Antibacterial nanoparticle-loaded pH-responsive collagen microneedle patch as well as preparation method and application of antibacterial nanoparticle-loaded pH-responsive collagen microneedle patch

Abstract

The invention relates to the technical field of biological materials, in particular to a pH response collagen microneedle patch loaded with antibacterial nanoparticles as well as a preparation method and application of the pH response collagen microneedle patch loaded with the antibacterial nanoparticles, and the microneedle patch is prepared by loading the antibacterial nanoparticles, aldehyde modified F127 and recombinant three-type collagen COL3 on a polymer matrix. When the microneedle patch is applied to a chronic wound of a diabetic patient, the microneedle penetrates through the skin in a minimally invasive mode and directly reaches a wound focus area. Under the irradiation of 808nm near-infrared light, the mesoporous polydopamine absorbs light energy, so that the loaded lyase LysAB2 is quickly released, bacterial cell walls are specifically recognized and cracked, and bacteria are efficiently killed; meanwhile, the collagen hydrogel constructs an appropriate microenvironment at the wound, cell adhesion, proliferation and migration are promoted, and the wound healing process is accelerated. Compared with a traditional diabetes mellitus chronic bacterial infection wound treatment means, the microneedle patch provided by the invention provides an efficient and safe new strategy for treating the chronic bacterial infection wound of a diabetes mellitus patient.

Description

Technical Field

[0001] This application relates to the field of biomaterials technology, and in particular to a pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles, its preparation method, and its application. Background Technology

[0002] Chronic bacterial wound infections in diabetic patients, especially those caused by Pseudomonas aeruginosa, have become a serious challenge in global public health. Diabetes leads to metabolic disorders and impaired immune function, significantly reducing wound healing ability and greatly increasing the risk of bacterial infection. These chronically infected wounds not only severely impact patients' quality of life and prolong hospital stays, but can also lead to serious complications such as sepsis, and even endanger life. Currently, routine treatments include local debridement, antibiotic therapy, and dressing coverage. However, long-term use of antibiotics easily leads to bacterial resistance, significantly reducing treatment effectiveness; local debridement and dressing changes are painful and frequent, resulting in poor patient compliance; furthermore, traditional treatment methods are difficult to effectively promote wound tissue regeneration, failing to meet the urgent clinical need for efficient and safe treatment methods.

[0003] With the development of biomedical technology, microneedling technology has shown great potential in wound treatment due to its ability to achieve efficient drug delivery and localized therapy. Microneedles penetrate the stratum corneum of the skin through tiny needle tips, delivering drugs directly to the lesion site, avoiding drug loss during systemic circulation and improving drug bioavailability. However, conventional microneedling techniques have limitations in treating chronic diabetic wound infections, including uncontrollable drug release and insufficient specific bacterial killing ability.

[0004] In recent years, photoresponsive and bioactive substance-loaded microneedle technology has gradually become a research hotspot. 808nm near-infrared light has excellent tissue penetration, capable of activating specific materials to produce photothermal effects or promoting drug release; while loading bioactive substances such as antibacterial lysins can achieve precise bacterial killing. However, how to effectively combine multiple functional materials to construct a microneedle patch that can respond to near-infrared light for controlled drug release, specifically and efficiently kill Pseudomonas aeruginosa, and simultaneously promote diabetic wound healing, while ensuring the stability and synergistic effect of each component in the microneedle system, remains a key technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] This application provides a pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles, its preparation method, and its application. A biodegradable microneedle patch made of physically inert polymer PVA is developed by combining mesoporous polydopamine loaded with antibacterial lysin LysAB2 with collagen hydrogel. Combined with the 808nm near-infrared light response characteristics, a microneedle patch that can efficiently kill bacteria and promote wound healing is obtained.

[0006] To address the aforementioned technical problems, in a first aspect, embodiments of this application provide a pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles. The microneedle patch is prepared by loading antibacterial nanoparticles, aldehyde-modified F127, and recombinant type III collagen COL3 onto a polymer matrix.

[0007] In some exemplary embodiments, the antibacterial nanoparticles are composite nanoparticles of mesoporous polydopamine loaded with the lysin LysAB2, namely LysAB2@MPDA.

[0008] In some exemplary embodiments, the aldehyde-modified F127 is prepared by Dess-Martin high-iodine reagent oxidation method, which oxidizes the primary alcohol at the end of Pluronic F127 to an aldehyde group.

[0009] In some exemplary embodiments, the polymer matrix is ​​an aqueous solution of polyvinyl alcohol; the polymer matrix is ​​prepared by dissolving polyvinyl alcohol in deionized water to prepare a 20% PVA solution, which is then stored at room temperature.

