A double-layer microneedle patch, a preparation method and application thereof

Abstract

The present application relates to a kind of double-layer microneedle patch and its preparation method and application, including drug delivery unit and adhering unit, drug delivery unit is GCM-MN microneedle array, and adhering unit is CSH hydrogel patch, when using, first GCM-MN microneedle array is through pressure on wound, then CSH hydrogel patch is covered on GCM-MN microneedle array.The CSH hydrogel patch in the present application is conducive to creating a moist healing environment, has excellent adhesive stability and skin adaptability, provides adherent barrier for wound, GCM-MN microneedle array has good cell compatibility, cell affinity, blood compatibility and biodegradability.With the degradation of microneedle array in wound, encapsulated GCM nanoparticles are continuously released, and in-depth wound site.It can be seen that the double-layer microneedle patch of the present application has great clinical application potential in the field of infectious wound treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to a double-layer microneedle patch and a preparation method and application thereof. BACKGROUND

[0002] Skin tissue is susceptible to various external factors (such as trauma, surgery and burns, etc.) or internal factors (such as diabetes and vascular diseases, etc.), leading to the destruction of its anatomical continuity, thus damaging the skin barrier and making it easy for bacteria to enter deeper tissues. Bacterial infection of open wounds significantly delays wound healing, and by prolonging the inflammatory phase and causing cell damage, it further inhibits angiogenesis. It can be seen that the colonization of bacteria at the wound site poses a major challenge to wound healing, and therefore the development of strategies that can effectively control wound infection and accelerate wound healing is a key task in the field of biomedicine.

[0003] In recent years, metal ions, metal oxides and metal hydroxides (such as silver, zinc, copper, cobalt, cerium and gallium) have been used as an alternative or complementary means to antibiotics for the prevention and control of infection. MOFs are a class of porous hybrid coordination polymers formed by self-assembly of metal ions and organic ligands. Compared with traditional metals and metal oxides, MOFs are more gentle as a repository of metal ions, and in addition, the structural features of the MOFs organic ligands are also important determinants of their antibacterial activity. In addition, the pore structure of MOFs is often considered to be its most remarkable and valuable feature, providing elastic pores superior to molecular sieves and higher loading capacity than lipids or polymers. For example, Chinese invention patent No. CN202411099352.7 (publication No. CN118873734A) “Cu-MOF-based composite antibacterial hydrogel wound dressing and preparation method thereof”, Chinese invention patent No. CN202410217827.1 (publication No. CN118079075A) “Nanoreactor hydrogel for promoting diabetic wound healing and preparation method and application thereof”, etc. Further, metformin (Met) is a natural product extracted from the legume plant Galega officinalis, and is often used as an oral hypoglycemic drug for the treatment of type 2 diabetes. Recent studies have shown that metformin also has anti-inflammatory effects and regulates the proliferation, migration and angiogenesis of vascular cells such as endothelial cells by activating the AMPK pathway.

[0004] However, the therapeutic efficacy of the multifunctional MOF platform is often limited by its tissue penetration, and it is usually only in contact with the epidermis of the wound, while for deeper trauma, the successful regeneration of the dermis is more challenging than the restoration of the epidermis. This is because the repair of infected wounds not only requires the restoration of the epidermis, but also the removal of bacteria in the dermis and the complete regeneration of skin appendages (such as cysts, glands and blood vessels, etc.). SUMMARY

[0005] The first technical problem to be solved by the present application is to provide a double-layer microneedle patch that can deliver drugs directly to the deep part of a wound.

[0006] The second technical problem to be solved by the present application is to provide a double-layer microneedle patch that can deliver drugs directly to the deep part of a wound and has good applicability.

[0007] The third technical problem to be solved by the present application is to provide a double-layer microneedle patch that can effectively control wound infection.

[0008] The fourth technical problem to be solved by the present application is to provide a double-layer microneedle patch that can effectively control wound infection and effectively promote wound healing.

[0009] The fifth technical problem to be solved by the present application is to provide a preparation method of the double-layer microneedle patch.

[0010] The sixth technical problem to be solved by the present application is to provide an application of the double-layer microneedle patch.

[0011] The technical solution adopted by the present application to solve at least one of the above technical problems is a double-layer microneedle patch, characterized in that it comprises a drug delivery unit and an adhesive unit, wherein the drug delivery unit is a GCM-MN microneedle array, and the adhesive unit is a CSH hydrogel patch, and in use, the GCM-MN microneedle array is first applied to the wound site by pressure to ensure that the microneedles can penetrate the skin of the wound site, and then the CSH hydrogel patch is covered on the GCM-MN microneedle array to ensure a moist healing environment.

[0012] Further, the GCM-MN microneedle array uses γ-PGA as the matrix material of the needle tip to load GCM nanoparticles, and uses PVP as the backing layer material, which has good biological safety, mechanical strength and rapid solubility. In the present application, γ-polyglutamic acid (γ-PGA) and PVP are biocompatible macromolecules with good biodegradability and hygroscopicity, which enable the GCM-MN to absorb interstitial fluid and degrade, thereby promoting the release of GCM nanoparticles.

[0013] Further, the GCM-MN microneedle array has pyramidal tips, and each tip has a length of 640 μm, a base diameter of 320 μm, and a spacing of 550 μm between adjacent tips. The outermost layer of the skin, the avascular epidermis, is about 100 μm thick, while the underlying dermis, which is rich in blood vessels, is typically between 1000 and 2000 μm thick. Therefore, the 640 μm long GCM-MN is sufficient to penetrate the epidermis and reach the dermis, facilitating the delivery of the drug.

[0014] To further solve the fifth technical problem, the technical solution adopted is: a preparation method of the double-layer microneedle patch, characterized in that it comprises the following steps: (1) synthesis of GCM; (2) preparation of GCM-MN microneedle array; (3) preparation of CSH hydrogel patch.

[0015] Further, the synthesis of GCM in step (1) is as follows: Ga(NO3)3 and Met are mixed in water, and a Ga 3+ and Met coordination complex is generated by reaction; then, a carbenicillin solution is added, and a Ga-Car-MOF complex is formed with Ga 3+ After stirring and reaction, the precipitate is collected and washed to remove unreacted substances; finally, white GCM nanoparticles are obtained by freeze-drying.

[0016] Further, the preparation of the GCM-MN microneedle array in step (2) is as follows: a two-step template replication method is used, first, the GCM nanoparticles prepared in step (1) are dispersed in water and mixed with γ-PGA, and the mixture is obtained by vacuum and low-speed centrifugation; then, the mixture is filled into the tips of the needle mold, after removing the excess material, pure PVP pre-gel is added to form the GCM-MN microneedle array.

[0017] Further, the preparation of the CSH hydrogel patch in step (3) is as follows: first, CSH is formed by covalently binding hydrogenated caffeic acid modified chitosan; then, the CSH solution is crosslinked in a mold to prepare the CSH hydrogel patch.

[0018] To further solve the sixth technical problem, the technical solution adopted is: the application of the double-layer microneedle patch in the preparation of an infectious wound healing medical device.

[0019] Compared with the prior art, the advantages of the present application are that the double-layer microneedle patch (GCM-MN-CSH) of the present application comprises a drug delivery unit and an adhesion unit, wherein the drug delivery unit is a GCM-MN microneedle array, and the adhesion unit is a CSH hydrogel patch. When the double-layer microneedle patch is applied to a bacterial infectious wound, the CSH hydrogel patch is beneficial to create a moist healing environment, exhibits seamless skin adhesion, has excellent adhesion stability and skin adaptability, provides an adhesive barrier for the wound, and at the same time, the GCM-MN microneedle array of the present application has good cell compatibility, cell affinity, blood compatibility and biodegradability. And with the degradation of the microneedle array at the wound site, the encapsulated GCM nanoparticles are continuously released and penetrate into the wound site.

