Preparation and application of biomimetic mineralized nanoparticles

Abstract

The application belongs to the technical field of antibacterial infection materials, and particularly relates to preparation and application of a biomimetic mineralization nanoparticle. x S y / AZM, which is obtained by fixing glucose oxidase (GOx) in FexSy nanoparticles hybridized with azithromycin (AZM) through a biomimetic mineralization strategy, and the nanoparticle GOx@Fe x S y / AZM serves as a therapeutic drug to catalyze a large amount of active oxygen free radicals (ROS) to improve inflammatory response in order to rapidly kill infectious microorganisms in the early stage of bacterial infection of a wound surface; and in the subsequent healing period, H2S released by the material and AZM will jointly promote the polarization of macrophages to M2 macrophages of a repair type to effectively mediate the tissue repair after a high inflammatory response, so that the ordered treatment of a high blood sugar infection wound surface is achieved.

Description

Technical Field

[0001] This invention belongs to the field of antibacterial infection materials technology, specifically relating to the preparation and application of biomimetic mineralized nanoparticles. Background Technology

[0002] Diabetes is a complex metabolic disease that affects the health of millions of people worldwide. Currently, there are approximately 285 million adults with diabetes, and experts predict this number will increase to 439 million by 2030.

[0003] Bacterial infection is the most common complication of diabetic ulcers and a major cause of poor tissue regeneration in patients with chronic diabetes. The hyperglycemic state in diabetic patients alters the immune response, reducing resistance to infection. Combined with prolonged open wounds, this makes diabetic wounds highly susceptible to infection. Studies indicate that infections of diabetic ulcers are often complex, involving both Gram-positive and Gram-negative bacteria, including Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Incomplete eradication of these microorganisms can lead to wound recurrence and persistent local inflammation. This results in a prolonged high level of inflammatory factors, extending the inflammatory phase and causing chronic wounds.

[0004] Current clinical treatments for chronic diabetic wounds include skin infusion, infection control, metabolic management, treatment of complications, and local wound care. However, these standard treatments remain limited in their effectiveness in promoting diabetic wound healing. Traditional diabetic wound treatment relies primarily on wound dressings, a lengthy process prone to secondary damage and adverse psychological and physiological effects on patients. While antibiotic treatment is crucial for controlling diabetic ulcer infections, the diversity of infecting bacteria and the formation of bacterial biofilms present new challenges. Although techniques for promoting diabetic tissue repair exist, such as topical drug therapy (e.g., drugs, peptides, and growth factors), cell therapy (e.g., stem cells and fibroblasts), and nanomaterial-based therapies, the complex etiology of diabetic wounds means that single therapies using one or two substances are insufficient to accelerate healing. Therefore, a more comprehensive approach could be considered, combining different needs of the wound healing process by releasing multiple substances at different stages of wound development to regulate specific biological functions within the complex pathological microenvironment, thus achieving orderly treatment of diabetic wound infections. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes a method for preparing and applying biomimetic mineralization nanoparticles. A biomimetic mineralization strategy is used to immobilize glucose oxidase (GOx) within azithromycin (AZM) hybridized FexSy nanoparticles, yielding GOx@Fe nanoparticles. x S y / AZM, these nanoparticles, act as therapeutic agents. In the early stages of bacterial infection in wounds, they catalyze glucose to produce a large number of reactive oxygen species (ROS), enhancing the inflammatory response to rapidly kill the infecting microorganisms. During the subsequent healing period, the H2S released by the material and AZM work together to promote the polarization of macrophages into repair-type M2 macrophages, effectively mediating tissue repair after the hyperinflammatory response and achieving orderly treatment of hyperglycemic wounds.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0007] This invention provides a method for preparing biomimetic mineralized nanoparticles, comprising the following steps:

[0008] S1: Add glucose oxidase to FeCl2·4H2O solution and stir, then add azithromycin ethanol solution and continue stirring to obtain a mixed solution;

[0009] S2: Add NaS solution to the mixed solution to react and obtain a black precipitate. After washing and drying, the nanoparticles GOx@Fe are obtained. x S y / AZM.

[0010] Preferably, the mass ratio of glucose oxidase, FeCl2·4H2O solution, azithromycin ethanol solution, and NaS solution is 2:5:10:3;

[0011] Preferably, in step S1, glucose oxidase is added to FeCl2·4H2O solution and stirred for 3 min, then azithromycin ethanol solution is added and stirred at low temperature for 3 min.