[0010] Secondly, embodiments of this application also provide a method for preparing a pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles, comprising the following steps: Step 1, preparing a hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3; Step 2, preparing antibacterial nanoparticles and mixing them with the hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3 to obtain a drug-loaded hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3; Step 3, placing the drug-loaded hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3 on a microneedle PDMS mold, pressing it into the needle holes of the mold as microneedle tips, centrifuging and vacuum drying the mold to obtain a mold loaded with microneedle patches; Step 4, pouring a polymer matrix onto the mold loaded with microneedle patches, drying it, and then peeling the microneedle patches off the mold to obtain the microneedle patches.

[0011] In some exemplary embodiments, in step one, preparing a hydrogel crosslinked with aldehyde-modified F127 and recombinant type III collagen COL3 includes:

[0012] Weigh out aldehyde-modified F127, add deionized water to dissolve it, and obtain aldehyde-modified F127 solution;

[0013] Weigh out recombinant type III collagen COL3, add deionized water to dissolve it, and obtain a recombinant type III collagen solution;

[0014] The aldehyde-modified F127 solution and the recombinant type III collagen solution were mixed and allowed to stand at 37°C for 24 hours to obtain a hydrogel crosslinked with aldehyde-modified F127 and recombinant type III collagen COL3.

[0015] In some exemplary embodiments, in step two, mesoporous polydopamine loaded with LysAB2 is dispersed in a solvent to form a uniform dispersion, and then added to a hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3, and stirred thoroughly to obtain a drug-loaded hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3.

[0016] In some exemplary embodiments, in step three, the drug-loaded aldehyde-modified F127 and recombinant type III collagen COL3 crosslinked hydrogel is pressed into the needle hole of the mold as a microneedle tip using a medicine spoon, and the mold is centrifuged at 2200 rpm for 15 minutes; then the pressed mold is placed in a vacuum drying oven and vacuumed three times to obtain a mold loaded with microneedle patches.

[0017] In some exemplary embodiments, in step four, polyvinyl alcohol is dissolved in deionized water to prepare a 20% PVA solution; then the 20% PVA solution is poured onto a mold carrying a microneedle patch, the mold is placed in a ventilated place to dry for one day, and the microneedle patch is peeled off from the mold to obtain the microneedle patch.

[0018] Thirdly, embodiments of this application also provide the application of the pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles described in the above embodiments in the preparation of patch formulations for bacterial infected wounds.

[0019] The technical solution provided in this application has at least the following advantages:

[0020] This application provides a pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles, its preparation method, and its application. The microneedle patch is prepared by loading antibacterial nanoparticles, aldehyde-modified F127, and recombinant type III collagen COL3 onto a polymer matrix. When applied to chronic wounds in diabetic patients, the microneedles penetrate the skin minimally invasively, reaching the wound lesion area. Under 808nm near-infrared light irradiation, mesoporous polydopamine absorbs light energy, prompting the rapid release of the loaded lysin LysAB2, which specifically recognizes and lyses bacterial cell walls, efficiently killing bacteria. Simultaneously, the collagen hydrogel constructs a suitable microenvironment at the wound site, promoting cell adhesion, proliferation, and migration, accelerating the wound healing process. Compared with traditional treatments for chronic bacterial infections in diabetic patients, the microneedle patch of this application provides a highly efficient and safe new strategy for treating chronic bacterial infections in diabetic patients. Attached Figure Description

[0021] One or more embodiments are illustrated by way of example with reference to the accompanying drawings. These illustrations do not constitute a limitation on the embodiments, and unless otherwise stated, the figures in the drawings are not to be limited by scale.

[0022] Figure 1 This diagram illustrates the preparation and use of pH-responsive collagen microneedle patches loaded with antibacterial nanoparticles, as provided in the embodiments of this application.

[0023] Figure 2 Bright-field images of aldehyde-modified F127 and recombinant type III collagen crosslinked hydrogels before and after gelation, provided in the embodiments of this application.

[0024] Figure 3 The injectability of the hydrogel provided in the embodiments of this application was verified using a syringe.

[0025] Figure 4 The NMR spectra of F127 and aldehyde-modified F127 provided in the embodiments of this application.

[0026] Figure 5 FTIR spectra of F127 and aldehyde-modified F127 provided in the embodiments of this application.

[0027] Figure 6 SEM image of the collagen hydrogel provided in the embodiments of this application.

[0028] Figure 7 SEM image of mesoporous polydopamine provided in the embodiments of this application.

[0029] Figure 8 Bright field images and SEM images of collagen microneedles provided in the embodiments of this application.

