A glucose-responsive antibacterial nanocomposite microneedle patch, its preparation method and application

Abstract

This invention relates to a glucose-responsive antibacterial nanocomposite microneedle patch, its preparation method, and its application, belonging to the field of biomedical materials technology. The glucose-responsive antibacterial nanocomposite microneedle patch is an integral soluble microneedle. The functional component of the microneedle material is a nanocomposite CIP / GOx@ZIF-8, which uses ZIF-8 as a nanocarrier, co-loading ciprofloxacin hydrochloride and glucose oxidase. This invention synthesizes CIP / GOx@ZIF-8 using a one-step self-assembly method, achieving co-loading of CIP HCl and GOx. Furthermore, this invention further prepares CIP / GOx@ZIF-8 into microneedles. The preparation method is simple, easy to use, and allows for self-administration. Topical application to skin wounds is safer and more effective than systemic administration, further improving drug delivery efficiency and delivering therapeutic agents deep into the wound, promoting wound healing. This invention combines multiple therapeutic mechanisms, significantly enhancing the antibacterial properties of the microneedles and achieving the treatment of bacterially infected diabetic wounds.

Description

Technical Field

[0001] This invention relates to a glucose-responsive antibacterial nanocomposite microneedle patch, its preparation method, and its application, belonging to the field of biomedical materials technology. Background Technology

[0002] Chronic wounds heal slowly over a long period, with complex pathological microenvironments and numerous interacting factors. They are primarily characterized by four stages: coagulation, inflammation, proliferation, and remodeling. Due to the diverse types of chronic wounds and their prevalence across various population groups, chronic wounds pose a significant challenge to global healthcare systems. Therefore, there is a strong need to improve treatment methods for different types of chronic wounds, enhance treatment outcomes, and develop more effective treatment approaches to improve quality of life and reduce the burden on healthcare.

[0003] Diabetes is a chronic disease characterized by high blood sugar, which can lead to complications such as cardiovascular disease, kidney disease, and cancer, endangering life. Wounds caused by diabetes are difficult to heal and treat. The high-sugar microenvironment of diabetic wounds provides suitable growth conditions for bacteria, making them prone to bacterial and biofilm infections. This is a serious risk factor for wound healing and treatment; if infection is not effectively controlled, the wound may ulcerate, and amputation may even be necessary.

[0004] Glucose oxidase (GOx) is a natural glycoprotein that catalyzes the conversion of glucose into gluconic acid and H2O2 in the presence of O2. Therefore, GOx can directly break down glucose, cutting off the supply of nutrients to bacteria and starving them. Appropriate levels of H2O2 not only have antibacterial and hemostatic effects but also promote cytokine secretion, angiogenesis, and tissue regeneration, which is beneficial for wound healing.

[0005] Treatment methods for diabetic wounds include antibacterial therapy, anti-inflammatory therapy, blood sugar-lowering therapy, and immunotherapy. Among these, antibacterial therapy is an effective method for wound healing. By applying antibacterial drugs systemically or locally to the wound site, bacteria and their biofilm infections are rapidly eliminated, thereby promoting wound healing. Because single-drug antibacterial therapy has many limitations, multiplex antibacterial combination therapy has become a more ideal option. For example, the combined application of antibacterial nanomaterials can improve treatment efficacy and reduce bacterial resistance. Summary of the Invention

[0006] To address the shortcomings and deficiencies of existing treatments for bacterial infectious diabetic wounds and improve therapeutic efficacy, this invention presents a glucose-responsive antibacterial nanocomposite microneedle patch that combines multiple therapeutic mechanisms to promote the healing of bacterial infectious diabetic wounds, potentially offering a new option for the treatment of such wounds.

[0007] A glucose-responsive antibacterial nanocomposite microneedle patch, wherein the microneedle patch is an integral soluble microneedle, and the functional component of the microneedle material is a nanocomposite CIP / GOx@ZIF-8, wherein the nanocomposite uses ZIF-8 as a carrier, and ciprofloxacin hydrochloride and glucose oxidase are co-loaded on the carrier.

[0008] The ZIF-8 described in this invention is a zeolite imidazole ester framework material, which can be synthesized by the following method: 30-50 mg / mL of the mixture is added at a volume ratio of 1:1 to 1:5. -1 Add zinc source solution to a concentration of 50–250 mg / mL. -1 The reaction was carried out in a 2-methylimidazole solution at room temperature, followed by centrifugation, washing, and drying to obtain ZIF-8.

[0009] Preferably, the mass ratio of 2-methylimidazole to zinc source is 4:1 to 15:1.

[0010] Both ciprofloxacin hydrochloride (CIP HCl) and glucose oxidase (GOx) described in this invention are commercially available.