[0020] Specifically, the positive charge surface of the GCM nanoparticles (wherein the amino group in the Met molecule increases the positive charge of the GCM) enhances its adsorption to bacteria, increases contact and promotes lysis through an acid reaction, Ga 3+ The synergistic effect of carbenicillin and carbenicillin enhances the antibacterial activity. Metformin further enhances the proliferation, migration, angiogenesis and collagen deposition of endothelial cells, and at the same time increases the infiltration of M2 macrophages in the tissue. It is verified through experiments that the double-layer microneedle patch of the present application has strong antibacterial effect (including in vivo and in vitro antibacterial effect), can effectively reduce the inflammatory response, activates the protective immune response, and promotes epithelial regeneration and new blood vessel formation.

[0021] The above effects collectively promote infection control, reduce inflammation, stimulate tissue proliferation and promote tissue remodeling, ultimately support programmed wound healing, and thus accelerate wound healing. It can be seen that the double-layer microneedle patch (GCM-MN-CSH) of the present application has great clinical application potential in the field of infectious wound treatment. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1For synthesis and characterization of GCM nanoparticles: (A) Schematic illustration of one-pot synthesis process of GCM nanoparticles; (B) FE-SEM image of GCM nanoparticles; (C) FE-SEM image of GC nanoparticles; (D) Particle size distribution of GC and GCM nanoparticles; (E) FTIR spectra of Met, Car, GC and GCM; (F) XRD patterns of GC and GCM; (G) In vitro release profile of carbenicillin (Car) from GCM nanoparticles in PBS solution at pH 5.5 and 7.4; (H-I) Effect of GCM nanoparticles at different concentrations on the survival rate of S. aureus (H) and P. aeruginosa (I) under different pH conditions; (J) Surface zeta potential of GC (I), GCM (II), S. aureus (III), S. aureus co-cultured with GC (IV), S. aureus co-cultured with GCM (V), P. aeruginosa (VI), P. aeruginosa co-cultured with GC (VII), P. aeruginosa co-cultured with GCM (VIII) measured using dynamic light scattering (DLS); (K) SEM images of S. aureus and P. aeruginosa samples after treatment with GC nanoparticles and GCM nanoparticles;

[0023] Figure 2 For synthesis and characterization of GCM-MN-CSH: (A) Optical image of GCM-MN-CSH patch; (B) Schematic illustration of the preparation process of double-layered GCM-MN-CSH patch; (C) Field emission scanning electron microscope (FE-SEM) image of GCM-MN, scale bar: 300 pm; (D) Local magnified FE-SEM image of GCM-MN, scale bar: 100 pm; (E) Fluorescence microscope image of GCM-MN tip labeled with rhodamine B, scale bar: 50 pm; (F) Photograph of GCM-MN applied on pig skin and stained with 0.4% trypan blue; (G) Morphological images under microscope showing the moisture absorption of GCM-MN at different time points at room temperature in a 100% humidity chamber, scale bar: 200 pm; (H) In vitro release profile of GCM-MN in PBS at pH 5.5 and 7.4; (I) Optical images of CSH for joints, skin, and adhesion to skin while bearing a 3 g weight; (J) Comparison of adhesion strength of CSH and GCM-MN-CSH;

[0024] Figure 3In vitro antibacterial ability of GCM-MNs: (A) Bacterial viability of S. aureus and P. aeruginosa after different treatment groups; (B) Statistical bar graph showing colony counts of S. aureus and P. aeruginosa after different combination treatments; (C) Standard plate images of S. aureus and P. aeruginosa under different treatment groups; (D) Fluorescence images of S. aureus and P. aeruginosa after different treatment groups using SYTO 9 / PI staining under fluorescence microscope, scale bar: 50 pm;

[0025] Figure 4 Evaluation of biocompatibility and pro-angiogenic ability of GCM nanoparticles (NPs) and GCM-MNs in vitro: (A) Cell viability of HUVEC cells after 24 hours exposure to different concentrations of GCM NPs and GCM-MNs; (B) Hemolysis rate of different concentrations of GCM NPs; (C) Hemolysis rate of different concentrations of GCM-MNs; (D) Microscope images of HUVECs after treatment with GCM NPs and GCM-MNs, showing cell migration at 0 hours and 12 hours, scale bar: 200 pm; (E) Representative quantitative analysis of migration rate; (F) Microscope images of HUVECs performing endothelial tube formation experiment, scale bar: 200 pm; (G) Representative quantitative analysis of total tube length of each group; (H) Representative quantitative analysis of node number of each group;

[0026] Figure 5 GCM-MN-CSH promotes infected wound healing in vivo: (A) Schematic diagram of treating infected wound model in vivo by GCM-MN-CSH; (B-D) Dynamic changes of wound healing in different treatment groups: sequential photographs (B), wound bed closure trajectory (C), statistical analysis of wound area (D), observed at 0, 2, 4, 6 and 8 days, including control group (I), blank microneedle (II), Met-MN (III), GC-MN (IV), GCM-MN (V) and GCM-MN-CSH (VI) groups; (E-F) Photographs (E) and quantitative analysis (F) of residual bacteria on LB medium plates at the wound site on day 8 after treatment; (G) Body weight change rate of mice in different treatment groups during wound healing;

[0027] Figure 6For the study of tissue regeneration, angiogenesis and immunofluorescence staining after treatment: (A) H&E staining, Masson's trichrome staining and immunofluorescence staining images of the wound at day 8 in different groups: control group (I), blank-MN group (II), Met-MN group (III), GC-MN group (IV), GCM-MN group (V), GCM-MN-CSH group (VI) (B) Representative quantitative analysis of the epidermal thickness of the wound; (C) Representative quantitative analysis of the relative collagen deposition level; (D-E) Representative quantitative analysis of the relative fluorescence intensity: IL-1β (D) and IL-6 (E). (F-G) Representative quantitative analysis of the mean fluorescence intensity of CD 86 (F) and CD 206 (G); (H-I) Representative quantitative analysis of the mean fluorescence intensity of a-SMA (H) and CD 31 (I);

[0028] Figure 7 For the analysis of the loading of Met in GCM by high-performance liquid chromatography (HPLC): (A) Standard curve of the concentration of Met determined by HPLC with UV detection versus the peak area at 233 nm; (B) Loading rate of Met in GCM nanoparticles;

[0029] Figure 8 For the antibacterial effect of GCM: (A) Activity of S. aureus after treatment with GCM nanoparticles at different concentrations and time points; (B) Activity of P. aeruginosa after treatment with GCM nanoparticles at different concentrations and time points;

[0030] Figure 9 For the results of bacterial activity inhibition: (A) Bacterial activity; (B) Quantitative analysis using standard plate counting method; (C) Standard plate images of S. aureus and P. aeruginosa after treatment with different components;

[0031] Figure 10 For the fluorescence microscopy images of S. aureus and P. aeruginosa stained with SYTO9 / PI after treatment with different components, scale bar: 50 pm;

[0032] Figure 11 For the verification of the formation and binding degree of covalent bonds in CSH: (A) UV-vis absorption spectra of salicylic acid and CSH; (B) FTIR spectra of CS, salicylic acid and CSH; (C) Linking efficiency of salicylic acid in CSH;

[0033] Figure 12 For the optical photograph of GCM-MN;

[0034] Figure 13Optical images and adhesion of CSH hydrogel patch: (A) Optical images of CSH hydrogel patch; (B) Adhesion of CSH hydrogel to skin;

[0035] Figure 14 EDS element mapping of C, N, O, S and Ga elements in GCM-MN array, scale: 100 μm;

[0036] Figure 15 Typical force-displacement curves during single tip compression of Blank-MN and GCM-MN array;

[0037] Figure 16 Dissolution process of GCM-MN after insertion into mouse skin wound;

[0038] Figure 17 Cell viability after treatment of NIH / 3T3 cells with different concentrations of GCM nanoparticles (GCM NPs) and GCM-MN for 24 h;

[0039] Figure 18 Effect of GCM nanoparticles (GCM NPs) and GCM-MN on NIH-3T3 cell migration: (A) Representative images of scratch wound migration, scale: 200 μm; (B) Quantitative analysis of NIH-3T3 cell migration rate after 24 h;

[0040] Figure 19 HE staining of the effect of GCM-MN-CSH on mouse major organs. DETAILED DESCRIPTION

[0041] Example 1: Test materials and methods

[0042] 1.1. Materials and chemicals

[0043] Gallium nitrate (Ga(N03)3), carbenicillin (Car), high molecular weight gamma-polyglutamic acid (gamma-PGA, Mw>700,000) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); metformin (Met) and Hydrocaffeic acid were purchased from Adamas-Beta Pharm Co., Ltd. (Shanghai, China); Chitosan (CS, 200 kDa, degree of deacetylation >95%) was purchased from Macklin Biochemical Co., Ltd. (Shanghai, China); Rhodamine B was purchased from Bide Pharmaceutical Co., Ltd. (Shanghai, China).