[0012] Preferably, in step S2, the black precipitate is washed twice with boiled deionized water and then freeze-dried to obtain nanoparticles GOx@FexSy / AZM.

[0013] Another object of the present invention is to provide biomimetic mineralized nanoparticles GOx@Fe prepared by the above method. x S y Application of / AZM in the systematic treatment of hyperglycemic infected wounds.

[0014] The beneficial effects of this invention are:

[0015] The biomimetic mineralized nanoparticles GOx@Fe prepared in this invention x Sy / AZM, as a therapeutic agent, can catalyze the production of large amounts of ROS from glucose in the early stages of bacterial infection of wounds, thereby enhancing the inflammatory response and rapidly killing the infecting microorganisms. During the subsequent healing period, the H2S released by the material, together with AZM, will promote the polarization of macrophages towards the repair-oriented M2, effectively mediating tissue repair after the hyperinflammatory response, achieving orderly treatment of hyperglycemic wounds. Furthermore, due to the fixation of enzymes by the obtained material and the interaction between AZM and Fe... 2+ Coordination between GOx and Fe 2+ It is effectively protected and can be stored at room temperature for a long time without deterioration, thus providing GOx@Fe x S y / AZM provides important support for the orderly management of infected wounds mediated by therapeutic drugs. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments 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.

[0017] Figure 1 The image shows the TEM characterization of the nanoparticles in Example 1.

[0018] Figure 2 In Example 1, GOx@Fe x S y / AZM's element mapping graph;

[0019] Figure 3 Fe in Example 1 x S y and GOx@Fe x S y DLS diagram of / AZM;

[0020] Figure 4 In Example 1, GOx@Fe x S y Infrared spectra of different components during the preparation of / AZM;

[0021] Figure 5 This is a standard curve of the contents of GOx, AZM, and Na2S in Example 1;

[0022] Figure 6 Fe in Example 2 x S y with GOx@Fe x S y XPS spectrum corresponding to / AZM;

[0023] Figure 7 Fe in Example 2 x S y GOx@Fe x S y GOx@Fe x S y Stability images of / AZM at 0, 1, 2, 3, 4, and 5 days, where the left tube is Fe. x S y , intermediate tube GOx@Fe x S y The right tube is GOx@Fe x S y / AZM;

[0024] Figure 8 For example, Fe was placed on the third day in Example 2. x S y Powder morphology diagram of GOx@FexSy / AZM after oxidation;

[0025] Figure 9 In Example 2, GOx@Fe x S y / Schematic diagram of the protective effect of AZM on GOx;

[0026] Figure 10 In Example 3, GOx@Fe x S y Electron micrographs of AZM in solutions at pH 7.4, 6, and 5;

[0027] Figure 11 In Example 3, GOx@Fe x S y / AZM H2S release curve;

[0028] Figure 12 It is AZM and Fe in Example 3 2+ Ion release curve;

[0029] Figure 13 It is the 200 μg / mL GOx@Fe in Example 4 x S y / AZM glucose concentration change at different reaction times and pH change at different reaction times;

[0030] Figure 14 Different concentrations of GOx@Fe in Example 4 x S y The graph shows the changes in glucose concentration over 12 hours under AZM and the changes in pH of the catalytic glucose solution over 3 hours.

[0031] Figure 15 It is GOx@Fe in Example 4 x S y / AZM, 5μg / mL GOx@Fe x S y / AZM+Glu and 50μg / mLGOx@Fe x S y ESR spectrum of / AZM+Glu;

[0032] Figure 16 In Example 4, Fe was used in different solutions. 2+ UV-Vis absorption spectra and images of MB after degradation by Fenton-like mediated reaction;

[0033] Figure 17 Different concentrations of Fe in Example 4 2+ UV-Vis absorption spectra and images of MB after degradation by Fenton-like mediated reaction;

[0034] Figure 18 Different Fe contents in Example 5 x S y AZM@Fe x S y GOx@Fe x S y and GOx@Fe x S y Turbidity diagrams of S. aureus and E. coli cultures in AZM;