[0011] Preferably, the GOx loading rate in the CIP / GOx@ZIF-8 nanocomposite is 1-8%, and the drug loading rate of CIP HCl is 1-10%.

[0012] Preferably, the average particle size of the nanocomposite CIP / GOx@ZIF-8 is below 380 nm, and the carrier ZIF-8 is at the nanoscale.

[0013] The microneedle material of the present invention is prepared by combining the CIP / GOx@ZIF-8 nanocomposite with a soluble matrix material.

[0014] The aforementioned soluble matrix material can be any matrix material disclosed in the prior art that can be used to prepare soluble microneedles, such as PVP.

[0015] Another object of the present invention is to provide a method for preparing the above-mentioned glucose-responsive antibacterial nanocomposite microneedle patch.

[0016] A method for preparing a glucose-responsive antibacterial nanocomposite microneedle patch, the method comprising the step of synthesizing a CIP / GOx@ZIF-8 nanocomposite, specifically:

[0017] 5–10 mg / mL -1 CIP HCl solution and 5–20 mg / mL -1 Add GOx solution to a concentration of 150–250 mg / mL. -1 The corresponding solution was obtained by adding 30–50 mg·mL⁻¹ of 2-methylimidazole solution at a volume ratio of 1:1 to 1:5. -1The zinc source solution was added to a 2-methylimidazole mixed solution containing CIP, HCl, and GOx to react and obtain a dispersion. The dispersion was centrifuged, washed, and dried to obtain CIP / GOx@ZIF-8.

[0018] In the above technical solution, the volume ratio of CIP HCl solution to GOx solution is 1:1, and the volume ratio of CIP HCl solution to 2-methylimidazole solution is 1:3 to 1:12.

[0019] Preferably, the mass ratio of 2-methylimidazole to zinc source is 4:1 to 15:1.

[0020] Preferably, the zinc source is zinc nitrate hexahydrate or zinc acetate.

[0021] Preferably, the reaction is carried out at room temperature for 15 to 60 minutes.

[0022] The method of the present invention includes the step of microneedle preparation, specifically: mixing CIP / GOx@ZIF-8 nanocomposite and soluble matrix material uniformly in water to obtain a mixed solution; then pouring it into a microneedle mold, centrifuging, filling the groove with liquid, drying, demolding, and obtaining nanocomposite microneedles.

[0023] Preferably, the soluble matrix material is PVP, and the concentration of PVP in the mixed solution is 15-30% (w / v); the concentration of the CIP / GOx@ZIF-8 nanocomposite in the mixed solution is 0.6-1.2 mg·mL. -1 .

[0024] Another object of the present invention is to provide a glucose-responsive antibacterial nanocomposite microneedle patch for the treatment of bacterially infected diabetic wounds.

[0025] The beneficial effects of this invention are as follows: This invention synthesizes CIP / GOx@ZIF-8 using a one-step self-assembly method with an aqueous phase as the reaction system. The synthesis method is simple, green, safe, and the reaction conditions are mild, with a short reaction time, small reaction volume, and low cost, achieving co-loading of CIP HCl and GOx. Furthermore, this invention further prepares CIP / GOx@ZIF-8 into microneedles. The preparation method is simple and easy to use, allowing for self-drug administration. Topical application to skin wounds is safer and more effective than systemic administration, further improving drug delivery efficiency and delivering therapeutic agents deep into the wound, which is beneficial for wound healing. This invention combines multiple therapeutic mechanisms, significantly enhancing the antibacterial properties of the microneedles and achieving the treatment of bacterially infected diabetic wounds. Attached Figure Description

[0026] Figure 1The images show the XRD patterns of CIP / GOx@ZIF-8 synthesized in Example 1, CIP@ZIF-8 synthesized in Comparative Example 3, ZIF-8 synthesized in Comparative Example 1, and a ZIF-8 standard card.

[0027] Figure 2 Thermogravimetric curves of CIP / GOx@ZIF-8 synthesized in Example 1, CIP@ZIF-8 synthesized in Comparative Example 3, and ZIF-8 synthesized in Comparative Example 1 are shown.

[0028] Figure 3 A and 3B are SEM images of CIP / GOx@ZIF-8 synthesized in Example 1, with a scale bar of 500 nm. Figure 3 C is the SEM mapping image of Zn element synthesized by CIP / GOx@ZIF-8 in Example 1, with a scale bar of 500 nm. Figure 3 D is the SEM mapping image of F element synthesized by CIP / GOx@ZIF-8 in Example 1, with a scale bar of 500 nm. Figure 3 E is the SEM mapping image of O element of CIP / GOx@ZIF-8 synthesized in Example 1, with a scale bar of 500 nm. Figure 3 F is the SEM mapping image of CIP / GOx@ZIF-8 synthesized in Example 1 for element S, with a scale bar of 500 nm.