[0044] Cell counting kit (CCK-8) was purchased from Thermo Fisher Scientific (Pittsburgh, USA); trypticase soy broth (TSB), Luria-Bertani (LB) medium and agar premix powder, Live / Dead BacLight bacterial viability kit (L7012) were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China).

[0045] High glucose Dulbecco's modified medium (HG-DMEM) and fetal bovine serum (FBS) were purchased from Biological Industries (Israel); growth factor reduced (GFR) Matrigel was purchased from Corning (New York, USA).

[0046] 1.2. Synthesis of GCM and GC

[0047] GCM was synthesized by existing single-pot stirring method, the specific steps are as follows:

[0048] 10 mmol of Ga(N03)3and Met were mixed in 12 mL of water, followed by slowly adding 6 mL of 5 mmol carbenicillin (Car) solution. The mixture was continuously stirred at room temperature for 48 hours, then separated by centrifugation (12,000 x g) to obtain a white precipitate, washed with deionized water for three times to remove unreacted chemicals, and freeze-dried at -60°C for 12 hours. The synthesis process of GC was similar to the above steps, the only difference was that Met was not added.

[0049] To quantify the Met in GCM, the supernatant after synthesis was collected, filtered through a 0.22 pm membrane, and analyzed by high performance liquid chromatography (HPLC, Thermo UltiMate 3000, USA). Hypersil GOLD-C18 column (250 x 4.6 mm, 5 pm) and Vanquish variable wavelength detector were used. The sample (20 pL) was eluted with a mixture of 85:15 (v / v) 10 mM sodium phosphate dibasic (pH 3.5) and methanol at a flow rate of 1 mL / min, and the detection wavelength was 233 nm. The concentration of Met was quantified by standard curve (R 2 = 0.999) with a concentration range of 0.03125 mg / mL to 0.5 mg / mL.

[0050] 1.3. Characterization of GCM

[0051] The morphology of GCM and GC particles was observed by field emission scanning electron microscopy (FE-SEM) (Carl Zeiss, Oberkochen, Germany) at 5 kV, and the particle size was measured by ImageJ software. Fourier transform infrared spectroscopy (FTIR) was performed on a Nicolet iS10 FTIR spectrometer (Thermo, Massachusetts, USA) at 2 cm -1The resolution of the diffractograms was 0.02° 2Q. Structural features were scanned by a Bruker D8 X-ray diffractometer (copper target) from 5° to 40° 2Q at a speed of 2° / min. The zeta potential was determined by a Malvern Zetasizer Nano ZS90 (Malvern Instruments Ltd, Worcestershire, UK).

[0052] To evaluate the release of carbenicillin (Car) and metformin (Met) in the GCM nanocomposite, 12 mg of GCM was dispersed in 12 mL of 0.05 M PBS at pH 5.5 and 7.4, respectively, and kept at room temperature. At certain time intervals (0, 1, 3, 6, 9, 12, 24, 48 hours), 300 μL of supernatant sample was collected. For Car, 20 μL of sample was analyzed under isocratic conditions using a 65:35 mixture of 0.05 M KH2PO4(pH 3.35) and methanol at a flow rate of 1 mL / min. Car concentration was quantified using two standard curves, concentration vs. peak area relationship (R2= 0.999) at pH 5.5 and pH 7.4, respectively. The concentration range of these curves was 15.625 μg / mL to 500 μg / mL. 2

[0053] 1.4. Formation and characterization of CSH hydrogel patches

[0054] Firstly, chitosan was modified by covalently binding chitosan with salicylic acid hydrogen amine, the specific steps were as follows: chitosan with a concentration of 0.058 mol / L was dissolved in 21.25 mL of water at pH 1.6. Then, the pH was adjusted to 5.4 with 1 M NaOH, and 1.5 mL of 1.08 mol / L salicylic acid hydrogen amine solution was added. Next, 0.6224 g of EDC-HCl (dissolved in 25 mL of 50% ethanol solution) was added step by step, the pH was adjusted to 4.6 with 1 M NaOH, and the reaction was stirred at room temperature for 12 hours. After the reaction, the reaction solution was dialyzed using a dialysis bag with a molecular weight cut-off of 14,000 Da, first in pure water at pH 5.0 for 2 days, then in pure water at pH 5.5 for 4 hours, and the dialysis solution was changed every 3-6 hours. After dialysis, CSH was obtained by freeze-drying.

[0055] To prepare the hydrogel patch, 67.5 mg of CSH was dissolved in 1.5 mL of PBS. The solution was centrifuged at 3,000 rpm for 5 minutes to remove air bubbles, then transferred to a mold with a size of 2 cm x 2 cm, and dried in a desiccator for 6 hours.

[0056] Synthesis verification: The UV-Vis absorption spectra of salicylic acid hydrogen amine and CSH were recorded by visible spectroscopy, and their chemical structure and functional groups were characterized by FTIR spectrometer.

[0057] ​Adhesion performance verification: The adhesion performance of the hydrogel patch was evaluated by applying it to human skin and observing whether there was any peeling phenomenon. In addition, an EP tube filled with water (about 3 grams) was attached to the hydrogel, and the adhesion strength was evaluated.

[0058] 1.5. Preparation of GCM-MN array

[0059] GCM powder at 10 mg / mL was dispersed in 0.9 mL of a y-PGA (390 mg / mL) solution and loaded into a PDMS mold. The solution was distributed into the pinholes by vacuum debubbling for 2 minutes and horizontal centrifugation (3,000 rpm) for 5 minutes. Then, 300 pL of a 20% polyvinylpyrrolidone (PVP) solution was added to form the backing layer of the array. After drying for 9 hours, careful demolding was performed and storage was performed for subsequent use and analysis. Control MNs were prepared by the same method, containing Met, Ga, and Car equivalent to GCM and GC at the same concentration as GCM.

[0060] 1.6. Characterization of GCM-MN-CSH

[0061] Microstructure characterization: The microstructure of GCM-MN was analyzed using a Hitachi SU8010 FE-SEM (enhanced metal spray coating), and elemental distribution was determined by EDX-assisted SEM (energy dispersive X-ray analysis).

[0062] Distribution of GCM in MNs: A distribution image of GCM nanoparticles labeled with rhodamine B in GCM-MN was taken using a Nexcope NIB620-FL fluorescence microscope.

[0063] Mechanical strength: To evaluate the skin insertion ability, GCM-MN arrays labeled with 0.4% trypan blue were applied to fresh pig skin, removed after 1 minute, and the micropores on the skin were examined using a digital microscope. In addition, the mechanical integrity of GCM-MN was evaluated using a mechanical tensile testing machine (AMETEK Inc, Leicester, UK). MNs were compressed on a horizontal sample table at a speed of 0.5 mm / min. Subsequently, a force displacement curve was plotted to evaluate the results.