[0035] Figure 19 In Example 5, S. aureus and E. coli were treated with 10 μg / mL PBS and Fe... x S y AZM@Fe x S y GOx@Fe x S and GOx@Fe x S y / Image of colonies formed after AZM treatment;

[0036] Figure 20 In Example 5, S. aureus and E. coli were treated with 10 μg / mL PBS and Fe... x S y AZM@Fe x S y GOx@Fe x S y and GOx@Fe x S y / Quantitative results of colonies formed after AZM treatment;

[0037] Figure 21 In Example 5, confocal fluorescence microscopy was used to analyze PBS and Fe... x S y AZM@Fe x S y GOx@Fe x S y and GOx@Fe x S y Schematic diagram of live / dead staining analysis of S. aureus and E. coli incubated with AZM;

[0038] Figure 22 It is the same as that in Example 5 with PBS and Fe x S y AZM@Fe x S y GOx@Fe x S y and GOx@Fe x S y SEM images of S. aureus and E. coli bred together with AZM;

[0039] Figure 23 These are the corresponding eradication effects of different treatments on Staphylococcus aureus biofilm in Example 6;

[0040] Figure 24 These are three-dimensional confocal fluorescence microscopy images of Staphylococcus aureus biofilms after different treatments in Example 6;

[0041] Figure 25 These are different concentrations of GOx@Fe in Example 7. x S y Schematic diagram of the percentage of hemolysis of red blood cells by AZM;

[0042] Figure 26 This is a schematic diagram of the quantitative analysis of RAW264.7 cell viability using the CCK-8 assay in Example 7;

[0043] Figure 27 This is a schematic diagram of the quantitative analysis of L929 activity using the CCK-8 assay in Example 7;

[0044] Figure 28 This is a flow cytometry plot of CD86 (M1 marker) and CD206 (M2 marker) expression after different treatments in Example 7;

[0045] Figure 29This is a schematic diagram of the immunofluorescence staining used in Example 7 to evaluate the macrophage phenotypes of each group: Arg-1 (red, M2 macrophage marker), iNOS (green, M1 macrophage marker), and Hoechst (blue, cell nucleus).

[0046] Figure 30 This is an ELISA result of macrophage phenotypes Arg-1 (M2 macrophage marker), iNOS (M1 macrophage marker), Hoechst (cell nucleus), and TNF-α in RAW264.7 supernatant in Example 7.

[0047] Figure 31 This is an ELISA result of macrophage phenotypes Arg-1 (M2 macrophage marker), iNOS (M1 macrophage marker), Hoechst (cell nucleus), and IL-6 in RAW264.7 supernatant in Example 7.

[0048] Figure 32 This is an ELISA result of macrophage phenotypes Arg-1 (M2 macrophage marker), iNOS (M1 macrophage marker), Hoechst (cell nucleus), and CD206 in RAW264.7 supernatant in Example 7.

[0049] Figure 33 This is an ELISA result of macrophage phenotypes Arg-1 (M2 macrophage marker), iNOS (M1 macrophage marker), Hoechst (cell nucleus), and IL-12 in RAW264.7 supernatant in Example 7.

[0050] Figure 34 These are scratch assay images of L929 cells cultured in macrophage conditioned medium for 0 and 24 hours in Example 7;

[0051] Figure 35 This is a quantitative analysis diagram of the relative scratch area of ​​L929 cells cultured in macrophage conditioned medium for 0 and 24 hours in Example 7;

[0052] Figure 36 These are representative images of Staphylococcus aureus-infected wounds in mice after different treatments in Example 8;

[0053] Figure 37 This is a quantitative graph showing the percentage of wound healing area at different time points in Example 8;

[0054] Figure 38 This is a diagram showing the bacterial survival of Pseudomonas aeruginosa in the skin or spleen of mice in Example 8;

[0055] Figure 39This is a quantitative graph showing the bacterial survival of Pseudomonas aeruginosa in mouse skin or spleen infected in Example 8;

[0056] Figure 40 This is a schematic diagram of the detection of TNF-α levels in granulation tissue using the ELISA method in Example 8;

[0057] Figure 41 This is a schematic diagram of the detection of IL-6 levels in granulation tissue using the ELISA method in Example 8;

[0058] Figure 42 These are H&E and Masson's staining images of wound tissues from different treatment groups at 12 days in Example 8;