[0030] Figure 9 The particle size and potential of MPDA, LysAB2, and LysAB2@MPDA provided in the embodiments of this application.

[0031] Figure 10 This is a schematic diagram of the photothermal effect of collagen microneedles provided in the embodiments of this application.

[0032] Figure 11 The in vitro release of the collagen hydrogel provided in the embodiments of this application under two different environments.

[0033] Figure 12 The in vivo release of collagen microneedles provided in this application embodiment after acting on the back of a mouse.

[0034] Figure 13 The diagram shows the results of a biocompatibility experiment provided in the embodiments of this application.

[0035] Figure 14 The image shows the results of a cell migration experiment provided in an embodiment of this application.

[0036] Figure 15 The image shows the results of animal experiments provided in the embodiments of this application.

[0037] Figure 16 The image shows the H&E and Masson staining results of mouse dorsal skin provided in the embodiments of this application.

[0038] Figure 17 An immunofluorescence image of mouse dorsal skin provided in an embodiment of this application. Detailed Implementation

[0039] As the background technology shows, how to effectively combine multiple functional materials to construct a microneedle patch that can respond to near-infrared light to achieve controlled drug release, specifically and efficiently kill Pseudomonas aeruginosa, and simultaneously promote the healing of diabetic wounds, while ensuring the stability and synergistic effect of each component in the microneedle system, remains a key technical problem that urgently needs to be solved in this field.

[0040] To address the aforementioned technical problems, this application provides a pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles, its preparation method, and its application. The microneedle patch is prepared by loading antibacterial nanoparticles, aldehyde-modified F127, and recombinant type III collagen COL3 onto a polymer matrix. This application also combines mesoporous polydopamine loaded with the antibacterial lysin LysAB2 with collagen hydrogel to develop a biodegradable microneedle patch made of physically inert polymer PVA. Combined with its 808nm near-infrared light response characteristics, this results in a microneedle patch capable of efficiently killing bacteria and promoting wound healing.

[0041] The embodiments of this application will now be described in detail with reference to the accompanying drawings. However, those skilled in the art will understand that many technical details have been provided in the embodiments of this application to facilitate a better understanding of the application. However, the technical solutions claimed in this application can be implemented even without these technical details and various variations and modifications based on the following embodiments.

[0042] This application provides a pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles. The microneedle patch is prepared by loading antibacterial nanoparticles, aldehyde-modified F127, and recombinant type III collagen COL3 onto a polymer matrix.

[0043] In some embodiments, the antibacterial nanoparticles are composite nanoparticles of mesoporous polydopamine loaded with the lysin LysAB2, namely LysAB2@MPDA.

[0044] In some embodiments, aldehyde-modified F127 is prepared by Dess-Martin high-iodine reagent oxidation method, which oxidizes the primary alcohol at the end of Pluronic F127 to an aldehyde group.

[0045] In some embodiments, the polymer matrix is ​​an aqueous solution of polyvinyl alcohol; the polymer matrix is ​​prepared by dissolving polyvinyl alcohol in deionized water to prepare a 20% PVA solution, which is then stored at room temperature.

[0046] Specifically, the polymer substrate is composed of a biocompatible, physically inert polymer, PVA. The preparation method is as follows: 4g of PVA is dissolved in 20mL of deionized water to prepare a 20% PVA solution, which is then stored at room temperature for later use.

[0047] This application also provides a method for preparing a pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles, comprising the following steps: Step 1, preparing a hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3; Step 2, preparing antibacterial nanoparticles and mixing them with the hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3 to obtain a drug-loaded hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3; Step 3, placing the drug-loaded hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3 on a microneedle PDMS mold, pressing it into the needle hole of the mold as a microneedle tip, centrifuging and vacuum drying the mold to obtain a mold loaded with microneedle patches; Step 4, pouring a polymer matrix onto the mold loaded with microneedle patches, drying it, and then peeling the microneedle patches off the mold to obtain the microneedle patches. Figure 1 The diagram illustrates the preparation and use process of the microneedle patch of this application.

[0048] Specifically, the aldehyde-modified F127 used in this application is prepared by chemical oxidation of the primary alcohol at the end of Pluronic F127 to an aldehyde group. The specific steps are as follows:

[0049] First, 8g of Pluronic F127 was added to 200mL of anhydrous dichloromethane and stirred until completely dissolved. Under ice-water bath conditions, 1.072g of Dess-Martin high-iodine reagent was added in small portions to the above solution and stirred for 30min. Then, the temperature was raised to 40℃ and the reaction was stirred at 600rpm for 24h. The insoluble reduction product was removed by vacuum filtration, and the filtrate was evaporated by rotary evaporation to obtain a viscous liquid. The viscous liquid was added dropwise to 100-150mL of ice-cold diethyl ether and stirred in an ice-water bath for 1h. The precipitate was collected by centrifugation at 9000rpm for 5min. The precipitate was dried under vacuum at 40℃ for 12h to obtain aldehyde-modified F127.

