Collagen microneedle patches loaded with copper-based antibacterial nanoparticles, their preparation methods and applications

Abstract

This application relates to the field of hydrogel microneedle preparation technology, and particularly to a collagen microneedle patch loaded with copper-based antibacterial nanoparticles, its preparation method, and its application. The microneedle patch is prepared from copper-based antibacterial nanoparticles, recombinant type XVII collagen, and sodium alginate. The copper-based antibacterial nanoparticles are Cu-CpG@PDA composite nanoparticles; the recombinant type XVII collagen is rCol XVII. The microneedle patch is prepared by first preparing Cu-CpG@PDA nanoparticles using antibacterial copper ions, the nucleic acid adjuvant CpG oligonucleotide, and polydopamine (PDA); then, the Cu-CpG@PDA nanoparticles are doped into recombinant type XVII collagen (rCol XVII) and sodium alginate to prepare a collagen hydrogel loaded with Cu-CpG@PDA nanoparticles; finally, the hydrogel is laid in a mold, and after vacuum treatment, centrifugation, and drying, the collagen microneedle patch loaded with copper-based antibacterial nanoparticles is obtained. The collagen microneedle patch of this application has good photothermal conversion ability, antibacterial properties and skin regeneration ability, and can be used to treat bacterial infections and promote wound closure.

Description

Technical Field

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

[0002] Since the introduction of antibiotics in the 1930s, the threat of bacterial infections has significantly decreased. However, multidrug resistance (MDR) due to antibiotic overuse is on the rise. In this context, there is an urgent need to develop innovative non-antibiotic antibacterial methods. In recent years, with advancements in nanotechnology, metal-based nanoparticles such as gold, silver, copper (copper oxide), and zinc oxide have been widely used in the treatment of wounds infected with multidrug-resistant bacteria, achieving significant therapeutic effects. Among these, copper-based nanoparticles possess excellent bactericidal activity, good biocompatibility, and biosafety, and are widely used in dental antibacterial coatings and viral disinfection.

[0003] While nanoparticles of antibacterial drugs have demonstrated excellent antibacterial effects, their subcutaneous penetration remains a significant hurdle to overcome. Microneedles (MNs), as a promising drug delivery platform, enable effective transdermal drug transport through minimally invasive skin lesions. However, for applications requiring prolonged or sustained drug release, existing microneedle technologies still face limitations in controlling drug release rates and durations. Currently, the types of materials used to construct microneedle patches are limited, and these materials still have limitations in terms of biocompatibility, degradability, and functionality. Consequently, some microneedle systems may fail to achieve uniform and stable drug release, impacting therapeutic efficacy. Summary of the Invention

[0004] This application provides a collagen microneedle patch loaded with copper-based antibacterial nanoparticles, its preparation method, and its application. In this application, copper-based antibacterial nanoparticles (Cu-CpG@PDA) are doped into recombinant type XVII collagen hydrogel to obtain Cu-CpG@PDA loaded with Cu-CpG@PDA. Finally, the collagen microneedle patch loaded with copper-based antibacterial nanoparticles is prepared using a mold. The microneedle patch of this application exhibits good photothermal conversion ability, antibacterial properties, and skin regeneration ability, and can be used to treat bacterial infections and promote wound closure.

[0005] To address the aforementioned technical problems, in a first aspect, embodiments of this application provide a collagen microneedle patch loaded with copper-based antibacterial nanoparticles. The microneedle patch is prepared from copper-based antibacterial nanoparticles, recombinant type XVII collagen, and sodium alginate; wherein the copper-based antibacterial nanoparticles are Cu-CpG@PDA composite nanoparticles; and the recombinant type XVII collagen is rCol XVII.

[0006] In some exemplary embodiments, the mass ratio of recombinant type XVII collagen to sodium alginate is 7:3; the concentration range of Cu-CpG@PDA composite nanoparticles in the recombinant type XVII collagen and sodium alginate system is 0 mM to 20 mM.

