This is an embodiment of the present invention: DGH-YB-cut bacterial DNA gel electrophoresis.

[0028] Figure 7 This invention demonstrates the DNA cleavage activity of DGH-YB in this embodiment.

[0029] Figure 8 This invention relates to the DGH-YB catalytic activity of Glu in generating hydroxyl radicals.

[0030] Figure 9 These are the UV-Vis spectral analysis results of RhB-DNase I solutions of different concentrations in the embodiments of the present invention; where (A) is the UV-Vis spectrum of RhB-DNase I, and (B) is the standard curve of absorbance value of RhB-DNase I at 554 nm.

[0031] Figure 10 The UV-Vis spectral analysis results of RhB-GOx solutions of different concentrations in this embodiment of the invention; wherein (A) is the UV-Vis spectrum of RhB-GOx, and (B) is the standard curve of absorbance value of RhB-GOx at 554 nm.

[0032] Figure 11 These are the UV-Vis spectral analysis results of Rhb-HRP solutions of different concentrations in the embodiments of the present invention; where (A) is the UV-Vis spectrum of Rhb-HRP, and (B) is the standard curve of absorbance value of Rhb-HRP at 554 nm.

[0033] Figure 12 This is a comparison of the catalytic performance of Free DGH and DGH-YB with the same substrate concentration in the embodiments of the present invention; wherein (A) is the change of the catalytic rate of Free DGH and DGH-YB over time; and (B) is the conversion number of Glu catalyzed by Free DGH and DGH-YB.

[0034] Figure 13 This is an example of how the DNA cleavage activity of DGH-YB and Free DGH changes over time in this invention.

[0035] Figure 14 This invention relates to the DNA cleavage activity of DGH-YB and Free DGH after different storage days in embodiments of the present invention.

[0036] Figure 15 This is an example of how the activity of DGH-YB changes with the number of recycling cycles in this invention.

[0037] Figure 16 This is a crystal violet-stained biofilm from an embodiment of the present invention.

[0038] Figure 17 This is an embodiment of the present invention. MRSA AM staining of biomembranes.

[0039] Figure 18 Different incubation times in embodiments of the present invention MRSA AM staining of biomembranes.

[0040] Figure 19 These are the MIC and MBC of DGH-YB and GH-YB in embodiments of the present invention.

[0041] Figure 20 These are confocal microscopy images of different materials interacting with MRSA biofilms after AM / PI staining, as described in an embodiment of the present invention.

[0042] Figure 21 These are photographs of wound sizes at different times of day in an embodiment of the present invention.

[0043] Figure 22 This is an optimization of the glucose oxidase concentration in DGH-YB in the embodiments of the present invention; where (A)-(E) are ultraviolet-visible spectra, and (F) is a TMB colorimetric photograph taken under sunlight.

[0044] Figure 23 This is an optimization of the DNase I enzyme concentration in DGH-YB of the present invention; where (A)-(C) are ultraviolet-visible spectra, and (D) is the DNA cleavage activity.

[0045] Figure 24 This invention relates to the antibacterial effect of a rat bone defect biofilm infection model; wherein (A) represents bacterial colonies and turbid liquid in the infected implant; (B) represents the quantification of turbid liquid by OD value; (C) represents the quantification of bacterial colonies; and (D) represents the release of copper ions over time.

[0046] Figure 25 These are live / dead fluorescence microscopy images of DGH-YB and GH-YB co-cultured with L929 cells according to embodiments of the present invention.

[0047] Figure 26 This embodiment of the invention evaluates the biocompatibility of DGH-YB and GH-YB through a hemolysis test; wherein (A) is a photo of the added red blood cell suspension, (B) is a photo of the supernatant, and (C) is the hemolysis rate.

[0048] Figure 27 The embodiments of this invention use PBS (control), Free GH, GH-YB, and DGH-YB for treatment. MRSA and E. coli Survival rate. Detailed Implementation

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

[0050] Basic concept of the embodiments of the present invention: Conventional antibiotics struggle to penetrate biofilms and thus fail to achieve bactericidal effects. Bacterial biofilms are composed of eDNA, polysaccharides, proteins, and lipids. While nucleases are the first thought for disrupting eDNA, they are naturally occurring enzymes susceptible to environmental interference and have poor resistance to complex matrices. Furthermore, nucleases can only disrupt biofilms and do not possess bactericidal activity. This invention employs GOx and HRP as model enzymes, cascading to generate ROS for bactericidal action. Through mineralization, the stability of DNase I is enhanced, and bactericidal enzymes are fused together to form DGH-YB, resulting in a material that improves nuclease stability while also possessing bactericidal activity.