[0064] Solubility test: GCM-MN was exposed to a sealed bottle with 100% relative humidity, and changes in morphology were recorded at different time points (0, 15, 30, 45, 60, and 75 minutes) using an optical microscope. To perform in vivo solubility testing, GCM-MN was applied to the skin of anesthetized mice, allowed to penetrate, and photographed at different time points (0, 5, 10, 15, 20, 25, 30 minutes) to observe the dissolution of the microneedles.

[0065] Adhesion test: Freshly dried pigskin was fixed on the base of a mechanical testing machine, and CSH patches were adhered to both sides of the pigskin and stretched until detachment occurred. The maximum force at the instant of detachment was recorded to calculate the adhesive stress. For GCM-MN-CSH samples, GCM-MN was inserted into the pigskin first, and then CSH patches were attached.

[0066] GCM release profile determination: Individual GCM-MN were immersed in 3 mL of PBS at pH 5.5 and 7.4, respectively. The absorbance of the supernatant was measured every 10, 20, 30, 60, 120, and 180 minutes with a UV-2700 spectrometer (Shimadzu, Kyoto, Japan) at a wavelength of 250 nm. The concentration of GCM was quantified by constructing a standard curve with known concentrations of GCM PBS solution.

[0067] 1.7. Anti-bacterial test

[0068] 1.7.1. Bacterial culture preparation

[0069] Gram-positive S. aureus (ATCC 29213) and Gram-negative P. aeruginosa (ATCC 27853) were cultured in TSB and LB media, respectively, at 37 °C for 12 hours. After culture, the bacterial suspension was centrifuged at 10,000 x g for 1 minute at 4 °C, washed three times with PBS to remove residual culture medium, and then resuspended in PBS to obtain the desired concentration for the experiment.

[0070] 1.7.2. Evaluation of the antibacterial activity of GCM nanoparticles

[0071] Different concentrations of GCM nanoparticles (0, 10, 30, 50, 70 μg / mL) were mixed with 1 x 10 6 CFU / mL of bacteria in PBS / media at pH 5.5 and 7.4, respectively. In 96-well plates, the bacteria were incubated at 37 °C. The proliferation of bacteria was evaluated by the CCK-8 method, and the count was taken after 1, 2, 3, 4, 5 hours of incubation. In addition, the colony counting method was also performed. The control concentrations included Met at 16.97 μg / mL, Car at 30.25 μg / mL, Ga at 2.78 μg / mL, and GC at 50 μg / mL. SEM was used for bacterial morphology analysis before and after GCM and GC treatment. The zeta potential of GCM and GC mixed with bacteria was determined by Malvern Zetasizer Nano ZS90.

[0072] 1.7.3. Anti-bacterial activity of MN

[0073] Different MNs (blank-MN, Met-MN, Car-MN, Ga-MN, GC-MN, and GCM-MN) were co-cultured with 1 x 10 6 CFU / mL of bacterial solution at pH 5.5. The bacteria proliferation was evaluated by CCK-8 method and the bactericidal rate was quantified by colony counting after 3 hours of incubation at 37 °C in microplates. The bacterial activity was evaluated by SYTO 9 (live cells) and PI (dead cells) staining.

[0074] 1.8. In vitro cell experiments

[0075] 1.8.1. Cell proliferation evaluation

[0076] Mouse embryonic fibroblasts (NIH / 3T3) and human umbilical vein endothelial cells (HUVECs) were used to evaluate the cytotoxicity of GCM nanoparticles and GCM-MN, respectively. Both cell types were cultured in high glucose DMEM medium supplemented with 10% fetal bovine serum at 37 °C in a 5% CO2 atmosphere. Cells were seeded in 96-well plates at 1 x 10 4 cells per well. GCM nanoparticles and GCM-MN were UV-sterilized for 15 minutes and then placed on the cell layer. After 24 hours of incubation, CCK-8 reagent was added and incubated for another 2 hours. The absorbance was measured by a microplate reader at 450 nm and the cell viability was calculated.

[0077] 1.8.2. Cell migration experiments

[0078] The in vitro wound healing assay was used to evaluate the chemotactic effect of GCM and GCM-MN on HUVECs and NIH / 3T3 cell migration. Cells were seeded in 6-well plates at a density of 1 x 10 6 and cultured until approximately 90% confluence. A sterile pipette tip was used to create a scratch in the cell monolayer. GCM and GCM-MN were UV-irradiated for 15 minutes and then added to the culture medium at a concentration of 50 pg / mL. 2 mL of DMEM containing 2% fetal bovine serum was used as a control. The control, GCM, and GCM-MN groups were incubated with the cells for 12 and 24 hours. After incubation, cell migration was observed using a bright-field microscope and the scratch gap was measured. The scratch closure was quantified using ImageJ software, and the migration rate was calculated using the formula: migration rate = (initial scratch area - final scratch area) / initial scratch area.

[0079] 1.8.3. Tube formation analysis

[0080] The pro-angiogenic effect of GCM and GCM-MN on HUVECs was evaluated using an in vitro tube formation assay. Each well of a 48-well plate was coated with 150 μL of growth factor-reduced Matrigel, refrigerated overnight, and then incubated at 37 °C for 30 min to form a gel. 1 x 105HUVECs were seeded onto the solidified gel and treated with serum-free DMEM containing 50 μg / mL of GCM and GCM-MN, or left untreated as a control. After 6 h of incubation, cell images were taken using a bright field microscope. The number of branch points and the average tube length were quantified by ImageJ software to analyze tube formation. 5 7

[0081] 1.9. In vivo wound healing evaluation

[0082] 1.9.1. Full-thickness infected wound model in rats

[0083] An infected wound model was established in BALB / c female mice (13-15 g, 5 weeks old). The mice were first injected intraperitoneally with 0.2 mL / 10 g of 1.25% bromoethanol solution, then shaved, disinfected with medical alcohol and iodophor, and a circular incision was made on the back using a 6 mm diameter biopsy punch. The wound was inoculated with 20 μL of S. aureus suspension (1 x 105CFU / mL). After 24 h, the mice were randomly divided into six groups: PBS (control), Blank-MN, Met-MN, GC-MN, GCM-MN, and GCM-MN-CSH. The MN array was applied under anesthesia and changed every day for the first four days. The mice were fed under standard conditions, ensuring adequate food and water. Wound healing progression was recorded by taking pictures, and the wound area was quantified on days 0, 2, 4, 6, and 8, analyzed using Image-Pro Plus software. The formula for calculating the wound area rate is: wound area rate = (wound area / initial wound area) x 100%. On day 8 after treatment, the wound tissue was collected, and the bacterial clearance was evaluated using the standard plate counting method.

[0084] ​​After 8 days of treatment, all mice were euthanized and skin tissue containing the wound area was collected. The tissue was fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and sectioned into 5 pm thin slices for histological examination. Granulation tissue formation and collagen deposition were evaluated by HE (Hematoxylin-Eosin) staining and Masson’s trichrome staining. Immunohistochemical staining was used to evaluate inflammation and immune response, with IL-1 b (inflammation), IL-6 (inflammation), CD206 (M2 type macrophage), CD86 (M1 type macrophage) markers. Neovascularization was analyzed by CD31 (endothelial cell marker) and a-SMA (fibroblast marker) staining. In addition, major organs (including heart, liver, spleen, lung, and kidney) were collected from all groups and subjected to HE staining to evaluate systemic effects.