[0007] In some exemplary embodiments, the microneedle patch includes a 30×30 array of circular needles; each circular needle has a bottom diameter of 300 μm and a height of 600 μm.

[0008] In some exemplary embodiments, the preparation method of copper-based antibacterial nanoparticles is as follows: firstly, Cu-CpG composite nanoparticles are prepared by co-assembly of divalent copper ions and nucleic acid immunoadjuvant CpG oligonucleotides through coordination and electrostatic interaction; then, polydopamine PDA is coated on the surface of Cu-CpG composite nanoparticles by in-situ polymerization to prepare Cu-CpG@PDA composite nanoparticles.

[0009] Secondly, this application also provides a method for preparing a collagen microneedle patch loaded with copper-based antibacterial nanoparticles, comprising the following steps: Step 1, dissolving recombinant type XVII collagen in a solvent to obtain a recombinant type XVII collagen solution, dispersing copper-based antibacterial nanoparticles into the recombinant type XVII collagen solution, mixing and stirring for 2h to 6h to obtain dissolved collagen; wherein, the copper-based antibacterial nanoparticles are Cu-CpG@PDA composite nanoparticles; the recombinant type XVII collagen is rColXVII; Step 2, adding sodium alginate to the dissolved collagen and stirring evenly, centrifuging to obtain a hydrogel; Step 3, spreading the hydrogel into a mold, pressing it with a scraper, vacuuming or centrifuging to ensure the gel fully fills the needle tip, scraping off excess mother liquor with a scraper, and drying in a fume hood for 1h to 4h; Step 4, adding polyvinyl alcohol solution to the mold to form a microneedle substrate, and air-drying at room temperature to obtain a microneedle patch.

[0010] In some exemplary embodiments, in step one, the solvent is a phosphate buffer solution with pH=7, the dispersion concentration of rCol XVII is 112 mg / mL to 224 mg / mL, and the dispersion concentration of Cu-CpG@PDA composite nanoparticles is 0 mM to 20 mM.

[0011] In some exemplary embodiments, in step two, the dispersion concentration of sodium alginate is 24 mg / mL to 72 mg / mL.

[0012] In some exemplary embodiments, in step three, the centrifugation conditions for the hydrogel are: a rotation speed of 5000 rpm to 8000 rpm and a time of 2 min to 5 min, until all air bubbles in the gel are removed.

[0013] In some exemplary embodiments, in step four, the concentration percentage of the polyvinyl alcohol solution is 15% w / v to 20% w / v.

[0014] Furthermore, this application also provides an application of a collagen microneedle patch loaded with copper-based antibacterial nanoparticles as described in the above embodiments in the preparation of a patch formulation for bacterial infected wounds.

[0015] The technical solution provided in this application has at least the following advantages:

[0016] This application provides a collagen microneedle patch loaded with copper-based antibacterial nanoparticles, its preparation method, and its application. The microneedle patch is prepared from copper-based antibacterial nanoparticles, recombinant type XVII collagen, and sodium alginate. The copper-based antibacterial nanoparticles are Cu-CpG@PDA composite nanoparticles; the recombinant type XVII collagen is rCol XVII. This application prepares copper-based antibacterial nanoparticles Cu-CpG@PDA using a multi-level assembly technique, then dops Cu-CpG@PDA into a recombinant type XVII collagen hydrogel to obtain Cu-CpG@PDA loaded with Cu-CpG@PDA. Finally, a collagen microneedle patch loaded with copper-based antibacterial nanoparticles is prepared using a mold. The Cu-CpG@PDA nanoparticles provide antibacterial properties, the recombinant type XVII collagen promotes wound healing, and sodium alginate acts as a stabilizer and thickener to improve sample stability and facilitate gelation. The prepared antibacterial nanoparticle collagen microneedles have good biocompatibility, photothermal conversion effect, antibacterial properties and skin regeneration ability, and can be used for antibacterial treatment of bacterial wounds and skin healing. Attached Figure Description

[0017] 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.