[0059] Figure 43 These are H&E staining images of major organs of mice 12 days after different treatments in Example 8;

[0060] Figure 44 These are histological analysis images of mice in each group after treatment in Example 8;

[0061] Figure 45 This is a schematic diagram of the blood biochemistry and hematology examination of db / db mice 12 days after treatment in Example 8. Detailed Implementation

[0062] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0063] Example 1

[0064] Nanoparticles GOx@Fe x S y Synthesis and characterization of / AZM:

[0065] 10 mg of glucose oxidase was added to FeCl2·4H2O (5 mg / mL, 5 mL) solution and stirred for 3 min. Then, azithromycin (0.1 g / mL, 500 μL) was dissolved in ethanol and stirred at low temperature for 3 min. Finally, NaS (3 mg / mL, 5 mL) was added and reacted for 30 min to obtain a black precipitate. The precipitate was washed twice with boiled deionized water and freeze-dried to obtain mineralized nanoparticles GOx@Fe x S y / AZM, GOX@Fe x S y TEM characterization of / AZM as follows Figure 1 As shown.

[0066] Fe was then synthesized using the same method.x S y TEM characterization of FexSy as follows Figure 1 As shown in A. To further determine GOx@Fe x S y / AZM elemental composition, scanned GOx@Fe x S y / AZM analyzed the elemental distributions of S, Fe, C, O, and N. The elemental distributions are as follows: Figure 2 As shown. Figure 3 As shown, the hydrated particle size (DLS) indicates Fe x S y and GOx@Fe x S y The diameters of / AZM are approximately 125.3 nm and 135.9 nm, respectively. When glucose oxidase (GOx) is coated with Fe... x S y After being combined with azithromycin (AZM), its particle size was larger than that of Fe alone. x S y The nanoparticles increased in size by approximately 15 nm, further confirming that GOx was successfully coated with Fe. x S y And AZM. For example Figure 4 As shown, with Fe x S y In comparison, GOx@Fe x S y / AZM at 1537cm -1 and 2969cm -1 The characteristic absorption peaks at these locations are attributed to the C=O stretching vibration in the amide group of GOx and the CH contraction in AZM, respectively, confirming the presence of GOx@Fe x S y Successful synthesis of / AZM.

[0067] To further confirm GOx@Fe x S y The percentage contents of GOx and AZM loaded in AZM were calculated using standard curves via the Coomassie Brilliant Blue method (Bradford method) and spectrophotometry, respectively. The loadings of GOx and AZM were 12.1 w / w% and 29 w / w%, respectively. Figure 5 As shown. To better quantify the Fenton reaction catalyzed by Fe ions, Fe was measured by ICP. x S y and GOx@Fe x S y The Fe content in / AZM, the results showed Fe x S y and GOx@Fe x Sy The Fe content in / AZM was 44.3% and 17.4%, respectively.

[0068] Example 2

[0069] GOx@Fe x S y / AZM for Fe 2+ The protective effect of GOx:

[0070] Study of Fe using X-ray photoelectron spectroscopy x S y and GOx@Fe x S y / AZM iron valence state during storage. Preparation of GOx@Fe x S y Fe2p of / AZM nanoparticles 3 / 2 The binding energy is 712.3 eV, equivalent to pure FexSy. After being left for 3 days, Fe... x S y Approximately 61.64% of FexSy was oxidized to Fe3O4 ( Figure 6 (As shown in Figure A), while GOx@Fe x S y / AZM contains only 22.62% Fe x S y It is oxidized to Fe3O4 ( Figure 6 (As shown). Furthermore, Fe... x S y GOx@Fe x S y and GOx@Fe x S y / AZM solution exposed to air showed results even on the fifth day GOX@Fe x S y / AZM still shows no obvious color change (e.g. Figure 7 (As shown). This phenomenon indicates that the enzyme is immobilized by the obtained material and that AZM reacts with Fe. 2+ The coordination between them makes Fe 2+ It can be effectively protected, maintaining the stability of particles in the solution. Meanwhile, freeze-dried Fe... x S y and GOx@Fe x S y AZM powder also showed similar Fe after being left for 15 days. 2+ The protective effect (such as) Figure 8 (As shown). To further investigate GOx@Fe x S yThe protective effect of AZM nanoparticles on GOx: We will combine free GOx and GOx@Fe x S y / AZM was placed in a high-temperature environment of 60°C to evaluate GOX and GOX@Fe x S y The amount of glucose consumed by / AZM was used to assess the activity of GOx, and the results showed that GOx@Fe x S y / AZM can effectively prevent the rapid deactivation of GOX in high-temperature environments (e.g., Figure 9 (As shown).

