Soluble double-layer microneedle patch loaded with LL37 ZIF-8 and Prussian blue nano-enzyme pharmaceutical composition and application of soluble double-layer microneedle patch loaded with LL37 ZIF-8 and Prussian blue nano-enzyme pharmaceutical composition

Abstract

The invention discloses a soluble double-layer microneedle patch loaded with an LL37-coated ZIF-8 and Prussian blue nano-enzyme pharmaceutical composition and application, and belongs to the technical field of biomedical materials. The soluble double-layer microneedle patch provided by the invention comprises a needle body layer and a backing layer, the needle body layer comprises a pullulan polysaccharide matrix, LL37 (at) ZIF-8 and a Prussian blue nano enzyme; and the backing layer comprises a pullulan polysaccharide matrix. According to the preparation method, pullulan, LL37 ZIF-8 and Prussian blue nano enzyme (PB) are combined for use, so that the soluble double-layer microneedle patch integrating efficient antibiosis, continuous antioxidation, healing promotion and biocompatibility can be obtained, and the application performance is good; and the preparation method has the advantages of mild reaction conditions, simple preparation, low raw material price, easiness in batch production and manufacturing and the like, and is easy for large-scale production.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly to a soluble bilayer microneedle patch loaded with LL37@ZIF-8 and Prussian blue nanozyme drug composition and its application. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to enhance the overall understanding of the present invention, and is not necessarily regarded as an admission or an indication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] As the largest organ of the human body, the skin plays a core role in resisting the invasion of external pathogens, maintaining the stability of the internal environment, and tissue repair. However, factors such as diabetes, immunodeficiency, and trauma often lead to impaired skin regeneration ability. Bacterial infection is one of the factors that cause delayed wound healing. Foreign bacteria can rapidly multiply at the wound site and release pathogenic proteases to damage the wound microenvironment, resulting in local inflammation or even sepsis. Moreover, foreign bacteria can also upregulate the content of reactive oxygen species (ROS) through their own metabolites, inducing host oxidative stress and further aggravating the damage. The excessive release of ROS will also cause damage to extracellular matrix proteins and initiate apoptosis. These reactions subsequently lead to an increase in the level of inflammatory cytokines and further exacerbate inflammation. Eventually, the wound is repeatedly infected, posing a serious threat to the quality of life and physical health of the patient.

[0004] Currently, the commonly used wound dressings in clinics are mainly divided into three categories: traditional dressings (such as alginate, hydrocolloid) can provide a physical barrier, but lack antibacterial and antioxidant activities; dressings containing antibiotics have the risk of drug resistance and cannot neutralize excessive ROS; nano-material dressings (such as silver-based materials) have broad-spectrum antibacterial properties, but have cytotoxicity and long-term safety hazards. As an emerging drug delivery system, microneedle technology has attracted attention due to the advantages of its soluble microneedle type in avoiding mechanical damage and biological residues. However, existing products still have the following limitations: functional singularity: most microneedles only load a single drug, making it difficult to simultaneously address the dual challenges of infection and oxidative stress. Moreover, the activities of multiple drugs may interfere with each other due to differences in physical and chemical properties, and the stability of the carrier may decrease; drug stability: natural active ingredients such as antimicrobial peptides are easily inactivated by enzymatic hydrolysis, and traditional embedding methods result in insufficient sustained-release performance; tissue compatibility: some synthetic materials may cause immune rejection reactions and delay the healing process.

[0005] Therefore, how to provide a new type of wound dressing that integrates efficient antibacterial, continuous antioxidant, wound healing promotion, and biocompatibility to meet the urgent clinical needs for the treatment of multifunctional infectious wounds is an urgent problem to be solved. Summary of the Invention

[0006] In view of this, the present invention provides a soluble bilayer microneedle patch loaded with LL37@ZIF-8 and Prussian blue nanozyme drug composition and its application. The soluble bilayer microneedle patch provided by the present invention has a killing effect on Gram-negative bacteria and Gram-positive bacteria, and it exhibits good antioxidant capacity, biocompatibility and cell migration promoting ability, and can accelerate the healing of chronic infectious wounds.

[0007] In a first aspect, the present invention provides a soluble bilayer microneedle patch loaded with LL37@ZIF-8 and Prussian blue nanozyme drug composition, comprising a needle body layer and a backing layer;

[0008] The needle body layer comprises pullulan polysaccharide matrix, LL37@ZIF-8 and Prussian blue nanozyme; the backing layer comprises pullulan polysaccharide matrix.