[0029] Figure 4 A is a TEM image of CIP / GOx@ZIF-8 synthesized in Example 1 in 0 mM glucose solution. B is a TEM image of CIP / GOx@ZIF-8 synthesized in Example 1 in 20 mM glucose solution. Scale bars are both 500 nm.

[0030] Figure 5 The changes in glucose concentration consumed by CIP / GOx@ZIF-8 synthesized in Example 1 at different time points.

[0031] Figure 6 The H2O2 concentration changes at different time points for CIP / GOx@ZIF-8 synthesized in Example 1.

[0032] Figure 7 This is a photograph of a 96-well plate containing Staphylococcus aureus and Escherichia coli treated with CIP / GOx@ZIF-8 synthesized in Example 1 of the MIC experiment.

[0033] Figure 8Plate photographs of Staphylococcus aureus and Escherichia coli treated with CIP / GOx@ZIF-8 synthesized in Example 1, ZIF-8 synthesized in Comparative Example 1, GOx@ZIF-8 synthesized in Comparative Example 2, and CIP@ZIF-8 synthesized in Comparative Example 3 in the plate coating experiment.

[0034] Figure 9 SEM images of Staphylococcus aureus and Escherichia coli treated with CIP / GOx@ZIF-8 synthesized in Example 1, ZIF-8 synthesized in Comparative Example 1, GOx@ZIF-8 synthesized in Comparative Example 2, and CIP@ZIF-8 synthesized in Comparative Example 3, with a scale bar of 2 μm.

[0035] Figure 10 A is a camera image of the CIP / GOx@ZIF-8MNs prepared in Example 2. B is an optical microscope image of the CIP / GOx@ZIF-8MNs prepared in Example 2. C is a SEM image of the CIP / GOx@ZIF-8MNs prepared in Example 2, with a scale bar of 200 μm.

[0036] Figure 11 A is an image of unstained ex vivo mouse skin after puncture with CIP / GOx@ZIF-8MNs prepared in Example 2. B is an image of ex vivo mouse skin stained with trypan blue after puncture with CIP / GOx@ZIF-8MNs prepared in Example 2. C is an image of H&E staining of ex vivo mouse skin stained with trypan blue after puncture with CIP / GOx@ZIF-8MNs prepared in Example 2. D is an optical microscope image of CIP / GOx@ZIF-8MNs prepared in Example 2 after dissolution.

[0037] Figure 12 The in vitro drug release curve of CIP / GOx@ZIF-8MNs prepared in Example 2 is shown.

[0038] Figure 13 Photographs of plates containing Staphylococcus aureus and Escherichia coli treated with CIP / GOx@ZIF-8MNs prepared in Example 2 of the plate coating experiment.

[0039] Figure 14 SEM images of Staphylococcus aureus and Escherichia coli treated with CIP / GOx@ZIF-8MNs prepared in Example 2, with a scale bar of 2 μm.

[0040] Figure 15 Hemolysis experiments were conducted on the CIP / GOx@ZIF-8 synthesized in Example 1, the BMNs prepared in Comparative Example 4, and the CIP / GOx@ZIF-8MNs prepared in Example 2.

[0041] Figure 16 Cytotoxicity experiments were conducted on CIP / GOx@ZIF-8 synthesized in Example 1, BMNs prepared in Comparative Example 4, and CIP / GOx@ZIF-8MNs prepared in Example 2.

[0042] Figure 17 This describes the in vivo treatment process of the CIP / GOx@ZIF-8MNs prepared in Example 2.

[0043] Figure 18 Changes in rat body weight during in vivo treatment with CIP / GOx@ZIF-8MNs prepared in Example 2.

[0044] Figure 19 This study investigates the in vivo biosafety of CIP / GOx@ZIF-8MNs prepared in Example 2. Detailed Implementation

[0045] The following non-limiting embodiments are intended to enable those skilled in the art to more fully understand the invention, but do not limit the invention in any way.

[0046] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; the reagents and materials described are commercially available unless otherwise specified.

[0047] One of the specific implementation methods:

[0048] A glucose-responsive antibacterial nanocomposite microneedle patch is composed of a zeolite imidazole ester framework material ZIF-8 (CIP / GOx@ZIF-8) co-loaded with ciprofloxacin hydrochloride (CIP HCl) and GOx, and polyvinylpyrrolidone (PVP) (CIP / GOx@ZIF-8MNs). The microneedles exhibit intact morphology, rapid dissolution, effective skin penetration, and strong antibacterial properties. Under the high glucose levels of the diabetic wound microenvironment, the microneedles efficiently release the drug.