[0085] 1.10. In vitro blood compatibility test

[0086] Hemolysis rate was used to evaluate the blood compatibility of GCM and GCM-MN. The blood of BALB / c mice was separated into red blood cells by centrifugation (4 °C, 15 min) and washed with normal saline until the supernatant was colorless. The red blood cell suspension was diluted to 4% (v / v). Different concentrations of GCM and GCM-MN extract (6.25, 12.5, 25, 50 and 100 pg / mL) were added to equal volume of red blood cell suspension, respectively. Normal saline and ultrapure water were used as negative and positive controls, respectively, each mixed with equal volume of 2% red blood cell suspension. All groups were incubated at 37 °C for 4 hours, and after centrifugation at 1500 x g for 5 minutes, 100 pL of supernatant was measured for absorbance at 540 nm. The formula for calculating hemolysis rate is:

[0087] Hemolysis rate = (supernatant absorbance - control group absorbance) / (positive control absorbance - negative control absorbance) x 100%.

[0088] 1.11. Statistical analysis

[0089] All data were derived from three or more independent experiments, and the results were expressed as mean ± standard deviation (SD). Data analysis was performed using GraphPad Prism 8 software (GraphPad Software, California, USA). Statistical significance between two groups was determined by Student’s t-test, while differences among multiple groups were assessed by one-way analysis of variance (ANOVA). The level of statistical significance was set at p-value less than 0.05. Statistically significant results were represented as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns represents no significant difference.

[0090] Example 2: Test results

[0091] 2.1. Synthesis and Characterization of GCM Nanoparticles

[0092] GCM nanoparticles were synthesized using an existing single-pot method, the synthesis process of which is as follows: Figure 1 As shown in Figure A, firstly, Ga(NO3)3 and Met are mixed in water, and the reaction produces Ga. 3+ The complex was then formed with Met. Subsequently, carbenicillin (Car) solution was added to react with Ga. 3+ A Ga-Car-MOF complex was formed. After 48 hours of stirring, the precipitate was collected and washed with deionized water to remove unreacted substances. Finally, white GCM nanoparticles were obtained by freeze-drying.

[0093] The loading of Met in GCM was analyzed by high performance liquid chromatography (HPLC). The results showed that the loading efficiency of Met in GCM was 34.0 ± 2.2%. Figure 7 The morphology of GCM was observed using scanning electron microscopy (SEM), revealing that the particles were spherical with a diameter of approximately 75.1 ± 12.1 nm. Figure 1 B. Figure 1 D), and the particle size distribution is relatively uniform. In contrast, the average diameter of GC particles is 49.0 ± 7.9 nm. Figure 1 C Figure 1 (D) This particle size difference may be related to the addition of Met, whose larger molecules lead to an increase in the size of GCM particles. Fourier transform infrared spectroscopy (FTIR) analysis further confirmed the successful synthesis of GCM. Figure 1 The FTIR spectrum in E shows that at 1380 cm⁻¹ -1 2970cm -1 and 1770cm -1 Significant absorption peaks appeared at all locations, corresponding to the C=C stretching vibration of the aromatic ring, the bending vibration of the -C(CH3)2 group, and the C=O stretching vibration of carbenicillin, respectively. Compared with GC, GCM showed a significant absorption peak at 1153 cm⁻¹. -1 and 1228cm -1 The absorption peak at this location is significantly enhanced, indicating that Met has been embedded in the MOF framework, a result consistent with HPLC data. X-ray diffraction (XRD) patterns of GCM and GC show characteristic diffraction peaks consistent with the known structure of Ga-Car-MOF. Figure 1 F). Specifically, the diffraction peak at 18.1° indicates that the crystal structure of GCM is preserved. Furthermore, the zeta potential of the GCM particles increases from 8.21 ± 0.40 mV for GC to 11.17 ± 0.42 mV. Figure 1J), which indicates that the introduction of Met enhances the positive charge of the particles. The amino groups in the Met molecule increase the positive charge of GCM, which can also enhance its interaction with the negatively charged bacterial surface, thereby improving the antibacterial performance. In summary, the characterization methods such as SEM, FTIR and XRD show that the GCM nanoparticles with the expected morphology and structure are successfully prepared, and further verify the effective loading of Met in the MOF.

[0094] Considering the slightly acidic pH value of the wound microenvironment, which is usually between 5 and 6, and the wound pH value tends to be alkaline as the clinical infection progresses. In addition, previous studies have shown that the coordination bond between carbenicillin (Car) and Ga 3+ Therefore, the present application evaluates the Car concentration released by GCM in PBS solutions at pH 5.5 and 7.4 to study the pH-responsive degradation ability of GCM. The research results of the present application show that about 392.08 ± 7.82 μg / mL of Car is released under the condition of pH 5.5 after 48 hours, while only 205.41 ± 26.02 μg / mL of Car is released under the condition of pH 7.4 Figure 1 G). This result shows that GCM retains the pH-sensitive characteristics of GC and can efficiently release most of the drug-loaded substances in an acidic environment. Specifically, the release of Car in GCM increases as the pH value decreases, and shows a slower and lower release rate under neutral conditions.

[0095] Wounds exposed to the external environment are susceptible to infection by pathogenic bacteria, such as Staphylococcus spp., Enterococcus spp., Enterobacteriaceae, and Pseudomonas spp. These bacteria can cause sepsis and lead to potentially life-threatening complications, so it is crucial to eliminate these bacteria during the wound healing process. In order to evaluate the antibacterial effect of GCM, the present application uses CCK-8 method and colony counting method to test its bacteriostatic and bactericidal effects on gram-positive S. aureus and gram-negative P. aeruginosa, respectively. Preliminary tests show that when the concentration of GCM exceeds 50 μg / mL, the survival rate of S. aureus after 1 hour of co-incubation with GCM is almost zero Figure 8 A), and the survival rate of P. aeruginosa after 3 hours of co-incubation with GCM also reaches a similar level Figure 8 B). Considering the acidic microenvironment of acute trauma, the present application further studies the antibacterial effect of GCM under the conditions of pH 5.5 and 7.4. As Figure 1As shown in FIG. H, GCM significantly reduced the survival rate of bacteria at pH 5.5 more than at pH 7.4, indicating that the antibacterial activity of GCM depends on pH, and the antibacterial effect is enhanced when the pH is reduced. Notably, 30 pg / mL of GCM completely inhibited the activity of S. aureus at pH 5.5, while 50 pg / mL was required to inhibit P. aeruginosa. This result is consistent with the mechanism of action of carbenicillin (Car), which is a beta-lactam antibiotic that inhibits the synthesis of mucopolysaccharide synthetase, disrupts the synthesis of the bacterial cell wall, and causes structural defects. For P. aeruginosa, the partial impermeability of the outer membrane glycoprotein phospholipid bilayer to carbenicillin makes it less permeable, so carbenicillin has a poor antibacterial effect on it.

[0096] Further, the present application used equal amounts of free Met, Ga, Car, and GC and GCM to conduct a detailed study of the inhibition of bacterial activity. The results of the CCK-8 experiment Figure 9 A) showed that the Met treatment group had little effect on the survival rate of the two bacteria. In contrast, the Ga treatment group had no effect on the survival rate of S. aureus, but significantly reduced the survival rate of P. aeruginosa to 13.6%. This reduction can be due to Ga's ability to interfere with iron uptake and interfere with iron signaling through the transcriptional regulator pvdS. Carbenicillin (Car) had a stronger inhibitory effect on S. aureus than on P. aeruginosa. Both GC and GCM treatment reduced the survival rate of S. aureus and P. aeruginosa to about 95%, and there was no significant difference between the two. Further, the present application also evaluated the cell membrane permeability of S. aureus and P. aeruginosa after GCM nanoparticle treatment by SYTO9 and PI double staining. SYTO9 can stain all bacterial DNA, while PI selectively stains the DNA of bacteria with damaged cell membranes, indicating cell death, as PI cannot penetrate intact cell membranes. The observation after treatment showed that the red fluorescence intensity of the GCM and GC treatment groups increased significantly Figure 10 ), indicating that the GCM group was significantly superior to the other groups in terms of antibacterial effect. The above results show that the antibacterial effect of GCM is exerted through the synergistic effect of Ga and carbenicillin (Car) release, rather than relying on Met, thereby effectively inhibiting the proliferation of bacteria. This finding was further confirmed by colony counting on agar plates Figure 9 B-C), confirming the strong antibacterial properties of GCM.