[0018] Figure 1 Scanning electron microscope (SEM) images of blank MN and Cu-CpG@PDA rCol-MN prepared in Examples 1 and 2.

[0019] Figure 2 This is a super depth-of-field microscope image of Cu-CpG@PDA rCol-MN prepared in Example 2.

[0020] Figure 3A The image shows a transmission electron microscope (TEM) image of the copper-based composite nanoparticles prepared in Example 2.

[0021] Figure 3B The image shows the size distribution of the copper-based composite nanoparticles prepared in Example 2.

[0022] Figure 4A schematic diagram of the mechanical properties of the blank MN gel and the rCol XVII hydrogel loaded with Cu-CpG@PDA NP prepared in Example 3.

[0023] Figure 5 This is a schematic diagram of the in vivo degradation performance of blank MN and Cu-CpG@PDA rCol-MN prepared in Example 4.

[0024] Figure 6 The images and quantitative data of Staphylococcus aureus and Escherichia coli incubated with different samples prepared in Examples 2 and 3 under conditions of no or near-infrared irradiation are schematic diagrams.

[0025] Figure 7 This is a schematic diagram illustrating the cytotoxicity of different gels prepared in Example 6 on L929 cells.

[0026] Figure 8 The gelation of samples prepared in different proportions in Example 7.

[0027] Figure 9 The effects of copper-based composite nanoparticles prepared in Example 2 and rCol XVII hydrogel loaded with Cu-CpG@PDA on the migration of L929 cells were investigated. Detailed Implementation

[0028] As can be seen from the background technology, for situations requiring long-term or continuous drug release, existing microneedle technology still has certain limitations in controlling the drug release rate and release time.

[0029] Currently, the types of materials used to construct microneedle patches are limited, and these materials still have certain limitations in terms of biocompatibility, degradability, and functionality. Some microneedle systems may not be able to achieve uniform and stable drug release, affecting therapeutic effects. Collagen has been widely used in the field of biomaterials due to its excellent biological functions (promoting cell adhesion and proliferation), biocompatibility, low immunogenicity, rapid hemostasis, and biodegradability. In particular, type XVII collagen (Col XVII), as a transmembrane protein, constitutes hemiliposomes, mediates the interaction between stem cells and surrounding cells and matrix, and regulates skin homeostasis, aging, and wound repair. Compared with other types of collagen, the multiple repeating collagen and non-collagenous domains of Col XVII give it greater flexibility and diversity. It has special intermolecular interactions, such as intramolecular chain structure and intermolecular crosslinks, thereby enhancing the stability and mechanical strength of the extracellular matrix. In addition, Col XVII has unique cell adhesion sites and signal transduction functions, which can regulate cell migration, proliferation, and differentiation. Therefore, this application uses recombinant type XVII collagen (rCol XVII) to construct hydrogel microneedle patches for loading copper-based antibacterial nanoparticles and treating skin infection wounds.

[0030] To address the aforementioned technical problems, this application provides a collagen microneedle patch loaded with copper-based antibacterial nanoparticles, its preparation method, and its application. The microneedle patch is prepared from copper-based antibacterial nanoparticles, recombinant type XVII collagen, and sodium alginate; wherein the copper-based antibacterial nanoparticles are Cu-CpG@PDA composite nanoparticles; and the recombinant type XVII collagen is rCol XVII. This application provides a collagen microneedle patch loaded with copper-based antibacterial nanoparticles, its preparation method, and its application. In this application, copper-based antibacterial nanoparticles (Cu-CpG@PDA) are doped into a recombinant type XVII collagen hydrogel to obtain Cu-CpG@PDA loaded with Cu-CpG@PDA. Finally, a collagen microneedle patch loaded with copper-based antibacterial nanoparticles is prepared using a mold. The microneedle patch of this application exhibits good photothermal conversion ability, antibacterial properties, and skin regeneration ability, and can be used to treat bacterial infections and promote wound closure.

[0031] 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.