[0051] Example 1

[0052] A method for preparing a mineralizing enzyme composite powder (DGH-YB) for disrupting biofilms: like Figure 1 As shown, 8 μL of DNase I (50000 U / mL), 250 μL of HRP (2 mg / mL), and 16.6 μL of GOx (30 mg / mL) were added to a 1.5 mL EP tube, and 1X PBS (phosphate buffer with a concentration of 1M) was added to bring the volume to 960 μL to obtain an enzyme solution. The enzyme solution contained 400 U / mL of natural deoxyribonuclease (DNase I), 0.5 mg / mL of glucose oxidase (GOx), and 0.5 mg / mL of horseradish peroxidase (HRP). Add 40 μL CuSO4 (200 mM) to the enzyme solution, mix thoroughly, and incubate in a constant temperature metal bath (25 ℃) for 24 h. Remove the centrifuge tube, centrifuge (6000 rpm, 6 min), discard the supernatant, wash 2-3 times with PBS, and resuspend the resulting precipitate in PBS buffer.

[0053] DNase I, as a protease, has very poor stability in complex biological environments and has no bactericidal activity, so it cannot be used as an antibacterial material. In this invention, DNase I is mineralized to remove biofilms, thus solving the problem of drug resistance caused by biofilms and improving the stability of DNase I. In addition, enzymes with bactericidal activity are fused together.

[0054] This invention introduces GOx and HRP. In the presence of glucose, GOx and HRP undergo a cascade reaction, catalyzing the generation of hydroxyl radicals from Glu, thus achieving sterilization. Because both GOx and HRP are natural enzymes, they possess good biocompatibility. Therefore, by mineralizing these three enzymes through biomineralization, not only can biofilms be disrupted, but bacteria within them can also be killed.

[0055] DGH-YB represents a hybrid nanoparticle mineralized with three enzymes: DNase I, GOx, and HRP (DGH being the first letters of the three enzymes). Electron microscopy revealed that the surface of the hybrid material has pores, resembling a yarn ball, hence the name DGH-YB.

[0056] The morphology of the DGH-YB prepared in Example 1 was observed using a scanning electron microscope, and the results are as follows: Figure 2 As shown, the size of DGH-YB is 9 μm. EDX energy dispersive spectroscopy analysis yielded the following results: Figure 3 As shown, DGH-YB contains C, P, O, and Cu elements, as well as the characteristic protein element N, indicating the successful synthesis of DGH-YB. DNase I, GOx, and HRP were stained with three different fluorescent dyes, and the results were observed using a confocal microscope. The results are as follows: Figure 4 As shown, the distribution of three enzymes can be observed in DGH-YB, and the fluorescence of the three enzymes is not located at the same position. The zeta potential of DGH-YB was measured using dynamic light scattering (DLS) technology, and the results are as follows... Figure 5 As shown, free DNase I, GOx, and HRP exhibit negative charges. These negative charges are amplified after the formation of DGH-YB. These results demonstrate the successful synthesis of DGH-YB.

[0057] Sample preparation: PBS is a 1M phosphate buffer solution.

[0058] Free DNase I is unmineralized DNase I with the same activity concentration as in Example 1.

[0059] Free GH is a mixture of unmineralized GOx and HRP, with the same mass concentration as in Example 1.

[0060] Free DGH is a mixture of unmineralized DNase I, GOx, and HRP, with the same mass concentrations as in Example 1. The specific steps are as follows: DNase I, GOx, and HRP are mixed and dissolved in phosphate buffer in a certain proportion to obtain an enzyme solution. The activity concentration of DNase I in the enzyme solution is 400 U / mL, the mass concentration of GOx is 0.5 mg / mL, and the mass concentration of HRP is 0.5 mg / mL. The step of unmineralization is not performed.

[0061] D-YB consists of particles formed by mineralizing DNase I alone. Except for the absence of GOx and HRP, the preparation steps are the same as in Example 1.