[0071] Example 3

[0072] GOx@Fe x S y In vitro degradation and drug release of AZM:

[0073] Due to GOx@Fe x S y / AZM can cause drug disintegration by generating H2S gas in the acidic microenvironment of diabetic wounds, via GOx@Fe x S y / AZM showed gradual disintegration as the pH decreased in pH values ​​of 7.4, 6, and 5 using electron microscopy (e.g., ...). Figure 10 (As shown). By measuring at the same concentration of GOx@Fe x S y / AZM under different pH conditions, GOx, AZM and Fe 2+ The cumulative release (e.g.) Figure 11 and Figure 12 (As shown).

[0074] The results showed that as pH decreased, GOx, AZM, and Fe... 2+ The release rate of GOx@Fe increased significantly, indicating that x S y / AZM's pH responsiveness allows for rapid H2S gas generation and drug release targeting the wound.

[0075] Example 4

[0076] GOx@Fe x S y Catalytic activity test of / AZM:

[0077] Because GOx can specifically catalyze the oxidation of glucose to gluconic acid and H2O2, the generated H2O2 can be reacted with Fe... 2+The Fenton-like mediated reaction catalyzes the generation of ·OH, which is antibacterial. Therefore, GOx@Fe was evaluated by measuring glucose consumption, gluconic acid production, and ·OH generation. x S y The catalytic ability of / AZM. First, the amount of glucose consumed during the reaction was assessed using a blood glucose meter. Over time, GOx@Fe x S y / AZM was incubated at 200 μg / mL with glucose gradually reduced ( Figure 13 Within the same 24-hour reaction time, glucose concentration decreased with increasing GOx@Fe x S y / The concentration of AZM nanoparticles increases and decreases ( Figure 14 Then, the production of gluconic acid was assessed by detecting pH changes. The solution changed from neutral (~7.4) to acidic (~3.24) over 24 hours. Figure 13 The pH value decreased rapidly in the first three hours, then changed slowly. At a fixed reaction time of 24 hours, the pH of the solution decreased with increasing GOx@Fe x S y / ZAM concentration increases and decreases ( Figure 14 ).

[0078] Subsequently, the formation of ·OH was measured by ESR spectroscopy using the spin trapping agent 5,5-dimethyl-1-pyrrolline N-oxide (DMPO). The ESR spectra clearly showed the characteristic quartet signal (1:2:2:1) of DMPO-OH, indicating that the ·OH species are generated via GOx / Glu / Fe. 2+ mediated catalytic production ( Figure 15 Methylene blue (MB) is a dye that can be degraded by ·OH and has been selected as an indicator of ·OH generation. Figure 16 As shown, when MB and GOx@Fe x S y When AZM and Glu were incubated in NaHCO3 / CO2 buffer for 1 h, a significant decrease in MB absorbance was observed, while no significant change in MB absorbance was observed after the same treatment with other groups in the solution. Next, we will explore different concentrations of GOx@Fe x S y The ability of / AZM to catalyze the production of ·OH was found to be influenced by GOx@Fe. x S y / AZM concentration increases and gradually fades ( Figure 17 This proves that GOx@Fe x S y / AZM can generate ·OH by consuming Glu.

[0079] Example 5

[0080] GOx@Fe x S y / AZM's in vitro antibacterial activity:

[0081] Due to impaired glucose metabolism, wound conditions in diabetic patients are often complex. Chronic diabetic wounds are characterized by long-term inflammation and recurrent bacterial infections. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) infections are the most common bacterial infections in diabetic wounds. Therefore, to evaluate the synergistic antibacterial effect of nanocomposite materials, viability assays, dilution plate assays, live / dead staining, and bacterial morphology observations were performed on S. aureus and E. coli. Figure 18 As shown, the poor water solubility of free AZM during high glucose culture leads to AZM@Fe x S y Its antibacterial effect is limited in high-sugar environments. GOx@Fe x S y The antibacterial activity of the group was improved, but it still had certain limitations at low concentrations. However, GOx@Fe x S y / AZM showed a significant improvement in antibacterial activity, and even at low concentrations (7.5ug / ulGOx@Fe x S y / AZM) can significantly reduce the number of viable bacteria. When GOx@Fe x S y / AZM at a concentration of only 7.5 μg / mL showed good antibacterial effects, therefore subsequent experiments used GOx@Fe x S y The optimized dose for AZM was 10 μg / mL. A blank control and Fe were also included. x S y AZM@Fe x S y and GOx@Fe x S y Group.