[0032] See Figure 1This application provides a collagen microneedle patch loaded with copper-based antibacterial nanoparticles. The microneedle patch is prepared from copper-based antibacterial nanoparticles, recombinant type XVII collagen, and sodium alginate. The copper-based antibacterial nanoparticles are Cu-CpG@PDA composite nanoparticles, and the recombinant type XVII collagen is rCol XVII.

[0033] The microneedle patch prepared in this application is composed of Cu-CpG@PDA composite nanoparticles, recombinant type XVII collagen (rColXVII), and sodium alginate (SA). Using divalent copper ions (Cu(II)), the nucleic acid adjuvant CpG oligonucleotide, and dopamine (DA) as raw materials, Cu-CpG@PDA antibacterial nanoparticles were prepared through DNA assembly and in-situ surface polymerization. Then, the Cu-CpG@PDA nanoparticles were doped into a hydrogel of recombinant type XVII collagen (rCol XVII) and sodium alginate (SA) to prepare a collagen hydrogel loaded with Cu-CpG@PDA nanoparticles. Finally, the mixed hydrogel was laid in a polydimethylsiloxane (PDMS) mold, and after vacuum treatment, centrifugation, and drying, a collagen microneedle patch (Cu-CpG@PDA rCol-MN) loaded with copper-based antibacterial nanoparticles was obtained.

[0034] The copper-loaded antibacterial nanoparticle collagen microneedle patch prepared in this application has an inner Cu(II) layer that can penetrate bacterial cell membranes, cause oxidative damage, and hinder bacterial metabolism, thereby bringing about antibacterial effects; CpG can bind to Toll-like receptors (TLR9) and activate innate immune responses; polydopamine (PDA) has structural stabilizing effects and photothermal conversion properties, and generates heat through near-infrared light irradiation to enhance the antibacterial effect; rCol XVII, as a transmembrane protein, can form hemiliposomes, mediating the interaction between stem cells and surrounding cells and matrix, and providing functions in regulating skin homeostasis, aging, and wound repair; sodium alginate acts as a stabilizer and thickener.

[0035] In some embodiments, the mass ratio of recombinant type XVII collagen to sodium alginate is 7:3; the concentration range of Cu-CpG@PDA composite nanoparticles in the recombinant type XVII collagen and sodium alginate system is 0 mM to 20 mM.

[0036] In some embodiments, the microneedle patch includes a 30×30 array of circular needles; each circular needle has a bottom diameter of 300 μm and a height of 600 μm.

[0037] In some embodiments, the preparation method of copper-based antibacterial nanoparticles is as follows: firstly, Cu-CpG composite nanoparticles are prepared by co-assembly of divalent copper ions and nucleic acid immunoadjuvant CpG oligonucleotides through coordination and electrostatic interaction; then, polydopamine PDA is coated on the surface of Cu-CpG composite nanoparticles by in-situ polymerization to prepare Cu-CpG@PDA composite nanoparticles.

[0038] In addition, this application also provides a method for preparing a collagen microneedle patch loaded with copper-based antibacterial nanoparticles, comprising the following steps:

[0039] Step 1: Dissolve recombinant type XVII collagen in a solvent to obtain a recombinant type XVII collagen solution. Disperse copper-based antibacterial nanoparticles into the recombinant type XVII collagen solution, mix and stir for 2 to 6 hours to obtain dissolved collagen. Among them, the copper-based antibacterial nanoparticles are Cu-CpG@PDA composite nanoparticles; the recombinant type XVII collagen is rCol XVII.

[0040] Step 2: Add sodium alginate to the dissolved collagen and stir until homogeneous. Centrifuge to obtain a hydrogel.

[0041] Step 3: Pour the hydrogel into the mold, press it with a scraper, vacuum or centrifuge to ensure the gel fully fills the needle tip, scrape off the excess mother liquor with a scraper, and dry in a fume hood for 1 to 4 hours.

[0042] Step 4: Add polyvinyl alcohol (PVA) solution to the mold to form a microneedle (MN) substrate, and let it air dry at room temperature to obtain a microneedle patch.