[0049] The CIP / GOx@ZIF-8 described in this invention is a nanomedicine delivery system. ZIF-8, acting as a nanomedicine carrier, co-loads CIP HCl and GOx, exhibiting good enzyme activity and strong antibacterial properties. It is sensitive to high glucose levels; upon degradation, CIP / GOx@ZIF-8 releases Zn. 2+ CIP HCl, GOx.

[0050] The various therapeutic mechanisms described in this invention involve GOx consuming glucose and Zn, nutrients required for bacterial growth. 2+H2O2 and CIP HCl kill bacteria through different antibacterial mechanisms. When used in combination, they have a strong antibacterial effect against Staphylococcus aureus and Escherichia coli, and can successfully treat bacterial infectious diabetic wounds.

[0051] A method for preparing a glucose-responsive antibacterial nanocomposite microneedle patch involves first co-loading CIPHCl and GOx into ZIF-8, and then preparing soluble microneedles containing CIP / GOx@ZIF-8 using a soluble material as a matrix. The specific steps include:

[0052] (1) Dissolve 2-methylimidazole and zinc source in water to obtain 2-methylimidazole solution and zinc source solution respectively;

[0053] (2) Dissolve CIP HCl and GOx in water to obtain CIP HCl solution and GOx solution respectively;

[0054] (3) Add CIP HCl solution and GOx solution to 2-methylimidazole solution according to a certain volume ratio to obtain a 2-methylimidazole mixed solution containing CIP HCl and GOx;

[0055] (4) Add the zinc source solution to a 2-methylimidazole mixed solution containing CIP HCl and GOx at a certain volume ratio, and react at room temperature to obtain a dispersion;

[0056] (5) Centrifuge the dispersion, wash and dry it to obtain CIP / GOx@ZIF-8;

[0057] (6) Dissolve the soluble matrix material in water to obtain a soluble matrix material solution;

[0058] (7) Add CIP / GOx@ZIF-8 to the soluble matrix material solution, mix well, and let stand to obtain a soluble matrix material mixed solution containing CIP / GOx@ZIF-8;

[0059] (8) Pour the soluble matrix material mixture containing CIP / GOx@ZIF-8 into the microneedle mold, centrifuge, fill the groove with liquid, dry, demold, and obtain CIP / GOx@ZIF-8MNs.

[0060] In step (1), preferably, the mass ratio of 2-methylimidazole to zinc source is 4:1 to 15:1.

[0061] In step (1), preferably, the concentration of the 2-methylimidazole solution is 150–250 mg·mL. -1 .

[0062] In step (1), preferably, the concentration of the zinc source solution is 30–50 mg / mL. -1 .

[0063] In step (1), preferably, the zinc source is zinc nitrate hexahydrate or zinc acetate.

[0064] In step (2), preferably, the concentration of the CIP HCl solution is 5–10 mg / mL. -1 .

[0065] In step (2), preferably, the concentration of the GOx solution is 5–20 mg / mL. -1 .

[0066] In step (3), preferably, the volume ratio of CIP HCl solution, GOx solution and 2-methylimidazole solution is 1:1:3 to 1:1:12.

[0067] In step (4), preferably, the volume ratio of the zinc source solution and the 2-methylimidazole mixed solution containing CIP HCl and GOx is 1:1 to 1:5.

[0068] In step (4), preferably, the reaction time is 15 to 60 minutes.

[0069] In step (5), preferably, the centrifugation speed is 9000-12000 rpm and the centrifugation time is 10-20 min.

[0070] In step (5), preferably, the cleaning method is water washing or alcohol washing.

[0071] In step (5), the drying method is preferably freeze drying.

[0072] In step (6), the soluble matrix material is preferably PVP.

[0073] In step (6), preferably, the concentration of PVP in the mixed solution is 15-30% (w / v).

[0074] In step (7), preferably, the concentration of the CIP / GOx@ZIF-8 nanocomposite in the mixed solution is 0.6-1.2 mg·mL. -1 .

[0075] In step (8), preferably, the centrifugation speed is 2500-3500 rpm and the centrifugation time is 5-15 min.