[0082] Then, the dilution plate assay was used to further quantify the antibacterial effects of different treatments on *S. aureus* and *E. coli*. For example... Figure 19 As shown, the control group and Fe x S y The colony count in the treatment group remained almost unchanged. (Compared to AZM@Fe) x S y and GOx@Fe x Compared to Group S, GOx@Fe x S y The colony count in the / AZM group was significantly lower, indicating that GOx@Fex S y / AZM nanoparticles exhibit the best antibacterial effect under hyperglycemic conditions, as shown in the quantitative results. Figure 20 Then, the SYTO9 / PI live / dead fluorescence staining method was used to further analyze the effects of different treatments on the survival of *S. aureus* and *E. coli*. Figure 21 As shown, the fluorescence intensity of SYTO9 (green) and PI (red) in the six groups was consistent with the results of the bacterial survival test. Control group, Fe... x S y Almost no bacteria stained with PI (red fluorescence) were observed in the treatment group. Compared with the AZM@FexS and GOx@FexSy groups, GOx@Fe x S y The enhanced red fluorescence in the / AZM group indicates that GOx@Fe x S y / AZM nanoparticles exhibit rapid antibacterial effects in hyperglycemic environments. To more intuitively observe GOx@Fe x S y To assess the antibacterial effect of AZM nanoparticles, we also used inhibition zones to evaluate the drug's antibacterial efficacy. The results showed that GOx@Fe x S y The AZM group showed the largest inhibition zone compared to other groups.

[0083] To further study GOX@Fe x S y The antibacterial process of / AZM nanoparticles was observed using scanning electron microscopy, revealing structural changes in bacteria. Figure 22 As shown, the control group and Fe x S y The incubated *S. aureus* and *E. coli* maintained their intact bacterial morphology. Notably, the cytoplasm of bacteria incubated with AZM@FexSy and GOx@FexS formed aggregates, indicating cell damage. However, GOx@FexS... x S y After treatment with AZM, most of the bacterial membrane dissolves, leading to the loss of the integrity of the bacterial cell wall structure.

[0084] In summary, these results indicate that GOx@Fe x S y / AZM interacts strongly with bacteria by disrupting the integrity of bacterial cell walls and membranes. GOx@Fe x S y / AZM nanoparticles are significantly more effective than any single treatment.

[0085] Example 6

[0086] In vitro antibacterial biofilm activity of GOx@FexSy / AZM:

[0087] The effects of different treatments on the disruption of Staphylococcus aureus biofilm were assessed using crystal violet staining. For example... Figure 23 As shown, treatment at high concentrations (500 μg / mL) resulted in significant biofilm disruption. Notably, GOx@Fe... x S y The AZM scale exhibits the strongest biofilm disruption capability. Furthermore, confocal microscopy was used to directly observe changes in biofilm thickness and density, further verifying the biofilm disruption effects of different treatments on Staphylococcus aureus. Figure 24 As shown, Fe x S y AZM@Fe x S y The treated bacterial biofilm had an intact and compact structure, similar to the control. GOx@Fe x S y The treatment effectively eradicated bacterial biofilms to some extent. In contrast, GOx@Fe x S y The bacterial biofilms treated with / AZM are the thinnest and sparsest.