[0062] GH-YB is a particle formed from mineralized GOx and HRP. Except for the absence of DNase I, the preparation steps are the same as in Example 1.

[0063] DGH-YB was prepared in Example 1.

[0064] Verification of DGH-YB DNA cleavage activity: Bacterial eDNA is homologous to bacterial genome DNA; therefore, bacterial genome DNA was extracted to verify whether DGH-YB has cleavage activity. MRSA bacterial genome DNA was extracted using a bacterial genome DNA extraction kit (Sangon Biotech) for subsequent experiments. 9 µL of the sample (lanes 1-6: Marker (molecular weight standard reference), PBS, Free DNase I + bacterial genome DNA, D-YB + bacterial genome DNA, DGH-YB + bacterial genome DNA, GH-YB + bacterial genome DNA, respectively) was thoroughly mixed with 1 µL of 6× loading buffer. Electrophoresis was performed at 90V for 40 min, and the gel was imaged using a ChemiDoc XRS gel imaging system. The bacterial genome DNA content was the same in lanes 1-6, and the results are as follows. Figure 6 As shown, Free DNase I can cleave bacterial DNA, and the DGH-YB synthesized in Example 1 retains its cleavage activity against bacterial DNA.

[0065] In a 100 μL reaction buffer system, experimental groups GH-YB, DGH-YB, and Free DNase I were set up. First, 2 μL of 10 μM DNA (labeled with TAMRA and BHQ) solution was added, where TAMRA refers to tetramethylrhodamine and BHQ refers to the quencher; then, 2 μL of ultrapure water and 8 μL of buffer were added, and finally 8 μL of powder particles (GH-YB, DGH-YB, and Free DNase I for the three experimental groups, respectively) were added. After incubation in a constant temperature metal bath at 37°C for 0.5 h, 80 μL of ultrapure water was added, and fluorescence in the 555 nm-670 nm range was collected under 545 nm excitation. The results are as follows. Figure 7 As shown, when the initial amount of enzyme added is kept the same, the cleavage activity of DGH-YB is not significantly different from that of Free DNase I, indicating that DGH-YB has good DNA cleavage activity.

[0066] Verification of the DGH-YB catalytic activity in generating hydroxyl radicals from Glu: To investigate the catalytic effect of the synthesized DGH-YB on Glu, the TMB colorimetric method was used. In the presence of hydroxyl radicals, TMB is oxidized to oxTMB. Free GH, GH-YB, and DGH-YB were added to 3',3',5',5'-tetramethylbenzidine (TMB) indicator, and Glu was added simultaneously. The final concentrations of Free GH, GH-YB, and DGH-YB were 0.08 mg / mL, the final concentration of TMB was 0.6 mg / mL, and the final concentration of glucose was 10 mM. The mixture was incubated at 25°C for 1 h, and the UV absorbance at 650 nm was measured using a UV spectrophotometer. The results are as follows: Figure 8 As shown, when the initial amount of enzyme added is kept the same, the catalytic activity of DGH-YB is slightly lower than that of Free GH.

[0067] DGH-YB enzyme loading assay: To prepare rhodamine B-labeled protein, the fluorescent dye rhodamine B (Rhb) was added to the protein solution, and the mixture was stirred in the dark for 12 hours. Afterward, the solution was strongly dialyzed with distilled water (3000 Da) for 24 hours. The product was lyophilized to obtain Rhb-DNase I, Rhb-GOx, and Rhb-HRP, and DGH-YB was prepared from the above-mentioned fluorescent dye-rich protein using the steps of Example 1. The initial concentration of dye was taken, and the UV absorption at 400 nm–700 nm was measured, showing a linear change within a certain concentration range. The DGH-YB supernatant was taken, and the UV absorption at 400 nm–700 nm was measured, and the enzyme content in the supernatant was calculated. The results are as follows: Figures 9-11As shown in Figures (A) and (B), the loading rates of DNase I, GOx, and HRP in DGH-YB were 30.09%, 7.19%, and 5.68%, respectively.

[0068] Figure 9 The legend (400 U / mL, etc.) represents the activity concentration of DNase I after mixing. The mass concentration of GOx in the mixture is 0.5 mg / mL, and the concentration of HRP is 0.5 mg / mL. Figure 10 The legend (0.5 mg / mL, etc.) represents the concentration of glucose oxidase (GOx) after mixing, and the concentration of DNase I 400 U / mL and HRP 0.5 mg / mL in the mixture; Figure 11 The legend (0.5 mg / mL, etc.) represents the concentration of HRP, and the mass concentration of DNase I 400 U / mL and GOx in the mixture is 0.5 mg / mL.