[0043] In some embodiments, in step one, the solvent is a phosphate buffered saline (PBS) solution with pH=7, the dispersion concentration of rCol XVII is 112 mg / mL to 224 mg / mL, and the dispersion concentration of Cu-CpG@PDA composite nanoparticles is 0 mM to 20 mM.

[0044] In some embodiments, in step two, the dispersion concentration of sodium alginate (SA) is 24 mg / mL to 72 mg / mL. Specifically, 24 mg to 72 mg of sodium alginate is weighed, added to rCol XVII solution, stirred until homogeneous, and then centrifuged.

[0045] In some embodiments, in step three, the centrifugation conditions for the hydrogel are: a rotation speed of 5000 rpm to 8000 rpm and a time of 2 min to 5 min, until all air bubbles in the gel are removed. The hydrogel is then laid into a polydimethylsiloxane (PDMS) mold, and after vacuuming, centrifugation, and drying, a collagen microneedle patch (Cu-CpG@PDA rCol-MN) loaded with copper-based antibacterial nanoparticles is obtained.

[0046] In some embodiments, in step four, the concentration percentage of the polyvinyl alcohol solution is 15% w / v to 20% w / v.

[0047] Furthermore, this application also provides an application of the collagen microneedle patch loaded with copper-based antibacterial nanoparticles as described in the above embodiments in the preparation of patch formulations for bacterial infected wounds. The collagen microneedle patch (Cu-CpG@PDA rCol-MN) prepared in this application is used to prepare patch formulations for bacterial infected wounds. The Cu-CpG@PDA rCol-MN of this application has good photothermal conversion ability, antibacterial properties, and skin regeneration ability, and can be used to treat bacterial infections and promote wound closure.

[0048] Example 1: Preparation of Blank MN

[0049] Weigh 112 mg of recombinant type XVII collagen and dissolve it in 1 mL of pH 7.0 PBS by sonication. Weigh 48 mg of sodium alginate (SA) and add it to the dissolved collagen, stirring until homogeneous to obtain a hydrogel. Centrifuge (8000 rpm, 2 min) to remove air bubbles and form rCol XVII gel. Place the gel into a mold (30*30 array, outer diameter 30*30 mm, thickness 8 mm). Press the gel with a spatula, vacuum-seal to ensure the gel fully fills the needle tip, scrape off excess mother liquor, and dry in a fume hood for 1 h. Add 2 mL of 20% PVA solution to the mold and air-dry at room temperature to obtain blank MN (Blank MN). Figure 1 As shown.

[0050] Example 2: Preparation of Cu-CpG@PDArCol-MN

[0051] (1) Mix 45 μL CuCl2 (200 mM), 40 μL CpG (200 μM), and 15 μL Tris buffer + Mg 2+ Solution (Tris buffer concentration 20mM, Mg) 2+ Cu-CpG nanoparticles were prepared by mixing a 10 mM solution and heating the mixture at 95 °C for 2 h. The nanoparticles were then dispersed in a 0.2 mg / mL DA Tris buffer solution (10 mM, pH = 8.5), mixed, stirred in the dark for 3 h, centrifuged (12000 rpm, 15 min) with water, washed 3 times, and freeze-dried to obtain Cu-CpG@PDA nanoparticles.