[0097] It can be seen that due to the presence of lipopolysaccharide and phospholipopeptide in the bacterial cell wall, its surface is usually negatively charged. The positively charged GCM can be "adsorbed" to the negatively charged S. aureus and P. aeruginosa surface by electrostatic attraction. Figure 1 J). Subsequently, the pH-responsive released carbenicillin (Car) rapidly penetrates the bacterial cell wall, precisely inhibiting the further cleavage of peptidoglycan by transpeptidase, leading to bacterial lysis and death, which is also confirmed by the observed morphology of the bacterial cell wall by scanning electron microscopy (SEM) Figure 1 K). S. aureus and P. aeruginosa in the control group (i.e. untreated group) exhibit a relatively spherical and smooth morphology, with intact cell walls. In contrast, in the GCM-treated bacteria, the cell surface was observed to become wrinkled and cracked. These findings indicate that the bacterial wall was significantly damaged, significantly affecting the permeability of the bacteria. The released Ga 3+ ions enter the bacteria, interfere with the iron metabolism of the bacteria, and use the iron stress in the internal environment to inhibit bacterial growth. Finally, in the slightly acidic environment of bacterial infection, GCM can release drug-loaded substances by pH response, improve the utilization efficiency of antibiotics, and exhibit a synergistic antibacterial effect in vitro.

[0098] 2.2. Synthesis and characterization of GCM-MN-CSH

[0099] The double-layer microneedle patch GCM-MN-CSH prepared in the present application Figure 2 A) is composed of a drug delivery unit (GCM-MN) and an adhesive hydrogel patch (CSH). As shown in Figure 2 B, since γ-polyglutamic acid (γ-PGA) has strong mechanical properties after drying, biocompatible γ-polyglutamic acid (γ-PGA) was selected as the matrix material of the GCM-MN needle tip to load GCM nanoparticles. PVP was selected as the backing layer material of GCM-MN due to its good biosafety, mechanical strength and rapid solubility. GCM-MN was prepared by a two-step template replication method, in which GCM nanoparticles were dispersed in water and mixed with γ-PGA. The mixture was filled into the needle tip of the needle mold by vacuum and low-speed centrifugation, and after removing the excess material, pure PVP pre-gel was added to form a GCM-MN microneedle array. For the CSH hydrogel patch, first, the chitosan was modified by covalently binding hydrocaffeic acid to form CSH. The formation of covalent bonds and the degree of binding were verified by Fourier transform infrared spectroscopy (FTIR) and ultraviolet spectroscopy (UV), as shown in Figure 11The CSH solution was then crosslinked in the mold to produce the hydrogel patch. Both components, GCM-MN and CSH, achieved 100% demolding rate, with GCM-MN microneedles intact and uniformly arranged in a 12 x 12 array Figure 12 ), and CSH showing a consistent thickness Figure 13 ).

[0100] SEM images verified the pyramidal shape of GCM-MN tip, with each needle being 640 pm long, base diameter of 320 pm, and tip-to-tip distance of 550 pm Figure 2 The outermost layer of skin, the avascular epidermis, is approximately 100 pm thick, while the underlying vascular-rich dermis is typically between 1000 and 2000 pm thick. Therefore, the 640 pm long GCM-MN is sufficient to penetrate the epidermis and reach the dermis layer. Fluorescence microscopy images of GCM labeled with rhodamine B showed uniform red fluorescence at the needle tips Figure 2 E), indicating uniform distribution of GCM within the needles. Additional elemental mapping Figure 14 ) further supported the uniform embedding of GCM, highlighting the successful fabrication of GCM-MN and their use for transdermal drug delivery. The sharp pyramidal structure of GCM-MN facilitates rapid and non-invasive insertion into the dermis. This was verified in tests performed on pig skin to assess the transdermal delivery potential of GCM-MN. As shown in Figure 2 F, upon penetration of the skin by the microneedles, the puncture channels stained with trypan blue could be clearly observed. Furthermore, the mechanical strength of GCM-MN was evaluated using an electronic tensile tester, and the results confirmed that the addition of GCM nanoparticles did not significantly compromise the structural integrity of the microneedles (p > 0.05). The test results showed that the mechanical strength of the microneedles was 0.52 N per needle, sufficient to effectively pierce the skin without breaking, Figure 15 further confirming this.

[0101] Existing studies have shown that gamma-polyglutamic acid (y-PGA) and PVP, as biocompatible macromolecules, have good biodegradability and hygroscopicity. These properties enable GCM-MN to absorb interstitial fluid and degrade, thereby facilitating the release of GCM nanoparticles. To assess the in vitro degradation of GCM-MN, they were placed in a sealed box with 100% relative humidity, and morphological changes were recorded. Within 15 minutes, the tips of GCM-MN began to absorb water and dissolve, and after 75 minutes they were completely dissolved Figure 2 G). In vivo, when GCM-MN were applied to S. aureus-infected wounds in mice, the contact surface of the patch became wet immediately, indicating that the tips of GCM-MN had begun to dissolve Figure 17). Within 30 min, the GCM-MNs in contact with the wound almost completely dissolved. The dissolution rate of the GCM-MN tips in the wound was faster than that in the sealed box, which could be due to the increased temperature of the skin in the wound, thus promoting the acceleration of dissolution. In addition, the present invention also investigated the release kinetics of GCM in PBS at pH 5.5 and 7.4. The release profile showed a sustained release pattern, with cumulative release amounts of 83.19 ± 10.93 pg / mL and 76.24 ± 3.68 pg / mL at pH 5.5 and pH 7.4, respectively, after 2 h Figure 2 H) These results highlight the hygroscopic and biodegradable properties of GCM-MNs and confirm their pH-responsive behavior, further emphasizing their potential as an effective platform for the rapid transdermal delivery of therapeutic drugs.

[0102] The present invention also evaluated the adhesive properties of the CSH hydrogel patch to assess its suitability as a skin protective layer. The CSH hydrogel patch exhibited seamless skin adhesion, maintaining contact even in the presence of a 3-gram weight force on a flexed finger joint, indicating its excellent adhesive stability and skin adaptability Figure 2 I and Figure 13 B) Further investigations focused on whether the addition of GCM-MNs affected the tissue adhesive properties of the CSH hydrogel patch. Freshly prepared CSH hydrogel patches and GCM-MN-CSH were attached to porcine skin and subjected to vertical stretching until detachment. The stress-displacement curves measured in the tensile adhesion test indicated that the addition of GCM-MNs did not change the adhesive strength Figure 2 J) Thus, GCM-MN-CSH exhibited strong adhesive properties, effectively maintaining attachment to the skin tissue.

[0103] 2.3. In vitro antibacterial ability of GCM-MNs

[0104] Staphylococcus aureus and Pseudomonas aeruginosa are widely present in chronic wounds, and their expressed virulence factors and surface proteins can hinder wound healing. S. aureus is usually colonized in the superficial layer of chronic wounds, while P. aeruginosa is commonly found in deeper wounds. Wounds infected with these bacteria are larger and heal more slowly than uninfected wounds, highlighting the critical role of microneedles (MNs) in targeting the surface and deep layers of wounds. To verify the in vitro antibacterial activity of GCM-MNs, the present invention tested the co-culture effects of blank-MNs and Met-, Ga-, Car-, GC-, and GCM-loaded MNs with S. aureus and P. aeruginosa in PBS. As shown in Figure 3As shown, after 3 hours of incubation, the cell viability of GCM-MN and GC-MN groups was significantly reduced compared to the control group, blank microneedle group and Met-, Ga-, Car-loaded microneedle groups Figure 3 A) and colony count reduction Figure 3 B-C). Notably, the red fluorescence of GCM-MN and GC-MN groups was significantly enhanced, indicating bacterial death Figure 3 D). This result is consistent with the antibacterial effect of GCM and GC nanoparticles, proving that embedding GCM nanoparticles into microneedles can retain their antibacterial properties. Importantly, GCM-MN exhibited antibacterial activity from the epidermis layer to the dermis layer, which is crucial for accelerating wound healing.