[0009] Preferably, the height of the needle body layer of the soluble bilayer microneedle patch is 850-900 μm, the bottom diameter of the needle body of the needle body layer is 250-350 μm, and the distance between adjacent two needle bodies is 800-850 μm.

[0010] Preferably, the particle size of LL37@ZIF-8 is 600-900 nm; the particle size of Prussian blue nanozyme is 60-90 nm.

[0011] In a second aspect, the present invention provides a preparation method of the above soluble bilayer microneedle patch, comprising the following steps:

[0012] Adding the needle body layer solution into a mold, centrifuging to remove bubbles, drying and then adding the backing layer matrix solution, centrifuging to remove bubbles, and drying to obtain the soluble bilayer microneedle patch;

[0013] Wherein, the needle body layer solution comprises pullulan polysaccharide matrix solution, LL37@ZIF-8 and Prussian blue nanozyme; the backing layer matrix solution is pullulan polysaccharide matrix solution.

[0014] Preferably, the material of the mold is polydimethylsiloxane; the rotation speed of the centrifugation is 3000-5000 rpm, and the centrifugation time is 40-80 min.

[0015] Preferably, the pullulan polysaccharide matrix solution is a 25-35 wt% aqueous solution of pullulan polysaccharide.

[0016] Preferably, in the needle body layer solution, the concentration of LL37@ZIF-8 in the pullulan polysaccharide matrix solution is 120-280 μg / mL.

[0017] Preferably, in the needle body layer solution, the concentration of Prussian blue nanozyme in the pullulan polysaccharide matrix solution is 80-120 μg / mL.

[0018] Preferably, the preparation method of the LL37@ZIF-8 is as follows: dissolve the antimicrobial peptide LL37 in a dimethylimidazole solution, then add a zinc salt, and purify after the reaction to obtain the product.

[0019] Furthermore, the dosage ratio of the antimicrobial peptide LL37, dimethylimidazole, and zinc salt is 2 mg:(0.7 - 0.9) mmol:(0.15 - 0.25) mmol; the temperature of the reaction is 10 - 40 °C, and the time of the reaction is 1.5 - 5 h.

[0020] Preferably, in the step of adding the backing layer matrix solution after drying, the drying temperature is 15 - 30 °C, and the drying time is 8 - 15 h.

[0021] Preferably, in the step of obtaining the soluble double-layer microneedle patch after drying, the drying temperature is 15 - 30 °C, and the drying time is 20 - 30 h.

[0022] In a third aspect, the present invention provides the use of the above soluble double-layer microneedle patch or the soluble double-layer microneedle patch prepared by the above preparation method in the preparation of a drug for treating infectious wound healing.

[0023] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0024] (1) The soluble double-layer microneedle patch provided by the present invention encapsulates the antimicrobial peptide LL37 in situ into the metal-organic framework ZIF-8. On the one hand, it can effectively solve the problems of the short half-life of the antimicrobial peptide LL37, instability in biological media, susceptibility to protein hydrolysis, and potential toxicity. On the other hand, it greatly improves the antibacterial ability; moreover, it can utilize the pH-responsive property of ZIF-8 (degradation triggered by the slightly acidic environment of the wound) to achieve targeted release at the infection site; at the same time, it can physically isolate LL37 from the Prussian blue (PB) nanozyme to avoid mutual interference of antioxidant-antibacterial activities. In addition, the presence of the PB nanozyme can also reduce the ROS induced by ZIF-8 and improve the biocompatibility of the soluble double-layer microneedle patch.

[0025] (2) The soluble double-layer microneedle patch provided by the present invention has a double-layer structure. The needle body layer contains active ingredients, while the backing layer does not contain active ingredients. This design can prevent drug waste to a certain extent and avoid side effects caused by excessive addition of active ingredients.

[0026] (3) The soluble bilayer microneedle patch provided by the present invention has excellent antibacterial and antioxidant properties, enabling it to effectively kill bacteria in wounds, prevent further aggravation of infections, regulate the wound microenvironment, and promote wound healing. By combining pullulan, LL37@ZIF-8, and Prussian blue nanozyme (PB), the present invention can obtain a soluble bilayer microneedle patch that combines high-efficiency antibacterial, continuous antioxidant, wound-healing promotion, and biocompatibility, with good application performance.