[0050] In some embodiments, in step one, preparing a hydrogel crosslinked with aldehyde-modified F127 and recombinant type III collagen COL3 includes:

[0051] Weigh out aldehyde-modified F127, add deionized water to dissolve it, and obtain aldehyde-modified F127 solution;

[0052] Weigh out recombinant type III collagen COL3, add deionized water to dissolve it, and obtain a recombinant type III collagen solution;

[0053] The aldehyde-modified F127 solution and the recombinant type III collagen solution were mixed and allowed to stand at 37°C for 24 hours to obtain a hydrogel crosslinked with aldehyde-modified F127 and recombinant type III collagen COL3.

[0054] Specifically, the collagen hydrogel is formed by cross-linking aldehyde-modified F127 with recombinant type III collagen through a Schiff base reaction. Taking a 25% collagen hydrogel as an example: 400 mg of aldehyde-modified F127 is weighed and dissolved in 4 mL of deionized water, and 200 mg of recombinant type III collagen is weighed and dissolved in 1 mL of deionized water. The two solutions are mixed evenly, and a Schiff base reaction is carried out under suitable temperature and pH conditions to obtain a hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen.

[0055] In some embodiments, in step two, mesoporous polydopamine loaded with LysAB2 is dispersed in a solvent to form a uniform dispersion, and then added to a hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3, and stirred thoroughly to obtain a drug-loaded hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3.

[0056] Specifically, in step two, the antibacterial nanoparticles are LysAB2@MPDA composite nanoparticles of mesoporous polydopamine loaded with the lysin LysAB2. To prepare the antibacterial nanoparticles, firstly, MPDA was synthesized: F127 (360 mg), tris(hydroxymethyl)aminomethane (90 mg), and 3,3',5,5'-tetramethylbenzidine (0.42 mL) were dissolved in 120 mL of deionized water / ethanol (1 / 1, v / v) and mixed at 37 °C for 30 min. Then, dopamine hydrochloride (60 mg) was dissolved in 10 mL of deionized water and added dropwise to the mixture. The mixture was stirred for 24 h, and the precipitate was collected by centrifugation. The precipitate was then washed with ethanol / acetone (2 / 1, v / v) using sonication for 30 min, and finally washed with ethanol to obtain MPDA. Then, LysAB2@MPDA was prepared. The specific steps are as follows: MPDA (1 mg) and LysAB2 (100 μL) are suspended in 1 mL of deionized water, and after 24 h, MPDA loaded with LysAB2 is collected.

[0057] In some embodiments, in step three, the drug-loaded aldehyde-modified F127 and recombinant type III collagen COL3 crosslinked hydrogel is pressed into the pinhole of the mold using a spatula to serve as the microneedle tip. The mold is then centrifuged at 2200 rpm for 15 minutes. The pressed mold is then placed in a vacuum drying oven and repeatedly vacuumed three times to obtain a mold containing the microneedle patch. It should be noted that in step three, 50 mg of drug-loaded collagen hydrogel is placed on the microneedle PDMS mold, and the drug-loaded collagen hydrogel is pressed into the pinhole of the mold using a small spatula.

[0058] In some embodiments, in step four, polyvinyl alcohol is dissolved in deionized water to prepare a 20% PVA solution; then the 20% PVA solution is poured onto a mold carrying a microneedle patch, the mold is placed in a ventilated place to dry for one day, the microneedle patch is carefully peeled off, and the resulting microneedle patch is stored in a sealed well plate for further study.

[0059] Furthermore, this application also provides an application of the pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles described in the above embodiments in the preparation of patch formulations for bacterial infected wounds. The pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles of this application consists of a hydrogel formed by cross-linking aldehyde-modified F127 with recombinant type III collagen, encapsulating drug-loaded nanoparticles LysAB2@MPDA and a polymer substrate. When applied to chronic wounds in diabetic patients, the microneedles penetrate the skin in a minimally invasive manner, directly reaching the wound lesion area. Under 808nm near-infrared light irradiation, the mesoporous polydopamine absorbs light energy, prompting the rapid release of the loaded lysin LysAB2, which specifically recognizes and lyses the bacterial cell wall, efficiently killing bacteria; simultaneously, the collagen hydrogel constructs a suitable microenvironment at the wound site, promoting cell adhesion, proliferation, and migration, accelerating the wound healing process. Compared with traditional treatments for chronic bacterial infections in diabetic patients, the microneedle patch of this application provides a highly efficient and safe new strategy for treating chronic bacterial infections in diabetic patients.