[0076] Example 1

[0077] (1) Dissolve 750 mg of 2-methylimidazole in 3 mL of water and dissolve 50 mg of zinc nitrate hexahydrate in 1 mL of water to obtain 2-methylimidazole solution and zinc nitrate hexahydrate solution;

[0078] (2) Dissolve 10 mg of CIP HCl in 1 mL of water to obtain a CIP HCl solution; dissolve 5 mg of GOx in 1 mL of water to obtain a GOx solution;

[0079] (3) Add 1 mL of CIP HCl solution and 1 mL of GOx solution to 3 mL of 2-methylimidazole solution to obtain a 2-methylimidazole mixed solution containing CIP HCl and GOx;

[0080] (4) Add 1 mL of zinc nitrate hexahydrate solution to 5 mL of 2-methylimidazole mixed solution containing CIP HCl and GOx, react at room temperature for 30 min to obtain a dispersion;

[0081] (5) Centrifuge the dispersion at 11,000 rpm for 10 min, wash with water 3 times, freeze dry to obtain CIP / GOx@ZIF-8.

[0082] Comparative Example 1

[0083] (1) Dissolve 750 mg of 2-methylimidazole in 3 mL of water and dissolve 50 mg of zinc nitrate hexahydrate in 1 mL of water to obtain 2-methylimidazole solution and zinc nitrate hexahydrate solution;

[0084] (2) Add 1 mL of zinc nitrate hexahydrate solution to 3 mL of 2-methylimidazole solution and react at room temperature for 30 min to obtain a dispersion;

[0085] (3) Centrifuge the dispersion at 11000 rpm for 10 min, wash with water 3 times, and freeze-dry to obtain ZIF-8. Comparative Example 2

[0086] (1) Dissolve 750 mg of 2-methylimidazole in 3 mL of water and dissolve 50 mg of zinc nitrate hexahydrate in 1 mL of water to obtain 2-methylimidazole solution and zinc nitrate hexahydrate solution;

[0087] (2) Dissolve 5 mg of GOx in 1 mL of water to obtain a GOx solution;

[0088] (3) Add 1 mL of GOx solution to 3 mL of 2-methylimidazole solution to obtain a 2-methylimidazole mixed solution containing GOx;

[0089] (4) Add 1 mL of zinc nitrate hexahydrate solution to 4 mL of 2-methylimidazole mixed solution containing GOx, react at room temperature for 30 min to obtain a dispersion;

[0090] (5) Centrifuge the dispersion at 11,000 rpm for 10 min, wash with water 3 times, freeze dry to obtain GOx@ZIF-8.

[0091] Comparative Example 3

[0092] (1) Dissolve 750 mg of 2-methylimidazole in 3 mL of water and dissolve 50 mg of zinc nitrate hexahydrate in 1 mL of water to obtain 2-methylimidazole solution and zinc nitrate hexahydrate solution;

[0093] (2) Dissolve 10 mg of CIP HCl in 1 mL of water to obtain a CIP HCl solution;

[0094] (3) Add 1 mL of CIP HCl solution to 3 mL of 2-methylimidazole solution to obtain a mixed solution of 2-methylimidazole containing CIP HCl;

[0095] (4) Add 1 mL of zinc nitrate hexahydrate solution to 4 mL of 2-methylimidazole mixed solution containing CIP HCl, react at room temperature for 30 min to obtain a dispersion;

[0096] (5) Centrifuge the dispersion at 11,000 rpm for 10 min, wash with water 3 times, freeze dry to obtain CIP@ZIF-8.

[0097] The test results are as follows:

[0098] (1) The crystal structures of CIP / GOx@ZIF-8 synthesized in Example 1, CIP@ZIF-8 synthesized in Comparative Example 3, and ZIF-8 synthesized in Comparative Example 1 were evaluated by X-ray diffraction (XRD). Figure 1 As shown, the XRD diffraction peak positions of each group of samples are similar, indicating that each group of samples has a good crystal structure.

[0099] (2) The drug loading rate of the CIP / GOx@ZIF-8 synthesized in Example 1 was evaluated using thermogravimetric analysis (TGA) and a BCA protein concentration assay kit. Figure 2 As shown, based on the weight loss rate of each group of samples at 700℃ and the results of the BCA protein concentration assay kit, the drug loading rate of CIP HCl is approximately 8.36%, and the drug loading rate of GOx is approximately 4.27%.

[0100] (3) The morphology and elemental distribution of the CIP / GOx@ZIF-8 synthesized in Example 1 were investigated using SEM. Figure 3 As shown, the particle size of CIP / GOx@ZIF-8 is less than 380 nm, the morphology is rhombic dodecahedron, and it contains Zn, F, S and O elements.