[0027] (4) The preparation method of the soluble bilayer microneedle patch provided by the present invention has the advantages of mild reaction conditions, simple preparation, low raw material prices, easy mass production, etc., and is easy to scale up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings forming a part of this specification are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. Obviously, for those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0029] Figure 1 It is the SEM image of the PB nanozyme prepared in Example 2 of the present invention;

[0030] Figure 2 It is the schematic preparation process diagram of the soluble bilayer microneedle patch in Example 3 of the present invention;

[0031] Figure 3 It is the morphology diagram of the soluble bilayer microneedle patch in Example 3 of the present invention. Among them, Figures a and b are the overall and partial views of the soluble bilayer microneedle patch taken by a digital camera; Figures c, d, and e are SEM images;

[0032] Figure 4 It is the SEM-EDS test diagram of the soluble bilayer microneedle patch in Example 3 of the present invention;

[0033] Figure 5 It is the HE staining image of the soluble bilayer microneedle patch inserted into the skin of SD rats in Example 3 of the present invention;

[0034] Figure 6 It is the SEM image and TEM image of ZIF-8 prepared in Comparative Example 1 of the present invention and LL37@ZIF-8 prepared in Example 1;

[0035] Figure 7 It is the MBC test result diagram of LL37 of the present invention and LZ in Example 1 against Staphylococcus aureus and Escherichia coli;

[0036] Figure 8Inhibition effect diagrams of Example 3 (PB+LZ@MN), Comparative Example 2 (MN), Comparative Example 3 (PB@MN), and Comparative Example 4 (LZ@MN) of the present invention against Staphylococcus aureus and Escherichia coli; a is the image of the inhibition zone; b is the test image of the colony plate counting method; c and d are the colony plate counting statistical diagrams of Staphylococcus aureus and Escherichia coli.

[0037] Figure 9 Quantitative analysis test results of the ability of PB nanozyme with different concentrations of the present invention to scavenge ROS; a and b are the test images and statistical diagrams of the ability of different concentrations of PB to scavenge ROS by confocal laser scanning microscopy (CLSM); c and d are the test images and statistical diagrams of the ability of different concentrations of PB to scavenge ROS by flow cytometry;

[0038] Figure 10 Biocompatibility test results of the soluble bilayer microneedle patch of Example 3 of the present invention; a is the statistical diagram of the MTT experiment test; b is the statistical diagram of the hemolysis ability test;

[0039] Figure 11 Test and statistical diagram of the ability of the soluble bilayer microneedle patch of Example 3 of the present invention to promote cell migration;

[0040] Figure 12 Effect diagrams of the soluble bilayer microneedle patches of Example 3 (PB+LZ@MN), Comparative Example 2 (MN), Comparative Example 3 (PB@MN), and Comparative Example 4 (LZ@MN) of the present invention in the wound healing of a full-thickness skin defect infection model caused by Staphylococcus aureus infection in SD rats. Among them, a is the wound healing effect diagram after using the soluble bilayer microneedle patch in the full-thickness skin defect infection model of SD rats; b is the wound contraction simulation diagram processed by Image J software; c is the statistical diagram of the wound healing rate on the 3rd, 7th, 10th, and 14th days after using the soluble bilayer microneedle patch in the full-thickness skin defect infection model of SD rats. Detailed implementation manners

[0041] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0042] The present invention provides a soluble bilayer microneedle patch loaded with a drug composition of LL37@ZIF-8 and Prussian blue nanozyme, comprising a needle body layer and a backing layer;

[0043] The needle body layer comprises pullulan polysaccharide matrix, LL37@ZIF-8, and Prussian blue nanozyme; the backing layer comprises pullulan polysaccharide matrix.

[0044] In the soluble bilayer microneedle patch of the present invention, the pullulan polysaccharide matrix of the needle body layer is a natural polysaccharide material with high biocompatibility and rapid solubility, and the mechanical strength after film formation is sufficient to support the needle body to penetrate the skin; moreover, pullulan polysaccharide is beneficial to maintaining the homeostasis of skin cells, accelerating the wound healing process, and helping to lock in an appropriate amount of moisture.

[0045] Antimicrobial peptide LL37 has a variety of physiological functions, including antimicrobial activity, immunomodulation, promoting tissue repair, and antitumor effects. It can effectively combat a variety of bacteria, fungi, and viruses, and is not easily induced to develop drug resistance. However, the short half-life, instability in biological media, susceptibility to proteolytic hydrolysis, and potential toxicity of LL37 limit its therapeutic potential. In the present invention, LL37@ZIF-8 is used, and antimicrobial peptide LL37 is in-situ encapsulated by the ZIF-8 metal-organic framework. The pH-responsive property of ZIF-8 (disintegrating in the acidic environment of the wound) is utilized to achieve targeted release, while protecting LL37 from oxidative degradation. The two can also play a synergistic role in enhancing the antibacterial ability. At the same time, the framework structure of ZIF-8 physically isolates LL37 and Prussian blue nanozyme, avoiding interference between antioxidant and antibacterial activities. Prussian blue nanozyme (PB) has peroxidase-like activity and can scavenge excessive ROS. In addition, it is found in the present invention that when LL37@ZIF-8 is used alone as the active ingredient, the biocompatibility is poor, while when PB + LL37@ZIF-8 is used as the active ingredient, the biocompatibility can be further enhanced.