[0052] (2) Weigh 112 mg of recombinant type XVII collagen, add Cu-CpG@PDA nanoparticles, and mix in 600 μL of pH 7.0 PBS. Stir in an ice bath for 4 h. Then weigh 48 mg of sodium alginate (SA), add it to the above solution, stir evenly, and centrifuge (8000 rpm, 2 min) to remove air bubbles from the gel, obtaining rCol XVII hydrogel loaded with Cu-CpG@PDA NP. Spread it into a mold, press it with a scraper, vacuum or centrifuge to fully fill the needle tip with gel, scrape off excess mother liquor with a scraper, and dry in a fume hood for 1 h. Finally, add 2 mL of 20% PVA solution to the mold and air dry at room temperature to obtain collagen microneedle patch loaded with Cu-CpG@PDA (Cu-CpG@PDA rCol-MN). Figure 2 As shown. This microneedle patch consists of a 30×30 array of circular needles, each with a base diameter of 300 μm and a height of 600 μm. A super-depth-of-field microscope image of the Cu-CpG@PDArCol-MN prepared in Example 2 is shown below. Figure 2 As shown, the transmission electron microscope (TEM) images and size distribution diagrams of the copper-based composite nanoparticles are respectively as follows: Figure 3A and Figure 3B As shown.

[0053] Example 3: Collagen hydrogel gelation ratio test

[0054] Different amounts of recombinant type XVII collagen and sodium alginate (total 160 mg) were weighed to achieve mass ratios of 9:1, 8:2, 7:3, 6:4, and 5:5. rCol XVII was first dissolved in 1 mL of pH 7.0 PBS by sonication, followed by the addition of the corresponding amount of sodium alginate and stirring until homogeneous. The mixture was then centrifuged (8000 rpm, 2 min) to remove air bubbles, and the gelation process was observed. Gel formation failed when the ratio of rCol XVII to sodium alginate was 9:1 or 8:2. Since a 7:3 mass ratio was chosen to prepare the rCol XVII hydrogel, as it was necessary to ensure the highest possible content of rCol XVII under gelation conditions. Figure 4 The mechanical properties of the blank MN gel and the rCol XVII hydrogel loaded with Cu-CpG@PDANP prepared in Example 3 are shown, where (a) represents the rheological properties and (b) represents the viscosity as a function of shear rate.

[0055] Example 4: Mechanical Property Testing of Microneedle Hydrogels. Blank microneedle gels and Col XVII hydrogels loaded with Cu-CpG@PDA at a concentration of 8 mM were prepared. The rheological properties of the hydrogels were evaluated using a rheometer. Different pressures (0, 150, 300, 450 g) were applied to the blank microneedles, and the changes in the microneedle tips were observed under a bright-field inverted fluorescence microscope.

[0056] Example 5: Degradation Energy Test of Microneedle Hydrogel

[0057] Blank MN and Cu-CpG@PDArCol-MN were pressed onto the backs of ICR mice and fixed with wooden clips for different times (0, 5, 10, 15, 20, 30 min). The degradation degree of the microneedle tips was then observed and photographed under a bright-field inverted fluorescence microscope. The in vivo degradation performance of blank MN and Cu-CpG@PDArCol-MN prepared in Example 4 is as follows: Figure 5 As shown.

[0058] Example 6 Antibacterial coating plate experiment

[0059] Staphylococcus aureus and Escherichia coli were cultured overnight in tryptic soy peptone broth (TSB and LB) on a shaker (37°C, 250 rpm), and centrifuged to obtain bacteria in the logarithmic growth phase. Bacterial concentration was monitored by measuring the OD600 nm value. When the OD600 value of the bacteria was diluted to 0.1, the concentration of Staphylococcus aureus was 2 × 10⁻⁶. 8 CFU / mL. Then, take 50 μL of a concentration of 2 × 10⁻⁶ CFU / mL. 8 A CFU / mL suspension of *S. aureus* was mixed with 50 μL of different samples (2.5 mM Cu-CpG@PDA and rCol XVII hydrogel loaded with 2.5 mM Cu-CpG@PDA) in a 1.5 mL centrifuge tube. Sterile PBS was used as the control group (the phototherapy group was irradiated with 0.75 W 808 nm near-infrared light for 3 min). The mixture was then incubated at 37°C on a shaker at 250 rpm for 1 h. After incubation, the mixture of each sample group and bacteria was diluted 20,000 times, and 100 μL was evenly spread onto the corresponding bacterial culture medium plates. Three replicates were prepared for each group. The plates were incubated at 37°C for 16 h, and colony counting was performed.