[0060] The following specific embodiments further explain the pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles and its preparation method provided in this application. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Unless otherwise specified, specific conditions in the embodiments were performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without specified manufacturers are all commercially available conventional products.

[0061] Example 1: Preparation of aldehyde-modified F127

[0062] First, add 8.0 g of Pluronic F127 to 200 mL of anhydrous dichloromethane and stir until completely dissolved. Under ice-water bath conditions, slowly add 1.072 g of Dess-Martin high-iodine reagent in small portions to the above solution and stir continuously for 30 min.

[0063] The solution was then heated to 40°C and stirred at 600 rpm for 24 h. After the reaction was complete, insoluble reduction products were removed by vacuum filtration, and the resulting filtrate was rotary evaporated to obtain a viscous liquid. This viscous liquid was added dropwise to 100-150 mL of ice-cold diethyl ether and stirred in an ice-water bath for 1 h to induce the precipitation of a large amount of precipitate. Finally, the precipitate was collected by centrifugation at 9000 rpm for 5 min and dried under vacuum at 40°C for 12 h to obtain aldehyde-modified F127 (F127-CHO), which was stored at -20°C for later use. Figure 4 , Figure 5 The NMR and FTIR spectra of F127 and aldehyde-modified F127 are shown respectively. In the NMR spectrum, the aldehyde-modified F127, compared to F127, exhibits a new characteristic peak related to the aldehyde group at a specific chemical shift, indicating successful aldehyde group introduction. The FTIR spectrum shows that the aldehyde-modified F127 has a distinct absorption peak at the characteristic absorption peak position of the aldehyde group, while F127 does not have this peak, confirming successful aldehyde group grafting and demonstrating the advantages of structural modification.

[0064] Example 2: Packetization of LysAB2@MPDA

[0065] Synthesis of MPDA: 360 mg Pluronic F127, 90 mg tris(hydroxymethyl)aminomethane, and 0.42 mL of 3,3',5,5'-tetramethylbenzidine were added to 120 mL of deionized water / ethanol mixture (volume ratio 1:1) and stirred at 37 °C for 30 min to obtain mixture A; 60 mL of dopamine hydrochloride was weighed and dissolved in 10 mL of deionized water to obtain solution B. Solution B was added dropwise to mixture A and the reaction was continued for 24 h; after the reaction was completed, the precipitate of the reaction product was collected by centrifugation. The precipitate was first ultrasonically washed with ethanol / acetone mixture (volume ratio 2:1) for 30 min, then washed with ethanol, and dried to obtain MPDA.

[0066] Preparation of LysAB2@MPDA: 1 mg MPDA and 100 μL LysAB2 were suspended in 1 mL of deionized water. After 24 h, the MPDA loaded with LysAB2 was collected. Figure 6 , Figure 7 SEM images of collagen hydrogel and mesoporous polydopamine are shown. The SEM image of collagen hydrogel shows a uniform and interconnected three-dimensional network structure, exhibiting good structural uniformity and stability.

[0067] Example 3: Preparation of cross-linked hydrogels of aldehyde-modified F127 and recombinant type III collagen

[0068] Weigh 400 mg of aldehyde-modified F127 and dissolve it in 4 ml of deionized water. Then weigh 200 mg of recombinant human type III collagen and dissolve it in 1 ml of deionized water. Mix the two solutions evenly and allow them to undergo a Schiff base reaction under suitable temperature and pH conditions to obtain a hydrogel crosslinked with aldehyde-modified F127 and recombinant human type III collagen. Figure 2 Bright-field images of aldehyde-modified F127 and recombinant type III collagen crosslinked hydrogels before and after gelation are shown. Figure 2 It can be seen that before gelation, the hydrogel is a uniform, flowing, light-colored, transparent liquid without particles or sediment, indicating that the two components can be evenly dispersed after mixing. After gelation, it transforms into a gel state, indicating that a stable three-dimensional network structure has been formed through a cross-linking reaction. The injectability of the hydrogel was verified using a syringe, such as... Figure 3 As shown.