[0069] DGH-YB catalytic efficiency determination: With the concentrations of DGH-YB and Free DGH kept constant, different concentrations of Glu were added, and the Michaelis constants for Glu catalysis by DGH-YB and Free DGH were calculated. The results are as follows: Figure 12 As shown in Figures (A) and (B), while maintaining the same amount of enzyme, DGH-YB significantly improved the catalytic efficiency of GOx and HRP, as... Figure 12 As shown in Figure (A), maintaining the same substrate concentration, DGH-YB completely depleted the substrate in 300 s, while Free DGH took 1200 s to deplete it. The calculated transition number of DGH-YB was 7.2 times that of Free DGH.

[0070] DGH-YB stability test: In the above experiments, DNase I exhibited good cleavage activity in neutral buffer. When the pH was changed to 3, 4, 5, 6, 7, 8, and 9, both DGH-YB and the free enzyme showed good cleavage activity at pH 4-7. The results are as follows... Figure 13 As shown, at pH=9, the enzyme activity of Free DGH decreased significantly, but DGH-YB maintained good enzyme activity. After synthesizing DGH-YB, DGH-YB powder was obtained by freeze-drying. DGH-YB and Free DGH were placed in a constant temperature metal bath at 37℃ for 15 days. The enzyme activities of DGH-YB and Free DGH were measured on days 0, 5, 10, and 15, respectively. The results are as follows. Figure 14As shown, on day 5, DGH-YB activity decreased slightly, while Free DGH was almost inactivated. On day 15, DGH-YB still exhibited high activity. After one DNA cleavage experiment, DGH-YB was recovered by centrifugation for subsequent DNA cleavage experiments. The results are as follows... Figure 15 As shown, when the DGH-YB activity was recovered for the fifth time, it only decreased to 60% of the initial activity.

[0071] DGH-YB disrupts biomembranes in vitro: After pre-washing 14 mm glass discs with deionized water and ethanol respectively, a sterile 24-well plate was taken, and the cleaned glass discs were added to the wells. The plate was then sterilized under ultraviolet light in a clean bench for 30 minutes. Then, 1 mL of 1x10⁻⁶ glass discs was added to the wells. 5 CFU / mL MRSA Bacterial suspensions were kept humid by surrounding the suspension with TSB medium (without bacterial suspension). The suspensions were incubated at 37°C for 24 hours. After rinsing 2-3 times with PBS, loosely adhering bacteria were removed, leaving the bacterial biofilm on the surface of the wells. Once the biofilm formed, PBS, GH-YB, Free DGH, Free DNase I, D-YB, and DGH-YB (DNase I, GOx, and HRP levels were the same in different samples) were added, and the mixture was incubated at 37°C for 6 hours. After rinsing 2-3 times with PBS to remove free bacteria, 1% crystal violet staining solution was added and incubated for 0.5 hours. The crystal violet solution was then aspirated, and the suspensions were rinsed 2-3 times with PBS to remove unbound crystal violet. The suspensions were then air-dried. Finally, the crystal violet bound to the biofilm was redissolved with 33% acetic acid solution, and the absorbance at 595 nm was measured using a UV spectrophotometer. Results are as follows: Figure 16 As shown, the DGH-YB group showed the lightest crystal violet staining, indicating the strongest biofilm disruption. DGH-YB can disrupt mature biofilms by cutting eDNA.

[0072] After bacterial biofilm formation, PBS, GH-YB, Free DGH, Free DNase I, D-YB, and DGH-YB were added, and the mixture was incubated at 37°C for 6 h. The mixture was then washed 2-3 times with PBS to remove free bacteria. Calcein AM (calcein acetoxymethyl ester) was added, and the mixture was incubated for 0.5 h. Biofilm changes were observed using a confocal microscope. Results are as follows: Figure 17 As shown, DGH-YB can disrupt established mature biofilms.