[0101] (4) The glucose sensitivity of CIP / GOx@ZIF-8 synthesized in Example 1 was investigated using transmission electron microscopy (TEM). CIP / GOx@ZIF-8 was dissolved in 0 mM and 20 mM glucose solutions, respectively, and incubated at 37°C and 100 rpm for 24 h. The samples were then observed and photographed using TEM. Figure 4 As shown, 20 mM glucose solution can destroy the structure of CIP / GOx@ZIF-8, while 0 mM glucose solution cannot destroy the structure of CIP / GOx@ZIF-8, indicating that it has glucose sensitivity.

[0102] (5) The concentration of glucose consumed by the CIP / GOx@ZIF-8 synthesized in Example 1 was evaluated using a glucose assay kit. The detection principle of the glucose assay kit is based on the O-toluidine method, where glucose assay reagent undergoes a condensation reaction with glucose, and the detection wavelength is 630 nm. A standard curve for glucose was set up, and a standard of 300 μg / mL was prepared using 20 mM glucose solution. -1 The CIP / GOx@ZIF-8 assay kit was used to perform tests at specified time points, following the instructions for the glucose assay kit. The glucose concentration of the samples at different test time points was quantitatively calculated. Figure 5 As shown, glucose concentration decreases over time, indicating that CIP / GOx@ZIF-8 can consume glucose.

[0103] (6) The concentration of H2O2 generated by CIP / GOx@ZIF-8 synthesized in Example 1 was evaluated by the titanium sulfate colorimetric method. A standard curve for H2O2 was set up, and a standard curve of 300 μg·mL was prepared using 20 mM glucose solution. -1 The CIP / GOx@ZIF-8 assay was performed at specified time points, with titanium sulfate solution added for colorimetric reaction. The detection wavelength was 405 nm, and the H2O2 concentration of the sample at different detection time points was quantitatively calculated. Figure 6 As shown, the H2O2 concentration increases with time, indicating that CIP / GOx@ZIF-8 can generate H2O2.

[0104] (7) Referring to the CLSI standard, the minimum inhibitory concentration (MIC) of CIP / GOx@ZIF-8 synthesized in Example 1 was evaluated using the 2-fold dilution method. The bacterial strains were Staphylococcus aureus and Escherichia coli, and the bacterial suspension concentration was 10. 7 CFU·mL -1 The glucose concentration was 20 mM, and images were taken of the 96-well plate. Figure 7 As shown, the MIC for Staphylococcus aureus is 150 μg·mL. -1For Escherichia coli, the MIC is 18.75 μg·mL. -1 .

[0105] (8) The antibacterial properties of CIP / GOx@ZIF-8 synthesized in Example 1, ZIF-8 synthesized in Comparative Example 1, GOx@ZIF-8 synthesized in Comparative Example 2, and CIP@ZIF-8 synthesized in Comparative Example 3 were evaluated by plate coating. The bacterial strains were Staphylococcus aureus and Escherichia coli, and the bacterial suspension concentration was 10. 7 CFU·mL -1 The glucose concentration was 20 mM. After incubating the samples at the MIC concentration and the bacterial suspension together, the mixture was diluted to a suitable concentration, plated, and incubated at 37°C. The plates were photographed and the results recorded. Figure 8 As shown, no bacteria grew on the CIP / GOx@ZIF-8 plate, indicating the strongest antibacterial performance.

[0106] (9) The antibacterial properties of CIP / GOx@ZIF-8 synthesized in Example 1, ZIF-8 synthesized in Comparative Example 1, GOx@ZIF-8 synthesized in Comparative Example 2, and CIP@ZIF-8 synthesized in Comparative Example 3 were further evaluated by SEM. The bacterial strains were Staphylococcus aureus and Escherichia coli, and the bacterial suspension concentration was 10. 7 CFU·mL -1 The glucose concentration was 20 mM. Samples at each MIC concentration were incubated with the bacterial suspension, centrifuged at 4000 rpm for 10 min, and fixed with 2.5% glutaraldehyde for 6 h. After washing three times, the samples were dehydrated with ethanol of varying concentrations, dried at room temperature, sputter-coated with gold, and observed and photographed using SEM. Figure 9 As shown, except for the control group, all samples could destroy the bacterial structure, with CIP / GOx@ZIF-8 exhibiting the strongest antibacterial properties.

[0107] Example 2

[0108] (1) Dissolve 0.2 mg PVP in 1 mL of water, stir well, and let stand to obtain a 20% (w / v) PVP solution;

[0109] (1) Add 1.2 mg of CIP / GOx@ZIF-8 synthesized in Example 1 to 1 mL of 20% (w / v) PVP solution, mix well, and let stand to obtain a PVP mixed solution containing CIP / GOx@ZIF-8;

[0110] (2) PVP mixed solution containing CIP / GOx@ZIF-8 was poured into microneedle mold, centrifuged at 2500 rpm for 10 min, the liquid in the groove was filled, dried, and demolded to obtain CIP / GOx@ZIF-8MNs.