[0105] 2.4. In vitro biocompatibility evaluation of GCM-MN

[0106] Cell compatibility and cell affinity are the key to the successful application of transdermal drug delivery systems. Understanding the interaction of nanoparticles (NPs) and microneedles (MNs) with cells and their effects on cell growth and reproduction is crucial for evaluating their effects on biological tissues and ensuring the safety of in vivo applications, helping to prevent harmful effects that may inhibit wound healing or cause adverse reactions. The present invention evaluated the cytotoxicity of GCM nanoparticles and GCM-MN at different concentrations on NIH / 3T3 fibroblasts and human umbilical vein endothelial cells (HUVECs) using the CCK-8 method. The results showed that even at a high concentration (100 μg / mL), after 1 day of co-culture, neither GCM nanoparticles nor GCM-MN exhibited significant cytotoxicity, with cell viability remaining above 95% Figure 17 and Figure 4 A), demonstrating their good cell compatibility. In addition, considering the inevitable contact with blood during in vivo application, it is also necessary to evaluate the hemocompatibility. The hemolysis rate of GCM nanoparticles and GCM-MN was significantly lower than the positive control (water, inducing 100% hemolysis) and close to the negative control (normal saline). Even at a concentration as high as 100 μg / mL, the hemolysis rate was less than 2%, and there was no statistically significant difference between the test materials Figure 4 B-C). These studies show that GCM nanoparticles and GCM-MN will not cause adverse reactions in the biological system, confirming their safety in medical and biological applications. In summary, GCM-MN in the present invention exhibits excellent biocompatibility and has the potential to be used as a local transdermal delivery system for the treatment of infected wounds.

[0107] 2.5. In vitro cell migration and angiogenesis properties of GCM-MN

[0108] In this invention, at a concentration of 100 μg / mL, GCM nanoparticles significantly improved the survival rate of HUVECs, reaching 118.8 ± 5.5%. Figure 4 A) indicates that it promotes cell proliferation. This synergistic effect may be attributed to metformin (Met) in GCM nanoparticles. Studies have shown that Met downregulates autophagy in HUVECs by activating the Hedgehog signaling pathway, which is crucial for tissue maintenance, renewal, and regeneration. Furthermore, Met can alleviate oxidative stress, thereby further improving endothelial cell function. Subsequently, this invention used an in vitro scratch assay to evaluate the effects of GCM nanoparticles and GCM-MN on cell migration. Semi-quantitative analysis showed that, compared with the control group, fibroblasts and endothelial cells exposed to GCM nanoparticles and GCM-MN migrated to the wound area more rapidly after 24 hours and 12 hours of culture, respectively. Figure 18 , Figure 4 Specifically, the cell coverage rates in the wound space of the GCM nanoparticle and GCM-MN groups were 38.3±0.3% and 42.9±3.9%, respectively, significantly higher than that of the control group (26.7±3.29%). Notably, there was no significant difference between the GCM nanoparticle and GCM-MN groups (p>0.05). These results indicate that GCM nanoparticles and GCM-MN can effectively promote the growth and migration of NIH / 3T3 fibroblasts and HUVECs, promote the rapid closure of cell-free gaps, and may accelerate the wound healing process.

[0109] Angiogenesis and vascular remodeling are crucial for tissue healing because newly formed blood vessels provide nutrients and oxygen to the wound, accelerating wound closure. This invention evaluates the pro-angiogenic potential of GCM nanoparticles (NPs) and GCM-MN through in vitro angiogenesis experiments. Figure 4 As shown in Figure FH, HUVECs exposed to 50 μg / mL GCM nanoparticles and GCM-MN exhibited significantly enhanced lumen formation, with more branching points and longer lumen lengths compared to the control group. This enhanced angiogenesis was attributed to the activation of the AMPK signaling pathway and the upregulation of the RasGRP1-dependent VEGF signaling pathway by Met in GCM nanoparticles and GCM-MN. These signaling pathways are crucial for endothelial cell proliferation and migration (verified by cell proliferation experiments). These signaling pathways help regulate angiogenesis, promoting the formation of a robust vascular network, thereby facilitating efficient nutrient delivery and wound healing. Combined with the results of the HUVEC migration assay, GCM-MN demonstrates the ability to promote cell migration and lumen formation, supporting its application in subsequent in vivo experiments.

[0110] 2.6. Wound healing effect of GCM-MN-CSH double-layer microneedle patch in bacterial infected wounds

[0111] To demonstrate the clinical potential of the microneedle patch in the present application, the wound healing effect of the double-layer GCM-MN-CSH patch was evaluated using a S. aureus infected female BALB / c mouse model Figure 5 A). First, GCM-MN was applied directly on the wound under appropriate pressure to ensure that the microneedles could penetrate the skin, and then a CSH hydrogel patch was covered to maintain a moist healing environment, prevent dehydration, and protect the wound from secondary damage. The dynamic changes in wound morphology were observed on days 0, 2, 4, 6, and 8 as shown in Figure 5 B-C. By day 8, the wounds in the GCM-MN-CSH and GCM-MN groups were almost completely healed, with barely distinguishable wound borders, while the wounds in the blank-MN, Met-MN, and GC-MN groups were significantly slower to heal, with visible scabbing and depression on the wound surface. The wounds in the control group remained moist and unhealed, without scabbing. Quantitative analysis by digital imaging Figure 5 D) showed that the relative wound area in the GCM-MN-CSH and GCM-MN groups decreased to 26.10 ± 7.79% and 40.65 ± 16.31% by day 4, and further decreased to 9.53 ± 2.72% and 14.39 ± 8.65% by day 8, which was significantly better than the 47.43 ± 17.02% in the control group (p < 0.05). The complete removal of epidermal scabbing in the GCM-MN-CSH and GCM-MN groups indicated successful epidermal tissue regeneration. Previous studies have shown that the application of antimicrobial metal-organic framework microneedles (MOF-MN) can only accelerate the healing of wounds of similar size after 12 days, while the GCM-MN-CSH in this study achieved this effect in only 8 days. This enhancement in healing effect is attributed to the strong antibacterial effect of Ga and carbenicillin (Car), as well as the significant impact of Met on cell proliferation, migration, and angiogenesis, which was confirmed by in vitro experiments.

[0112] 2.7. In vivo antibacterial effect

[0113] The in vivo antibacterial effect of different groups was evaluated by collecting skin tissue containing the entire wound area at day 8. As shown in Figure 5As shown in FIGS. E-F, the results showed that microneedles loaded with GC and GCM nanoparticles (GC-MN, GCM-MN and GCM-MN-CSH) exhibited significant antibacterial effects, with a significant reduction in bacterial load in the wound area. This confirmed the strong antibacterial potential of these carriers in vivo, consistent with their antibacterial effects in vitro. In particular, the GCM-MN-CSH group showed very strong antibacterial effects, significantly better than the GCM-MN group (p<0.05). This enhanced effect can be attributed to the CSH hydrogel patch, which not only maintains a moist healing environment, but also protects the wound from external contamination, thereby further preventing additional damage and contamination of the wound.

[0114] 2.8. Systemic safety of GCM-MN-CSH double-layer microneedle patch

[0115] To further evaluate the safety of the GCM-MN-CSH double-layer microneedle patch, the present application conducted relevant biosafety tests in mice in vivo. Post-treatment observations showed that the body weight of the mice did not change significantly, suggesting that these treatments did not adversely affect the whole body of the mice Figure 5 G). In addition, pathological analysis of the major organs of the mice (including heart, liver, spleen, lung and kidney) by HE staining showed that these organs had no significant pathological abnormalities Figure 19 ). This further verified the safety of GCM-MN-CSH in vivo, indicating that it can be suitable for clinical applications.