[0073] PBS, GH-YB, Free DGH, Free DNase I, D-YB, and DGH-YB were added respectively. MRSABacterial suspensions were washed 2-3 times with PBS at 12 h, 24 h, 48 h, and 72 h to remove free bacteria, and then incubated with Calcein AM for 0.5 h. Biofilm changes were observed using a confocal microscope. Results are as follows: Figure 18 As shown, DGH-YB can inhibit the formation of biofilms.

[0074] Validation of DGH-YB's in vitro biofilm scavenging and bactericidal activities: MRSA and E. coli The bacteria were used as representative pathogens to study the bacterial eradication ability of DGH-YB. Its antibacterial properties were investigated using the minimum inhibitory concentration (MIC), absorbance at 600 nm (OD600), and agar plate counts. Bacteria were incubated with a series of final concentrations of DGH-YB. The systems contained Glu and 10... 6 CFU / mL bacteria were cultured at 37°C for 12 h. Then, the OD600 values ​​of the bacterial suspensions treated with different concentrations were recorded, and the MIC values ​​were calculated. The bacterial suspensions were then serially diluted and used for agar plate counting. The agar plates were incubated at 37°C for another 12 h, and the final colony count was obtained. Results are as follows: Figure 19 As shown, DGH-YB pairs MRSA and E. coli Both exhibited good bactericidal activity. The MIC of DGH-YB was 0.23 mg / mL, and that of MBC was 0.5 mg / mL.

[0075] Will MRSA The suspension (100 μL, 10⁸ CFU / mL) and LB, along with 800 μL of LB medium, were placed in a confocal microplate and incubated at 37 °C. After 24 hours, the medium was removed, and unattached bacteria were gently washed three times with sterile PBS. Then, 800 μL of LB medium, 100 μL of Glu, and 100 μL of DGH-YB were added, and the mixture was incubated at 37 °C for 5 h. AM / PI was then added, and the mixture was incubated for 0.5 h. The results were observed using a confocal laser scanning microscope. Figure 20 As shown, DGH-YB can not only effectively disrupt biofilms, but also kill the bacteria released from the disrupted biofilms.

[0076] Verification of DGH-YB's biofilm scavenging and bactericidal activities in vivo: A biofilm infection model of diabetic mice was established by randomly assigning all mice (n=3). After anesthesia with 3% sodium pentobarbital, pores with a diameter of approximately 0.5 cm were created on the epidermis. Then, 150 μL of... MRSA suspension (1×10 8CFU / mL was applied to the wound and incubated for 48 h. Afterwards, the wound was treated with PBS, GH-YB, Free DGH, and DGH-YB (with identical DNase I, GOx, and HRP levels in different samples). Nine days later, the treated wound was excised and fixed with 4% paraformaldehyde solution for histological hematoxylin-eosin (H&E), Masson staining, and CD31 staining analysis. Results are as follows: Figure 21 As shown, DGH-YB can effectively remove biofilms from wounds in diabetic mice, kill bacteria, promote increased CD31 expression, and reduce the expression of inflammatory factors (TNF-α, IL-6), thus synergistically promoting wound healing.

[0077] Figure 21 As shown: First, compared to the control group, the wound in the DGH-YB group completely healed by day seven, and on day two, the number of bacteria at the wound site was reduced by 94%, while other groups still had a large number of bacteria at the wound site and had not healed even after nine days. Second, compared to the GH-YB group, the DGH-YB group lacked DNase I, which prevented the disruption of the biofilm, significantly reducing the bactericidal effect of GH-YB and resulting in slower wound healing. Finally, compared to Free DGH, DGH-YB, due to the lack of mineralization, exhibited poor stability of the natural enzyme in complex biological environments, thus producing a poor effect. In summary, the mineralized DGH-YB of this invention not only improves the stability of DNase I but also enhances the bactericidal efficiency.

[0078] Diabetic wounds are extremely difficult to heal due to their microenvironment, characterized by high glucose levels, hypoxia, and susceptibility to bacterial growth. Compared to normal tissue (2 mM), diabetic wounds can contain up to 10 mM glucose. This high glucose level acts as a substrate for the cascade catalysis of GOx and HRP, generating hydroxyl radicals more efficiently for bactericidal effects. Simultaneously, it consumes glucose to generate gluconic acid, lowering the pH near the wound and synergistically promoting wound healing. The lyophilized powder prepared using the method described in this invention can be used as a topical medication or dressing for wounds, applicable to various bacterial infection environments (such as implant infections, catheter infections, or wound infections). Normal human tissue contains 1-2 mM of glucose, varying depending on the location. DGH-YB can efficiently disrupt biofilms, and even small amounts of glucose can achieve good bactericidal effects, while simultaneously releasing Cu... 2+ It can promote bone regeneration.