[0088] Example 7

[0089] GOx@Fe x S y In vitro evaluation of the immunomodulatory function of AZM:

[0090] In order to study GOX@Fe x S y The biological characteristics and biocompatibility of AZM are crucial prerequisites for the in vivo use of nanoparticles. Biocompatibility is first assessed through hemolysis and cytotoxicity tests. Figures 25 to 27 As shown, when GOx@Fe x S y / AZM was co-cultured with erythrocytes (RBCs). The hemolysis rate at an ultra-high experimental concentration (1600 μg mL⁻¹) was only 0.42%, indicating that GOx@Fe x S y / AZM showed negligible hemolysis. Cytotoxicity was determined by the CCK-8 assay. RAW264.7 and L929 cells were incubated with 30 μg / mL GOx@Fe x S y Cell viability under AZM culture remained above 85%, indicating good biocompatibility. Therefore, subsequent experiments used GOx@Fe x S yThe optimized dose for AZM was 10 μg / mL. A blank control and Fe were also included. x S y AZM@Fe x S and GOx@Fe x S y Group.

[0091] Macrophages play a crucial role in homeostasis, host defense, and tissue remodeling within the inflammatory immune microenvironment. Depending on the microenvironment, macrophages can differentiate into different subsets, such as classically activated M1 macrophages and alternately activated M2 macrophages, due to their high plasticity. Flow cytometry analysis showed that 9.84% of M2 macrophages in the Control group were CD206 positive; however, after GOx@Fe... x S y / AZM-treated macrophages M2 macrophages showed a CD206-positive cell level of 23.9% ( Figure 28 Simultaneously, immunofluorescence staining showed GOx@Fe x S y The fluorescence signal of Arg-1, the M2 marker in macrophages, was significantly enhanced in the AZM-treated group (Figure). Figure 29 ELISA results showed that after macrophage treatment with IL@ZIF, the secretion of pro-inflammatory cytokines TNF-α and IL-6 was significantly reduced, while the secretion of anti-inflammatory cytokines CD206 and IL-12 (P70) was significantly increased. Figures 30 to 33 These results demonstrate that GOx@Fe x S y / AZM can induce macrophages to polarize towards M2.

[0092] Subsequently, based on the results of macrophage ELISA, GOx@Fe was studied in vitro using macrophage conditioned culture. x S y / AZM promotes the regulation of cell migration. Scratch assay images and quantitative results show that GOx@Fe x S y The AZM group exhibited effective cell migration ability and reduced scratch area. Figures 34 to 35 ).

[0093] Example 8

[0094] Biological experiments promoting the healing of diabetic skin wounds:

[0095] Based on in vitro results, we further validated whether the composite nanoparticles could accelerate the healing process of diabetic infected wounds. We used a diabetic mouse skin wound model infected with *S. aureus* to evaluate the antibacterial and wound-healing effects of our strategy. Treatment was administered 24 hours after *S. aureus* infection. Representative images of the infected wounds are shown. Figure 36 (as shown) and the wound healing process as follows Figure 37 As shown, the wound area was also measured. (Using GOx@Fe) x S y Mice treated with AZM showed near-complete healing of their wounds after 12 days, while other treatments resulted in incomplete recovery. x S y AZM@Fe x S y and GOx@Fe x S y The group has a certain therapeutic effect on diabetic wounds. In addition, GOx@Fe x S y After AZM treatment, the number of S. aureus bacteria in the skin was significantly reduced. Figures 37 to 39 ) and inflammatory factors such as TNF-α and IL-6 were significantly reduced ( Figures 40 to 41 This further demonstrates that GOx@FexSy / AZM treatment reduces bacterial infection and inflammation in wounds.

[0096] To further investigate the histopathological changes within the wound, wound samples were collected on day 12, and sections were cut for H&E staining. Figure 42 and Figure 43 As shown, the epidermis in the infected wound remains incomplete. Necrotic cells and numerous hematoxylin-positive cells are visible in the wound area. Simultaneously, quantitative measurements of collagen deposition indicate that in GOx@Fe... x S y In the / AZM group, there was extensive (yellow circle) and new angiogenesis (yellow arrow). However, in GOx@Fe x S y In the AZM treatment group, epithelialization was complete, with the wound covered by a single, intact layer of epidermis. Furthermore, the number of necrotic cells and hematoxylin-positive cells was significantly reduced compared to infected wounds. Similarly, collagen deposition was another important factor in wound healing, as detected using Masson staining. Clearly, GOx@Fe x S y The / AZM group showed the highest collagen deposition density in the diabetic wound area at 14 days.