[0069] Example 4: Preparation of pH-responsive collagen microneedle patches

[0070] 50 mg of drug-loaded collagen hydrogel was placed on a microneedle PDMS mold. The gel was pressed into the needle holes of the mold using a small medicine spoon to serve as the microneedle tip. The mold was centrifuged at 2200 rpm for 15 minutes. The mold was then placed in a vacuum drying oven and vacuumed three times. Next, 20% PVA solution was poured onto the PDMS mold containing the needle tip until it completely covered the needle tip and just filled the mold to a thickness of about 0.5 mL. Finally, the covered mold was placed in a ventilated place to dry for one day. The microneedle patch was carefully peeled off and the obtained microneedle patch was stored in a sealed well plate for further study. Figure 8 Bright field images and SEM images of collagen microneedles are shown, both of which comprehensively reflect the morphological characteristics of collagen microneedles. Figure 9 The particle size and potential of MPDA, LysAB2, and LysAB2@MPDA are shown. Comparing the data of the three, it can be seen that the particle size of LysAB2@MPDA is larger than that of MPDA and LysAB2 when they exist alone, and its potential value is between that of MPDA and LysAB2. This result indicates that MPDA and LysAB2 have successfully combined through electrostatic adsorption to form the LysAB2@MPDA composite system. Figure 10 The photothermal effect of collagen microneedles is shown. From Figure 10 The temperature changes of collagen microneedles under light conditions can be observed. After about 5 minutes of irradiation, the temperature can reach the temperature required for angiogenesis.

[0071] Example 5: In vitro release capability

[0072] Collagen hydrogels containing Rhodamine B-stained MPDANPs were placed in 15 mL centrifuge tubes to simulate nanoparticle release. The tubes were divided into two groups, with 10 mL of pH 7.4 PBS solution added to one group and 10 mL of pH 5 PBS solution added to the other. The tubes were placed in a shaker at 37°C and shaken. 1 mL of sample was taken periodically and the same 1 mL solution was added to each sample. After 24 hours, the sample was transferred to a 96-well plate for in vivo imaging. Figure 11 The in vitro release of collagen hydrogel in two different environments is illustrated. Figure 11 The differences in release rate, release amount, and release duration of collagen hydrogel under two different environments can be clearly compared. This difference fully demonstrates that the collagen hydrogel has good pH responsiveness, enabling it to adjust its release behavior according to changes in environmental pH.

[0073] Example 6: In vivo release capacity

[0074] After pressing Rhodamine B-stained collagen microneedles on the backs of mice for 20 minutes, the release was recorded daily using an in vivo imaging system. Figure 12 The in vivo release of collagen microneedles after application to the back of mice is shown. Figure 13 , Figure 14 The results of biocompatibility and cell migration experiments are shown, indicating that the collagen-related materials have no obvious toxicity, good biocompatibility, and can effectively promote cell migration.

[0075] Example 7: In vivo antibacterial experiment

[0076] The specific experimental steps are as follows:

[0077] A bacterial infection wound model of diabetes in mice was established: Forty-two female 6-week-old ICR mice were fasted for 12 hours and then injected intraperitoneally with streptozotocin solution (dissolved in 0.1 mol / L citrate buffer, pH 4.2) at a dose of 50 mg / kg body weight for 5 consecutive days to induce a diabetes model. 72 hours after the last injection, blood glucose was measured by tail vein. Mice with a blood glucose level ≥16.7 mmol / L for 3 consecutive days were considered to have successfully developed a diabetes model.

[0078] Constructing a Pseudomonas aeruginosa infection wound model: Diabetic mice were anesthetized, and their backs were shaved (approximately 2cm × 3cm in area). The skin was disinfected with 75% ethanol. A full-thickness skin defect (reaching the fascia layer, with epidermis and dermis removed) was prepared in the center of the back using a 6mm diameter sterile biopsy punch. Immediately after hemostasis, 50μL of Pseudomonas aeruginosa bacterial suspension (concentration 1×10⁻⁶) was evenly dripped into the wound. 7 -1×10 8CFU / 10μL was collected and allowed to be absorbed. Mice were randomly divided into 6 groups: ①PBS group ②Blank MN group ③MPDAMN group ④LysAB2@MPDA MN group ⑤MPDA MN+NIR group ⑥LysAB2@MPDA MN+NIR group. Purulent discharge from the wounds of mice after modeling confirmed successful modeling.

[0079] (3) Animal grouping

[0080] ①PBS group: 200 μL PBS was administered to the back.

[0081] ② Blank MN group, back massage for 15 minutes.

[0082] ③MPDA MN group, back pressure for 15 minutes.

[0083] ④LysAB2@MPDAMN group, back pressure for 15 minutes.

[0084] ⑤MPDAMN+NIR group: 15 min of back pressure + 5 min of 808nm laser irradiation.

[0085] ⑥LysAB2@MPDA MN+NIR group, back pressure for 15 minutes + 808nm laser irradiation for 5 minutes.