[0079] Rat model of bone defect biofilm infection: 36 adult male SD rats (200–250 g) were randomly divided into four groups: pTA(Titanium implants containing only polytannic acid), Free DGH, GH-YB, and DGH-YB. To establish an implantation-related infection model, rats were anesthetized with 1.5% isoflurane, and cylindrical bone defects were surgically created at the lateral femoral condyle. Each defect was inoculated with 50 μL of [amount missing]. MRSA suspension (1×10 8 CFU·mL⁻¹). Twenty-four hours later, an engineered titanium alloy implant (3 mm in diameter, 5 mm in height) was implanted. pTA, Free DGH, GH-YB, and DGH-YB coatings were constructed on the implants (prostheses) of the four groups of rats, respectively, and implanted together with the prostheses. The rats were euthanized one week post-surgery to assess antibacterial efficacy, such as… Figure 24 As shown in Figures (A)-(D), the experimental results indicate that while the pTA group (antibacterial rate 0.92%) and the GH-YB group (antibacterial rate 17.65%) exhibited some antibacterial activity, they were unable to effectively disrupt the biofilm, resulting in a significant number of bacteria remaining on the prosthesis surface after one week. While free DNase I (Free DGH group, antibacterial rate 26.23%) could degrade the biofilm, its poor stability in the complex biological environment of bone tissue limited its antibacterial effect. The DGH-YB group, however, improved the stability of DNase I, enabling it to stably and efficiently disrupt the biofilm in the complex biological environment. It also enhanced the catalytic efficiency of the GOx and HRP cascade, significantly increasing the efficiency of hydroxyl radical generation and improving the bactericidal effect, achieving an antibacterial rate of 95% in the bone defect model.

[0080] Biosafety: Live / dead fluorescence microscopy images of DGH-YB and GH-YB co-cultured with L929 cells, such as Figure 25 As shown, the high cell survival rate indicates that the DGH-YB prepared in this embodiment of the invention has good biocompatibility.

[0081] The hemolysis test results of DGH-YB and GH-YB are as follows: Figure 26 As shown in Figures (A)-(C), the hemolysis rate (%) of DGH-YB is less than 4%, indicating good blood compatibility.

[0082] Finally, the samples were treated with PBS (control), Free GH, GH-YB, and DGH-YB. MRSA and E. coli Survival rate, experimental results are as follows Figure 27 As shown, the DGH-YB prepared in the embodiments of the present invention is effective against Escherichia coli and drug-resistant bacteria. MRSA The clearance rates were 94.78% and 97.45%, respectively.

[0083] Example 2

[0084] A method for preparing a mineralizing enzyme composite powder (DGH-YB) for disrupting biofilms: Add DNase I, HRP, and GOx to a 1.5 mL EP tube, and then add 1X PBS to bring the volume to 940 μL to obtain an enzyme solution. The enzyme solution contains DNase I with an activity concentration of 800 U / mL, GOx with a mass concentration of 0.05 mg / mL, and HRP with a mass concentration of 0.25 mg / mL. Add 60 μL CuSO4 (150 mM) to the solution, mix thoroughly, and place in a constant temperature metal bath (20 ℃) ​​for 48 h. Remove the centrifuge tube, centrifuge (5500 rpm, 4 min), discard the supernatant, wash 2-3 times with PBS, and resuspend the resulting precipitate in PBS buffer.

[0085] Example 3

[0086] A method for preparing a mineralizing enzyme composite powder (DGH-YB) for disrupting biofilms: Add DNase I, HRP, and GOx to a 1.5 mL EP tube, and then add 1X PBS to bring the volume to 980 μL to obtain an enzyme solution. The enzyme solution contains DNase I with an activity concentration of 600 U / mL, GOx with a mass concentration of 1 mg / mL, and HRP with a mass concentration of 0.5 mg / mL. Add 20 μL CuSO4 (180 mM) to the solution, mix thoroughly, and place in a constant temperature metal bath (22 ℃) for 12 h. Remove the centrifuge tube, centrifuge (5800 rpm, 5 min), discard the supernatant, wash 2-3 times with PBS, and resuspend the resulting precipitate in PBS buffer.