[0046] The backing layer of the present invention is a pure pullulan polysaccharide matrix, which only serves as a mechanical support layer, avoiding waste of drugs when not inserted into the skin, and at the same time avoiding side effects caused by excessive addition of active ingredients. By combining pullulan polysaccharide, LL37@ZIF-8, and Prussian blue nanozyme (PB), the present invention can obtain a soluble bilayer microneedle patch with high-efficiency antibacterial, continuous antioxidant, wound-healing promotion, and biocompatibility integrated, having good application performance.

[0047] In the present invention, the height of the needle body layer of the soluble bilayer microneedle patch is 850 - 900 μm, the bottom diameter of the needle body of the needle body layer is 250 - 350 μm, and the distance between adjacent two needle bodies is 800 - 850 μm. The above height design of the present invention ensures that the needle body penetrates the stratum corneum (50 - 100 μm) and the bacterial biofilm (200 - 500 μm) and reaches the dermis layer (>500 μm); the ratio of the bottom diameter to the height of the conical needle body (about 1:3) optimizes the mechanical distribution and prevents breakage during puncture; at the same time, the design of the needle body layer also ensures that there is enough drug loading capacity to meet the treatment requirements.

[0048] In the present invention, the particle size of LL37@ZIF-8 is 600 - 900 nm; the particle size of the Prussian blue nanozyme is 60 - 90 nm.

[0049] The present invention also provides a method for preparing the above-mentioned soluble bilayer microneedle patch, which comprises the following steps:

[0050] Add the solution of the needle body layer into a mold, perform centrifugation to remove bubbles, and after drying, add the matrix solution of the backing layer, perform centrifugation to remove bubbles, and the soluble bilayer microneedle patch is obtained after drying;

[0051] Among them, the solution of the needle body layer includes a pullulan polysaccharide matrix solution, LL37@ZIF-8, and Prussian blue nanozyme; the matrix solution of the backing layer is a pullulan polysaccharide matrix solution.

[0052] In the present invention, the material of the mold is polydimethylsiloxane; the rotation speed of the centrifugation is 3000-5000 rpm, and the centrifugation time is 40-80 min to ensure complete removal of bubbles.

[0053] In the present invention, the pullulan polysaccharide matrix solution is an aqueous solution of pullulan polysaccharide with a concentration of 25-35 wt%, which avoids the difficulty in forming caused by too dilute concentration or insufficient centrifugal filling or bubble removal caused by too high concentration.

[0054] In the solution of the needle body layer of the present invention, the concentration of LL37@ZIF-8 in the pullulan polysaccharide matrix solution is 120-280 μg / mL. When the concentration is too low, the antibacterial effect is insufficient.

[0055] In the present invention, in the solution of the needle body layer, the concentration of Prussian blue nanozyme in the pullulan polysaccharide matrix solution is 80-120 μg / mL. When the concentration is too low, the ROS scavenging efficiency is insufficient, and when the concentration is too high, it may cause self-aggregation of the nanozyme and reduce the catalytic activity.

[0056] In the present invention, the preparation method of the LL37@ZIF-8 is as follows: dissolve the antibacterial peptide LL37 in a dimethylimidazole solution, then add a zinc salt, and after reaction, purify to obtain it. Further, the dosage ratio of the antibacterial peptide LL37, dimethylimidazole, and zinc salt is 2 mg: (0.7-0.9) mmol: (0.15-0.25) mmol; the temperature of the reaction is 10-40 °C, and the reaction time is 1.5-5 h. The present invention does not impose special restrictions on the solvent of the dimethylimidazole solution, and common solvents in the art can be used; the present invention also does not impose special restrictions on the type of zinc salt, and common zinc salt types in the art can be used.

[0057] In the present invention, in the step of adding the matrix solution of the backing layer after drying, the drying temperature is 15-30 °C, and the drying time is 8-15 h.

[0058] In the present invention, in the step of obtaining the soluble double-layer microneedle patch after drying, the drying temperature is 15-30°C and the drying time is 20-30 h.