[0086] (4) Administer medication starting on day 0 after successful model establishment, with a three-day interval between doses. Take photos daily to record the condition of the wounds on the backs of the mice.

[0087] Observations showed that, with prolonged treatment, the LysAB2@MPDA MN+NIR group had the smallest wounds on day 4 after the start of treatment and gradually recovered, followed by the MPDA MN+NIR group. Results are as follows... Figure 15 As shown, the wound healing speed promoted by the drug-loaded microneedle patch of this application is significantly greater than that of the control group.

[0088] Example 8: Biocompatibility Experiment

[0089] The CCK-8 assay was used to investigate the toxicity of collagen hydrogels to L929 cells, thereby evaluating the biocompatibility of the collagen hydrogels. First, cells were seeded in 96-well microplates (10 cells per well). 4 Cells were cultured overnight. 2 g of LysAB2@MPDA-coated collagen hydrogel was added to 2 mL of DMEM medium and extracted overnight. The extract was then co-incubated with the overnight cultured cells. After 24 h of incubation, cell viability was assessed by measuring absorbance at 490 nm using a microplate reader to evaluate the biocompatibility of the collagen hydrogel. Results are shown below. Figure 13 The results showed that collagen hydrogels do not produce toxicity to cells.

[0090] Example 9: Cell Migration Experiment

[0091] First, draw three horizontal lines along a ruler on the bottom of the six-well plate as marker lines. Then, divide the plate into six groups: Control, Blank MN, MPDA MN, LysAB2@MPDA MN, MPDA MN+NIR, and LysAB2@MPDA MN+NIR. Add 2 mL of pre-prepared cell suspension to each well, with a cell density of 6 × 10⁻⁶ cells / well. 6 Divide the cells into approximately 100 cells per well and spread them evenly. Once the cells have reached confluence, using a ruler as a guide, draw two perpendicular lines with a 200µL pipette tip perpendicular to the marked lines on the well plate. The scratches should intersect the marked lines, creating several intersection points to serve as fixed detection points. Discard the old culture medium and gently wash the cells two to three times with PBS until all cells are removed. Add either drug-treated or serum-free culture medium according to the cell group. After scratching, washing, and adding culture medium, take photographs at different magnifications using a microscope as a 0h control. Incubate at 37°C in a 5% CO2 incubator. At the desired time points, remove the cells and observe the scratch width at the same location under a microscope, taking photographs as well. Finally, analyze the migration area using ImageJ software. Figure 16 The results of H&E and Masson staining of mouse dorsal skin are shown, reflecting the skin tissue healing status, collagen fiber distribution, and repair effect. Figure 17 The results of immunofluorescence on mouse dorsal skin were shown, and CD31, CD206, iNOS and Ki67 were detected. These results can reflect the formation of new blood vessels, inflammation regulation and cell proliferation in the skin, providing molecular evidence for the mechanism of collagen microneedles in promoting skin repair.

[0092] Compared with existing technologies, the pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles, its preparation method, and its application provided in this application have the following advantages:

[0093] (1) The pH-responsive collagen hydrogel contained in this application is formed by cross-linking Schiff bases. The Schiff bases are broken by the acidic pH environment at the chronic wound site of the skin, thereby releasing the encapsulated nanoparticles into the wound and providing a new way for wound healing.

[0094] (2) This application uses 808nm near-infrared light to trigger the release of mesoporous polydopamine lyase loaded with LysAB2, which has high targeting and controllability. Compared with traditional antibiotic treatment, it can avoid the problem of bacterial resistance caused by systemic drug administration, accurately act on bacteria, significantly improve bactericidal efficiency, and reduce toxic side effects on normal tissues.

[0095] (3) The microneedle patch of this application is simple to prepare and low in cost, which lays the foundation for large-scale preparation.

[0096] (4) This application combines antibacterial and healing-promoting functions. While the lysin LysAB2 specifically kills bacteria, the hydrogel component promotes wound tissue regeneration. The multi-effect synergistic effect can more effectively treat chronic bacterial wounds in diabetic patients, shorten the wound healing cycle, and improve patient prognosis compared to single-function treatments.

[0097] Based on the above technical solutions, this application provides a pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles, its preparation method, and its application. The microneedle patch is prepared by loading antibacterial nanoparticles, aldehyde-modified F127, and recombinant type III collagen COL3 onto a polymer matrix. When this microneedle patch is applied to chronic wounds in diabetic patients, the microneedles penetrate the skin in a minimally invasive manner, directly reaching the wound lesion area. Under 808nm near-infrared light irradiation, mesoporous polydopamine absorbs light energy, prompting the rapid release of the loaded lysin LysAB2, which specifically recognizes and lyses bacterial cell walls, efficiently killing bacteria. Simultaneously, the collagen hydrogel constructs a suitable microenvironment at the wound site, promoting cell adhesion, proliferation, and migration, accelerating the wound healing process. Compared with traditional treatments for chronic bacterial infections in diabetic patients, the microneedle patch of this application provides a highly efficient and safe new strategy for treating chronic bacterial infections in diabetic patients.