[0087] The experimental data for Examples 2 and 3 are shown in [reference needed]. Figures 22-23 . Figure 22 In Figure (A), the concentration of horseradish peroxidase (HRP) is 0.25 mg / mL; Figure 22 The concentration of horseradish peroxidase (HRP) in (B)-(F) was 0.5 mg / mL; Figure 23 The concentration of horseradish peroxidase (HRP) in diagrams (A)-(D) is 0.5 mg / mL.

[0088] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. A mineralizing enzyme composite powder for disrupting biofilms, characterized in that, The composite powder is a hybrid nanoparticle prepared by one-step biomineralization, and the composite powder contains nuclease and bactericidal active enzyme; The nuclease is one or more of deoxyribonuclease, ribonuclease or nonspecific nuclease in any proportion; The bactericidal active enzyme is a single enzyme or multiple enzymes, wherein the multiple enzyme cascade generates hydroxyl radicals.

2. The mineralizing enzyme composite powder for disrupting biofilms according to claim 1, characterized in that, The nuclease is a natural deoxyribonuclease, and the bactericidal active enzymes are glucose oxidase and horseradish peroxidase. The activity concentration of the natural deoxyribonuclease is 400-800 U / mL, the mass concentration of glucose oxidase is 0.05-1 mg / mL, and the mass concentration of horseradish peroxidase is 0.25-0.5 mg / mL. The deoxyribonuclease in the hybrid nanoparticles can effectively disrupt biofilms under alkaline conditions or in complex biological environments, exposing bacteria. Glucose oxidase consumes glucose to create an acidic environment, while the generated H2O2 serves as a substrate for horseradish peroxidase, continuing the reaction to generate hydroxyl radicals for synergistic bactericidal action.

3. The mineralizing enzyme composite powder for disrupting biofilms according to claim 2, characterized in that, The composite powder achieved a 94.78% clearance rate against Escherichia coli and a 97.45% clearance rate against methicillin-resistant Staphylococcus aureus, shortening wound healing time to 7 days.

4. A method for preparing a mineralizing enzyme composite powder for disrupting biofilms, characterized in that, Includes the following steps: S1, Natural deoxyribonuclease, glucose oxidase and horseradish peroxidase are mixed and dissolved in phosphate buffer in a certain proportion to obtain an enzyme solution. The activity concentration of natural deoxyribonuclease in the enzyme solution is 400-800 U / mL, the mass concentration of GOx is 0.05-1 mg / mL, and the mass concentration of HRP is 0.25-0.5 mg / mL. S2. The enzyme solution obtained in S1 is mixed with the metal salt solution at a volume ratio of 94-98:2-6. The mixture is thoroughly mixed and reacted in a constant temperature metal bath at 20-25℃ for 12-48 hours. During the synthesis process, inorganic components grow around the enzyme to form enzyme-based hybrid nanoparticles, which improve the stability and catalytic efficiency of the enzyme.

5. The method for preparing a mineralizing enzyme composite powder for disrupting biofilms according to claim 4, characterized in that, Also includes: The three-dimensional flower-like structure was allowed to stand, washed, and collected. The resulting precipitate was then resuspended in phosphate buffer.

6. The method for preparing a mineralizing enzyme composite powder for disrupting biofilms according to claim 4, characterized in that, The concentration of the phosphate buffer solution is 0.5-2M.

7. The method for preparing a mineralizing enzyme composite powder for disrupting biofilms according to claim 4, characterized in that, The metal salt solution is a 100-200 mM CuSO4 solution.

8. The application of a mineralizing enzyme composite powder for disrupting biofilms as described in any one of claims 1-3 in an antibacterial product, wherein the antibacterial product is a wound dressing, a wound topical drug, or a drug loaded onto a prosthesis.

9. The application of the mineralizing enzyme composite powder for disrupting biofilms according to claim 8 in antibacterial products, characterized in that, The glucose concentration in the application environment is 1mM-10mM.

10. The application of the mineralizing enzyme composite powder for disrupting biofilms according to claim 8 in antibacterial products, characterized in that, The antimicrobial product is used to treat or prevent infections associated with bacterial biofilms, including implant infections, catheter infections, or wound infections.

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