[0059] The present invention also provides the use of the above-mentioned soluble double-layer microneedle patch or the soluble double-layer microneedle patch prepared by the above-mentioned preparation method in the preparation of a drug for treating infectious wound healing. The soluble double-layer microneedles provided by the present invention have excellent antibacterial and antioxidant properties, enabling them to effectively kill bacteria in the wound, prevent further infection exacerbation, regulate the wound microenvironment, and promote wound healing.

[0060] The technical solution of the present invention will be further described below in conjunction with specific embodiments. The present invention places no special restrictions on the sources of the reagents used in the following examples, and commercially available products well-known to those skilled in the art can be used.

[0061] In the following examples, the PDMS microneedle mold has a size of 1.5×1.5 cm; the number of needle bodies is 15×15; the needle bodies are conical with a height of 880 microns and a bottom diameter of 300 microns, and the distance between adjacent two needle bodies is 830 microns.

[0062] Example 1

[0063] This example provides a preparation method of LL37@ZIF-8 (hereinafter abbreviated as LZ).

[0064] First, 2 mg of LL37 was dissolved in 5 mL of 160 mM dimethylimidazole aqueous solution. Immediately afterwards, 5 mL of 40 mM zinc acetate solution was prepared. Subsequently, these two solutions were fully mixed and allowed to stand at room temperature (25±3°C) for 2 hours. After standing, the precipitate was separated by centrifugation, and the precipitate was washed three times with deionized water, and the precipitate was freeze-dried to obtain LZ nanoparticles. At the same time, the supernatant was retained for determining the content of unencapsulated LL37, and then the encapsulation efficiency (EE%) and drug loading (LC%) were calculated using a BCA kit. The calculation of the encapsulation efficiency (EE%) and drug loading efficiency (LC%) of LL37 was based on the following formulas (1) and (2):

[0065] EE% = (amount of initial LL37 - amount of free LL37) / amount of initial LL37 × 100%;

[0066] LC% = (amount of initial LL37 - amount of free LL37) / total weight of the composite material × 100%.

[0067] Calculated by the BCA method, the encapsulation efficiency of ZIF-8 for LL37 was 89.1%, and its drug loading efficiency was 6.3%.

[0068] Example 2

[0069] This example provides a method for preparing Prussian blue (PB) nanozyme.

[0070] First, 1 mmol of ferric chloride (FeCl3) and 20 mmol of polyvinylpyrrolidone (PVP) were dissolved in 20 mL of deionized water to form solution A. Subsequently, 1 mmol of potassium ferrocyanide (K4[Fe(CN)6]) was dissolved in 20 mL of deionized water to prepare solution B. Then, solution B was mixed with solution A and stirred at 60 °C for 30 minutes. After it was cooled to room temperature (25 ± 3 °C), the mixture was centrifuged at a speed of 9500 revolutions per minute for 20 minutes. After that, the precipitate was collected and washed three times with acetone. Finally, the precipitate was treated by freeze-drying to obtain PB nanozyme.

[0071] Figure 1 This is the SEM image of the PB nanozyme prepared in this example. It can be seen that the PB nanozyme presents a relatively uniform square structure.

[0072] Example 3

[0073] This example provides a method for preparing a dissolvable bilayer microneedle patch (PB+LZ@MN) loaded with LZ and PB.

[0074] (1) Preparation of the needle matrix solution: Prepare a 30% w / w pullulan polysaccharide solution using ultrapure water, and add LZ and PB to it so that the concentration of LZ in the matrix solution reaches 256 μg / mL and the concentration of PB in the matrix solution reaches 100 μg / mL.

[0075] (2) Preparation of the backing layer matrix solution: Prepare a 30% w / w pullulan polysaccharide solution using ultrapure water.

[0076] (3) Preparation of the dissolvable bilayer microneedle patch loaded with LZ and PB: Add the needle matrix solution to the PDMS microneedle mold. Centrifuge at 4200 rpm for 60 min. After centrifugation, observe whether the bubbles are completely removed. If not, centrifuge again until the bubbles are completely removed. Use a spatula to scrape off the excess matrix solution. After drying the needle at room temperature (25 ± 3 °C) for 10 hours, drop the backing layer matrix solution again to prepare the backing layer, and repeat the above centrifugation operation until the bubbles are completely removed. Then, after drying at room temperature (25 ± 3 °C) for 24 hours, demold to obtain the dissolvable bilayer microneedle patch loaded with LZ and PB.