[0116] 2.9. Evaluation of tissue regeneration, inflammatory response and angiogenesis

[0117] To further evaluate the ability of the GCM-MN-CSH double-layer microneedle patch to regenerate tissue after treatment, the present application evaluated re-epithelialization and collagen production of the wound by histological analysis. HE staining was used to evaluate wound contraction, epithelial gap, granulation tissue formation and tissue remodeling. As shown in FIGS. Figure 6 A, the tissues of the control group, blank-MN group, Met-MN group and GC-MN group showed obvious rupture and inflammatory cell infiltration, while the tissues of the GCM-MN and GCM-MN-CSH groups showed significantly reduced inflammatory cells, and the migration of epithelial tissue was significantly enhanced, resulting in a reduction in wound length and dermal gap. Further quantitative evaluation of the thickness of the new epithelial tissue Figure 6 B) showed that the thickness of the epidermis in the GCM-MN-CSH group was significantly increased, significantly higher than that in the control group (p<0.0001). The production and deposition of collagen were evaluated by Masson trichrome staining Figure 6A), GCM-MN and GCM-MN-CSH groups showed better collagen arrangement and enhanced deep blue staining, indicating more intense collagen synthesis. Quantitative analysis showed that the collagen coverage of GCM-MN-CSH group reached 66.15 ± 4.40%, which was significantly better than other groups Figure 6 C). These findings suggest that GCM-MN-CSH has a significant promoting effect on collagen deposition and tissue remodeling.

[0118] 2.10. Immunohistochemical analysis of inflammatory response and angiogenesis

[0119] Inflammatory response caused by infection can hinder the healing process of wound bed, in which macrophages play a crucial role in wound healing and tissue regeneration. In the early stage of wound healing, M1 type macrophages phagocytose necrotic / apoptotic neutrophils or damaged cells and produce pro-inflammatory cytokines such as IL-1β and IL-6, enhancing the inflammatory response. On the contrary, M2 type macrophages produce anti-inflammatory cytokines and growth factors to help restore damaged tissues. To further study the inflammatory response, the present invention analyzed the expression levels of pro-inflammatory cytokines (IL-1β and IL-6) and markers of M1 type (CD86) and M2 type (CD206) macrophages by immunohistochemical staining Figure 6 A). The results showed that the expression of IL-6 and CD86 was significantly higher in the control group, blank-MN group, Met-MN group and GC-MN group than in the GCM-MN and GCM-MN-CSH groups Figure 6 D-F), while the secretion of IL-1β was significantly increased in the control group and blank-MN group Figure 6 D). However, GCM-MN and GCM-MN-CSH groups showed a significant decrease in IL-1β, IL-6 and CD86 levels, while the expression of CD206 was significantly increased Figure 6 G). These results suggest that the inflammatory response is reduced in GCM-MN and GCM-MN-CSH groups, which helps macrophages transform from M1 type to M2 type, thereby improving wound healing capacity.

[0120] Angiogenesis is an important marker of healing, as it promotes wound repair by providing oxygen, nutrients and growth factors. The present invention analyzed mature angiogenesis by immunofluorescence staining to evaluate new blood vessel formation, labeling CD31 (red) for endothelial cells and α-SMA (green) for fibroblasts Figure 6 A). GC-MN, GCM-MN and GCM-MN-CSH groups showed more mature vascular-like structures, indicating strong angiogenic capacity Figure 6H-I). In contrast, in the control, blank-MN and Met-MN groups, fewer mature blood vessel-like structures were observed. This difference can be attributed to the lack of antibacterial capacity in these groups, and the persistent bacterial infection can have inhibited neovascularization at the wound site.

[0121] The above results show that the wound healing in the GCM-MN-CSH group was accelerated, and this rapid recovery capacity was attributed to the strong antibacterial effect of the microneedle patch, the reduction of inflammatory response, and the improvement of cell dynamics, including rapid proliferation and migration. In addition, this treatment promoted a high content of collagen fibers, the formation of granulation tissue, and significant vascular regeneration, which are important components of effective wound repair.

[0122] In summary, the multifunctional double-layer MOF-based microneedle patch (GCM-MN-CSH) in the present application exhibits excellent antibacterial performance, promotes tissue regeneration, and has good biocompatibility in the treatment of infected wounds. The microneedle array loads GCM nanoparticles into γ-PGA through rapid solubility. The chitosan hydrogel layer modified by salicylic acid amine provides good adhesion, and this double-layer microneedle patch has a regular morphology, uniform structure, and efficient transdermal penetration ability. Antibacterial experiments confirmed that GCM-MN exerts a synergistic antibacterial effect by interfering with iron metabolism, and by Ga 3+ and carbenicillin inhibit bacterial cell wall synthesis. In addition, it also enhances cell proliferation, migration, and angiogenesis. In a mouse wound model infected with S. aureus, the GCM-MN-CSH microneedle patch accelerated wound healing through minimal trauma, exhibiting strong antibacterial effect, effective epithelialization process, and granulation tissue formation. Mechanism analysis further revealed that the GCM-MN-CSH microneedle patch produced a significant therapeutic effect on infected wounds by reducing the levels of inflammatory cytokines, promoting the polarization of M2 macrophages, enhancing collagen deposition, and accelerating angiogenesis. Overall, the multifunctional performance of the GCM-MN-CSH double-layer microneedle patch in the present application makes it a promising candidate for clinical application in the treatment of infected wounds.

Claims

1. A dual-layer microneedle patch, characterized by, The application relates to a double-layer microneedle patch for treating infected wounds, which comprises a drug delivery unit and an adhesive unit, wherein the drug delivery unit is a GCM-MN microneedle array, the length of each needle tip is 640 microns, and the adhesive unit is a CSH hydrogel patch. In use, the GCM-MN microneedle array is first applied to a wound by pressure to ensure that the microneedles can penetrate the skin of the wound, and then the CSH hydrogel patch is covered on the GCM-MN microneedle array to ensure a moist healing environment. The preparation method comprises the following steps: (1) synthesis of GCM; (2) preparation of a GCM-MN microneedle array; (3) preparation of a CSH hydrogel patch. In step (1), the synthesis of GCM is as follows: Ga(NO3)3 and Met are mixed in water to generate Ga³⁺ and Met coordination complexes, wherein Met is metformin; then, a carbenicillin solution is added to form Ga-Car-MOF complexes with Ga³⁺; after stirring and reaction, the precipitate is collected and washed to remove unreacted substances; finally, white GCM nanoparticles are obtained through freeze-drying. In step (2), the preparation of the GCM-MN microneedle array is as follows: a two-step template replication method is adopted, first, the GCM nanoparticles prepared in step (1) are dispersed in water and mixed with gamma-PGA, then the mixture is filled into the needle tips of a needle mold through vacuum and low-speed centrifugation, after removing the excess material, pure PVP pre-gel is added to form a GCM-MN microneedle array. In step (3), the preparation of the CSH hydrogel patch is as follows: first, CSH powder is formed by covalently combining hydrogenated caffeic acid modified chitosan; then, the CSH solution is crosslinked in a mold to prepare a CSH hydrogel patch.

2. The dual-layer microneedle patch of claim 1, wherein, The GCM-MN microneedle array uses gamma-PGA as the matrix material of the needle tips and uses PVP as the backing layer material.

3. The dual-layer microneedle patch of claim 1, wherein, The needle tips of the GCM-MN microneedle array are all pyramid-shaped, the base diameter is 320 microns, and the spacing between adjacent needle tips is 550 microns.

4. Use of a double-layer microneedle patch according to claim 1 or 2 or 3, characterized in that, The application also discloses an application of the double-layer microneedle patch in the preparation of an infected wound healing medical device.

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