[0098] Those skilled in the art will understand that the above-described embodiments are specific examples of implementing this application, and in practical applications, various changes in form and detail may be made without departing from the spirit and scope of this application. Any person skilled in the art can make their own modifications and alterations without departing from the spirit and scope of this application; therefore, the scope of protection of this application should be determined by the scope defined in the claims.

Claims

1. A pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles, characterized in that, The microneedle patch is prepared by loading antibacterial nanoparticles, aldehyde-modified F127, and recombinant type III collagen COL3 onto a polymer matrix.

2. The pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles according to claim 1, characterized in that, The antibacterial nanoparticles are mesoporous polydopamine composite nanoparticles LysAB2@MPDA loaded with the lysin LysAB2.

3. The pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles according to claim 1, characterized in that, The aldehyde-modified F127 was prepared by Dess-Martin high-iodine reagent oxidation method, which oxidized the primary alcohol at the end of Pluronic F127 to an aldehyde group.

4. The pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles according to claim 1, characterized in that, The polymer matrix is ​​an aqueous solution of polyvinyl alcohol; The polymer matrix is ​​prepared by dissolving polyvinyl alcohol in deionized water to prepare a 20% PVA solution, which is then stored at room temperature.

5. A method for preparing a pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles, characterized in that, Includes the following steps: Step 1: Prepare a hydrogel crosslinked with aldehyde-modified F127 and recombinant type III collagen COL3; Step 2: Prepare antibacterial nanoparticles and mix them with a hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3 to obtain a drug-loaded hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3. Step 3: Place the drug-loaded aldehyde-modified F127 and recombinant type III collagen COL3 crosslinked hydrogel on the microneedle PDMS mold, press it into the needle hole of the mold as the microneedle tip, centrifuge the mold and vacuum dry it to obtain the mold loaded with microneedle patch. Step 4: Pour the polymer matrix onto the mold containing the microneedle patch. After drying, peel the microneedle patch off the mold to obtain the microneedle patch.

6. The method for preparing pH-responsive collagen microneedle patches loaded with antibacterial nanoparticles according to claim 5, characterized in that, In step one, a hydrogel crosslinked with aldehyde-modified F127 and recombinant type III collagen COL3 is prepared, including: Weigh out aldehyde-modified F127, add deionized water to dissolve it, and obtain aldehyde-modified F127 solution; Weigh out recombinant type III collagen COL3, add deionized water to dissolve it, and obtain a recombinant type III collagen solution; The aldehyde-modified F127 solution and the recombinant type III collagen solution were mixed and allowed to stand at 37°C for 24 hours to obtain a hydrogel crosslinked with aldehyde-modified F127 and recombinant type III collagen COL3.

7. The method for preparing pH-responsive collagen microneedle patches loaded with antibacterial nanoparticles according to claim 5, characterized in that, In step two, mesoporous polydopamine loaded with LysAB2 is dispersed in a solvent to form a uniform dispersion, and then added to a hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3. The mixture is stirred thoroughly to obtain a drug-loaded hydrogel cross-linked with aldehyde-modified F127 and recombinant type III collagen COL3.

8. The method for preparing pH-responsive collagen microneedle patches loaded with antibacterial nanoparticles according to claim 5, characterized in that, In step three, the drug-loaded aldehyde-modified F127 and recombinant type III collagen COL3 crosslinked hydrogel is pressed into the needle hole of the mold as the microneedle tip using a medicine spoon. The mold is then centrifuged at 2200 rpm for 15 minutes. The pressed mold is then placed in a vacuum drying oven and vacuumed three times to obtain a mold containing microneedle patches.

9. The method for preparing pH-responsive collagen microneedle patches loaded with antibacterial nanoparticles according to claim 5, characterized in that, In step four, polyvinyl alcohol is dissolved in deionized water to prepare a 20% PVA solution; then the 20% PVA solution is poured onto a mold containing microneedle patches, the mold is placed in a ventilated place to dry for one day, and then the microneedle patches are peeled off from the mold to obtain the microneedle patches.

10. The use of a pH-responsive collagen microneedle patch loaded with antibacterial nanoparticles as described in claim 1 in the preparation of a patch formulation for bacterial-infected wounds.

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