[0077] Figure 2 This is the schematic diagram of the preparation process of the dissolvable bilayer microneedle patch loaded with LZ and PB in this example. Observed with a digital camera, Figure 3Figures a and b show that the microneedle patch presents a regularly arranged conical microneedle array. As observed by SEM, as shown in the Figure 3 images c, d, and e in

[0078] Figure 4 reveal the sharp conical tips of the microneedles, where the average spacing between the microneedle tips is 830 μm, the average height of the microneedles is 880 μm, and the average width of their bases is 300 μm.

[0079] Figure 5 The HE staining images of

[0080] confirm that the soluble bilayer microneedle patch of this example has sufficient mechanical strength to successfully pierce the skin of rats.

[0081] Compared with Example 1, the difference in this comparative example is that LL37 is not added.

[0082] The SEM images and TEM images of ZIF-8 prepared in this comparative example and LL37@ZIF-8 prepared in Example 1 are as shown in Figure 6 It can be seen that ZIF-8 presents a hexagonal dodecahedron morphology, and the TEM image reveals that the edges of ZIF-8 are smooth. After directly fixing LL37 to ZIF-8, the edges of LZ appear more irregular compared to ZIF-8.

[0083] Comparative Example 2

[0084] Compared with Example 3, the difference in this comparative example is that the needle matrix solution does not contain LZ and PB, and MN is prepared.

[0085] Comparative Example 3

[0086] Compared with Example 3, the difference in this comparative example is that the needle matrix solution does not contain LZ, and PB@MN is prepared.

[0087] Comparative Example 4

[0088] Compared with Example 3, the difference in this comparative example is that the needle matrix solution does not contain PB, and LZ@MN is prepared.

[0089] Test Example

[0090] 1. Comparison of antibacterial tests between LL37 and LZ

[0091] Figure 7The MBC (minimum bactericidal concentration) test results of LL37 and LZ of Example 1 against Staphylococcus aureus and Escherichia coli are as follows. The MBC test result of LL37 against Staphylococcus aureus is 16 μg / mL; the MBC test result of LL37 against Escherichia coli is 256 μg / mL; the MBC test result of LZ against Staphylococcus aureus is 128 μg / mL; the MBC test result of LZ against Escherichia coli is 256 μg / mL. Both 16 μg / mL of LL37 and 128 μg / mL of LZ can kill 100% of Staphylococcus aureus. It is worth noting that according to the LC% (6.3%) of LL37 in LZ, the effective concentration of LL37 in 128 μg / mL of LZ is approximately 8.06 μg / mL, which is about half of its dosage when used alone. The MBC test results of Escherichia coli showed a similar trend. Both 128 μg / mL of LL37 and 256 μg / mL of LZ can kill 100% of Escherichia coli. According to the LC% (6.3%) of LL37 in LZ, the effective concentration of LL37 in 256 μg / mL of LZ is approximately 16.13 μg / mL. Compared with using LL37 alone, its dosage is greatly reduced. These results indicate that the prepared LZ not only has strong antibacterial activity against both Staphylococcus aureus and Escherichia coli, but also reduces the use of the antibacterial peptide LL37, reducing costs while avoiding the cytotoxicity problem caused by the large amount of LL37 used.

[0092] 2. Antibacterial ability test comparison of soluble bilayer microneedle patches

[0093] The inhibitory effects of Example 3 (PB + LZ@MN), Comparative Example 2 (MN), Comparative Example 3 (PB@MN), and Comparative Example 4 (LZ@MN) against Staphylococcus aureus and Escherichia coli were compared, as Figure 8 shown. Figure 8 Figure a shows the photos of the inhibition zones after co - culturing Staphylococcus aureus and Escherichia coli with various microneedles for 18 hours, with the group without microneedles as the control group (Control). PB + LZ@MN and LZ@MN showed significant inhibitory effects on both bacteria, and no colony growth was observed at the bottom and around the microneedle samples. In contrast, the antibacterial effect of PB@MN was slightly inferior. Further, the antibacterial effect of the microneedles was evaluated by the agar plate colony counting method (Figure b). The quantitative analysis results showed that the antibacterial rates of PB + LZ@MN against Staphylococcus aureus and Escherichia coli both reached 100%, while the antibacterial rates of LZ@MN against these two bacteria were 100% and 99.9 ± 0.1% respectively (Figures c and d). These results may benefit from the excellent antibacterial properties of LZ itself, and the addition of PB further enhanced the antibacterial efficacy of PB + LZ@MN.