[0060] Figure 6 The images and quantitative data of Staphylococcus aureus and Escherichia coli prepared in Examples 2 and 3, respectively, were obtained by incubating them with or without near-infrared irradiation. (a) is a colony image; (b) and (c) are quantitative data.

[0061] Example 7 Cytotoxicity Experiment

[0062] Frozen mouse fibroblasts (L929) were removed and thawed in sterile water at 37°C by agitation. 5 mL of DMEM serum medium was then transferred to a centrifuge tube, and the thawed cells were added. The tube was centrifuged (1000 rpm, 5 min) to remove the supernatant. The cell pellet at the bottom was resuspended, and the cell suspension was placed in a cell culture dish containing 6–7 mL of DMEM serum medium. Cells were passaged until optimal viability was achieved, and then cytotoxicity assays were performed. The counted cells were seeded into 96-well plates (1 × 10⁶ cells / well). 4 Cells were cultured in 96-well plates (100 μL / well) and then incubated in a 37°C cell culture incubator (5% CO2) for 12 h. 2.5 mM Cu-CpG@PDA and Cu-CpG@PDAMN groups were prepared using DMEM as solvent, and 100 μL of sample was added to each well. DMEM culture medium alone served as a control. After 24 h of culture, the upper layer of sample solution in the 96-well plate was aspirated, and 120 μL of DMEM / MTT solution (DMEM:MTT = 1:6) was added. The 96-well plate was then placed in a cell culture incubator in the dark for 4 h. After staining, the upper MTT solution was removed, and 150 μL of DMSO solution was added to the 96-well plate. The plate was then shaken until formazan was completely and uniformly dissolved in the DMSO. The absorbance at 490 nm was measured using a microplate reader to determine cell viability. The cytotoxicity of different gels prepared in Example 6 to L929 cells was as follows: Figure 7 As shown.

[0063] Example 8 L929 cell migration experiment

[0064] At the bottom of the six-well plate, use a marker to draw three horizontal lines along a ruler as reference lines. Then, plant L929 cells at a density of 6 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells. Once the cells had grown to confluence, two vertical lines were drawn in each well using a ruler and pipette tip, intersecting the reference line to form a fixed observation point. The old culture medium was discarded, and the wells were gently rinsed 2-3 times with PBS until all scraped cells were removed. Serum-free culture medium containing Cu-CpG@PDA nanoparticles or Cu-CpG@PDA-loaded rColXVII hydrogel was added, and the culture plate was incubated at 37°C and 5% CO2. Cells were observed under a microscope at the same scratch location at 0, 12, 24, and 48 hours, and photographs were taken to monitor the change in scratch width over time. The gelation of samples prepared in Example 7 at different proportions is shown below. Figure 8 As shown. The migration-promoting effect of the copper-based composite nanoparticles prepared in Example 2 and the rCol XVII hydrogel loaded with Cu-CpG@PDA on L929 cells is shown in the figure. Figure 9 As shown.

[0065] Based on the above technical solutions, this application provides a collagen microneedle patch loaded with copper-based antibacterial nanoparticles, its preparation method, and its application. The microneedle patch is prepared from copper-based antibacterial nanoparticles, recombinant type XVII collagen, and sodium alginate; wherein the copper-based antibacterial nanoparticles are Cu-CpG@PDA composite nanoparticles; and the recombinant type XVII collagen is rColXVII. This application prepares copper-based antibacterial nanoparticles Cu-CpG@PDA using a multi-level assembly technique, then dops Cu-CpG@PDA into a recombinant type XVII collagen hydrogel to obtain Cu-CpG@PDA loaded with Cu-CpG@PDA. Finally, a collagen microneedle patch loaded with copper-based antibacterial nanoparticles is prepared using a mold. The Cu-CpG@PDA nanoparticles provide antibacterial properties, the recombinant type XVII collagen promotes wound healing, and sodium alginate acts as a stabilizer and thickener to improve sample stability and facilitate gelation. The prepared antibacterial nanoparticle collagen microneedles have good biocompatibility, photothermal conversion effect, antibacterial properties and skin regeneration ability, and can be used for antibacterial treatment of bacterial wounds and skin healing.