[0097] The safety of the treatment was then further evaluated. We will use GOx@Fe x Sy When / AZM was applied to the skin of normal mice, GOx@Fe was found. x S y / AZM did not cause significant irritation or inflammation in normal epidermis. Furthermore, histological analysis of mice after treatment showed no significant organ damage or inflammatory lesions in any of the treatment groups. Figure 44 Furthermore, there were no significant changes in blood biochemistry and hematological examinations. Figure 45 The above results prove that GOx@Fe x S y The safety and high biocompatibility of / AZM in vivo demonstrate its great potential for future clinical applications.

[0098] In summary, the above embodiments utilize a biomimetic mineralization strategy to immobilize glucose oxidase (GOx) on azithromycin (AZM) hybrid Fe. x S y In the nanoparticles, named GOx@Fe x S y / AZM. This nanoparticle accelerates the healing process of chronic wounds in diabetic patients by rapidly killing bacteria and restoring macrophage function by reversing the hyperglycemic microenvironment. Specifically, in the early stage of bacterial infection in hyperglycemic wounds, it utilizes endogenous hyperglycemia to reverse the immunosuppressive microenvironment suitable for microbial growth and the rapid accumulation of large amounts of ROS, effectively clearing bacteria from hyperglycemic wounds. During the wound healing period, it mediates the restoration of tissue repair function by promoting the polarization of macrophages to M2 macrophages. In the skin infection lesions of diabetic patients, GOx@Fe x S y / AZM can catalyze the production of H2O2 and glucuronic acid from endogenous glucose, thus reducing blood glucose levels at the wound site. Meanwhile, GOx@Fe x S y / AZM responds to the slightly acidic environment of the wound by further disintegrating and protonating to release Fe 2+ AZM and H2S, through the Fenton reaction Fe 2+ The generated H2O2 is converted into highly toxic hydroxyl radicals (·OH) that interact with the released AZM to rapidly kill bacteria. Notably, AZM, as a long-lived drug, continues to exert broad-spectrum antibacterial activity in the later stages of wound healing, effectively preventing secondary infections caused by wound exposure. Subsequently, after the bacteria are killed, GOx@Fe x S y The H2S and AZM released by / AZM further induced the polarization of the anti-inflammatory phenotype in macrophage M2. M2 macrophages secreted abundant regeneration-related cytokines, reversing the pro-inflammatory microenvironment of a diabetic wound infection model and promoting the self-repair function of infected skin. This was due to the immobilization of enzymes by the obtained material and the interaction between AZM and Fe.2+ Coordination between GOx and Fe 2+ It is effectively protected. It can be stored at room temperature for extended periods without deterioration, and is also a GOx@Fe x S y / AZM-mediated orderly management of infected wounds provides important assurance. In vitro antibacterial experiments and in vivo skin infection experiments in diabetic mice indicate that GOx@Fe x S y / AZM exhibits excellent anti-infection capabilities and skin infection repair effects. This nanoparticle achieves ordered biological function regulation within complex pathological microenvironments, and holds promise for providing a novel treatment for complex diabetic infected wound diseases.

Claims

1. A method for preparing biomimetic mineralized nanoparticles, characterized in that, Includes the following steps: S1: Add glucose oxidase to FeCl2·4H2O solution and stir, then add azithromycin ethanol solution and continue stirring to obtain a mixed solution; S2: Add NaS solution to the mixed solution to react and obtain a black precipitate. After washing and drying, the nanoparticles GOx@Fe are obtained. x S y / AZM.

2. The method for preparing biomimetic mineralized nanoparticles according to claim 1, characterized in that, The mass ratio of glucose oxidase, FeCl2·4H2O solution, azithromycin ethanol solution, and NaS solution is 2:5:10:

3.

3. The method for preparing biomimetic mineralized nanoparticles according to claim 1, characterized in that, In step S1, glucose oxidase was added to FeCl2·4H2O solution and stirred for 3 min, followed by the addition of azithromycin ethanol solution and stirring at 4°C for 3 min.

4. The method for preparing biomimetic mineralized nanoparticles according to claim 1, characterized in that, The black precipitate in S2 was washed twice with boiled deionized water and freeze-dried to obtain the nanoparticle GOx@Fe x S y / AZM.

5. Biomimetic mineralized nanoparticles GOx@Fe prepared by the method according to any one of claims 1 to 4 x S y Application of / AZM in the preparation of drugs for the orderly treatment of hyperglycemic infected wounds.

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