[0094] 3. Antioxidant Capacity Test of PB Nanozyme

[0095] Figure 9 The quantitative analysis test results showing the ability of PB at different concentrations to scavenge ROS are presented. A DCFH-DA (2′,7′-dichlorodihydrofluorescein diacetate) kit was used to evaluate the antioxidant capacity of PB. DCFH-DA can penetrate cell membranes and is decomposed by esterase into DCFH after entering the cells. The latter cannot penetrate cell membranes and thus accumulates inside the cells. In the presence of ROS inside the cells, DCFH is oxidized to DCF (2′,7′-dichlorofluorescein), and DCF has strong fluorescence characteristics. By analyzing Figure 9 the fluorescence staining images in a, we found that when L929 cells were completely exposed to H2O2 without the protection of PB, their relative fluorescence intensity was approximately 48.0 ± 1.9%. However, with the increase in PB concentration, the fluorescence intensity decreased significantly. When the PB concentration reached 100 μg / mL, the fluorescence intensity decreased to 2.8 ± 0.9% (Figure b). Flow cytometry (FACS) tests further verified this result. The control group cells showed the highest average fluorescence intensity on the far right of the fluorescence intensity distribution graph. With the increase in PB concentration, the number of cells containing ROS decreased, the average fluorescence intensity of the cells decreased, and the cell peak shifted to the left (Figure c). The results indicate that the ROS scavenging ability of PB shows a concentration-dependent relationship within its measurement range, that is, the higher the concentration, the stronger the ability to scavenge ROS (Figure d).

[0096] 4. Biocompatibility Test:

[0097] Figure 10 The biocompatibility test results of the soluble bilayer microneedle patch in Example 3 are shown. Biocompatibility is crucial for ensuring the safety and effectiveness of biomaterials and medical devices in clinical use. L929 mouse fibroblasts were used for the test. The MTT experiment results shown in Figure a indicate that all microneedle patches exhibited good cell compatibility. It is worth noting that compared with other groups, the cell viability of the LZ@MN group and the PB+LZ@MN group decreased, especially the cell viability of the LZ@MN group was lower than that of the PB+LZ@MN group. This phenomenon may be related to the effect of ZIF-8 in LZ on cell function, especially related to the accumulation of intracellular zinc ion content. The increase in zinc ion content may lead to an increase in the intracellular ROS level, thereby causing cell necrosis to a certain extent. However, the addition of PB in PB+LZ@MN can reduce the ROS induced by ZIF-8, showing the positive role of PB in regulating cell responses.

[0098] Blood compatibility is a key factor in evaluating the safety and effectiveness of biomaterials in clinical applications, especially for medical devices and implants that need to come into direct or indirect contact with blood. Therefore, we used fresh rabbit blood to evaluate the blood compatibility of the soluble bilayer microneedle patch of Example 3 through an in vitro hemolysis experiment. The quantitative research results shown in Figure b indicate that the hemolysis rates of all microneedle patch groups are lower than 5%, meeting the safety usage standards for medical biomaterials.

[0099] 5. Cell Migration Experiment

[0100] Using L929 cells, we evaluated the promoting effect of various microneedle patch extracts on cell migration through a cell scratch assay, which mimics the mechanism of wound closure. After incubating L929 cells with different microneedle patch extracts for 24 hours, a cell scratch was made using a pipette. The results of the scratch assay are shown in Figure 11 Figure a. Specifically, the cell migration rates of the control group and the MN group were 15.5 ± 5.5% and 15.3 ± 2.7% respectively, the migration rate of the PB@MN group reached 27.6 ± 4.9%, while that of the LZ@MN group was 13.0 ± 1.4%. Notably, the cell migration rate of the PB+LZ group was significantly increased, reaching 40.9 ± 1.7% ( Figure 11 Figure b). These findings reveal the effectiveness of PB+LZ@MN in promoting cell migration and indicate its application prospects in accelerating the wound healing process.