[0111] Comparative Example 4

[0112] (1) Dissolve 0.2 mg PVP in 1 mL of water, mix well, and let stand to obtain a 20% (w / v) PVP solution;

[0113] (2) PVP solution was poured into the microneedle mold, centrifuged at 2500 rpm for 10 min, the liquid in the groove was filled, dried, and demolded to obtain blank microneedles (BMNs).

[0114] The test results are as follows:

[0115] (1) The morphology of the CIP / GOx@ZIF-8MNs prepared in Example 2 was evaluated using a camera, optical microscope, and SEM, and photographs were taken and recorded. For example... Figure 10 As shown, the microneedles have complete morphology, a needle length of 800 μm, and an array number of 10 × 10.

[0116] (2) The puncture performance of CIP / GOx@ZIF-8MNs prepared in Example 2 was evaluated using ex vivo mouse skin. Microneedles were applied to ex vivo mouse skin for 3 minutes, stained with trypan blue, and excess trypan blue was absorbed with filter paper. The skin after microneedle puncture was observed and photographed, and H&E staining experiments were performed. Figure 11 As shown in B and C, blue micropores appeared on both the surface and inside of the detached mouse skin, with a depth of about 80 μm, indicating that the microneedles have good skin puncture performance.

[0117] (3) The solubility of the CIP / GOx@ZIF-8MNs prepared in Example 2 was evaluated using a 3% agarose gel to simulate a skin wound. Microneedles were applied to the 3% agarose gel, and observations and photographs were taken at specified time points. Figure 11 As shown in D, the microneedles exhibit good dissolution properties.

[0118] (4) The in vitro drug release of CIP / GOx@ZIF-8MNs prepared in Example 2 was evaluated. A standard curve for CIP HCl was set up. The microneedles were placed in PBS (10mM) solutions containing 20mM glucose and 5mM glucose at pH 7.4, respectively, and incubated at 37°C and 100 rpm. Detection was performed at specified time points with a detection wavelength of 317 nm. The cumulative release of CIP HCl at different detection time points was quantitatively calculated. Figure 12 As shown, the microneedles exhibit glucose responsiveness and efficiently release drugs in 20 mM glucose solution.

[0119] (5) The antibacterial properties of CIP / GOx@ZIF-8MNs prepared in Example 2 were evaluated by plate coating. The bacterial strains were Staphylococcus aureus and Escherichia coli, and the bacterial suspension concentration was 10. 7CFU·mL -1 The glucose concentration was 20 mM. After incubating the microneedles and bacterial suspension together, the mixture was diluted to a suitable concentration and plated. The plates were incubated at 37°C, and photographs were taken to record the results. Figure 13 As shown, no bacteria grew on the CIP / GOx@ZIF-8MNs plates, while a large number of bacteria grew on the control group plates, indicating that CIP / GOx@ZIF-8MNs has strong antibacterial properties.

[0120] (6) The antibacterial properties of CIP / GOx@ZIF-8MNs prepared in Example 2 were further evaluated by SEM. The bacterial strains were Staphylococcus aureus and Escherichia coli, and the bacterial suspension concentration was 10. 7 CFU·mL -1 The glucose concentration was 20 mM. After incubating the microneedles and bacterial suspension together, the mixture was centrifuged at 4000 rpm for 10 min and fixed with 2.5% glutaraldehyde for 6 h. After washing three times, the samples were dehydrated with ethanol of varying concentrations, dried at room temperature, sputter-coated with gold, and observed and photographed using SEM. Figure 14 As shown, compared with the control group, CIP / GOx@ZIF-8MNs can disrupt bacterial structure, indicating that CIP / GOx@ZIF-8MNs has good antibacterial properties.

[0121] (7) The hemolysis test was used to evaluate the blood compatibility of the CIP / GOx@ZIF-8 synthesized in Example 1, the BMNs prepared in Comparative Example 4, and the CIP / GOx@ZIF-8MNs prepared in Example 2. Blood was collected from the orbital rimus, washed with sterile PBS (10 mM) until the supernatant was clear, and diluted to a red blood cell dispersion concentration of 5%. The sample and red blood cell dispersion were mixed and incubated at 37°C for 1 h. The detection wavelength was 540 nm, and the hemolysis rate of different samples was calculated. Figure 15 As shown, the hemolysis rate of each group of samples was much less than 5%, and no hemolysis occurred, indicating that the blood compatibility of each group of samples was good.