[0066] 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 collagen microneedle patch loaded with copper-based antibacterial nanoparticles, characterized in that, The microneedle patch is prepared from copper-based antibacterial nanoparticles, recombinant type XVII collagen, and sodium alginate; wherein... The copper-based antibacterial nanoparticles are Cu-CpG@PDA composite nanoparticles; The recombinant type XVII collagen is rCol XVII; The preparation method of the copper-based antibacterial nanoparticles is as follows: First, Cu-CpG composite nanoparticles are prepared by co-assembly of divalent copper ions and nucleic acid immunoadjuvant CpG oligonucleotides through coordination and electrostatic interaction; then, polydopamine PDA is coated on the surface of Cu-CpG composite nanoparticles by in-situ polymerization to prepare Cu-CpG@PDA composite nanoparticles.

2. The collagen microneedle patch loaded with copper-based antibacterial nanoparticles according to claim 1, characterized in that, The mass ratio of recombinant type XVII collagen to sodium alginate is 7:

3.

3. The collagen microneedle patch loaded with copper-based antibacterial nanoparticles according to claim 1, characterized in that, The microneedle patch comprises a 30×30 array of circular needles; each circular needle has a base diameter of 300 µm and a height of 600 µm.

4. A method for preparing a collagen microneedle patch loaded with copper-based antibacterial nanoparticles, characterized in that, Includes the following steps: Step 1: Dissolve recombinant type XVII collagen in a solvent to obtain a recombinant type XVII collagen solution. Disperse copper-based antibacterial nanoparticles into the recombinant type XVII collagen solution, mix and stir for 2 h to 6 h to obtain dissolved collagen. The copper-based antibacterial nanoparticles are Cu-CpG@PDA composite nanoparticles; the recombinant type XVII collagen is rCol XVII. The preparation method of the copper-based antibacterial nanoparticles is as follows: First, Cu-CpG composite nanoparticles are prepared by co-assembly of divalent copper ions and the nucleic acid adjuvant CpG oligonucleotides through coordination and electrostatic interactions. Then, polydopamine PDA is coated onto the surface of the Cu-CpG composite nanoparticles through in-situ polymerization to prepare Cu-CpG@PDA composite nanoparticles. Step 2: Add sodium alginate to the dissolved collagen obtained in Step 1, stir well, and centrifuge to obtain hydrogel; Step 3: Pour the hydrogel obtained in Step 2 into the mold, press it with a scraper, vacuum or centrifuge it to make the gel fully fill the needle tip, scrape off the excess mother liquor with a scraper, and dry it in a fume hood for 1 h to 4 h. Step 4: Add polyvinyl alcohol solution to the mold obtained in Step 3 to form a microneedle substrate, and let it air dry at room temperature to obtain a microneedle patch.

5. The method for preparing collagen microneedle patches loaded with copper-based antibacterial nanoparticles according to claim 4, characterized in that, In step one, the solvent is a phosphate buffer solution with pH=7, and the dispersion concentration of rCol XVII is 112 mg / mL ~224 mg / mL.

6. The method for preparing collagen microneedle patches loaded with copper-based antibacterial nanoparticles according to claim 4, characterized in that, In step two, the concentration of sodium alginate is 24 mg / mL to 72 mg / mL.

7. The method for preparing collagen microneedle patches loaded with copper-based antibacterial nanoparticles according to claim 4, characterized in that, In step three, the centrifugation conditions for the hydrogel are: 5000 rpm to 8000 rpm and 2 min to 5 min, until all air bubbles in the gel are removed.

8. The method for preparing collagen microneedle patches loaded with copper-based antibacterial nanoparticles according to claim 4, characterized in that, In step four, the concentration percentage of the polyvinyl alcohol solution is 15% w / v ~ 20% w / v.

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

Images