[0101] 6. Wound Healing Promotion Experiment

[0102] A full-thickness skin defect infection model of male SD rats (180 - 200 g, 6 - 8 weeks old) was used to evaluate wound healing. Thirty SD rats were randomly and evenly divided into 5 groups: blank control group, MN group, PB@MN group, LZ@MN group, and PB+LZ@MN group. After anesthesia with isoflurane, the back was depilated and disinfected, and a circular wound with a diameter of 8 mm was made. Immediately, 50 μL of a Staphylococcus aureus solution with a concentration of 1×10 6 CFU / mL was dropped onto the wound to induce infection. After 30 minutes, according to the grouping, the corresponding microneedle dressings were applied to the rat wounds, and the animals were given routine care. Wound images were taken regularly. Figure 12 Shows the effects of the soluble bilayer microneedle patches of Example 3 (PB+LZ@MN), Comparative Example 2 (MN), Comparative Example 3 (PB@MN), and Comparative Example 4 (LZ@MN) in the wound healing of a full-thickness skin defect infection model with Staphylococcus aureus infection in SD rats. Figure 12Both a and b in it show the change of wound area over time. After 3 days of treatment, except for the PB+LZ@MN group, the wounds in other groups shrank and were accompanied by pus secretion. It is worth noting that the LZ@MN group (59.7±2.6%) and the PB+LZ@MN group (64.4±2.6%) showed higher wound healing rates, which was probably due to the antibacterial effect of LZ on Staphylococcus aureus in the early stage of infection. By the 7th day, the inflamed wounds in each group had recovered to a certain extent. Compared with the control group, all the groups treated with microneedles showed more obvious wound closure. In particular, the PB+LZ@MN group had the highest healing rate (94.3±0.5%), followed by the PB@MN group (87.7±1.5%). The difference in this effect may be attributed to the positive effect of PB in promoting tissue growth and remodeling, as well as the antibacterial effect of LZ in the early infection stage. By the 10th day, the wound healing rate of the PB+LZ@MN group reached 98.8±0.5%, significantly higher than that of the control group (83.9±1.0%) (Figure c). By the 14th day after treatment, except for the control group, all the microneedle patch treatment groups showed a certain degree of healing.

[0103] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A soluble bilayer microneedle patch loaded with LL37@ZIF-8 and Prussian blue nanozyme pharmaceutical composition, characterized in that, It includes a needle body layer and a backing layer; The needle body layer includes pullulan polysaccharide matrix, LL37@ZIF-8 and Prussian blue nanozyme; the backing layer includes pullulan polysaccharide matrix.

2. The soluble bilayer microneedle patch according to claim 1, wherein, The height of the needle body layer of the soluble double-layer microneedle patch is 850 - 900 μm, the bottom diameter of the needle body of the needle body layer is 250 - 350 μm, and the distance between adjacent two needle bodies is 800 - 850 μm.

3. The soluble bilayer microneedle patch according to claim 1, wherein The particle size of the LL37@ZIF-8 is 600 - 900 nm; the particle size of the Prussian blue nanozyme is 60 - 90 nm.

4. The preparation method of the soluble bilayer microneedle patch according to any one of claims 1 to 3, characterized in that, It includes the following steps: Add the needle body layer solution into a mold, centrifuge to remove bubbles, and after drying, add the backing layer matrix solution, centrifuge to remove bubbles, and after drying, the soluble double-layer microneedle patch is obtained; Among them, the needle body layer solution includes pullulan polysaccharide matrix solution, LL37@ZIF-8 and Prussian blue nanozyme; the backing layer matrix solution is pullulan polysaccharide matrix solution.

5. The preparation method according to claim 4, characterized in that, The material of the mold is polydimethylsiloxane; the rotation speed of the centrifugation is 3000 - 5000 rpm, and the centrifugation time is 40 - 80 min.

6. The preparation method according to claim 4, characterized in that, The pullulan polysaccharide matrix solution is a 25 - 35 wt% aqueous solution of pullulan polysaccharide; in the needle body layer solution, the concentration of LL37@ZIF-8 in the pullulan polysaccharide matrix solution is 120 - 280 μg / mL; the concentration of Prussian blue nanozyme in the pullulan polysaccharide matrix solution is 80 - 120 μg / mL.

7. The preparation method according to claim 4, characterized in that, The preparation method of the LL37@ZIF-8 is as follows: dissolve the antimicrobial peptide LL37 in a dimethylimidazole solution, then add a zinc salt, and after reaction, purify to obtain it.

8. The preparation method according to claim 7, characterized in that, The dosage ratio of the antimicrobial peptide LL37, dimethylimidazole, and zinc salt is 2 mg:(0.7 - 0.9) mmol:(0.15 - 0.25) mmol; the temperature of the reaction is 10 - 40 °C, and the reaction time is 1.5 - 5 h.

9. The preparation method according to claim 4, characterized in that, In the step of adding the backing layer matrix solution after drying, the drying temperature is 15 - 30 °C, and the drying time is 8 - 15 h; in the step of adding the backing layer matrix solution after drying, the drying temperature is 15 - 30 °C, and the drying time is 8 - 15 h.

10. Use of the soluble double-layer microneedle patch according to any one of claims 1 - 3 or the soluble double-layer microneedle patch prepared by the preparation method according to any one of claims 4 - 6 in the preparation of a drug for treating infectious wound healing.

Images