[0122] (8) The cell compatibility of CIP / GOx@ZIF-8 synthesized in Example 1, BMNs prepared in Comparative Example 4, and CIP / GOx@ZIF-8MNs prepared in Example 2 was evaluated using the CCK-8 kit. The cell model was NH3T3 fibroblasts, and the number of cells per well was 10. 5 The cultured cells and samples were mixed and incubated at 37°C for 24 hours. CCK-8 staining was performed, and the detection wavelength was 450 nm. The cell viability of different samples was calculated. Figure 16 As shown, the cell survival rate of each group of samples was greater than 80%, indicating that the cell compatibility of each group of samples was good.

[0123] (9) A bacterial infection-induced diabetic wound model was established in SD rats. The CIP / GOx@ZIF-8MNs prepared in Example 2 were applied to the rat wounds, with PBS as the control group. The wounds were observed and photographed at specified time points, and the changes in rat body weight during the observation period were recorded. Figure 17 and 18 As shown, compared with the control group, CIP / GOx@ZIF-8MNs had a significant therapeutic effect on bacterial-infected diabetic wounds, with no significant change in body weight, and successfully treated bacterial-infected diabetic wounds.

[0124] (10) Collect major organs from rats for H&E staining. For example... Figure 19 As shown, no abnormalities were found in the heart, liver, spleen, lungs, and kidneys of the rats, indicating that CIP / GOx@ZIF-8MNs has good in vivo biocompatibility.

Claims

1. A glucose-responsive antibacterial nanocomposite microneedle patch, characterized by: The microneedle patch is an integrated dissolvable microneedle, and the functional component of the microneedle material is a nanocomposite CIP / GOx@ZIF-8, which is a nanocarrier of ZIF-8, and the nanocarrier is co-loaded with ciprofloxacin hydrochloride and glucose oxidase, The nanocomposite CIP / GOx@ZIF-8 is prepared by adding 5-10 mg·mL -1 of CIP HCl solution and 5-20 mg·mL -1 of GOx solution into 150-250 mg·mL -1 of 2-methylimidazole solution to obtain a corresponding solution; adding 30-50 mg·mL -1 of zinc source solution into the mixed solution of 2-methylimidazole containing CIP HCl and GOx to react to obtain a dispersion; centrifuging, washing and drying the dispersion to obtain CIP / GOx@ZIF-8, wherein the mass ratio of 2-methylimidazole to zinc source is 4:1-15:1; and the zinc source is zinc nitrate hexahydrate or zinc acetate.

2. The microneedle patch of claim 1, wherein: The drug loading rate of GOx in the nanocomposite CIP / GOx@ZIF-8 is 1-8%, and the drug loading rate of CIP HCl is 1-10%.

3. The microneedle patch of claim 1, wherein: The average particle size of the nanocomposite CIP / GOx@ZIF-8 is below 380 nm, and the carrier ZIF-8 is in nanoscale.

4. The microneedle patch of claim 1, wherein: The microneedle material is prepared from the CIP / GOx@ZIF-8 nanocomposite and a soluble matrix material.

5. The method of making glucose-responsive antimicrobial nanocomposite microneedle patch of claim 1, characterized by: The method comprises the step of synthesizing the CIP / GOx@ZIF-8 nanocomposite, specifically: 5~10 mg·mL -1 CIP HCl solution and 5~20 mg·mL -1 GOx solution was added into 150~250 mg·mL -1 2-methylimidazole solution to obtain a corresponding solution; 30~50 mg·mL -1 Zinc source solution was added into the 2-methylimidazole mixed solution containing CIP HCl and GOx to react to obtain a dispersion; the dispersion was centrifuged, washed and dried to obtain CIP / GOx@ZIF-8, wherein the mass ratio of the 2-methylimidazole to the zinc source was 4:1~15:1; and the zinc source was zinc nitrate hexahydrate or zinc acetate.

6. The method of claim 5, wherein: The reaction is carried out at room temperature, and the reaction time is 15-60 min.

7. The method of claim 5, wherein: The method comprises the step of preparing microneedles, specifically: uniformly mixing the CIP / GOx@ZIF-8 nanocomposite and the soluble matrix material in water to obtain a mixed solution; then pouring the mixed solution into a microneedle mold, centrifuging, filling the grooves with liquid, drying, demolding, and obtaining the nanocomposite microneedle.

8. The method according to claim 7, characterized in that the soluble matrix material is PVP, the concentration of PVP in the mixed solution is 15-30% (w / v); the concentration of CIP / GOx@ZIF-8 nanocomposite in the mixed solution is 0.6-1.2 mg-mL -1 .

9. Use of the glucose-responsive antibacterial nanocomposite microneedle patch of claim 1 in the preparation of a product for treating a bacterial infectious diabetic wound.

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